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UBE2C Induces Cisplatin Resistance via ZEB1/2-Dependent Upregulation of ABCG2 and ERCC1 in NSCLC Cells

UBE2C Induces Cisplatin Resistance via ZEB1/2-Dependent Upregulation of ABCG2 and ERCC1 in NSCLC... Hindawi Journal of Oncology Volume 2019, Article ID 8607859, 15 pages https://doi.org/10.1155/2019/8607859 Research Article UBE2C Induces Cisplatin Resistance via ZEB1/2-Dependent Upregulation of ABCG2 and ERCC1 in NSCLC Cells 1 2 3 1 2 4 Yan Wu, Dan Jin, Xiaohong Wang, Jing Du, Weihua Di, Jiajia An, 2 1 Cuijie Shao, and Jiwei Guo Cancer Research Institute, Binzhou Medical University Hospital, Binzhou 256603, China Department of Pain Medicine, Binzhou Medical University Hospital, Binzhou 256603, China Department of yTh roid and Breast Surgery, Binzhou Medical University Hospital, Binzhou 256603, China Department of Clinical Laboratory, Binzhou Medical University Hospital, Binzhou 256603, China Correspondence should be addressed to Jiwei Guo; guojiwei0510@163.com Received 5 July 2018; Revised 19 October 2018; Accepted 19 November 2018; Published 1 January 2019 Academic Editor: Reza Izadpanah Copyright © 2019 Yan Wu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objectives. Cisplatin (DDP) is one of the most commonly used chemotherapeutic drugs for several cancers, including non-small- cell lung cancer (NSCLC). However, resistance to DDP eventually develops, limiting its further application. New therapy targets are urgently needed to reverse DDP resistance. Methods. eTh mRNA expression of UBE2C, ZEB1/2, ABCG2,and ERCC1 was analyzed by reverse transcription-polymerase chain reaction. The protein levels of these molecules were analyzed by Western blotting and immunouo fl rescent staining. Cell proliferation was detected by CCK8 and MTT assays. Cell migration and invasion were analyzed by wound healing assay and Transwell assays. Promoter activities and gene transcription were analyzed by luciferase reporter assay. Results. In this study, we examined the effect of UBE2C and ZEB1/2 expression levels in DDP-resistant cells of NSCLC. We confirmed that aberrant expression of UBE2C and ZEB1/2 plays a critical role in repressing the DDP sensitivity to NSCLC cells. Additionally, knockdown of UBE2C significantly sensitize d resistant cells to DDP by repressing the expression of ZEB1/2. Mechanistic investigations indicated that UBE2C transcriptionally regulated ZEB1/2 by accelerating promoter activity. This study revealed that ZEB1/2 promotes the epithelial mesenchymal transition and expression of ABCG2 and ERCC1 to participate in UBE2C-mediated NSCLC DDP-resistant cell progression, metastasis, and invasion. Conclusion.UBE2C maybeanovel therapy target for NSCLC for sensitizing cells to the chemotherapeutic agent DDP. 1. Introduction UBE2C, also known as UBCH10, is an important mem- ber of the ubiquitin-conjugating enzyme family. UBE2C Lung cancer is very common and one of the leading causes of specifically interacts with the anaphase-promoting com- cancer mortality worldwide [1, 2]. Lung cancer is divided into plex/cyclostome (APC/C). er Th e are more than 55 substrates two histopathological groups: small-cell lung cancer (SCLC) degraded by APC/C, including 37 substrates involved in and non-small cell lung cancer (NSCLC). NSCLC accounts cell cycle phase S and M (cyclin A, cyclin B, p21, and for 80–85% of all lung cancer cases and is oen ft diagnosed securin), 11 substrates that are proteins related to the cell at locally advanced stages which are not amenable to sur- cycle (E2-C, E2F1, JNK, Skp2), and two substrates which gical resection [3, 4]. Cisplatin (DDP)-based chemotherapy are APC/C co-activated factors (CDC20 and Cdh1) [9–12]. has been widely applied to treat many type cancers in UBE2C plays a principle role in cell cycle progression and was the clinic, including NSCLC. In NSCLC patients, cisplatin recently found to be aberrantly expressed in various cancers generally shows good therapeutic effects in the early stage of including lung cancer, ovarian cancer, bladder cancer, and chemotherapy, but drug resistance seriously limits the further lymphoma [13–16]. Moreover, a recent study showed that application of cisplatin [5–8]. Therefore, new therapeutic UBE2C, as a regulatory factor of its target genes, promotes targets to reverse DDP-resistance are urgently needed. tumor occurrence and development in many human cancers. 2 Journal of Oncology Furthermore, decreased UBE2C expression enhances the 2.2. Cell Transfection. For cell transfection, recombinant chemosensitivity of dual drug-resistant breast cancer cells to pcDNA3.1 plasmid contained UBE2C, ZEB1 or ZEB2, UBE2C epirubicin and docetaxel [17], suggesting that UBE2C plays small interfere RNA (siUBE2C, GenePharma Co. Ltd., Shang- an important role in drug resistance. hai, China), ZEB1 small interfere RNA (siZEB1, GenePharma The zinc-finger E-box binding homeobox (ZEB) family Co. Ltd., Shanghai, China), ZEB2 small interfere RNA comprises sequence specific DNA-binding transcription fac- (siZEB2, GenePharma Co. Ltd., Shanghai, China), and neg- tors and two members: ZEB1 and ZEB2 [18]. The lix-loop- ative control oligonucleotides (NCO, GenePharma Co. Ltd.) helix motif of ZEB1 and ZEB2 has high specific binding were introduced into the A549, H1299, A549/DDP and activity with bipartite E-boxes in the E-cadherin promoter H1299/DDP cells by using lipofectamine 2000 (Invitrogen) region [19]. In NSCLC, ZEB1 expression is upregulated by according to the manufacturer’s instructions. eTh selected cyclooxygenase-2, which decreases E-cadherin gene tran- sequences for knockdown as follows: scription [20]. It is clear till the expression level of E-cadherin 󸀠 󸀠 siUBE2C were 5 -CCUGCAAGAAACCUACUCA-3 , and ZEB1 were signicfi antly correlated with sensitivity of 󸀠 󸀠 getfi inib, suggesting that they are useful for predicting to siZEB1 were 5 -GGAUCAACCACCAAUGGUU-3 , the sensitivity to epidermal growth factor receptor-tyrosine 󸀠 󸀠 siZEB2 were 5 -CGAGAUAUGUAAACUAAGGA-3 . kinase inhibitor therapy in lung cancer [21]. Furthermore, ZEB1 plays an important role in the resistance to chemother- 2.3. Immunofluorescent Staining. For analysis the protein apy drugs, such as paclitaxel [22], gefitinib [23], and tamox- levels of UBE2C, ZEB1, and ZEB2, A549 and A549/DDP cells ifen [24]. Abnormal expression of E-cadherin and ZEB1/2 were grown on coverslips in a 24-well plate overnight and results in epithelial mesenchymal transition (EMT), stem- aer ft 24 h, transfected with plasmid or siRNA and treated like cell character, resistance to therapeutic agents, and cancer with or without DDP. Aeft r 48 h, cells were fixed in 4% progression [25]. However, the relationship between ZEB1/2 formaldehyde for 30 min and permeabilized by incubation in and DDP resistance in NSCLC remains unclear. 3% BSA in PBS for 30 min. eTh coverslips were subsequently Various genes have been suggested as biomarkers of the incubated with UBE2C, ZEB1, and ZEB2 antibody at 1:1000 resistance to chemotherapeutic agents, such as ERCC1 [26, dilution in PBS containing 3% BSA. Secondary uo fl rescence 27] and ABCG2 [28, 29]. Classic chemotherapeutic drugs, antibodies at 1:1000 dilution in PBS are containing 3% BSA. such as platinum salts, are known to kill tumor cells by DAPI (3𝜇g/mL) was used for nuclear staining. Images were directly reducing DNA integrity [30]. Excision repair cross- obtained with Zeiss Axio Imager Z1 Fluorescent Microscope. complementary gene 1 (ERCC1) is an important member The detailed experiment process was described in Guo et al. oftheDNArepair-relatedgene system andcounteracts the [4]. DNA damaging eeff cts of chemotherapy and therefore is asso- ciated with drug resistance. ATP-binding cassette subfamily 2.4. RNA Isolation and RT-PCR. We used Trizol reagent G member 2 (ABCG2) was first cloned from multidrug- (TransGen Biotech, Beijing, China) to isolate total RNA from resistant breast cancer cell lines and conrfi med to be involved the samples and cells. RNA was reverse transcribed into rst fi - in the resistance to many chemotherapeutic agents, such as strand cDNA using a TransScript All-in-One First-Strand mitoxantrone, topotecan, and SN-38 [31–34]. ABCG2 was cDNA Synthesis Kit (TransGen Biotech). cDNAs were used reported to play an important role in stem cell biology [35]. in RT-PCR and qPCR assay with the human GAPDH gene as In this study, we aimed to examine the expression of UBE2C an internal control. The final qPCR reaction mix contained and ZEB1/2 in DDP-resistant NSCLC cell lines and the role 10𝜇lBestar  SYBR Green qPCR Master Mix. Amplification of UBE2C in mediating the resistance of A549/DDP and was performed as follows: a denaturation step at 94 Cfor H1299/DDP cells to DDP. 5 mins, followed by 40 cycles of amplification at 94 Cfor ∘ ∘ 30 sec, 58 Cfor 30sec,and72 C for 30 sec. eTh reaction was stopped at 25 C for 5 mins. eTh relative expression levels 2. Materials and Methods were detected and analyzed by ABI Prism 7900HT/FAST −ΔΔct 2.1. Cell Lines and Culture. HBEC, A549, H1299, Calu6, and (Applied Biosystems, USA) based on the formula of2 . H460 cell lines were obtained from American Type Culture We got the images of RT-PCR by Image LabTM Software Collection (ATCC; Manassas, VA) and maintained in RPMI (ChemiDocTM XRS+, BiO-RAD), and these images were media supplemented with 10% FBS (FBS; Hyclone, USA), TIF with reversal color format. eTh RT-PCR primers were as 10 mM of glutamic acid, and 1% penicillin/streptomycin follows. The reverse PCR primers are as follows: (normal media). The cisplatin-resistant subline, A549/DDP, was a gift from the Resistant Cancer Cell Line (RCCL) UBE2C forward primer: 5 -GGATTTCTGCCTTCC- collection (http://www.kent.ac.uk/stms/cmp/RCCL/RCCLa- CTGAA-3 , bout.html). Another cisplatin-resistant subline, H1299/DDP, UBE2C reverse primer: 5 -GATAGCAGGGCGTGA- had been established in our laboratory in 2016 by adapting GGAAC-3 , the growth of H1299 cells in the presence of increasing ZEB1 forward primer: 5 -GATGATGAATGCGAG- concentrations of cisplatin until a n fi al concentration of TCAGATGC-3 , 12𝜇g/ml, followed by cultivation in RPMI-1640 medium supplemented with 10% FBS additionally contained 2𝜇g/ml ZEB1 reverse primer: 5 -CTGGTCCTCTTCAGG- cisplatin. TGCC-3 , Journal of Oncology 3 󸀠 󸀠 ZEB2 forward primer: 5 -CTCTTCCCACACGCT- ZEB1 reverse primer: 5 - CAACCGTGGGCACTG- 󸀠 󸀠 TAGTT-3 , CTGAA-3 󸀠 󸀠 ZEB2 reverse primer: 5 -GGCCTAAGCTTACAG- ZEB2 forward primer: 5 - TTGGTGTACCAAGAG- 󸀠 󸀠 GC-3 TGTCATG-3 , E-cadherin forward primer: 5 -ACCATTAACAGG- ZEB2 reverse primer: 5 - CAACCCTGAAACAGA- 󸀠 󸀠 AACACAGG-3 , GG-3 ABCG2 forward primer: 5 -TCAGGCTAGCAA- E-cadherin reverse primer: 5 -CAGTCACTTTCA- GTGTGGTG-3 , GCATCCACTTTCTCAGA-3 ABCG2 reverse primer: 5 -TTATAAGCTTCAGGC- Vimentin forward primer: 5 -CGCCAACTACAT- AGCGCTGACACGAA-3 ’ CGACAAGGTGC-3 , ERCC1 forward primer: 5 -GGGTCTGATTGAGAT- Vimentin reverse primer: 5 -CTGGTCCACCTG- TTTGGGTC-3 CCGGCGCAG-3 , ERCC1 reverse primer: 5 -CCTTGTAAAACGTTG- GAPDH forward primer: 5 -CTCCTCCTGTTC- CCTTCACT-3 GACAGTCAGC-3 , GAPDH reverse primer: 5 -CCC AAT ACG ACC 2.8. SA-𝛽-Gal Staining. SA-𝛽-gal was detected using the AAA TCC GTT-3 . Senescence𝛽-Galactosidase Staining kit (C0602; Beyotime) following the manufacturer’s instructions: In brief, the cells 2.5. Western Blot. For western blotting analysis, proteins were washed twice with PBS and then xe fi d with PBS con- of each group cells were denatured at 100 Cboiled5min taining 2% formaldehyde and 0.2% glutaraldehyde for 10 min. with SDS loading buffer. The proteins were transferred ec Th ellswerethenincubated at 37 Cfor 12hwithstaining to PVDF transfer membrane. Membranes were incubated solution. Aer ft being washed twice with PBS, the SA- 𝛽-gal- with the indicated antibodies overnight at 4 C followed positive cells were observed under an optical microscope by immunoblotting analysis. Proteins were detected using (IX53; Olympus) and assessed using the ImageJ software. eTh enhanced chemiluminescence detection reagents (Amer- detailed experiment process was described in Jin et al. [36]. sham). Tubulin was internal control. The primary antibodies used in this study were 1:1000 rabbit anti-ABCG2 and ERCC1 2.9. Wound-Healing Assay. Cells were seeded in 6-well plats (Santa Cruz, Dallas, TX, USA), 1:1000 antibody of Tubulin, for 24 h. Cells were wounded by 200P micropipette tip, then UBE2C, ZEB1, ZEB2, vimentin, E-cadherin, and cleaved washed by PBS, and incubated in RPMI containing 2% FBS capase-3 (Abcam, Cambridge, UK). The gray intensity anal- with various DDP and/or relevant plasmid for different times. ysis of western blotting images was carried out by ImageJ Images were captured at thetimepointsof0and36h aeft r software. wounding.Therelativedistanceofthescratcheswasobserved under an optical microscope (IX53, Olympus, Tokyo, Japan) 2.6. CCK-8 Analysis. Cells were seeded in 96-well plates andassessedusing theImageJsoftware. with 5000cells/well andincubated for24h andthentreated with DDP or/and transfected plasmid and siRNA mimics 2.10. Transwell Assay. The cells (1x10 ) were seeded into for indicated time. Add 10𝜇l of CCK-8 (C0037, beyotime) 300𝜇l serum-free DMEM medium in the upper chamber solution to each welloftheplate andthenincubatetheplate (with 8-𝜇m pore size Transwell inserts (Corning, USA)) for 4 hours in the incubator. Measure the absorbance at which was coated with Matrigel.10% FBSDMEMwas 450 nm using a microplate reader (Infinite  F50; Tecan Group added to the lower chamber. eTh cells were cultured for Ltd., Manne ¨ dorf, Switzerland). 48 h. The cells remaining on the upper surface of the membrane were erased and the chambers were fixed with 2.7. Luciferase Reporter Assay. ZEB1/2 promoter region was 4% paraformaldehyde. eTh chambers were stained with 0.1% cloned into the pGL3 vector (Promega). For the luciferase crystal violet solution for 20 min (cat. no. E607309; Sangon assay, A549 and H1299 cells were cotransfected with ZEB1/2 Biotech Co., Ltd., Shanghai, China) and washed with PBS promoter-pGL3 vector and UBE2C vector by using lipofec- and then photographed by Olympus light microscope (IX53, tamine 2000. 48 h later, luciferase reporter activities were Olympus, Tokyo, Japan). eTh detailed experiment process measured by using a Dual Luciferase Reporter Assay Kit was described in Guo et al. [37]. (Promega). The PCR primers were 2.11. Clonal Formation Assay. Cells were seeded into 12-well UBE2C forward primer: 5 - GATATGAACCTGTGT- with 300cells perwelland cultured for24hours followed TGT-3 by being exposed to drugs for another 24 hours. Cells were UBE2C reverse primer: 5 - GGCTCGGCTCAGCTC- then cultured in drug-free medium for another 10 to 15 days CTTTACGG-3 until clones of around 50 cells were formed. Cell clones were ZEB1 forward primer: 5 - GAAACCAGGCGTCCC- fixed with 4% paraformaldehyde for 0.5 hour and stained with TGG-3 crystal violet (Sangon) for 15 min before optical imaging. 4 Journal of Oncology 2.12. Human Lung Cancer Specimen Collection. Atotal of cell proliferation, we detected cell growth by CCK8 assay. Our 50 human lung cancer (NSCLC) with their corresponding results indicated that the IC values of DDP were 12.8, 8.5, normal lung specimens and a total of 40 NSCLC subjects and 4.9𝜇g/mL in A549 cells and 13.2, 10.1, and 5.4 𝜇g/mL in received anthracyclines-based neoadjuvant chemotherapy H1299 cells aer ft incubation for 24, 48, and 72 h, respectively werecollectedinAffiliatedHospitalofBinzhou Medical (Figures 1(b) and S1A). Similarly, the cellular growth was College with written consent of patients and the approval dose-dependently inhibited by DDP in A549 and H1299 from the Institute Research Ethics Committee. cells (Figures 1(b) and S1B). Moreover, DDP dose- (Figures 1(c)–1(e)) and time-dependent (Figures 1(f)–1(h)) reduced 2.13. Immunohistochemical Analysis. Tumor tissues were UBE2C andZEB1/2mRNAand proteinlevelsinA549and fixed in 4% paraformaldehyde overnight and then embedded H1299 cells according to the RT-PCR, western blotting, and in paraffin wax. Four-micrometer thick sections were and qPCR assay results. Furthermore, the cellular survival rate stainedusing hematoxylinandeosin(H&E)forhistological of DDP-resistant cells (A549/DDP and H1299/DDP) was analysis. signica fi ntly higher than that of their parent cells (A549 and H1299) (Figure 1(i)) following DDP treatment. The 2.14. Ethics Approval and Consent to Participate. The experi- RT-PCR, western blotting, and qPCR results revealed that mental protocol was approved by the Research Ethics Com- the mRNA and protein levels of ERCC1 and ABCG2 were mittee of Binzhou Medical University, China (No. 2017-016- significantly increased in DDP-resistant cells compared to 01 for human lung cancer specimen and No. 2017-009-09 in their parent cells (Figures 1(j)–1(l)). es Th e results indicate for mouse experiments in vivo) and the written informed that the expression of UBE2C and ZEB1/2 was higher in lung consent was obtained from all subjects. Informed consent cancer cells and downregulated by treatment with DDP. was obtained from all individual participants included in the study. All patients were staged based on the criteria of the 7th 3.2. UBE2C and ZEB1/2 Are Involved in DDP Resistance in Edition of the AJCC Cancer Staging Manual: Stomach (2010) Lung Cancer Cells. To further explore the underlying role of UBE2C and ZEB1/2 in DDP-resistant NSCLC cells, the 2.15. Spearman’s Rank Correlation Analysis. To examine the transcriptional activity of UBE2C and ZEB1/2 was analyzed correlation between the relative expression levels between by luciferase reporter assay. Our results showed that their ZEB1/2 and ABCG2/ERCC1, we first performed a normality transcriptional activities were higher in A549/DDP and test, which indicated that there was a correlation between H1299/DDP cells than in their parent cells (Figure 2(a)). them and that the data were nonparametric. eTh correla- The mRNA and protein levels of UBE2C and ZEB1/2 were tion of the relative expression levels between ZEB1/2 and also higher in A549/DDP and H1299/DDP cells than in ABCG2/ERCC1 was then assessed using Spearman’s rank DDP-sensitive cells by RT-PCR, immunoblotting, qPCR, correlation coefficient based on the results of western blot and immunouo fl rescent assays (Figures 2(b)–2(e)). We also analysis. observed that the cell morphology was dramatically altered, with shape appearing from flat to shuttle type with apiciform 2.16. Statistical Analysis. Each experiment was repeated at pseudopodium, in A549/DDP cells and H1299/DDP cells least three times. Data were analyzed using GraphPad Prism compared to their parent cells, A549 and H1299 cells, which 5 (GraphPad, La Jolla, CA, USA) and are presented as the contribute to cell drug resistance, migration, and invasion means± SD. eTh statistical analyses of the experiment data (Figure 2(f)). Moreover, the immunohistochemistry assay were performed by using a two-tailed Student’s paired T-test indicated that the protein levels of UBE2C and ZEB1/2 were and one-way ANOVA. Statistical significance was assessed obviously increased in human DDP-resistant lung cancer at least three independent experiments and significance was tissues compared to in nonresistant lung cancer tissues considered at either p-value< 0.05 was considered statisti- (Figures 2(g) and 2(h)). Together, these results suggest that cally significant and highlighted an asterisk in the figures, UBE2C and ZEB1/2 were overexpressed in DDP-resistant while p-values< 0.01 were highlighted using two asterisks NSCLC cells and tissues. and p-values< 0.001 highlighted using three asterisks in the figures. 3.3. UBE2C Upregulates the Expression of ZEB1/2 in DDP- Resistant NSCLC Cells. After demonstrating that both of UBE2C and ZEB1/2 expression levels were downregulated 3. Results by DDP treatment, we examined the relationship between 3.1. UBE2C and ZEB1/2 Were Abnormally Activated in Lung UBE2C and ZEB1/2 using specific siRNAs or plasmids to Cancer Cells and Were Downregulated by Treatment with silence or overexpress UBE2C (Figure 3(a)), ZEB1 (Fig- DDP. To investigate the roles of UBE2C and ZEB1/2, we first ure 3(b)), or ZEB2 (Figure 3(c)) in A549/DDP cells. examined the endogenous mRNA and protein expression of For UBE2C silencing, cells were transfected with siRNA UBE2C and ZEB1/2 in human lung cancer cells by reverse (siUBE2C-1 and siUBE2C-2) and UBE2C was overexpressed transcription-polymerase chain reaction (RT-PCR), western using pcDNA-UBE2C. We found that more UBE2C was blotting, and qPCR analysis. We found that UBE2C mRNA knocked down using siUBE2C-2 (Figures S2A, S2D, and and protein levels were higher in human lung cancer cells S2G), and thus this siRNA was used in these experiments. than in normal control human bronchial epithelial cells Similar results were obtained when siZEB1-1 or siZEB2-2 were (Figure 1(a)). To further examine the effect of DDP on A549 used to knock down ZEB1 or ZEB2, respectively (Figures S2B, Journal of Oncology 5 ∗∗ ∗∗ ∗∗ ∗∗ 1.2 3 1.2 UBE2C UBE2C WB 0.8 2 0.8 ZEB1 WB 0.6 0.6 ZEB1 1 0.4 0.4 ZEB2 WB ZEB2 0.2 0.2 Tubulin WB 0 0 0 0369 12 GAPDH 024 h 36 h48 h60 h Time g/ml 24 h UBE2C ZEB1 ZEB2 3 g/ml 48 h g/ml 72 h g/ml (a) (b) DDP (g/ l) DDP (g/ l) DDP (g/ l) DDP (g/ l) 1.2 1.2 13 9 0.9 13 9 13 9 ∗∗ 13 9 0.9 ∗∗ UBE2C UBE2C UBE2C WB UBE2C WB 0.6 0.6 ZEB1 ZEB1 ZEB1 WB ZEB1 WB ∗∗ ∗∗ 0.3 0.3 ZEB2 WB ZEB2 ZEB2 ZEB2 WB Tubulin WB GAPDH GAPDH Tubulin WB 0 0 DDP A549 DDP H1299 H1299 A549 A549 H1299 UBE2C UBE2C ZEB1 ZEB1 ZEB2 ZEB2 (c) (d) (e) 1.2 DDP (g/ l) 1.2 DDP (g/ l) DDP (g/ l) DDP (g/ l) ∗∗ ∗∗ 0.9 0.9 UBE2C WB UBE2C WB UBE2C UBE2C 0.6 0.6 ZEB1 WB ZEB1 WB ∗∗ ZEB1 ∗∗ ZEB1 0.3 0.3 ZEB2 WB ZEB2 WB ZEB2 ZEB2 Tubulin WB Tubulin WB 0 GAPDH GAPDH 0 0h 24h 48h DDP DDP 0h 24h 48h A549 H1299 A549 H1299 A549 H1299 UBE2C UBE2C ZEB1 ZEB1 ZEB2 ZEB2 (g) (f) (h) ∗∗ 1.6 ∗∗ 1.4 1.4 1.2 1.2 1 1 ABCG2 ABCG2 WB 0.8 0.8 0.6 0.6 ERCC1 ERCC1 WB 0.4 0.4 0.2 0.2 Tubulin WB GAPDH 0 0 control DDP control DDP ABCG2 ERCC1 (j) (i) (k) (l) Figure 1: Aberrant activation of UBE2Cand ZEB1/2in lung cancer cells anddownregulated by treatment with DDP. (a) Gel-based RT- PCR, immunoblotting with densitometric quantitation and qPCR demonstrating elevated mRNA and protein expression of UBE2C in human lung cancer cells compared with their normal control cell HBEC. (b) A549 cells were incubated with DDP at various concentrations for 24, 48, and 72 h (left panel) and various times at 3, 6 and 9 𝜇g/ml (right panel). eTh n cell viability was assessed (%). (c–e) DDP dose-dependently repressed UBE2C and ZEB1/2 in mRNA and protein level in A549 and H1299 cells analyzed by RT-PCR (c), Western blot (d) and qPCR (e) assay. (f–h) DDP time-dependently repressed UBE2C and ZEB1/2 in mRNA and protein level in A549 and H1299 cells analyzed by RT-PCR (f), Westernblot(g)andqPCR(h) assay. (i)Survivalratesof A549/DDP,H1299/DDP andtheircontrolcellstreatedwithDDPat6𝜇g/ml for 48 h. (j–l) eTh mRNA and protein expression levels were analyzed by RT-PCR (j), Western blot (k) and qPCR (l) assay. Results were presented as mean± SD, and the error bars represent the SD of three independent experiments.∗p<0.05;∗∗p<0.01 versus control group. A549 HBEC H460 Calu6 g/ml 3g/ml H1299 1g/ml 1g/ml H1299/DDP A549 g/ml g/ml A549/DDP A549/DDP A549 H1299 H1299 H1299/DDP 48h 48h 48h 48h A549 24h 24h 24h 24h H1299 H1299 HBEC A549 H1299 Calu6 H460 HBEC A549 H1299 0h Calu6 H460 0h 0h 0h AH1299/DDP H1299/DDP A549/DDP A549/DDP A549 mRNA mRNA Relative OD value mRNA mRNA Relative OD value mRNA Protein mRNA Protein Protein Relative mRNA level Protein Protein Protein Relative OD value Relative mRNA level Relative mRNA level Relative mRNA level Relative OD value Relative mRNA level Relative mRNA level 6 Journal of Oncology 3.6 ∗∗ ∗∗ UBE2C Luc ZEB1 Luc 3.5 ZEB2 Luc 3 4.5 3.5 ∗∗ ∗∗ ∗∗ ∗∗ 2.4 ∗∗ 3 ∗∗ 3.5 2.5 2.5 1.8 2.5 2 1.2 UBE2C UBE2C 2 1.5 1.5 1.5 0.6 1 ZEB1 ZEB1 0.5 0.5 0.5 ZEB2 ZEB2 GAPDH GAPDH UBE2C ZEB1 ZEB2 (a) (b) (c) A549 A549/DDP Hoechst Merge Hoechst Merge ∗∗ ∗∗ ∗∗ 5 m UBE2C WB UBE2C WB ZEB1 WB ZEB1 WB Th ZEB2 WB ZEB2 WB UBE2C ZEB1 ZEB2 Tubulin WB Tubulin WB A549 A549/DDP (d) (e) A549 A549/DDP ∗∗ ∗∗ ∗∗ 10 10 10 UBE2C ZEB1 ZEB2 5 5 5 H1299 H1299/DDP Th Th Th 0 0 (f) (g) (h) Figure 2: UBE2Cand ZEB1/2were involved in DDP resistance in lung cancer cells. (a) eTh luciferase reporter assay indicated that the transcriptional activity of UBE2C, ZEB1, and ZEB2 was higher in A549/DDP and H1299/DDP cells than their parent cells. (b, c) RT-PCR (b) and qPCR (c) demonstrating elevated mRNA level of UBE2C, ZEB1, and ZEB2 in DDP NSCLC resistant cell lines, A549/DDP and H1299/DDP, compared with their parent cells. (d, e) Immunoblotting (d) and immunou fl orescent staining (e) assay demonstrating increased protein level of UBE2C, ZEB1, and ZEB2 in DDP NSCLC resistant cell lines, A549/DDP and H1299/DDP cells. (f) Cellular morphology of DDP NSCLC resistant cell lines and their parent cells was analyzed by phase contrast microscope assay. (g) Immunohistochemical staining shows that the protein levels of UBE2C, ZEB1, and ZEB2 were higher in human DDP resistance lung cancer tissues than in nonresistance cancer tissues with anthracyclines-based neoadjuvant chemotherapy. (h) Statistical analysis of the protein level of UBE2C, ZEB1, and ZEB2 in DDP non/resistance lung cancer tissues (n=20). Results were presented as mean± SD, and the error bars represent the SD of three independent experiments. ∗∗p<0.01 versus control group. S2E, and S2H and Figures S2C, S2F, and S2I). To determine UBE2C increased these levels, which were detected by RT- whether the ZEB1/2 expression level was regulated through PCR, western blotting, and qPCR assay (Figures 3(d) and UBE2C, we used a control (cotransfection of pcDNA vector 3(e)) and immunou fl orescent staining (Figures 3(f) and 3(g)). and si control), siUBE2C (cotransfection of siRNA of UBE2C To further explore whether ZEB1 and ZEB2 are the direct and pcDNA vector), and Flag-UBE2C (cotransfection of targets of UBE2C, we performed a luciferase reporter assay pcDNA Flag-UBE2C and si control) in the same experiment in A549/DDP and H1299/DDP cells. We cloned the promoter (Figures 3(d) and 3(e)). These approaches were also used of ZEB1 or ZEB2 into the dual luciferase reporter vector and for ZEB1 and ZEB2. Knockdown of UBE2C decreased the then transfected these vectors into cells. ZEB1/2 promoter mRNA and protein levels of ZEB1/2, while overexpression of activity was obviously reduced in the UBE2C knockdown H1299/DDP H1299/DDP H1299/DDP H1299/DDP A549 A549 A549 A549 A549/DDP A549/DDP /DD A549 P A549/DDP H1299 H1299 H1299 H1299 Resistance Resistance Resistance Non-resistance Non-resistance Non-resistance A549 A549 H1299 H1299 A549/DDP A549/DDP H1299/DDP AH1299/DDP Protein Relative luciferase activity Protein Relative luciferase activity Resistance Non-resistance Relative luciferase activity ZEB2 ZEB1 UBE2C mRNA e relative protein level of UBE2C mRNA e relative protein level of ZEB1 Relative mRNA level e relative protein level e relative protein level of ZEB2 ∗∗ ∗∗ control control si UBE2C si UBE2C UBE2C UBE2C Journal of Oncology 7 ∗∗ 3.5 4 ∗∗ 2.8 3 2.1 1.4 ZEB2 WB ZEB2 0.7 ∗ UBE2C WB ∗ UBE2C Tubulin WB 0 GAPDH Tubulin WB GAPDH (a) (e) 3.5 ∗∗ UBE2C (g/ ml) UBE2C (g/ ml) 4.8 2.8 2.1 ∗∗ 12 3 1 ZEB1 1.4 ZEB1 WB UBE2C 3.2 0.7 UBE2C WB GAPDH ∗ Tubulin WB 2.4 ZEB1 ZEB1 WB 1.6 ZEB2 ZEB2 WB (b) 0.8 GAPDH Tubulin WB ∗∗ UBE2C g/ml 3g/ml 9g/ml UBE2C ZEB1 ZEB2 ZEB2 ZEB2 WB (i) GAPDH Tubulin WB UBE2C (2g/ ml) UBE2C (2g/ ml) 4.2 ∗∗ 3.5 (c) 2.8 UBE2C UBE2C WB ∗∗ 2.1 ZEB1 WB ZEB1 1.4 ZEB2 WB ZEB2 0.7 ZEB1 ZEB1 WB Tubulin WB GAPDH 1 0 GAPDH Tubulin WB ∗ UBE2C 24h 48h 72h UBE2C ZEB1 ZEB2 (j) (d) UBE2C ZEB1 Hoechst Merge UBE2C ZEB2 Hoechst Merge ZEB1 luc 4 ZEB1 luc ZEB2 luc ZEB2 luc 5 4 ∗∗ ∗∗ ∗∗ ∗∗ 5m 4 5m 3 3 0 0 The relative protein A549/DDP H1299/DDP level of ZEB2 (h) (f ) (g) Figure 3: UBE2Cupregulates the expression of ZEB1/2in DDP-resistant NSCLC cells. (a–c) Overexpression and knockdown of UBE2C (a), ZEB1 (b), and ZEB2 (c) were examined by RT-PCR, Western blot, and qPCR in the A549/DDP cells. (d–g) RT-PCR, Western blot, qPCR, and immunou fl orescence staining assay demonstrating that knockdown or overexpression of UBE2C using siRNA and pcDNA3.1-UBE2C could decrease or increase the ZEB1in the A549/DDP cells (d, f) and ZEB2 (e, g) expression in A549/DDP cells. (i, j) RT-PCR and Western blot result show that UBE2C dose-and time-dependently increased the mRNA and protein level of ZEB1 and ZEB2 in the A549/DDP cells. (h) eTh transcriptional activity of ZEB1/2 regulated by UBE2C was analyzed by luciferase reporter assay. Results were presented as mean ± SD, and the error bars represent the SD of three independent experiments.∗p<0.05;∗∗p<0.01 versus control group. group but enhanced in the UBE2C overexpression group 3.4. Knockdown of UBE2C Sensitizes DDP-Resistant NSCLC (Figure 3(h)). Moreover, UBE2C increased the mRNA and Cells to Cisplatin via Decreasing the Expression of ZEB1/2. protein levels of ZEB1 and ZEB2 in dose- and time-dependent To confirm whether UBE2C promotes cell proliferation by manners in A549/DDP cells (Figures 3(i) and 3(j)). Collec- targeting ZEB1/2, we cotransfected UBE2C and siZEB1 or tively, these data indicate that ZEB1 and ZEB2 expression was siUBE2C and ZEB1 into A549/DDP cells. We also sepa- upregulated by UBE2C in DDP-resistant NSCLC cells. rately transfected UBE2C or siUBE2C into A549/DDP cells The relat ive protein level of ZEB1 control control control siUBE2C UBE2C UBE2C UBE2C control control siZEB1 ZEB1 siZEB2 ZEB2 siUBE2C siUBE2C Control Control ZEB1 si ZEB2 ZEB2 Control ZEB1 si ZEB2 ZEB2 Control Control Control Control Control Control si UBE2C si UBE2C UBE2C UBE2C si ZEB1 si ZEB1 si UBE2C UBE2C si UBE2C UBE2C si UBE2C UBE2C si UBE2C UBE2C Control 48 h 72 h 48 h 72 h 24 h 24 h siUBE2C Control UBE2C mRNA mRNA mRNA mRNA Protein Protein Protein Protein UBE2C siUBE2C Control Relative mRNA level Relative mRNA level Relative mRNA Relative mRNA level level of ZEB1 mRNA mRNA mRNA Relative luciferase activity Protein Protein Protein control si UBE2C UBE2C Relative luciferase activity control si UBE2C UBE2C Relative mRNA Relative luciferase activity level of ZEB2 Relative mRNA level Relative mRNA level control si UBE2C UBE2C Relative luciferase activity control si UBE2C UBE2C 8 Journal of Oncology and conducted RT-PCR, western blotting, and qPCR to of ZEB1/2 inhibited UBE2C-mediated cell migration and detect UBE2C and ZEB1. The ZEB1 level was increased by invasive growth of DDP-resistant NSCLC cells, we examined UBE2C transfection and decreased by siUBE2C. However, whether UBE2C regulates EMT marker proteins via ZEB1/2. the opposite effects were observed for each of these factors We cotransfected siUBE2C and ZEB1/2 or UBE2C and in A549/DDP cells ectopically transfected with siZEB1 or siZEB1/2 into cells and measured E-cadherin and vimentin overexpression of ZEB1 (Figure 4(a)). The protein expression mRNA and protein levels. RT-PCR, western blotting, and of ZEB2 showed a similar result as ZEB1 (Figure 4(b)). To qPCR assays indicated that the E-cadherin level was increased furtherinvestigatetheroleofUBE2CandZEB1/2inDDP- by siUBE2C transfection and decreased by UBE2C over- resistant cell proliferation, A549/DDP and H1299/DDP cells expression, while the opposite effects were observed for were cotransfected with UBE2C and siZEB1/2 or siUBE2C each of these factors in A549/DDP cells ectopically trans- and ZEB1/2. As shown in Figures 4(c) and 4(e), the CCK- fected with ZEB1/2 or siZEB1/2. In contrast, vimentin was 8 assay revealed that the cell proliferation capacity was decreased by siUBE2C transfection and increased by UBE2C markedly increased in A549/DDP and H1299/DDP cells overexpression, while the opposite effects were observed by overexpression of UBE2C and ZEB1/2, but drastically for each of these factors in A549/DDP cells ectopically decreased by introduction of siUBE2C or siZEB1/2. As transfected with ZEB1/2 or siZEB1/2 (Figures 5(e), 5(f), S3A, ZEB1/2 was regulated by UBE2C, we explored whether and S3B). Similar RT-PCR and western blotting results of knockdown of UBE2C inhibited cell proliferation in DDP- E-cadherin and vimentin were obtained for H1299/DDP resistant cells following treatment with DDP. A549/DDP and cell lines cotransfected with UBE2C and ZEB1/2 (data not H1299/DDP cells were cotransfected with UBE2C and siZEB1 shown). es Th e results suggest that knockdown of ZEB1/2 or siUBE2C and ZEB1 and then treated with DDP. Our data inhibits UBE2C-dependent cellular growth and invasiveness showed that resistance to DDP induced by UBE2C overex- and UBE2C-mediated EMT progress by promoting ZEB1/2 pression was remarkably decreased when ZEB1 or ZEB2 was expression in DDP-resistant NSCLC cells. knocked down in A549/DDP and H1299/DDP cells (Figures 4(d) and 4(f)). In contrast, overexpression of ZEB1 or ZEB2 3.6. UBE2C Increases ABCG2 and ERCC1 Expression via Reg- rescued the cell growth inhibited by knockdown of UBE2C in ulating ZEB1/2 in DDP-Resistant NSCLC Cells. To determine DDP-resistant NSCLC cells treated with DDP. es Th e results the molecular mechanism by which UBE2C and ZEB1/2 were conrfi med by Ki67 immunohistochemistry staining reverses DDP resistance in lung cancer cells, we conducted in A549/DDP cells treated with DDP (Figure 4(g)). The RT-PCR to confirm whether ZEB1/2 regulates the drug protein level of active caspase-3 determined by western blot resistance genes HER2, MRP1, KRAS, BRCA1, and MDR1 assay showed that treatment with DDP significantly induced in A549/DDP cells. However, ZEB1/2 did not regulate the apoptosis in A549/DDP cells transfected with siUBE2C, expression of these genes in A549/DDP cells according to our and this process was reversed by overexpression of ZEB1/2 RT-PCR and qPCR assays (Figures S4A and S4B). We con- (Figure 4(h)). Analysis of protein expression of Annexin V ducted RT-PCR, western blotting, and qPCR assays to further showedsimilarresultsforactivecaspase-3 by immunoufl o- investigate whether UBE2C, ZEB1, and ZEB2 regulate the rescent staining assay (Figure 4(i)). es Th e data indicate that drug resistance genes ABCG2 and ERCC1. eTh mRNA and knockdown of UBE2C sensitizes DDP-resistant NSCLC cells protein levels of ABCG2 and ERCC1 were measured at 48 h to cisplatin by decreasing the expression of ZEB1/2. aer ft UBE2C and ZEB1/2 transfection. The results showed that the mRNA and protein levels of ABCG2 and ERCC1 3.5. Knockdown of ZEB1/2 Inhibits UBE2C-Dependent Cellular were significantly upregulated aeft r UBE2C, ZEB1, and ZEB2 Growth, Invasiveness, and EMT in DDP-Resistant NSCLC overexpression. These inhibitory effects were suppressed Cells. Recent studies showed that UBE2C not only promotes by downregulation of UBE2C, ZEB1, and ZEB2 expression cell proliferation, but also is positively correlated with metas- (Figures 6(a)–6(c) and S3C–S3E). The immunoblotting assay tasis in many types of cancer cells [38, 39]. We showed that indicated that ZEB1 and ZEB2 increased ABCG2 and ERCC1 UBE2C regulates ZEB1/2 expression to promote NSCLC cell protein levels in a dose-and time-dependent manner in resistance to DDP. To further investigate whether UBE2C- A549/DDP cells (Figures 6(d) and 6(e)). Similar results were regulated ZEB1/2 expression aeff cts DDP-resistant cell senes- obtained by western blot analysis of the H1299/DDP cell cence, invasiveness, and EMT in DDP-resistant NSCLC cells, lines (data not shown). Moreover, the ABCG2/ERCC1 pro- we cotransfected UBE2C and siZEB1 or siUBE2C and ZEB1 moter and ZEB1 cotransfection into A549/DDP cells resulted into A549/DDP cells and then treated the cells with DDP and in significantly increased luciferase activity compared to conducted colony formation (Figure 5(a)), SA-𝛽-gal staining cotransfection with the control vector. Compared to the (Figure 5(b)), wound-healing (Figure 5(c)), and Matrigel control group, luciferase activity was decreased following invasion assays (Figure 5(d)). We found that overexpression cotransfection with siZEB1. Similar results were obtained of UBE2C dramatically increased colony formation ability aer ft cotransfection with the ABCG2/ERCC1 promoter and (Figure 5(a)), inhibited cell senescence (Figure 5(b)), and pro- ZEB2 or siZEB2 (Figure 6(f)). Furthermore, Spearman’s rank moted cell migration (Figure 5(c)) and invasion (Figure 5(d)), correlation analysis revealed significant positive correlations which were partially inhibited by knockdown of ZEB1. between ZEB1/2 and ABCG2/ERCC1 protein levels based on Cotransfection of UBE2C and siZEB2 or siUBE2C and ZEB2 western blot assays (Figure 6(g)). siUBE2C inhibited DDP- into A549/DDP cells showed similar results as cotransfection resistant NSCLC cell proliferation, migration, invasion, and of UEE2C and ZEB1 (data not shown). Because knockdown EMT, which contributed to reversing the DDP resistance Journal of Oncology 9 DDP 4.8 DDP 3.2 #∗∗ ∗∗ 2.4 1.6 #∗∗ 0.8 UBE2C UBE2C WB ∗ ZEB1 ZEB1 WB GAPDH Tubulin WB DDP UBE2C ZEB1 (a) DDP DDP 6 5 ∗∗ ∗∗ ∗∗ ∗∗ #∗∗ ∗∗ 4 3.2 2.4 1.6 UBE2C WB #∗∗ UBE2C 1 ∗ 0.8 ∗ ∗ ∗ ∗ ZEB2 WB ZEB2 0 0 Tubulin WB GAPDH A549/DDP DDP UBE2C ZEB2 (b) (c) 4 6 7 ∗∗ 3 4 ∗∗ ∗∗ 6 ∗∗ ∗∗ ∗∗ 5 ∗∗ 5 3 ∗∗ #∗∗ #∗∗ #∗∗ 2 NS 3 NS #∗∗ 1 NS #∗∗ 1 NS ∗ 1 #∗∗ #∗∗ 1 #∗∗ ∗ ∗ ∗ ∗ ∗ 1 ∗ ∗ 0 0 0 0 H1299/DDP DDP DDP DDP DDP A549/DDP H1299/DDP (d) (e) (f) DDP Annexin V PI Merge ∗∗ DDP Caspase-3 cleaved WB ∗∗ Control Si UBE2C Si UBE2C+ZEB1 UBE2C UBE2C+siZEB1 Tubulin WB #∗∗ #∗∗ 4 2 ∗∗ NS 3 #∗∗ 5m 1 #∗∗ NS #∗∗ #∗∗ DDP DDP DDP (g) (h) (i) Figure 4: Knockdownof UBE2C sensitizes DDP-resistant NSCLC cells to cisplatin via decreasing the expression of ZEB1/2.A549/DDP or H1299/DDP cells were transfected with UBE2C or siUBE2C. ZEB1 or ZEB2 were used for upregulated the protein level of UBE2C target genes and then treated with DDP 6𝜇g/ml for 48 h, respectively. (a, b) eTh mRNA and protein expression levels of UBE2C and ZEB1 (a) or UBE2C and ZEB2 (b) were analyzed by RT-PCR and immunoblotting assay. (c–f) The cellular growth was analyzed by CCK8 assay with or without treatment of DDP at 6𝜇g/ml for 48 h in A549/DDP (c, d) or H1299/DDP (e, f) cells. (g) eTh protein of Ki67 was analyzed by immunouo fl rescent staining. (h) eTh protein of active caspase-3 was analyzed by Western blot. (i) eTh protein of Annexin V was analyzed by immunouo fl rescent staining. Results were presented as mean ± SD, and the error bars represent the SD of three independent experiments. ∗p<0.05;∗∗p<0.01 versus control group. PBS PBS PBS PBS Si UBE2C Si UBE2C Si UBE2C Si UBE2C Control Control Control Control Si UBE2C+ZEB1 Si UBE2C+ZEB2 Si UBE2C+ZEB1 Si UBE2C+ZEB2 PBS UBE2C+siZEB1 UBE2C+siZEB2 UBE2C+siZEB1 UBE2C+siZEB2 control UBE2C UBE2C UBE2C UBE2C siUBE2C siUBE2C+ZEB2 UBE2C UBE2C+siZEB2 control siUBE2C siUBE2C+ZEB1 UBE2C UBE2C+siZEB1 Control Si UBE2C UBE2C si ZEB1 Control ZEB1 Si UBE2C UBE2C si ZEB1 ZEB1 Control Si UBE2C UBE2C si ZEB2 Control ZEB2 Si UBE2C UBE2C si ZEB2 ZEB2 Control Si UBE2C Si UBE2C+ZEB1 UBE2C UBE2C+siZEB1 control siUBE2C siUBE2C+ZEB1 UBE2C UBE2C+siZEB1 Control Si UBE2C Si UBE2C+ZEB1 UBE2C UBE2C+siZEB1 PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS UBE2C+siZEB1 Control Control Control Si UBE2C Si UBE2C Si UBE2C+ZEB2 Si UBE2C+ZEB1 UBE2C UBE2C UBE2C+siZEB2 UBE2C+siZEB1 Control Control Si UBE2C Si UBE2C Si UBE2C+ZEB2 Si UBE2C+ZEB1 UBE2C UBE2C UBE2C+siZEB2 UBE2C+siZEB1 Si UBE2C UBE2C Si UBE2C+ZEB1 Hoechst Merge Ki67 Relative OD value mRNA Relative OD value mRNA Protein The relative protein level of Ki67 Relative OD value Protein Relative OD value Relative mRNA level The relative protein level Relative mRNA level Relative OD value Relative OD value UBE2C Si UBE2C + Si UBE2C UBE2C +ZEB1 Control Relative OD value Relative OD value The relative protein level of Annex V 10 Journal of Oncology ∗∗ DDP Control Si UBE2C+ZEB1 UBE2C Si UBE2C UBE2C+siZEB1 2 #∗∗ NS #∗∗ DDP (a) ∗∗ #∗∗ NS 1 #∗∗ DDP (b) DDP Control Si UBE2C Si UBE2C+ZEB1 UBE2C UBE2C+siZEB1 DDP (c) DDP DDP DDP Control Si UBE2C Si UBE2C+ZEB1 UBE2C UBE2C+siZEB1 ∗∗ UBE2C #∗∗ UBE2C NS ZEB1 #∗∗ ZEB2 Th 0 E-cadherin E-cadherin Vimentin Vimentin GAPDH GAPDH DDP Invasion Migration mRNA (d) (e) DDP DDP UBE2C WB UBE2C WB ZEB1 WB ZEB2 WB E-cadherin WB E-cadherin WB Vimentin WB Vimentin WB Tubulin WB Tubulin WB Protein (f) Figure 5: Knockdownof ZEB1/2 inhibits UBE2C-dependent cellular growth, invasiveness, and EMT in DDP-resistant NSCLC cells. (a) Colony formation assay demonstrating that ectopic expression of UBE2C significantly enhanced colony formation density, which was blocked by knockdown ZEB1 in A549/DDP cells with treatment of DDP at 6𝜇g/ml for 48 h. (b) SA-𝛽-Gal assay was performed to detect the cell senescence of A549/DDP cells treated with DDP at 6𝜇g/ml for 48 h and transfected with UBE2C, siUBE2C alone, or the combination of siZEB1/2 and ZEB1/2 plasmid for 48 h. (c, d) Scratch assay (c) and Matrigel invasion assay (d) indicated that UBE2C promote cell migration and invasion via regulating ZEB1 in A549/DDP cells with treatment of DDP at 6𝜇g/ml for 36 h. (e, f) UBE2C significantly decreased E- cadherin and increased vimentin in mRNA (e) and protein (f) levels by regulating ZEB1/2 in the A549/DDP cells with treatment of DDP at 6𝜇g/ml for 48 h. Results were presented as mean ± SD, and the error bars represent the SD of three independent experiments.∗p<0.05; ∗∗p<0.01 versus control group. PBS PBS PBS PBS Si UBE2C Si UBE2C Control Control Si UBE2C+ZEB1 Si UBE2C+ZEB1 Control Control Si UBE2C Si UBE2C Si UBE2C+ZEB1 Si UBE2C+ZEB1 UBE2C+siZEB1 UBE2C+siZEB1 UBE2C+siZEB1 UBE2C+siZEB1 UBE2C UBE2C UBE2C UBE2C PBS PBS PBS PBS PBS PBS PBS UBE2C+siZEB1 UBE2C+ iZEB2 UBE2C+siZEB1 UBE2C+siZEB2 Si UBE2C Si UBE2C Si UBE2C Si UBE2C UBE2C UBE2C UBE2C UBE2C Control Control Control Control Si UBE2C+ZEB1 Si UBE2C+ZEB2 Si UBE2C+ZEB1 Si UBE2C+ZEB2 Migration Invasion 36 h 0 h e relative cell number The relative cell number The relative cell number Journal of Oncology 11 UBE2C ZEB2 ZEB1 ABCG2 ABCG2 ABCG2 ERCC1 ERCC1 ERCC1 GAPDH GAPDH GAPDH ZEB1 WB UBE2C WB ZEB1 WB ABCG2 WB ABCG2 WB ABCG2 WB ERCC1 WB ERCC1 WB ERCC1 WB Tubulin WB Tubulin WB Tubulin WB (a) (b) (c) ZEB1 ZEB1 (2g) ZEB2 (2g) ZEB2 ZEB2 WB ZEB2 WB ZEB1 WB ZEB1 WB ABCG2 WB ABCG2 WB ABCG2 WB ABCG2 WB ERCC1 WB ERCC1WB ERCC1 WB ERCC1WB Tubulin WB Tubulin WB Tubulin WB Tubulin WB (d) (e) ∗∗ ∗∗ ∗∗ ∗∗ R = 0.9408   14 P<0.001 R = 0.9179 R = 0.9196 P<0.001 R = 0.9203 P<0.001 P<0.001 10 12 2 6 1 4 ∗ ∗ 2 0 0 0 0 0 0 0 12 0 0 12 0 12 ABCG2 ERCC1 ABCG2 ERCC1 ZEB2 protein expression ZEB1 protein expression ZEB1 protein expression ZEB2 protein expression control control ZEB1 ZEB2 si ZEB1 si ZEB2 (g) (f) Figure 6: Continued. Control Si UBE2C UBE2C Control Si UBE2C UBE2C Control SiZEB1 ZEB1 Control Si ZEB1 ZEB1 Control SiZEB2 ZEB2 Control Si ZEB2 ZEB2 24 h 48 h 72 h 24 h 48 h 72 h Relative luciferase activity Protein g Protein mRNA g g Relative luciferase activity Protein mRNA ABCG2 protein expression Protein Protein ERCC1 protein expression 1 g 2 g g mRNA Protein ABCG2 protein expression Protein ERCC1 protein expression 12 Journal of Oncology −− + − − −− + − − Si UBE2C Si UBE2C −− + − − −− + − − Si UBE2C Si UBE2C UBE2C −− − − + UBE2C −− − − + UBE2C −− − − + UBE2C −− − − + control +− − − − control +− − − − control +− −− − control +− − − − −− − + + −− − + + Si ZEB2 Si ZEB2 −− − + + Si ZEB1 −− − + + Si ZEB1 −+ + − − −+ + − − ZEB2 ZEB2 −+ + − − −+ + − − ZEB1 ZEB1 UBE2C UBE2C UBE2C UBE2C ZEB2 ZEB2 ZEB1 ZEB1 ABCG2 ABCG2 ABCG2 ABCG2 ERCC1 ERCC1 ERCC1 ERCC1 GAPDH Tubulin GAPDH Tubulin (h) (i) (j) Figure 6: UBE2C increases the expression of ABCG2 and ERCC1via regulating ZEB1/2in DDP-resistant NSCLC cells. (a–c) A549/DDP cells were overexpressed or knockdown of UBE2C (a), ZEB1 (b), and ZEB2 (c), respectively. The mRNA and protein levels of ABCG2 and ERCC1 were analyzed by RT-PCR and western blot assay. (d, e) RT-PCR and Western blot result show that ZEB1 (d) or ZEB2 (e) dose-and time-dependently increased the protein level of ABCG2 and ERCC1 in A549/DDP cells. (f) eTh transcriptional activity of ABCG2 and ERCC1 regulated by ZEB1/2 was analyzed by luciferase reporter assay in A549/DDP cells. (g) eTh relationship between protein expression levels of ZEB1/2, ERCC1, and ABCG2 in 15 independent repetitive A549/DDP cells was analyzed based on western blot assay. (h, i) UBE2C increased the expression of ABCG2 and ERCC1 via regulation of ZEB1 (h) and ZEB2 (i) by RT-PCR and Western blot assay. (j) Schematic diagram of the mechanisms of UBE2C increased the drug resistance gene expression, ABCG2 and ERCC1, and the EMT progression via regulating ZEB1/2 expression in DDP-resistant NSCLC cells. Results were presented as mean± SD, and the error bars represent the SD of three independent experiments.∗p<0.05;∗∗p<0.01 versus control group. by regulating ABCG2 and ERCC1 (Figures 4–6). siZEB1/2 irinotecan treatment in patients with colorectal cancer [45]. played a similar important role in reversing DDP resistance We previously reported that UBE2C selectively represses in DDP-resistant NSCLC cells (Figure 5). Moreover, UBE2C autophagy in NSCLC, and disruption of UBE2C-mediated upregulated the expression of ZEB1/2 (Figure 3) and ZEB1/2 autophagy repression attenuates cell proliferation, clono- directly targeted ABCG2 and ERCC1 (Figure 6). eTh refore, genicity, and invasive growth of NSCLC [46]. Furthermore, we predicted that siUBE2C reverses DDP resistance by our previous research showed that the miR 495-UBE2C- regulating ABCG2 and ERCC1 by directly targeting ZEB1/2. ABCG2/ERCC1 axis reverses cisplatin resistance by down- To evaluate this hypothesis, we separately transfected ZEB1 regulating drug resistance genes in cisplatin-resistant non- or siZEB1 into the A549/DDP cell line. eTh ABCG2 or small-cell lung cancer cells, highlighting the mechanism of ERCC1 mRNA and protein levels were increased by ZEB1 how microRNA 495 downregulates UBE2C [37]. In this study, and reduced by siZEB1 transfection. However, the opposite we examined the expression of UBE2C in DDP-sensitive and effects were observed for each factor in A549/DDP cells DDP-resistant cells and the role of UBE2C in mediating the ectopically transfected with siUBE2C to ZEB1 or UBE2C resistance of A549/DDP and H1299/DDP cells to DDP. eTh to siZEB1 by RT-PCR, western blotting, and qPCR assays results showed that, compared to wild-type cells (A549 and (Figures 6(h) and S3F). Similar results were obtained by RT- H1299), the mRNA and protein expression of UBE2C were PCR and western blot analysis of ZEB2 in A5499/DDP cell significantly increased in DDP-resistant cells (A549/DDP and lines (Figures 6(i) and S3G). es Th e data indicate that ZEB1/2- H1299) and UBE2C was significantly decreased following mediated siUBE2C reverses DDP resistance by regulating DDP treatment in A549 and H1299 cells (Figures 1(c)- ABCG2 and ERCC1 in DDP-resistant NSCLC cells. 1(d)). UBE2C or ZEB1/2 deficiency was found to signifi- cantly increase sensitivity to DDP, prevent cell proliferation and colony formation ability, and promote cell senescence 4. Discussion in DDP-resistant cells. Reintroduction of ZEB1/2 notably The emergence of drug resistance is unavoidable and severely rescued the phenotypes induced by UBE2C knockdown. limits the curative effect of chemotherapy drug [5, 40, 41], Moreover, our results showed that ZEB1/2 regulated the including cisplatin. us, Th useful biomarkers are needed to expression of the drug-resistant genes ABCG2 and ERCC1 predict and overcome DDP resistance to treat patients with at the transcriptional level. We also found that knockdown NSCLC. UBE2C is highly expressed in many types of human of UBE2C significantly sensitized lung cancer cells to the carcinomas including NSCLCs and strongly associated with chemotherapeutic agent DDP by repressing ABCG2/ERCC1 tumor grade/poor prognosis [40–42]. The UBE2C protein expression through downregulation of ZEB1/2 in vitro.Based plays a critical role in activating the M-phase check point on these results, we demonstrated that knockdown of UBE2C by specifically binding to APC/C [43, 44]. Knockdown of is a potential strategy for reversing DDP resistance in NSCLC UBE2C enhances the chemosensitivity of epirubicin and cells. docetaxel to dual drug-resistant breast cancer cells [17]. EMT plays a critical role in accelerating cisplatin resis- The UBE2C expression level can indicate the sensitivity to tance, and mesenchymal-like cancers are more prone to mRNA Protein mRNA Protein Journal of Oncology 13 developing drug resistance [47]. eTh hallmark event of EMT Authors’ Contributions is downregulation of E-cadherin protein. At the transcrip- Jiwei Guo designed the experiments. Yan Wu, Dan Jin, tional level, E-cadherin gene expression is repressed by many Xiaohong Wang, Jing Du, Weihua Di, Jiajia An, Cuijie Shao, factors, such as ZEB1, ZEB2, Snail, and slug [48–50]. EMT andJiweiGuoperformed thework.JiweiGuo,Yan Wu,and promotes cancer metastasis and invasion and thus accelerates Dan Jin analyzed the data and competed the gfi ures. Jiwei the emergence of drug resistance. We previously reported Guo wrote the manuscript. Yan Wu and Dan Jin contributed that UBE2C promoted EMT by regulating E-cadherin and equally to this work vimentin. ZEB1 promotes tumorigenesis and metastasis, and its expression is correlated with poor outcomes in cancer, including resistance to chemotherapy [51, 52]. In the cur- Acknowledgments rent study, we found that the mRNA and protein levels of ZEB1/ZEB2 were significantly downregulated in NSCLC cells We appreciate Professor Sichuan Xi (National Institutes following treatment with DDP (Figures 1(c)-1(d)). of Health, USA) for critical reading of the manuscript. We also found that ZEB1/2 mediates UBE2C regula- The present study was supported by National Natural Sci- tion EMT, cell proliferation, migration, and invasion in ence Foundation of China (no. 31801085), the Natural Sci- DDP-resistant cells (Figures 5(a)–5(d)). Importantly, ZEB1/2 ence Foundation of Shandong Province (ZR2018QH004, upregulated the expression of ABCG2 and ERCC1 (Figures ZR2016HB55, ZR2017LH072, and ZR2017PH067), and Shan- 6(b)-6(c)) and repressed E-cadherin gene transcription in dong Provincial Pharmaceutical Technology Development DPP-resistant NSCLC cells. Accordingly, UBE2C upregulated Plan (2017WS154) and the Research Foundation of Binzhou the expression of ZEB1/2 by increasing their promoter activ- Medical University (BY2015KYQD25 and BY2015KJ14). ity in DDP-resistant NSCLC cells (Figure 3(h)). siUBE2C downregulated ABCG2 and ERCC1 by suppressing ZEB1/2 Supplementary Materials expression and thus enhancing cisplatin sensitivity in DDP- resistant NSCLC cells. These results demonstrate that UBE2C Supplementary 1. SupplementaryFigure S1: H1299 cells expression levels are useful for predicting the response or were incubated with DDP at various concentrations for 24, resistance to DDP in NSCLC cells. However, the under- 48, and 72 h (A) and various times at 3, 6, and 9𝜇g/ml (B). lying mechanisms regulating UBE2C have not been well- Then cell viability was assessed (%). characterizedinNSCLC orotherchemotherapeuticagent- resistant cancers. u Th s, our future studies will focus on Supplementary 2. SupplementaryFigure S2: A549/DDP these mechanisms. In summary, UBE2C plays a critical cells were transfected with siUBE2C-1/2, siZEB1-1/2, or role in decreasing the sensitivity to cisplatin, inhibiting cell siZEB2-1/2. (A-F) eTh mRNA and protein levels of UBE2C senescence, and promoting cell proliferation, migration, and (A, D), ZEB1 (B, E), and ZEB2 (C, F) were analyzed by RT- invasion via promoting the promoter activity of ZEB1/2; PCR, immunoblotting, and qPCR assay. (G-I) the cellular ZEB1/2 upregulates the expression of antidrug genes, ABCG2 proliferation was analyzed by CCK8 assay in A549/DDP and ERCC1, to induce DDP resistance in NSCLC cells cell with transfection with siUBE2C-1/2 (G), siZEB1-1/2 (H), (Figure 6(j)). Collectively, our results indicate that UBE2C- or siZEB2-1/2 (I). Results were presented as mean ± SD, ZEB1/2-ABCG2/ERCC1 reverses DDP resistance by down- and the error bars represent the SD of three independent regulating antidrug genes and reducing EMT in cisplatin- experiments.∗p<0.05;∗∗p<0.01 versus control group. resistant NSCLC cells. Supplementary 3. SupplementaryFigure S3: (A, B) A549/ DDP cells were transfected with UBE2C or siUBE2C. ZEB1 Abbreviations (A)orZEB2(B)wereused forupregulatingtheprotein level of UBE2C target genes, respectively. eTh mRNA levels DDP: Cis-diamminedichloroplatinum (II) (cisplatin) of UBE2C, ZEB1, ZEB2, E-cadherin, and vimentin were ABCG2: ATP-binding cassette subfamily G member 2 analyzed by qPCR. (C-E) A549/DDP cells were overexpressed ERCC1: Excision repair cross-complementation group 1 or knockdown of UBE2C (C), ZEB1 (D), and ZEB2 (E), UBE2C: Ubiquitin-conjugating enzyme E2 C EMT: Epithelial-to-mesenchymal transition respectively. eTh mRNA and protein levels of ABCG2 and ERCC1 were analyzed by qPCR. (F, G) A549/DDP cells were HBEC: Human bronchial epithelial cells transfected with UBE2C or siUBE2C. ZEB1 (F) or ZEB2 (G) NSCLC: Non-small-cell lung cancer were used for upregulating the protein level of UBE2C target siRNA: Short interfering RNA. genes, respectively. eTh mRNA levels of UBE2C, ZEB1, ZEB2, ABCG2, and ERCC1 were analyzed by qPCR. Results were Data Availability presented as mean± SD,and theerrorbarsrepresenttheSD of three independent experiments.∗p<0.05;∗∗p<0.01 versus ed Th ata usedtosupportthefindingsofthisstudy are control group. included within the article. Supplementary 4. 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UBE2C Induces Cisplatin Resistance via ZEB1/2-Dependent Upregulation of ABCG2 and ERCC1 in NSCLC Cells

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Copyright © 2019 Yan Wu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Abstract

Hindawi Journal of Oncology Volume 2019, Article ID 8607859, 15 pages https://doi.org/10.1155/2019/8607859 Research Article UBE2C Induces Cisplatin Resistance via ZEB1/2-Dependent Upregulation of ABCG2 and ERCC1 in NSCLC Cells 1 2 3 1 2 4 Yan Wu, Dan Jin, Xiaohong Wang, Jing Du, Weihua Di, Jiajia An, 2 1 Cuijie Shao, and Jiwei Guo Cancer Research Institute, Binzhou Medical University Hospital, Binzhou 256603, China Department of Pain Medicine, Binzhou Medical University Hospital, Binzhou 256603, China Department of yTh roid and Breast Surgery, Binzhou Medical University Hospital, Binzhou 256603, China Department of Clinical Laboratory, Binzhou Medical University Hospital, Binzhou 256603, China Correspondence should be addressed to Jiwei Guo; guojiwei0510@163.com Received 5 July 2018; Revised 19 October 2018; Accepted 19 November 2018; Published 1 January 2019 Academic Editor: Reza Izadpanah Copyright © 2019 Yan Wu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objectives. Cisplatin (DDP) is one of the most commonly used chemotherapeutic drugs for several cancers, including non-small- cell lung cancer (NSCLC). However, resistance to DDP eventually develops, limiting its further application. New therapy targets are urgently needed to reverse DDP resistance. Methods. eTh mRNA expression of UBE2C, ZEB1/2, ABCG2,and ERCC1 was analyzed by reverse transcription-polymerase chain reaction. The protein levels of these molecules were analyzed by Western blotting and immunouo fl rescent staining. Cell proliferation was detected by CCK8 and MTT assays. Cell migration and invasion were analyzed by wound healing assay and Transwell assays. Promoter activities and gene transcription were analyzed by luciferase reporter assay. Results. In this study, we examined the effect of UBE2C and ZEB1/2 expression levels in DDP-resistant cells of NSCLC. We confirmed that aberrant expression of UBE2C and ZEB1/2 plays a critical role in repressing the DDP sensitivity to NSCLC cells. Additionally, knockdown of UBE2C significantly sensitize d resistant cells to DDP by repressing the expression of ZEB1/2. Mechanistic investigations indicated that UBE2C transcriptionally regulated ZEB1/2 by accelerating promoter activity. This study revealed that ZEB1/2 promotes the epithelial mesenchymal transition and expression of ABCG2 and ERCC1 to participate in UBE2C-mediated NSCLC DDP-resistant cell progression, metastasis, and invasion. Conclusion.UBE2C maybeanovel therapy target for NSCLC for sensitizing cells to the chemotherapeutic agent DDP. 1. Introduction UBE2C, also known as UBCH10, is an important mem- ber of the ubiquitin-conjugating enzyme family. UBE2C Lung cancer is very common and one of the leading causes of specifically interacts with the anaphase-promoting com- cancer mortality worldwide [1, 2]. Lung cancer is divided into plex/cyclostome (APC/C). er Th e are more than 55 substrates two histopathological groups: small-cell lung cancer (SCLC) degraded by APC/C, including 37 substrates involved in and non-small cell lung cancer (NSCLC). NSCLC accounts cell cycle phase S and M (cyclin A, cyclin B, p21, and for 80–85% of all lung cancer cases and is oen ft diagnosed securin), 11 substrates that are proteins related to the cell at locally advanced stages which are not amenable to sur- cycle (E2-C, E2F1, JNK, Skp2), and two substrates which gical resection [3, 4]. Cisplatin (DDP)-based chemotherapy are APC/C co-activated factors (CDC20 and Cdh1) [9–12]. has been widely applied to treat many type cancers in UBE2C plays a principle role in cell cycle progression and was the clinic, including NSCLC. In NSCLC patients, cisplatin recently found to be aberrantly expressed in various cancers generally shows good therapeutic effects in the early stage of including lung cancer, ovarian cancer, bladder cancer, and chemotherapy, but drug resistance seriously limits the further lymphoma [13–16]. Moreover, a recent study showed that application of cisplatin [5–8]. Therefore, new therapeutic UBE2C, as a regulatory factor of its target genes, promotes targets to reverse DDP-resistance are urgently needed. tumor occurrence and development in many human cancers. 2 Journal of Oncology Furthermore, decreased UBE2C expression enhances the 2.2. Cell Transfection. For cell transfection, recombinant chemosensitivity of dual drug-resistant breast cancer cells to pcDNA3.1 plasmid contained UBE2C, ZEB1 or ZEB2, UBE2C epirubicin and docetaxel [17], suggesting that UBE2C plays small interfere RNA (siUBE2C, GenePharma Co. Ltd., Shang- an important role in drug resistance. hai, China), ZEB1 small interfere RNA (siZEB1, GenePharma The zinc-finger E-box binding homeobox (ZEB) family Co. Ltd., Shanghai, China), ZEB2 small interfere RNA comprises sequence specific DNA-binding transcription fac- (siZEB2, GenePharma Co. Ltd., Shanghai, China), and neg- tors and two members: ZEB1 and ZEB2 [18]. The lix-loop- ative control oligonucleotides (NCO, GenePharma Co. Ltd.) helix motif of ZEB1 and ZEB2 has high specific binding were introduced into the A549, H1299, A549/DDP and activity with bipartite E-boxes in the E-cadherin promoter H1299/DDP cells by using lipofectamine 2000 (Invitrogen) region [19]. In NSCLC, ZEB1 expression is upregulated by according to the manufacturer’s instructions. eTh selected cyclooxygenase-2, which decreases E-cadherin gene tran- sequences for knockdown as follows: scription [20]. It is clear till the expression level of E-cadherin 󸀠 󸀠 siUBE2C were 5 -CCUGCAAGAAACCUACUCA-3 , and ZEB1 were signicfi antly correlated with sensitivity of 󸀠 󸀠 getfi inib, suggesting that they are useful for predicting to siZEB1 were 5 -GGAUCAACCACCAAUGGUU-3 , the sensitivity to epidermal growth factor receptor-tyrosine 󸀠 󸀠 siZEB2 were 5 -CGAGAUAUGUAAACUAAGGA-3 . kinase inhibitor therapy in lung cancer [21]. Furthermore, ZEB1 plays an important role in the resistance to chemother- 2.3. Immunofluorescent Staining. For analysis the protein apy drugs, such as paclitaxel [22], gefitinib [23], and tamox- levels of UBE2C, ZEB1, and ZEB2, A549 and A549/DDP cells ifen [24]. Abnormal expression of E-cadherin and ZEB1/2 were grown on coverslips in a 24-well plate overnight and results in epithelial mesenchymal transition (EMT), stem- aer ft 24 h, transfected with plasmid or siRNA and treated like cell character, resistance to therapeutic agents, and cancer with or without DDP. Aeft r 48 h, cells were fixed in 4% progression [25]. However, the relationship between ZEB1/2 formaldehyde for 30 min and permeabilized by incubation in and DDP resistance in NSCLC remains unclear. 3% BSA in PBS for 30 min. eTh coverslips were subsequently Various genes have been suggested as biomarkers of the incubated with UBE2C, ZEB1, and ZEB2 antibody at 1:1000 resistance to chemotherapeutic agents, such as ERCC1 [26, dilution in PBS containing 3% BSA. Secondary uo fl rescence 27] and ABCG2 [28, 29]. Classic chemotherapeutic drugs, antibodies at 1:1000 dilution in PBS are containing 3% BSA. such as platinum salts, are known to kill tumor cells by DAPI (3𝜇g/mL) was used for nuclear staining. Images were directly reducing DNA integrity [30]. Excision repair cross- obtained with Zeiss Axio Imager Z1 Fluorescent Microscope. complementary gene 1 (ERCC1) is an important member The detailed experiment process was described in Guo et al. oftheDNArepair-relatedgene system andcounteracts the [4]. DNA damaging eeff cts of chemotherapy and therefore is asso- ciated with drug resistance. ATP-binding cassette subfamily 2.4. RNA Isolation and RT-PCR. We used Trizol reagent G member 2 (ABCG2) was first cloned from multidrug- (TransGen Biotech, Beijing, China) to isolate total RNA from resistant breast cancer cell lines and conrfi med to be involved the samples and cells. RNA was reverse transcribed into rst fi - in the resistance to many chemotherapeutic agents, such as strand cDNA using a TransScript All-in-One First-Strand mitoxantrone, topotecan, and SN-38 [31–34]. ABCG2 was cDNA Synthesis Kit (TransGen Biotech). cDNAs were used reported to play an important role in stem cell biology [35]. in RT-PCR and qPCR assay with the human GAPDH gene as In this study, we aimed to examine the expression of UBE2C an internal control. The final qPCR reaction mix contained and ZEB1/2 in DDP-resistant NSCLC cell lines and the role 10𝜇lBestar  SYBR Green qPCR Master Mix. Amplification of UBE2C in mediating the resistance of A549/DDP and was performed as follows: a denaturation step at 94 Cfor H1299/DDP cells to DDP. 5 mins, followed by 40 cycles of amplification at 94 Cfor ∘ ∘ 30 sec, 58 Cfor 30sec,and72 C for 30 sec. eTh reaction was stopped at 25 C for 5 mins. eTh relative expression levels 2. Materials and Methods were detected and analyzed by ABI Prism 7900HT/FAST −ΔΔct 2.1. Cell Lines and Culture. HBEC, A549, H1299, Calu6, and (Applied Biosystems, USA) based on the formula of2 . H460 cell lines were obtained from American Type Culture We got the images of RT-PCR by Image LabTM Software Collection (ATCC; Manassas, VA) and maintained in RPMI (ChemiDocTM XRS+, BiO-RAD), and these images were media supplemented with 10% FBS (FBS; Hyclone, USA), TIF with reversal color format. eTh RT-PCR primers were as 10 mM of glutamic acid, and 1% penicillin/streptomycin follows. The reverse PCR primers are as follows: (normal media). The cisplatin-resistant subline, A549/DDP, was a gift from the Resistant Cancer Cell Line (RCCL) UBE2C forward primer: 5 -GGATTTCTGCCTTCC- collection (http://www.kent.ac.uk/stms/cmp/RCCL/RCCLa- CTGAA-3 , bout.html). Another cisplatin-resistant subline, H1299/DDP, UBE2C reverse primer: 5 -GATAGCAGGGCGTGA- had been established in our laboratory in 2016 by adapting GGAAC-3 , the growth of H1299 cells in the presence of increasing ZEB1 forward primer: 5 -GATGATGAATGCGAG- concentrations of cisplatin until a n fi al concentration of TCAGATGC-3 , 12𝜇g/ml, followed by cultivation in RPMI-1640 medium supplemented with 10% FBS additionally contained 2𝜇g/ml ZEB1 reverse primer: 5 -CTGGTCCTCTTCAGG- cisplatin. TGCC-3 , Journal of Oncology 3 󸀠 󸀠 ZEB2 forward primer: 5 -CTCTTCCCACACGCT- ZEB1 reverse primer: 5 - CAACCGTGGGCACTG- 󸀠 󸀠 TAGTT-3 , CTGAA-3 󸀠 󸀠 ZEB2 reverse primer: 5 -GGCCTAAGCTTACAG- ZEB2 forward primer: 5 - TTGGTGTACCAAGAG- 󸀠 󸀠 GC-3 TGTCATG-3 , E-cadherin forward primer: 5 -ACCATTAACAGG- ZEB2 reverse primer: 5 - CAACCCTGAAACAGA- 󸀠 󸀠 AACACAGG-3 , GG-3 ABCG2 forward primer: 5 -TCAGGCTAGCAA- E-cadherin reverse primer: 5 -CAGTCACTTTCA- GTGTGGTG-3 , GCATCCACTTTCTCAGA-3 ABCG2 reverse primer: 5 -TTATAAGCTTCAGGC- Vimentin forward primer: 5 -CGCCAACTACAT- AGCGCTGACACGAA-3 ’ CGACAAGGTGC-3 , ERCC1 forward primer: 5 -GGGTCTGATTGAGAT- Vimentin reverse primer: 5 -CTGGTCCACCTG- TTTGGGTC-3 CCGGCGCAG-3 , ERCC1 reverse primer: 5 -CCTTGTAAAACGTTG- GAPDH forward primer: 5 -CTCCTCCTGTTC- CCTTCACT-3 GACAGTCAGC-3 , GAPDH reverse primer: 5 -CCC AAT ACG ACC 2.8. SA-𝛽-Gal Staining. SA-𝛽-gal was detected using the AAA TCC GTT-3 . Senescence𝛽-Galactosidase Staining kit (C0602; Beyotime) following the manufacturer’s instructions: In brief, the cells 2.5. Western Blot. For western blotting analysis, proteins were washed twice with PBS and then xe fi d with PBS con- of each group cells were denatured at 100 Cboiled5min taining 2% formaldehyde and 0.2% glutaraldehyde for 10 min. with SDS loading buffer. The proteins were transferred ec Th ellswerethenincubated at 37 Cfor 12hwithstaining to PVDF transfer membrane. Membranes were incubated solution. Aer ft being washed twice with PBS, the SA- 𝛽-gal- with the indicated antibodies overnight at 4 C followed positive cells were observed under an optical microscope by immunoblotting analysis. Proteins were detected using (IX53; Olympus) and assessed using the ImageJ software. eTh enhanced chemiluminescence detection reagents (Amer- detailed experiment process was described in Jin et al. [36]. sham). Tubulin was internal control. The primary antibodies used in this study were 1:1000 rabbit anti-ABCG2 and ERCC1 2.9. Wound-Healing Assay. Cells were seeded in 6-well plats (Santa Cruz, Dallas, TX, USA), 1:1000 antibody of Tubulin, for 24 h. Cells were wounded by 200P micropipette tip, then UBE2C, ZEB1, ZEB2, vimentin, E-cadherin, and cleaved washed by PBS, and incubated in RPMI containing 2% FBS capase-3 (Abcam, Cambridge, UK). The gray intensity anal- with various DDP and/or relevant plasmid for different times. ysis of western blotting images was carried out by ImageJ Images were captured at thetimepointsof0and36h aeft r software. wounding.Therelativedistanceofthescratcheswasobserved under an optical microscope (IX53, Olympus, Tokyo, Japan) 2.6. CCK-8 Analysis. Cells were seeded in 96-well plates andassessedusing theImageJsoftware. with 5000cells/well andincubated for24h andthentreated with DDP or/and transfected plasmid and siRNA mimics 2.10. Transwell Assay. The cells (1x10 ) were seeded into for indicated time. Add 10𝜇l of CCK-8 (C0037, beyotime) 300𝜇l serum-free DMEM medium in the upper chamber solution to each welloftheplate andthenincubatetheplate (with 8-𝜇m pore size Transwell inserts (Corning, USA)) for 4 hours in the incubator. Measure the absorbance at which was coated with Matrigel.10% FBSDMEMwas 450 nm using a microplate reader (Infinite  F50; Tecan Group added to the lower chamber. eTh cells were cultured for Ltd., Manne ¨ dorf, Switzerland). 48 h. The cells remaining on the upper surface of the membrane were erased and the chambers were fixed with 2.7. Luciferase Reporter Assay. ZEB1/2 promoter region was 4% paraformaldehyde. eTh chambers were stained with 0.1% cloned into the pGL3 vector (Promega). For the luciferase crystal violet solution for 20 min (cat. no. E607309; Sangon assay, A549 and H1299 cells were cotransfected with ZEB1/2 Biotech Co., Ltd., Shanghai, China) and washed with PBS promoter-pGL3 vector and UBE2C vector by using lipofec- and then photographed by Olympus light microscope (IX53, tamine 2000. 48 h later, luciferase reporter activities were Olympus, Tokyo, Japan). eTh detailed experiment process measured by using a Dual Luciferase Reporter Assay Kit was described in Guo et al. [37]. (Promega). The PCR primers were 2.11. Clonal Formation Assay. Cells were seeded into 12-well UBE2C forward primer: 5 - GATATGAACCTGTGT- with 300cells perwelland cultured for24hours followed TGT-3 by being exposed to drugs for another 24 hours. Cells were UBE2C reverse primer: 5 - GGCTCGGCTCAGCTC- then cultured in drug-free medium for another 10 to 15 days CTTTACGG-3 until clones of around 50 cells were formed. Cell clones were ZEB1 forward primer: 5 - GAAACCAGGCGTCCC- fixed with 4% paraformaldehyde for 0.5 hour and stained with TGG-3 crystal violet (Sangon) for 15 min before optical imaging. 4 Journal of Oncology 2.12. Human Lung Cancer Specimen Collection. Atotal of cell proliferation, we detected cell growth by CCK8 assay. Our 50 human lung cancer (NSCLC) with their corresponding results indicated that the IC values of DDP were 12.8, 8.5, normal lung specimens and a total of 40 NSCLC subjects and 4.9𝜇g/mL in A549 cells and 13.2, 10.1, and 5.4 𝜇g/mL in received anthracyclines-based neoadjuvant chemotherapy H1299 cells aer ft incubation for 24, 48, and 72 h, respectively werecollectedinAffiliatedHospitalofBinzhou Medical (Figures 1(b) and S1A). Similarly, the cellular growth was College with written consent of patients and the approval dose-dependently inhibited by DDP in A549 and H1299 from the Institute Research Ethics Committee. cells (Figures 1(b) and S1B). Moreover, DDP dose- (Figures 1(c)–1(e)) and time-dependent (Figures 1(f)–1(h)) reduced 2.13. Immunohistochemical Analysis. Tumor tissues were UBE2C andZEB1/2mRNAand proteinlevelsinA549and fixed in 4% paraformaldehyde overnight and then embedded H1299 cells according to the RT-PCR, western blotting, and in paraffin wax. Four-micrometer thick sections were and qPCR assay results. Furthermore, the cellular survival rate stainedusing hematoxylinandeosin(H&E)forhistological of DDP-resistant cells (A549/DDP and H1299/DDP) was analysis. signica fi ntly higher than that of their parent cells (A549 and H1299) (Figure 1(i)) following DDP treatment. The 2.14. Ethics Approval and Consent to Participate. The experi- RT-PCR, western blotting, and qPCR results revealed that mental protocol was approved by the Research Ethics Com- the mRNA and protein levels of ERCC1 and ABCG2 were mittee of Binzhou Medical University, China (No. 2017-016- significantly increased in DDP-resistant cells compared to 01 for human lung cancer specimen and No. 2017-009-09 in their parent cells (Figures 1(j)–1(l)). es Th e results indicate for mouse experiments in vivo) and the written informed that the expression of UBE2C and ZEB1/2 was higher in lung consent was obtained from all subjects. Informed consent cancer cells and downregulated by treatment with DDP. was obtained from all individual participants included in the study. All patients were staged based on the criteria of the 7th 3.2. UBE2C and ZEB1/2 Are Involved in DDP Resistance in Edition of the AJCC Cancer Staging Manual: Stomach (2010) Lung Cancer Cells. To further explore the underlying role of UBE2C and ZEB1/2 in DDP-resistant NSCLC cells, the 2.15. Spearman’s Rank Correlation Analysis. To examine the transcriptional activity of UBE2C and ZEB1/2 was analyzed correlation between the relative expression levels between by luciferase reporter assay. Our results showed that their ZEB1/2 and ABCG2/ERCC1, we first performed a normality transcriptional activities were higher in A549/DDP and test, which indicated that there was a correlation between H1299/DDP cells than in their parent cells (Figure 2(a)). them and that the data were nonparametric. eTh correla- The mRNA and protein levels of UBE2C and ZEB1/2 were tion of the relative expression levels between ZEB1/2 and also higher in A549/DDP and H1299/DDP cells than in ABCG2/ERCC1 was then assessed using Spearman’s rank DDP-sensitive cells by RT-PCR, immunoblotting, qPCR, correlation coefficient based on the results of western blot and immunouo fl rescent assays (Figures 2(b)–2(e)). We also analysis. observed that the cell morphology was dramatically altered, with shape appearing from flat to shuttle type with apiciform 2.16. Statistical Analysis. Each experiment was repeated at pseudopodium, in A549/DDP cells and H1299/DDP cells least three times. Data were analyzed using GraphPad Prism compared to their parent cells, A549 and H1299 cells, which 5 (GraphPad, La Jolla, CA, USA) and are presented as the contribute to cell drug resistance, migration, and invasion means± SD. eTh statistical analyses of the experiment data (Figure 2(f)). Moreover, the immunohistochemistry assay were performed by using a two-tailed Student’s paired T-test indicated that the protein levels of UBE2C and ZEB1/2 were and one-way ANOVA. Statistical significance was assessed obviously increased in human DDP-resistant lung cancer at least three independent experiments and significance was tissues compared to in nonresistant lung cancer tissues considered at either p-value< 0.05 was considered statisti- (Figures 2(g) and 2(h)). Together, these results suggest that cally significant and highlighted an asterisk in the figures, UBE2C and ZEB1/2 were overexpressed in DDP-resistant while p-values< 0.01 were highlighted using two asterisks NSCLC cells and tissues. and p-values< 0.001 highlighted using three asterisks in the figures. 3.3. UBE2C Upregulates the Expression of ZEB1/2 in DDP- Resistant NSCLC Cells. After demonstrating that both of UBE2C and ZEB1/2 expression levels were downregulated 3. Results by DDP treatment, we examined the relationship between 3.1. UBE2C and ZEB1/2 Were Abnormally Activated in Lung UBE2C and ZEB1/2 using specific siRNAs or plasmids to Cancer Cells and Were Downregulated by Treatment with silence or overexpress UBE2C (Figure 3(a)), ZEB1 (Fig- DDP. To investigate the roles of UBE2C and ZEB1/2, we first ure 3(b)), or ZEB2 (Figure 3(c)) in A549/DDP cells. examined the endogenous mRNA and protein expression of For UBE2C silencing, cells were transfected with siRNA UBE2C and ZEB1/2 in human lung cancer cells by reverse (siUBE2C-1 and siUBE2C-2) and UBE2C was overexpressed transcription-polymerase chain reaction (RT-PCR), western using pcDNA-UBE2C. We found that more UBE2C was blotting, and qPCR analysis. We found that UBE2C mRNA knocked down using siUBE2C-2 (Figures S2A, S2D, and and protein levels were higher in human lung cancer cells S2G), and thus this siRNA was used in these experiments. than in normal control human bronchial epithelial cells Similar results were obtained when siZEB1-1 or siZEB2-2 were (Figure 1(a)). To further examine the effect of DDP on A549 used to knock down ZEB1 or ZEB2, respectively (Figures S2B, Journal of Oncology 5 ∗∗ ∗∗ ∗∗ ∗∗ 1.2 3 1.2 UBE2C UBE2C WB 0.8 2 0.8 ZEB1 WB 0.6 0.6 ZEB1 1 0.4 0.4 ZEB2 WB ZEB2 0.2 0.2 Tubulin WB 0 0 0 0369 12 GAPDH 024 h 36 h48 h60 h Time g/ml 24 h UBE2C ZEB1 ZEB2 3 g/ml 48 h g/ml 72 h g/ml (a) (b) DDP (g/ l) DDP (g/ l) DDP (g/ l) DDP (g/ l) 1.2 1.2 13 9 0.9 13 9 13 9 ∗∗ 13 9 0.9 ∗∗ UBE2C UBE2C UBE2C WB UBE2C WB 0.6 0.6 ZEB1 ZEB1 ZEB1 WB ZEB1 WB ∗∗ ∗∗ 0.3 0.3 ZEB2 WB ZEB2 ZEB2 ZEB2 WB Tubulin WB GAPDH GAPDH Tubulin WB 0 0 DDP A549 DDP H1299 H1299 A549 A549 H1299 UBE2C UBE2C ZEB1 ZEB1 ZEB2 ZEB2 (c) (d) (e) 1.2 DDP (g/ l) 1.2 DDP (g/ l) DDP (g/ l) DDP (g/ l) ∗∗ ∗∗ 0.9 0.9 UBE2C WB UBE2C WB UBE2C UBE2C 0.6 0.6 ZEB1 WB ZEB1 WB ∗∗ ZEB1 ∗∗ ZEB1 0.3 0.3 ZEB2 WB ZEB2 WB ZEB2 ZEB2 Tubulin WB Tubulin WB 0 GAPDH GAPDH 0 0h 24h 48h DDP DDP 0h 24h 48h A549 H1299 A549 H1299 A549 H1299 UBE2C UBE2C ZEB1 ZEB1 ZEB2 ZEB2 (g) (f) (h) ∗∗ 1.6 ∗∗ 1.4 1.4 1.2 1.2 1 1 ABCG2 ABCG2 WB 0.8 0.8 0.6 0.6 ERCC1 ERCC1 WB 0.4 0.4 0.2 0.2 Tubulin WB GAPDH 0 0 control DDP control DDP ABCG2 ERCC1 (j) (i) (k) (l) Figure 1: Aberrant activation of UBE2Cand ZEB1/2in lung cancer cells anddownregulated by treatment with DDP. (a) Gel-based RT- PCR, immunoblotting with densitometric quantitation and qPCR demonstrating elevated mRNA and protein expression of UBE2C in human lung cancer cells compared with their normal control cell HBEC. (b) A549 cells were incubated with DDP at various concentrations for 24, 48, and 72 h (left panel) and various times at 3, 6 and 9 𝜇g/ml (right panel). eTh n cell viability was assessed (%). (c–e) DDP dose-dependently repressed UBE2C and ZEB1/2 in mRNA and protein level in A549 and H1299 cells analyzed by RT-PCR (c), Western blot (d) and qPCR (e) assay. (f–h) DDP time-dependently repressed UBE2C and ZEB1/2 in mRNA and protein level in A549 and H1299 cells analyzed by RT-PCR (f), Westernblot(g)andqPCR(h) assay. (i)Survivalratesof A549/DDP,H1299/DDP andtheircontrolcellstreatedwithDDPat6𝜇g/ml for 48 h. (j–l) eTh mRNA and protein expression levels were analyzed by RT-PCR (j), Western blot (k) and qPCR (l) assay. Results were presented as mean± SD, and the error bars represent the SD of three independent experiments.∗p<0.05;∗∗p<0.01 versus control group. A549 HBEC H460 Calu6 g/ml 3g/ml H1299 1g/ml 1g/ml H1299/DDP A549 g/ml g/ml A549/DDP A549/DDP A549 H1299 H1299 H1299/DDP 48h 48h 48h 48h A549 24h 24h 24h 24h H1299 H1299 HBEC A549 H1299 Calu6 H460 HBEC A549 H1299 0h Calu6 H460 0h 0h 0h AH1299/DDP H1299/DDP A549/DDP A549/DDP A549 mRNA mRNA Relative OD value mRNA mRNA Relative OD value mRNA Protein mRNA Protein Protein Relative mRNA level Protein Protein Protein Relative OD value Relative mRNA level Relative mRNA level Relative mRNA level Relative OD value Relative mRNA level Relative mRNA level 6 Journal of Oncology 3.6 ∗∗ ∗∗ UBE2C Luc ZEB1 Luc 3.5 ZEB2 Luc 3 4.5 3.5 ∗∗ ∗∗ ∗∗ ∗∗ 2.4 ∗∗ 3 ∗∗ 3.5 2.5 2.5 1.8 2.5 2 1.2 UBE2C UBE2C 2 1.5 1.5 1.5 0.6 1 ZEB1 ZEB1 0.5 0.5 0.5 ZEB2 ZEB2 GAPDH GAPDH UBE2C ZEB1 ZEB2 (a) (b) (c) A549 A549/DDP Hoechst Merge Hoechst Merge ∗∗ ∗∗ ∗∗ 5 m UBE2C WB UBE2C WB ZEB1 WB ZEB1 WB Th ZEB2 WB ZEB2 WB UBE2C ZEB1 ZEB2 Tubulin WB Tubulin WB A549 A549/DDP (d) (e) A549 A549/DDP ∗∗ ∗∗ ∗∗ 10 10 10 UBE2C ZEB1 ZEB2 5 5 5 H1299 H1299/DDP Th Th Th 0 0 (f) (g) (h) Figure 2: UBE2Cand ZEB1/2were involved in DDP resistance in lung cancer cells. (a) eTh luciferase reporter assay indicated that the transcriptional activity of UBE2C, ZEB1, and ZEB2 was higher in A549/DDP and H1299/DDP cells than their parent cells. (b, c) RT-PCR (b) and qPCR (c) demonstrating elevated mRNA level of UBE2C, ZEB1, and ZEB2 in DDP NSCLC resistant cell lines, A549/DDP and H1299/DDP, compared with their parent cells. (d, e) Immunoblotting (d) and immunou fl orescent staining (e) assay demonstrating increased protein level of UBE2C, ZEB1, and ZEB2 in DDP NSCLC resistant cell lines, A549/DDP and H1299/DDP cells. (f) Cellular morphology of DDP NSCLC resistant cell lines and their parent cells was analyzed by phase contrast microscope assay. (g) Immunohistochemical staining shows that the protein levels of UBE2C, ZEB1, and ZEB2 were higher in human DDP resistance lung cancer tissues than in nonresistance cancer tissues with anthracyclines-based neoadjuvant chemotherapy. (h) Statistical analysis of the protein level of UBE2C, ZEB1, and ZEB2 in DDP non/resistance lung cancer tissues (n=20). Results were presented as mean± SD, and the error bars represent the SD of three independent experiments. ∗∗p<0.01 versus control group. S2E, and S2H and Figures S2C, S2F, and S2I). To determine UBE2C increased these levels, which were detected by RT- whether the ZEB1/2 expression level was regulated through PCR, western blotting, and qPCR assay (Figures 3(d) and UBE2C, we used a control (cotransfection of pcDNA vector 3(e)) and immunou fl orescent staining (Figures 3(f) and 3(g)). and si control), siUBE2C (cotransfection of siRNA of UBE2C To further explore whether ZEB1 and ZEB2 are the direct and pcDNA vector), and Flag-UBE2C (cotransfection of targets of UBE2C, we performed a luciferase reporter assay pcDNA Flag-UBE2C and si control) in the same experiment in A549/DDP and H1299/DDP cells. We cloned the promoter (Figures 3(d) and 3(e)). These approaches were also used of ZEB1 or ZEB2 into the dual luciferase reporter vector and for ZEB1 and ZEB2. Knockdown of UBE2C decreased the then transfected these vectors into cells. ZEB1/2 promoter mRNA and protein levels of ZEB1/2, while overexpression of activity was obviously reduced in the UBE2C knockdown H1299/DDP H1299/DDP H1299/DDP H1299/DDP A549 A549 A549 A549 A549/DDP A549/DDP /DD A549 P A549/DDP H1299 H1299 H1299 H1299 Resistance Resistance Resistance Non-resistance Non-resistance Non-resistance A549 A549 H1299 H1299 A549/DDP A549/DDP H1299/DDP AH1299/DDP Protein Relative luciferase activity Protein Relative luciferase activity Resistance Non-resistance Relative luciferase activity ZEB2 ZEB1 UBE2C mRNA e relative protein level of UBE2C mRNA e relative protein level of ZEB1 Relative mRNA level e relative protein level e relative protein level of ZEB2 ∗∗ ∗∗ control control si UBE2C si UBE2C UBE2C UBE2C Journal of Oncology 7 ∗∗ 3.5 4 ∗∗ 2.8 3 2.1 1.4 ZEB2 WB ZEB2 0.7 ∗ UBE2C WB ∗ UBE2C Tubulin WB 0 GAPDH Tubulin WB GAPDH (a) (e) 3.5 ∗∗ UBE2C (g/ ml) UBE2C (g/ ml) 4.8 2.8 2.1 ∗∗ 12 3 1 ZEB1 1.4 ZEB1 WB UBE2C 3.2 0.7 UBE2C WB GAPDH ∗ Tubulin WB 2.4 ZEB1 ZEB1 WB 1.6 ZEB2 ZEB2 WB (b) 0.8 GAPDH Tubulin WB ∗∗ UBE2C g/ml 3g/ml 9g/ml UBE2C ZEB1 ZEB2 ZEB2 ZEB2 WB (i) GAPDH Tubulin WB UBE2C (2g/ ml) UBE2C (2g/ ml) 4.2 ∗∗ 3.5 (c) 2.8 UBE2C UBE2C WB ∗∗ 2.1 ZEB1 WB ZEB1 1.4 ZEB2 WB ZEB2 0.7 ZEB1 ZEB1 WB Tubulin WB GAPDH 1 0 GAPDH Tubulin WB ∗ UBE2C 24h 48h 72h UBE2C ZEB1 ZEB2 (j) (d) UBE2C ZEB1 Hoechst Merge UBE2C ZEB2 Hoechst Merge ZEB1 luc 4 ZEB1 luc ZEB2 luc ZEB2 luc 5 4 ∗∗ ∗∗ ∗∗ ∗∗ 5m 4 5m 3 3 0 0 The relative protein A549/DDP H1299/DDP level of ZEB2 (h) (f ) (g) Figure 3: UBE2Cupregulates the expression of ZEB1/2in DDP-resistant NSCLC cells. (a–c) Overexpression and knockdown of UBE2C (a), ZEB1 (b), and ZEB2 (c) were examined by RT-PCR, Western blot, and qPCR in the A549/DDP cells. (d–g) RT-PCR, Western blot, qPCR, and immunou fl orescence staining assay demonstrating that knockdown or overexpression of UBE2C using siRNA and pcDNA3.1-UBE2C could decrease or increase the ZEB1in the A549/DDP cells (d, f) and ZEB2 (e, g) expression in A549/DDP cells. (i, j) RT-PCR and Western blot result show that UBE2C dose-and time-dependently increased the mRNA and protein level of ZEB1 and ZEB2 in the A549/DDP cells. (h) eTh transcriptional activity of ZEB1/2 regulated by UBE2C was analyzed by luciferase reporter assay. Results were presented as mean ± SD, and the error bars represent the SD of three independent experiments.∗p<0.05;∗∗p<0.01 versus control group. group but enhanced in the UBE2C overexpression group 3.4. Knockdown of UBE2C Sensitizes DDP-Resistant NSCLC (Figure 3(h)). Moreover, UBE2C increased the mRNA and Cells to Cisplatin via Decreasing the Expression of ZEB1/2. protein levels of ZEB1 and ZEB2 in dose- and time-dependent To confirm whether UBE2C promotes cell proliferation by manners in A549/DDP cells (Figures 3(i) and 3(j)). Collec- targeting ZEB1/2, we cotransfected UBE2C and siZEB1 or tively, these data indicate that ZEB1 and ZEB2 expression was siUBE2C and ZEB1 into A549/DDP cells. We also sepa- upregulated by UBE2C in DDP-resistant NSCLC cells. rately transfected UBE2C or siUBE2C into A549/DDP cells The relat ive protein level of ZEB1 control control control siUBE2C UBE2C UBE2C UBE2C control control siZEB1 ZEB1 siZEB2 ZEB2 siUBE2C siUBE2C Control Control ZEB1 si ZEB2 ZEB2 Control ZEB1 si ZEB2 ZEB2 Control Control Control Control Control Control si UBE2C si UBE2C UBE2C UBE2C si ZEB1 si ZEB1 si UBE2C UBE2C si UBE2C UBE2C si UBE2C UBE2C si UBE2C UBE2C Control 48 h 72 h 48 h 72 h 24 h 24 h siUBE2C Control UBE2C mRNA mRNA mRNA mRNA Protein Protein Protein Protein UBE2C siUBE2C Control Relative mRNA level Relative mRNA level Relative mRNA Relative mRNA level level of ZEB1 mRNA mRNA mRNA Relative luciferase activity Protein Protein Protein control si UBE2C UBE2C Relative luciferase activity control si UBE2C UBE2C Relative mRNA Relative luciferase activity level of ZEB2 Relative mRNA level Relative mRNA level control si UBE2C UBE2C Relative luciferase activity control si UBE2C UBE2C 8 Journal of Oncology and conducted RT-PCR, western blotting, and qPCR to of ZEB1/2 inhibited UBE2C-mediated cell migration and detect UBE2C and ZEB1. The ZEB1 level was increased by invasive growth of DDP-resistant NSCLC cells, we examined UBE2C transfection and decreased by siUBE2C. However, whether UBE2C regulates EMT marker proteins via ZEB1/2. the opposite effects were observed for each of these factors We cotransfected siUBE2C and ZEB1/2 or UBE2C and in A549/DDP cells ectopically transfected with siZEB1 or siZEB1/2 into cells and measured E-cadherin and vimentin overexpression of ZEB1 (Figure 4(a)). The protein expression mRNA and protein levels. RT-PCR, western blotting, and of ZEB2 showed a similar result as ZEB1 (Figure 4(b)). To qPCR assays indicated that the E-cadherin level was increased furtherinvestigatetheroleofUBE2CandZEB1/2inDDP- by siUBE2C transfection and decreased by UBE2C over- resistant cell proliferation, A549/DDP and H1299/DDP cells expression, while the opposite effects were observed for were cotransfected with UBE2C and siZEB1/2 or siUBE2C each of these factors in A549/DDP cells ectopically trans- and ZEB1/2. As shown in Figures 4(c) and 4(e), the CCK- fected with ZEB1/2 or siZEB1/2. In contrast, vimentin was 8 assay revealed that the cell proliferation capacity was decreased by siUBE2C transfection and increased by UBE2C markedly increased in A549/DDP and H1299/DDP cells overexpression, while the opposite effects were observed by overexpression of UBE2C and ZEB1/2, but drastically for each of these factors in A549/DDP cells ectopically decreased by introduction of siUBE2C or siZEB1/2. As transfected with ZEB1/2 or siZEB1/2 (Figures 5(e), 5(f), S3A, ZEB1/2 was regulated by UBE2C, we explored whether and S3B). Similar RT-PCR and western blotting results of knockdown of UBE2C inhibited cell proliferation in DDP- E-cadherin and vimentin were obtained for H1299/DDP resistant cells following treatment with DDP. A549/DDP and cell lines cotransfected with UBE2C and ZEB1/2 (data not H1299/DDP cells were cotransfected with UBE2C and siZEB1 shown). es Th e results suggest that knockdown of ZEB1/2 or siUBE2C and ZEB1 and then treated with DDP. Our data inhibits UBE2C-dependent cellular growth and invasiveness showed that resistance to DDP induced by UBE2C overex- and UBE2C-mediated EMT progress by promoting ZEB1/2 pression was remarkably decreased when ZEB1 or ZEB2 was expression in DDP-resistant NSCLC cells. knocked down in A549/DDP and H1299/DDP cells (Figures 4(d) and 4(f)). In contrast, overexpression of ZEB1 or ZEB2 3.6. UBE2C Increases ABCG2 and ERCC1 Expression via Reg- rescued the cell growth inhibited by knockdown of UBE2C in ulating ZEB1/2 in DDP-Resistant NSCLC Cells. To determine DDP-resistant NSCLC cells treated with DDP. es Th e results the molecular mechanism by which UBE2C and ZEB1/2 were conrfi med by Ki67 immunohistochemistry staining reverses DDP resistance in lung cancer cells, we conducted in A549/DDP cells treated with DDP (Figure 4(g)). The RT-PCR to confirm whether ZEB1/2 regulates the drug protein level of active caspase-3 determined by western blot resistance genes HER2, MRP1, KRAS, BRCA1, and MDR1 assay showed that treatment with DDP significantly induced in A549/DDP cells. However, ZEB1/2 did not regulate the apoptosis in A549/DDP cells transfected with siUBE2C, expression of these genes in A549/DDP cells according to our and this process was reversed by overexpression of ZEB1/2 RT-PCR and qPCR assays (Figures S4A and S4B). We con- (Figure 4(h)). Analysis of protein expression of Annexin V ducted RT-PCR, western blotting, and qPCR assays to further showedsimilarresultsforactivecaspase-3 by immunoufl o- investigate whether UBE2C, ZEB1, and ZEB2 regulate the rescent staining assay (Figure 4(i)). es Th e data indicate that drug resistance genes ABCG2 and ERCC1. eTh mRNA and knockdown of UBE2C sensitizes DDP-resistant NSCLC cells protein levels of ABCG2 and ERCC1 were measured at 48 h to cisplatin by decreasing the expression of ZEB1/2. aer ft UBE2C and ZEB1/2 transfection. The results showed that the mRNA and protein levels of ABCG2 and ERCC1 3.5. Knockdown of ZEB1/2 Inhibits UBE2C-Dependent Cellular were significantly upregulated aeft r UBE2C, ZEB1, and ZEB2 Growth, Invasiveness, and EMT in DDP-Resistant NSCLC overexpression. These inhibitory effects were suppressed Cells. Recent studies showed that UBE2C not only promotes by downregulation of UBE2C, ZEB1, and ZEB2 expression cell proliferation, but also is positively correlated with metas- (Figures 6(a)–6(c) and S3C–S3E). The immunoblotting assay tasis in many types of cancer cells [38, 39]. We showed that indicated that ZEB1 and ZEB2 increased ABCG2 and ERCC1 UBE2C regulates ZEB1/2 expression to promote NSCLC cell protein levels in a dose-and time-dependent manner in resistance to DDP. To further investigate whether UBE2C- A549/DDP cells (Figures 6(d) and 6(e)). Similar results were regulated ZEB1/2 expression aeff cts DDP-resistant cell senes- obtained by western blot analysis of the H1299/DDP cell cence, invasiveness, and EMT in DDP-resistant NSCLC cells, lines (data not shown). Moreover, the ABCG2/ERCC1 pro- we cotransfected UBE2C and siZEB1 or siUBE2C and ZEB1 moter and ZEB1 cotransfection into A549/DDP cells resulted into A549/DDP cells and then treated the cells with DDP and in significantly increased luciferase activity compared to conducted colony formation (Figure 5(a)), SA-𝛽-gal staining cotransfection with the control vector. Compared to the (Figure 5(b)), wound-healing (Figure 5(c)), and Matrigel control group, luciferase activity was decreased following invasion assays (Figure 5(d)). We found that overexpression cotransfection with siZEB1. Similar results were obtained of UBE2C dramatically increased colony formation ability aer ft cotransfection with the ABCG2/ERCC1 promoter and (Figure 5(a)), inhibited cell senescence (Figure 5(b)), and pro- ZEB2 or siZEB2 (Figure 6(f)). Furthermore, Spearman’s rank moted cell migration (Figure 5(c)) and invasion (Figure 5(d)), correlation analysis revealed significant positive correlations which were partially inhibited by knockdown of ZEB1. between ZEB1/2 and ABCG2/ERCC1 protein levels based on Cotransfection of UBE2C and siZEB2 or siUBE2C and ZEB2 western blot assays (Figure 6(g)). siUBE2C inhibited DDP- into A549/DDP cells showed similar results as cotransfection resistant NSCLC cell proliferation, migration, invasion, and of UEE2C and ZEB1 (data not shown). Because knockdown EMT, which contributed to reversing the DDP resistance Journal of Oncology 9 DDP 4.8 DDP 3.2 #∗∗ ∗∗ 2.4 1.6 #∗∗ 0.8 UBE2C UBE2C WB ∗ ZEB1 ZEB1 WB GAPDH Tubulin WB DDP UBE2C ZEB1 (a) DDP DDP 6 5 ∗∗ ∗∗ ∗∗ ∗∗ #∗∗ ∗∗ 4 3.2 2.4 1.6 UBE2C WB #∗∗ UBE2C 1 ∗ 0.8 ∗ ∗ ∗ ∗ ZEB2 WB ZEB2 0 0 Tubulin WB GAPDH A549/DDP DDP UBE2C ZEB2 (b) (c) 4 6 7 ∗∗ 3 4 ∗∗ ∗∗ 6 ∗∗ ∗∗ ∗∗ 5 ∗∗ 5 3 ∗∗ #∗∗ #∗∗ #∗∗ 2 NS 3 NS #∗∗ 1 NS #∗∗ 1 NS ∗ 1 #∗∗ #∗∗ 1 #∗∗ ∗ ∗ ∗ ∗ ∗ 1 ∗ ∗ 0 0 0 0 H1299/DDP DDP DDP DDP DDP A549/DDP H1299/DDP (d) (e) (f) DDP Annexin V PI Merge ∗∗ DDP Caspase-3 cleaved WB ∗∗ Control Si UBE2C Si UBE2C+ZEB1 UBE2C UBE2C+siZEB1 Tubulin WB #∗∗ #∗∗ 4 2 ∗∗ NS 3 #∗∗ 5m 1 #∗∗ NS #∗∗ #∗∗ DDP DDP DDP (g) (h) (i) Figure 4: Knockdownof UBE2C sensitizes DDP-resistant NSCLC cells to cisplatin via decreasing the expression of ZEB1/2.A549/DDP or H1299/DDP cells were transfected with UBE2C or siUBE2C. ZEB1 or ZEB2 were used for upregulated the protein level of UBE2C target genes and then treated with DDP 6𝜇g/ml for 48 h, respectively. (a, b) eTh mRNA and protein expression levels of UBE2C and ZEB1 (a) or UBE2C and ZEB2 (b) were analyzed by RT-PCR and immunoblotting assay. (c–f) The cellular growth was analyzed by CCK8 assay with or without treatment of DDP at 6𝜇g/ml for 48 h in A549/DDP (c, d) or H1299/DDP (e, f) cells. (g) eTh protein of Ki67 was analyzed by immunouo fl rescent staining. (h) eTh protein of active caspase-3 was analyzed by Western blot. (i) eTh protein of Annexin V was analyzed by immunouo fl rescent staining. Results were presented as mean ± SD, and the error bars represent the SD of three independent experiments. ∗p<0.05;∗∗p<0.01 versus control group. PBS PBS PBS PBS Si UBE2C Si UBE2C Si UBE2C Si UBE2C Control Control Control Control Si UBE2C+ZEB1 Si UBE2C+ZEB2 Si UBE2C+ZEB1 Si UBE2C+ZEB2 PBS UBE2C+siZEB1 UBE2C+siZEB2 UBE2C+siZEB1 UBE2C+siZEB2 control UBE2C UBE2C UBE2C UBE2C siUBE2C siUBE2C+ZEB2 UBE2C UBE2C+siZEB2 control siUBE2C siUBE2C+ZEB1 UBE2C UBE2C+siZEB1 Control Si UBE2C UBE2C si ZEB1 Control ZEB1 Si UBE2C UBE2C si ZEB1 ZEB1 Control Si UBE2C UBE2C si ZEB2 Control ZEB2 Si UBE2C UBE2C si ZEB2 ZEB2 Control Si UBE2C Si UBE2C+ZEB1 UBE2C UBE2C+siZEB1 control siUBE2C siUBE2C+ZEB1 UBE2C UBE2C+siZEB1 Control Si UBE2C Si UBE2C+ZEB1 UBE2C UBE2C+siZEB1 PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS UBE2C+siZEB1 Control Control Control Si UBE2C Si UBE2C Si UBE2C+ZEB2 Si UBE2C+ZEB1 UBE2C UBE2C UBE2C+siZEB2 UBE2C+siZEB1 Control Control Si UBE2C Si UBE2C Si UBE2C+ZEB2 Si UBE2C+ZEB1 UBE2C UBE2C UBE2C+siZEB2 UBE2C+siZEB1 Si UBE2C UBE2C Si UBE2C+ZEB1 Hoechst Merge Ki67 Relative OD value mRNA Relative OD value mRNA Protein The relative protein level of Ki67 Relative OD value Protein Relative OD value Relative mRNA level The relative protein level Relative mRNA level Relative OD value Relative OD value UBE2C Si UBE2C + Si UBE2C UBE2C +ZEB1 Control Relative OD value Relative OD value The relative protein level of Annex V 10 Journal of Oncology ∗∗ DDP Control Si UBE2C+ZEB1 UBE2C Si UBE2C UBE2C+siZEB1 2 #∗∗ NS #∗∗ DDP (a) ∗∗ #∗∗ NS 1 #∗∗ DDP (b) DDP Control Si UBE2C Si UBE2C+ZEB1 UBE2C UBE2C+siZEB1 DDP (c) DDP DDP DDP Control Si UBE2C Si UBE2C+ZEB1 UBE2C UBE2C+siZEB1 ∗∗ UBE2C #∗∗ UBE2C NS ZEB1 #∗∗ ZEB2 Th 0 E-cadherin E-cadherin Vimentin Vimentin GAPDH GAPDH DDP Invasion Migration mRNA (d) (e) DDP DDP UBE2C WB UBE2C WB ZEB1 WB ZEB2 WB E-cadherin WB E-cadherin WB Vimentin WB Vimentin WB Tubulin WB Tubulin WB Protein (f) Figure 5: Knockdownof ZEB1/2 inhibits UBE2C-dependent cellular growth, invasiveness, and EMT in DDP-resistant NSCLC cells. (a) Colony formation assay demonstrating that ectopic expression of UBE2C significantly enhanced colony formation density, which was blocked by knockdown ZEB1 in A549/DDP cells with treatment of DDP at 6𝜇g/ml for 48 h. (b) SA-𝛽-Gal assay was performed to detect the cell senescence of A549/DDP cells treated with DDP at 6𝜇g/ml for 48 h and transfected with UBE2C, siUBE2C alone, or the combination of siZEB1/2 and ZEB1/2 plasmid for 48 h. (c, d) Scratch assay (c) and Matrigel invasion assay (d) indicated that UBE2C promote cell migration and invasion via regulating ZEB1 in A549/DDP cells with treatment of DDP at 6𝜇g/ml for 36 h. (e, f) UBE2C significantly decreased E- cadherin and increased vimentin in mRNA (e) and protein (f) levels by regulating ZEB1/2 in the A549/DDP cells with treatment of DDP at 6𝜇g/ml for 48 h. Results were presented as mean ± SD, and the error bars represent the SD of three independent experiments.∗p<0.05; ∗∗p<0.01 versus control group. PBS PBS PBS PBS Si UBE2C Si UBE2C Control Control Si UBE2C+ZEB1 Si UBE2C+ZEB1 Control Control Si UBE2C Si UBE2C Si UBE2C+ZEB1 Si UBE2C+ZEB1 UBE2C+siZEB1 UBE2C+siZEB1 UBE2C+siZEB1 UBE2C+siZEB1 UBE2C UBE2C UBE2C UBE2C PBS PBS PBS PBS PBS PBS PBS UBE2C+siZEB1 UBE2C+ iZEB2 UBE2C+siZEB1 UBE2C+siZEB2 Si UBE2C Si UBE2C Si UBE2C Si UBE2C UBE2C UBE2C UBE2C UBE2C Control Control Control Control Si UBE2C+ZEB1 Si UBE2C+ZEB2 Si UBE2C+ZEB1 Si UBE2C+ZEB2 Migration Invasion 36 h 0 h e relative cell number The relative cell number The relative cell number Journal of Oncology 11 UBE2C ZEB2 ZEB1 ABCG2 ABCG2 ABCG2 ERCC1 ERCC1 ERCC1 GAPDH GAPDH GAPDH ZEB1 WB UBE2C WB ZEB1 WB ABCG2 WB ABCG2 WB ABCG2 WB ERCC1 WB ERCC1 WB ERCC1 WB Tubulin WB Tubulin WB Tubulin WB (a) (b) (c) ZEB1 ZEB1 (2g) ZEB2 (2g) ZEB2 ZEB2 WB ZEB2 WB ZEB1 WB ZEB1 WB ABCG2 WB ABCG2 WB ABCG2 WB ABCG2 WB ERCC1 WB ERCC1WB ERCC1 WB ERCC1WB Tubulin WB Tubulin WB Tubulin WB Tubulin WB (d) (e) ∗∗ ∗∗ ∗∗ ∗∗ R = 0.9408   14 P<0.001 R = 0.9179 R = 0.9196 P<0.001 R = 0.9203 P<0.001 P<0.001 10 12 2 6 1 4 ∗ ∗ 2 0 0 0 0 0 0 0 12 0 0 12 0 12 ABCG2 ERCC1 ABCG2 ERCC1 ZEB2 protein expression ZEB1 protein expression ZEB1 protein expression ZEB2 protein expression control control ZEB1 ZEB2 si ZEB1 si ZEB2 (g) (f) Figure 6: Continued. Control Si UBE2C UBE2C Control Si UBE2C UBE2C Control SiZEB1 ZEB1 Control Si ZEB1 ZEB1 Control SiZEB2 ZEB2 Control Si ZEB2 ZEB2 24 h 48 h 72 h 24 h 48 h 72 h Relative luciferase activity Protein g Protein mRNA g g Relative luciferase activity Protein mRNA ABCG2 protein expression Protein Protein ERCC1 protein expression 1 g 2 g g mRNA Protein ABCG2 protein expression Protein ERCC1 protein expression 12 Journal of Oncology −− + − − −− + − − Si UBE2C Si UBE2C −− + − − −− + − − Si UBE2C Si UBE2C UBE2C −− − − + UBE2C −− − − + UBE2C −− − − + UBE2C −− − − + control +− − − − control +− − − − control +− −− − control +− − − − −− − + + −− − + + Si ZEB2 Si ZEB2 −− − + + Si ZEB1 −− − + + Si ZEB1 −+ + − − −+ + − − ZEB2 ZEB2 −+ + − − −+ + − − ZEB1 ZEB1 UBE2C UBE2C UBE2C UBE2C ZEB2 ZEB2 ZEB1 ZEB1 ABCG2 ABCG2 ABCG2 ABCG2 ERCC1 ERCC1 ERCC1 ERCC1 GAPDH Tubulin GAPDH Tubulin (h) (i) (j) Figure 6: UBE2C increases the expression of ABCG2 and ERCC1via regulating ZEB1/2in DDP-resistant NSCLC cells. (a–c) A549/DDP cells were overexpressed or knockdown of UBE2C (a), ZEB1 (b), and ZEB2 (c), respectively. The mRNA and protein levels of ABCG2 and ERCC1 were analyzed by RT-PCR and western blot assay. (d, e) RT-PCR and Western blot result show that ZEB1 (d) or ZEB2 (e) dose-and time-dependently increased the protein level of ABCG2 and ERCC1 in A549/DDP cells. (f) eTh transcriptional activity of ABCG2 and ERCC1 regulated by ZEB1/2 was analyzed by luciferase reporter assay in A549/DDP cells. (g) eTh relationship between protein expression levels of ZEB1/2, ERCC1, and ABCG2 in 15 independent repetitive A549/DDP cells was analyzed based on western blot assay. (h, i) UBE2C increased the expression of ABCG2 and ERCC1 via regulation of ZEB1 (h) and ZEB2 (i) by RT-PCR and Western blot assay. (j) Schematic diagram of the mechanisms of UBE2C increased the drug resistance gene expression, ABCG2 and ERCC1, and the EMT progression via regulating ZEB1/2 expression in DDP-resistant NSCLC cells. Results were presented as mean± SD, and the error bars represent the SD of three independent experiments.∗p<0.05;∗∗p<0.01 versus control group. by regulating ABCG2 and ERCC1 (Figures 4–6). siZEB1/2 irinotecan treatment in patients with colorectal cancer [45]. played a similar important role in reversing DDP resistance We previously reported that UBE2C selectively represses in DDP-resistant NSCLC cells (Figure 5). Moreover, UBE2C autophagy in NSCLC, and disruption of UBE2C-mediated upregulated the expression of ZEB1/2 (Figure 3) and ZEB1/2 autophagy repression attenuates cell proliferation, clono- directly targeted ABCG2 and ERCC1 (Figure 6). eTh refore, genicity, and invasive growth of NSCLC [46]. Furthermore, we predicted that siUBE2C reverses DDP resistance by our previous research showed that the miR 495-UBE2C- regulating ABCG2 and ERCC1 by directly targeting ZEB1/2. ABCG2/ERCC1 axis reverses cisplatin resistance by down- To evaluate this hypothesis, we separately transfected ZEB1 regulating drug resistance genes in cisplatin-resistant non- or siZEB1 into the A549/DDP cell line. eTh ABCG2 or small-cell lung cancer cells, highlighting the mechanism of ERCC1 mRNA and protein levels were increased by ZEB1 how microRNA 495 downregulates UBE2C [37]. In this study, and reduced by siZEB1 transfection. However, the opposite we examined the expression of UBE2C in DDP-sensitive and effects were observed for each factor in A549/DDP cells DDP-resistant cells and the role of UBE2C in mediating the ectopically transfected with siUBE2C to ZEB1 or UBE2C resistance of A549/DDP and H1299/DDP cells to DDP. eTh to siZEB1 by RT-PCR, western blotting, and qPCR assays results showed that, compared to wild-type cells (A549 and (Figures 6(h) and S3F). Similar results were obtained by RT- H1299), the mRNA and protein expression of UBE2C were PCR and western blot analysis of ZEB2 in A5499/DDP cell significantly increased in DDP-resistant cells (A549/DDP and lines (Figures 6(i) and S3G). es Th e data indicate that ZEB1/2- H1299) and UBE2C was significantly decreased following mediated siUBE2C reverses DDP resistance by regulating DDP treatment in A549 and H1299 cells (Figures 1(c)- ABCG2 and ERCC1 in DDP-resistant NSCLC cells. 1(d)). UBE2C or ZEB1/2 deficiency was found to signifi- cantly increase sensitivity to DDP, prevent cell proliferation and colony formation ability, and promote cell senescence 4. Discussion in DDP-resistant cells. Reintroduction of ZEB1/2 notably The emergence of drug resistance is unavoidable and severely rescued the phenotypes induced by UBE2C knockdown. limits the curative effect of chemotherapy drug [5, 40, 41], Moreover, our results showed that ZEB1/2 regulated the including cisplatin. us, Th useful biomarkers are needed to expression of the drug-resistant genes ABCG2 and ERCC1 predict and overcome DDP resistance to treat patients with at the transcriptional level. We also found that knockdown NSCLC. UBE2C is highly expressed in many types of human of UBE2C significantly sensitized lung cancer cells to the carcinomas including NSCLCs and strongly associated with chemotherapeutic agent DDP by repressing ABCG2/ERCC1 tumor grade/poor prognosis [40–42]. The UBE2C protein expression through downregulation of ZEB1/2 in vitro.Based plays a critical role in activating the M-phase check point on these results, we demonstrated that knockdown of UBE2C by specifically binding to APC/C [43, 44]. Knockdown of is a potential strategy for reversing DDP resistance in NSCLC UBE2C enhances the chemosensitivity of epirubicin and cells. docetaxel to dual drug-resistant breast cancer cells [17]. EMT plays a critical role in accelerating cisplatin resis- The UBE2C expression level can indicate the sensitivity to tance, and mesenchymal-like cancers are more prone to mRNA Protein mRNA Protein Journal of Oncology 13 developing drug resistance [47]. eTh hallmark event of EMT Authors’ Contributions is downregulation of E-cadherin protein. At the transcrip- Jiwei Guo designed the experiments. Yan Wu, Dan Jin, tional level, E-cadherin gene expression is repressed by many Xiaohong Wang, Jing Du, Weihua Di, Jiajia An, Cuijie Shao, factors, such as ZEB1, ZEB2, Snail, and slug [48–50]. EMT andJiweiGuoperformed thework.JiweiGuo,Yan Wu,and promotes cancer metastasis and invasion and thus accelerates Dan Jin analyzed the data and competed the gfi ures. Jiwei the emergence of drug resistance. We previously reported Guo wrote the manuscript. Yan Wu and Dan Jin contributed that UBE2C promoted EMT by regulating E-cadherin and equally to this work vimentin. ZEB1 promotes tumorigenesis and metastasis, and its expression is correlated with poor outcomes in cancer, including resistance to chemotherapy [51, 52]. In the cur- Acknowledgments rent study, we found that the mRNA and protein levels of ZEB1/ZEB2 were significantly downregulated in NSCLC cells We appreciate Professor Sichuan Xi (National Institutes following treatment with DDP (Figures 1(c)-1(d)). of Health, USA) for critical reading of the manuscript. We also found that ZEB1/2 mediates UBE2C regula- The present study was supported by National Natural Sci- tion EMT, cell proliferation, migration, and invasion in ence Foundation of China (no. 31801085), the Natural Sci- DDP-resistant cells (Figures 5(a)–5(d)). Importantly, ZEB1/2 ence Foundation of Shandong Province (ZR2018QH004, upregulated the expression of ABCG2 and ERCC1 (Figures ZR2016HB55, ZR2017LH072, and ZR2017PH067), and Shan- 6(b)-6(c)) and repressed E-cadherin gene transcription in dong Provincial Pharmaceutical Technology Development DPP-resistant NSCLC cells. Accordingly, UBE2C upregulated Plan (2017WS154) and the Research Foundation of Binzhou the expression of ZEB1/2 by increasing their promoter activ- Medical University (BY2015KYQD25 and BY2015KJ14). ity in DDP-resistant NSCLC cells (Figure 3(h)). siUBE2C downregulated ABCG2 and ERCC1 by suppressing ZEB1/2 Supplementary Materials expression and thus enhancing cisplatin sensitivity in DDP- resistant NSCLC cells. These results demonstrate that UBE2C Supplementary 1. SupplementaryFigure S1: H1299 cells expression levels are useful for predicting the response or were incubated with DDP at various concentrations for 24, resistance to DDP in NSCLC cells. However, the under- 48, and 72 h (A) and various times at 3, 6, and 9𝜇g/ml (B). lying mechanisms regulating UBE2C have not been well- Then cell viability was assessed (%). characterizedinNSCLC orotherchemotherapeuticagent- resistant cancers. u Th s, our future studies will focus on Supplementary 2. SupplementaryFigure S2: A549/DDP these mechanisms. In summary, UBE2C plays a critical cells were transfected with siUBE2C-1/2, siZEB1-1/2, or role in decreasing the sensitivity to cisplatin, inhibiting cell siZEB2-1/2. (A-F) eTh mRNA and protein levels of UBE2C senescence, and promoting cell proliferation, migration, and (A, D), ZEB1 (B, E), and ZEB2 (C, F) were analyzed by RT- invasion via promoting the promoter activity of ZEB1/2; PCR, immunoblotting, and qPCR assay. (G-I) the cellular ZEB1/2 upregulates the expression of antidrug genes, ABCG2 proliferation was analyzed by CCK8 assay in A549/DDP and ERCC1, to induce DDP resistance in NSCLC cells cell with transfection with siUBE2C-1/2 (G), siZEB1-1/2 (H), (Figure 6(j)). Collectively, our results indicate that UBE2C- or siZEB2-1/2 (I). Results were presented as mean ± SD, ZEB1/2-ABCG2/ERCC1 reverses DDP resistance by down- and the error bars represent the SD of three independent regulating antidrug genes and reducing EMT in cisplatin- experiments.∗p<0.05;∗∗p<0.01 versus control group. resistant NSCLC cells. Supplementary 3. SupplementaryFigure S3: (A, B) A549/ DDP cells were transfected with UBE2C or siUBE2C. ZEB1 Abbreviations (A)orZEB2(B)wereused forupregulatingtheprotein level of UBE2C target genes, respectively. eTh mRNA levels DDP: Cis-diamminedichloroplatinum (II) (cisplatin) of UBE2C, ZEB1, ZEB2, E-cadherin, and vimentin were ABCG2: ATP-binding cassette subfamily G member 2 analyzed by qPCR. (C-E) A549/DDP cells were overexpressed ERCC1: Excision repair cross-complementation group 1 or knockdown of UBE2C (C), ZEB1 (D), and ZEB2 (E), UBE2C: Ubiquitin-conjugating enzyme E2 C EMT: Epithelial-to-mesenchymal transition respectively. eTh mRNA and protein levels of ABCG2 and ERCC1 were analyzed by qPCR. (F, G) A549/DDP cells were HBEC: Human bronchial epithelial cells transfected with UBE2C or siUBE2C. ZEB1 (F) or ZEB2 (G) NSCLC: Non-small-cell lung cancer were used for upregulating the protein level of UBE2C target siRNA: Short interfering RNA. genes, respectively. eTh mRNA levels of UBE2C, ZEB1, ZEB2, ABCG2, and ERCC1 were analyzed by qPCR. Results were Data Availability presented as mean± SD,and theerrorbarsrepresenttheSD of three independent experiments.∗p<0.05;∗∗p<0.01 versus ed Th ata usedtosupportthefindingsofthisstudy are control group. included within the article. Supplementary 4. 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