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Hindawi Journal of Oncology Volume 2022, Article ID 1971559, 15 pages https://doi.org/10.1155/2022/1971559 Research Article microRNA-106b-5p Promotes Cell Growth and Sensitizes Chemosensitivity to Sorafenib by Targeting the BTG3/Bcl-xL/p27 Signaling Pathway in Hepatocellular Carcinoma 1,2 1,2 1,2 Bilegsaikhan Enkhnaran, Guang-Cong Zhang , Ning-Ping Zhang, 1,2 1,2 1,2 1,2 1,2 Hai-Ning Liu , Hao Wu, Shi Xuan, Xiang-Nan Yu , Guang-Qi Song , 1,2,3 1,2 4 1,2 Xi-Zhong Shen, Ji-Min Zhu , Xiu-Ping Liu , and Tao-Tao Liu Department of Gastroenterology and Hepatology, Zhongshan Hospital, Fudan University, Shanghai 200032, China Shanghai Institute of Liver Diseases, Zhongshan Hospital, Fudan University, Shanghai 200032, China Key Laboratory of Medical Molecular Virology, Shanghai Medical College of, Fudan University, Shanghai 200032, China Department of Pathology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China Correspondence should be addressed to Xiu-Ping Liu; xpliu1228@fudan.edu.cn and Tao-Tao Liu; liu.taotao@zs-hospital.sh.cn Received 23 August 2021; Revised 11 December 2021; Accepted 4 February 2022; Published 17 March 2022 Academic Editor: Dan Zhao Copyright © 2022 Bilegsaikhan Enkhnaran et al. 0is 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. microRNAs (miRNAs) and miRNA-mediated regulatory networks are promising candidates in the prevention and treatment of cancer, but the role of specific miRNAs involved in hepatocellular carcinoma (HCC) remains to be elusive. Herein, we found that miR-106b-5p is upregulated in both HCC patients’ tumor tissues and HCC cell lines. 0e miR-106b-5p expression level was positively correlated with α-fetoprotein (AFP), hepatitis B surface antigen (HBsAg), and tumor size. Overexpression of miR-106b- 5p promoted cell proliferation, migration, cell cycle G1/S transition, and tumor growth, while decreased miR-106b-5p expression had opposite effects. Mechanistic studies showed that B-cell translocation gene 3 (BTG3), a known antiproliferative protein, was a direct target of miR-106b-5p, whose expression level is inversely correlated with miR-106b-5p expression. Moreover, miR-106b- 5p positively regulates cell proliferation in a BTG3-dependent manner, resulting in upregulation of Bcl-xL, cyclin E1, and CDK2, as well as downregulation of p27. More importantly, we also demonstrated that miR-106b-5p enhances the resistance to sorafenib treatment in a BTG3-dependent manner. 0e in vivo findings showed that mice treated with a miR-106b-5p sponge presented a smaller tumor burden than controls, while the mice injected cells treated with miR-106b-5p had more considerable tumor burden than controls. Altogether, these data suggest that miR-106b-5p promotes cell proliferation and cell cycle and increases HCC cells’ resistance to sorafenib through the BTG3/Bcl-xL/p27 signaling pathway. free survival. 0erefore, comprehensive deciphering of the 1. Introduction mechanisms underlying rapid relapse and resistance to Hepatocellular carcinoma (HCC), the most prevalent sub- treatment in HCC remains established. type of liver cancer, has become the second leading cause of microRNAs (miRNAs) are small noncoding RNAs that cancer-related mortalities worldwide [1, 2]. Most patients regulate gene expression by directly binding to the 3′-un- progress to unresectable advanced stages due to a lack of translated region (3′-UTR) in targeting mRNAs [4]. Ac- accurate noninvasive surveillance, resulting in a dismal cumulating evidence has shown that miRNAs play survival [3]. Current therapeutic modalities, including ap- important roles in carcinogenesis, lipid metabolism, and proved multitarget inhibitor sorafenib, only slightly improve chemotherapy resistance by repressing the expression of median survival and hardly result in long-term progression- their targets [5, 6]. 0erefore, identifying tumor-associated 2 Journal of Oncology miRNAs and their target genes is critical for uncovering Transfection was performed using INTERFERin (Polyplus, miRNA mechanisms in tumor progression. Although pre- NY, USA). In contrast, transfection of plasmid was per- formed using a jetPrime reagent (Polyplus, NY, USA) vious studies have identified miRNAs as essential players of disease progression and resistance to conventional che- according to the manufacturer’s instructions. 0e miR- motherapies for various cancers, including HCC [7], further 106b-5p expression was enhanced by transfection of miR- investigation is still warranted to unravel the underlying 106b-5p mimics into PLC/PRF/5 and MHCC-97L cells and molecular mechanisms between miRNAs and HCC blocked by transfection of miR-106b-5p inhibitor into progression. MHCC-97H, HCC-LM3 cells. 0e transfection efficiency Our recently published study investigated the miRNA was evaluated by real-time quantitative PCR (qPCR). activation role of miR-93-5p in HCC [8]. Our previous study and others also suggested that oncogenic miR-106b-5p, 2.3. RNA Extraction and qPCR Analysis. Total RNAs were another member of the cluster, links with various types of extracted with a TRIzol reagent (Takara Bio, Kyoto, Japan). cancer [9–11], including HCC [12], along with several tumor For mRNA detection, complementary DNA (cDNA) was biological activities, including proliferation, apoptosis, and synthesized using the miRcute Plus miRNA First-Strand chemoresistance [13–15]. Chemoresistance constitutes a cDNA Synthesis Kit (Tiangen Biotech, Beijing, China). A significant malignant propensity to cancer development and miRcute Plus miRNA qPCR detection kit (Tiangen) was is a substantial obstacle to curing cancer. Recent data in- used for qPCR, with u6 as an internal control. 0e primers dicated changes in miRNA expressions in response to for hsa-miR-106b-5p and u6 were attained from Tiangen chemotherapy [5, 16]. Biotech. For mRNA detection, cDNA was synthesized using 0e purpose of this study is to investigate the significance a PrimeScript RT reagent kit (Takara) and qPCR was per- and underlying mechanisms of miR-106b-5p in cell pro- formed using the SYBR Premix Ex Taq II (Takara). liferation and chemotherapy resistance in HCC. Herein, we demonstrate the positive correlations of miR-106b-5p with BTG3 expression in a cohort of HCC tissue and cell lines. 2.4. Fluorescence In Situ Hybridization (FISH) Assay. A Furthermore, we validate that miR-106b-5p promotes G1/S specific miR-106b-5p FISH probe was synthesized and ap- cell-cycle transition and sensitizes HCC cells to sorafenib via plied in the experiment. Hybridization was carried overnight the BTG3/Bcl-xL/cyclinE1 pathway. with miR-106b-5p probes according to the manufacturer’s instructions of a fluorescence in situ hybridization kit (GenePharma, China). All fluorescence images were cap- 2. Materials and Methods tured by a confocal laser-scanning microscope (Leica; Wetzlar, Germany) 2.1. Cell Lines and HCC Tissues. HCC-LM3, PLC/PRF/5, MHCC-97L, and MHCC-97H were obtained from the Liver Cancer Institute, Zhongshan Hospital of Fudan University 2.5. Microarrays. Microarray analysis was performed as (Shanghai, China). A normal liver epithelial cell line (L02) described before. Briefly, Human miRNA Microarray Re- was obtained from the Cell Bank of the Chinese Academy of lease 21.0 (Agilent Technologies, Inc.) was used for screening Sciences (Shanghai, China). All cell lines were maintained in out the significantly differential expression of miRNAs. Dulbecco’s modified Eagle’s medium (DMEM, Corning, P< 0.05 and fold-change ≥2.0 were applied for the Lowell, MA, USA) supplemented with 10% fetal bovine threshold. Raw data used in the present study are accessible serum (FBS; Corning) and 1% penicillin/streptomycin through Gene Expression Omnibus (https://www.ncbi.nlm. (Corning). 0ese cells were cultured in a humidified incu- nih.gov/geo/query/acc.cgi?acc�GSE108724) [17]. bator at 37 C with 5% CO . Sorafenib was purchased from Sigma (Sigma, St. Louis, 2.6. Western Blotting. Total proteins were collected at 48 h MO, USA). 0is compound was dissolved in 100% DMSO after transfection and then electrophoresed using 10–12% (Sigma) and diluted with DMEM to 10 nM for studies. 0e SDS-PAGE gels, transferred onto polyvinylidene fluoride same concentration of DMSO was added to culture medium membrane (Millipore, Bedford, MA, USA), and blocked with as a control. A total of 90 pairs of HCC tissues and adjacent 5% nonfat dry milk in Tris-buffered saline containing 0.05% nontumor samples were obtained from patients who un- Tween 20. Membranes were incubated overnight at 4 C with derwent surgery at our institution. Written informed con- primary antibodies followed by the appropriate secondary sent was acquired from all individuals, and the study was antibody for 1 h at room temperature. 0e signals were de- approved by the Institute Ethics Committee. tected with electrogenerated chemiluminescence (ECL) de- veloper solution (Millipore). Primary antibodies were 2.2. Cell Transfection. 0e hsa-miR-106b-5p mimics, hsa- presented as follows: anti-β-actin (1 :1000; Cell Signaling miR-106b-5p inhibitor, and negative controls were attained Technology, Danvers, MA, USA), anti-Bcl-xL (1 :1000; CST), from RiboBio (Guangzhou, Guangdong, China). 0e overall anti-BTG3 (1 : 500; Sigma), anti-cyclinE1 (1 :1000, CST), anti- sequence of BTG3 was inserted into the pEX-3 vector P27 (1 :1000, Proteintech, Rosemont, IL, USA), and anti- (GenePharma, Shanghai, China) to construct the BTG3 CDK2 (1 :1000, Proteintech). 0e relative optical density of expression vector. Specific small interfering RNAs (siRNAs) bands was quantified 36 with a GelPro Analyzer (Media for BTG3 were designed by GenePharma (Shanghai, China). Cybernetics, Silver Spring, MD, USA). Journal of Oncology 3 2.7. Cell Proliferation Assay. According to the manufac- blocked with 5% bovine serum albumin (Amresco, USA). turer’s protocol, the cell viability was evaluated by a CCK-8 0e cells were then incubated with antibodies against Bcl-xL (CST, 2764) and BTG3 (Sigma-Aldrich, SAB4300958) assay (Beyotime). Briefly, cells were seeded on a 96-well plate at a density of 3 ×10 per well containing DMEM (100 mL) overnight at 4 C. 0e secondary antibodies, Alexa Fluor 488 in five replicates for each condition and maintained at 37 C and Alexa Fluor 594 (Yeasen, China), were incubated with with 5% CO . 0e CCK-8 solution (10 μL) was added to each the cells at 37 C for 1 h. 4′,6-Diamidino-2-phenylindole well and incubated for 2 h after transfected with indicated (DAPI) was stained to visualize the nucleus. A confocal constructs. 0e absorbance at 450 nm (with 620 nm as the laser-scanning microscope (Leica; Wetzlar, Germany) was reference) was measured using a spectrophotometer. used to capture fluorescence results. 2.8. Cell Cycle Analysis. Cell cycle analysis was carried out 2.13. Co-Immunoprecipitation (co-IP) Assay. Co-IP was using flow cytometry with propidium iodide staining (BD performed according to the instruction of G-agarose Biosciences, Franklin Lakes, NJ, USA). After transfection for (Millipore, Billerica, MA, USA). Briefly, cells were collected 48 hours, the cells were harvested and then fixed with 70% by immunoprecipitation lysis buffer with protease inhibi- ethanol at 4 C overnight. Next, after washing with PBS three tors. 0e target protein was immunoprecipitated with the times, the cells were resuspended in 500 μL propidium io- corresponding primary antibodies and evaluated by western dide and incubated at room temperature for 30 min. A total blotting. 0e primary antibodies were as follows: anti-Bcl-xL of 10,000 events were counted for each sample and analyzed (CST, 2764) and anti-myc (CST, 2276). with a FACScaliber Flow Cytometer (BD Biosciences). 2.14. Immunohistochemical Staining. Paraffin slides ob- 2.9. Wound-Healing Assay. Cells were seeded into a 6-well tained from in vivo experiments were used for immuno- plate. After 12 h of transfection, the cell layer was scratched histochemistry. 0e slides were dewaxed, rehydrated, and using a 200 μL pipette tip, and cells were cultured in a subjected to antigen retrieval. Subsequently, the tissues were DMEM with no FBS. Images of cells were captured at incubated with a panel of antibodies including anti-Bcl-xL initiation time and 72 h by a microscope (Olympus, Tokyo, (1 : 300, Cell Signaling Technology, Danvers, USA), anti- Japan). 0e migration abilities were quantified and nor- BTG3 (1 : 50, Sigma-Aldrich, St. Louis, MO, USA), anti- malized by a relative gap distance. cyclinE1 (1 : 50, Signalway Antibody, College Park, MD, USA), anti-PCNA (1 : 500, Proteintech, Rosemont, IL, USA), 2.10. Transwell Assay. 0e ability of cell to invasion or and anti-Ki67 (1 : 4000, Proteintech, Rosemont, IL, USA) migration was detected by Transwell (Corning) assay cov- overnight. After incubation with the corresponding sec- ered with or without Matrigel in the Transwell upper ondary antibodies, the samples were stained with dia- chambers. Briefly, after being transfected for 24 h, cell minobenzidine and hematoxylin. Immunohistochemically suspension (1 × 10^4 cells) in 200 μL serum-free DMEM was stained tissue sections were assessed independently by two seeded into the upper chambers, while 500 μL DMEM pathologists. containing 10% fetal bovine serum was placed in the lower chamber as a chemoattractant. Cells were incubated for 48 h. After removing the nonmigrating or noninvading cells, the 2.15. In Vivo Tumorigenesis in Nude Mice. All experimental remaining cells were fixed with 4% paraformaldehyde, procedures were approved by the Institutional Animal Care stained with 0.1% crystal violet (Beyotime, Shanghai, China), Committee. Five-week-old male BALB/c nude mice were and then counted under a light microscope (Olympus, purchased from the Shanghai SLAC Laboratory Animal Tokyo, Japan) in three random fields per well. 0e results (Shanghai, China). Briefly, 1 × 10^6 cells transfected with LV- were expressed as the average number of invasive cells per miR-106b-5p, LV-miR-106b-5p sponge, or LV-miR-NC in field. 0.2 mL DMEM without FBS were subcutaneously injected into the left flank of the mice (5-week-old, n � 4 for each group). Tumors were measured with a caliper every five days. 2.11. Luciferase Reporter Assay. Wild-type or mutant of 3′ Mice were anesthetized and sacrificed 25 days after tumor untranslated region (UTR) of BTG3 was cloned into a PHY- inoculation. 0e tumors were dissected from the body and 603 luciferase reporter vector (GeneChem, China). Subse- calculated volume as (length × width^2) × 0.5. quently, miR-106b-5p mimic or negative control combined with BTG3 wild-type or mutant reporter plasmid vector was cotransfected into HEK293T cells. Luciferase activity was 2.16. TCGA Data Acquisition. miR-106-5p expression and detected after transfection for 24 h and tested by an Orion II appropriate survival time in HCC and nontumor tissues microplate luminometer (Berthold, Germany). were acquired from UCSC Xena (https://xena.ucsc.edu/). Subsequently, the overall survival rate associated with high 2.12. Immunofluorescence Staining. Cells were inoculated on and low expression of miR-106-5p was analyzed in coverslips in 24-well plates, fixed with 4% paraformalde- GraphPad Prism 8.0.2 (GraphPad Prism, La Jolla, CA, hyde, permeabilized with 0.2% Triton X-100, and then USA). 4 Journal of Oncology assess the effect of miR-106b-5p on cell migration and in- 2.17. Statistical Analysis. Statistical analysis was performed using the SPSS 22.0 for Window (SPSS, Chicago, USA). Data vasion, respectively. Overexpression of miR-106b-5p pro- moted cell migration and invasion in PLC/PRF/5 and are presented as the mean± standard deviation (SD) from at least three independent studies. Student’s t-test was used to MHCC-97L cells. Conversely, suppression of miR-106b-5p analyze the differences between two groups, and one-way exhibited opposite effects in HCC-LM3 and MHCC-97H analysis of variance (ANOVA) was used to evaluate the cells (Figures 2(c) and 2(d)). Furthermore, the expression of differences between more than two groups. 0e chi-square E-cadherin/N-cadherin/vimentin serving as metastasis test was applied for examining the correlation between miR- markers was examined. We found that miR-106b-5p 106-5p expression and clinicopathological characteristics of overexpression upregulated E-cadherin expression and HCC patients. Statistical significance was set at P< 0.05 downregulated N-cadherin/vimentin expression in PLC/ (two-sided). PRF/5 and MHCC-97L cells, while miR-106b-5p suppres- sion exhibited opposite effects in HCC-LM3 and MHCC- 97H cells (Figure 2(e) and Supplementary Figure 2). 3. Results To determine whether miR-106b-5p could influence the sorafenib-induced inhibition of cell proliferation, we next 3.1. miR-106b-5p Is Overexpressed in Human HCC Tissues and investigated the effect of miR-106b-5p on cell viability in Cell Lines. Recently, our miRNA expression profiling HCC cell lines using a CCK-8 assay. 0e IC50 of sorafenib identified overexpression of miR-106-5p in 7 paired HCC following miR-106b-5p mimic treatment increased from tissues (fold change � 2.46, P< 0.001; Figure 1(a)) [17], 15.75 μM to 18.07 μM in PLC/PRF/5 cells and from 17.44 μM which is similar to the result of Yen et al. [18]. We examined to 23.27 μM in MHCC-97L cells. Moreover, the IC50 for miR-106b-5p expression in 90 pairs of clinical specimens sorafenib following miR-106b-5p inhibitor treatment de- using qPCR to corroborate this result. In general, the mean creased from 26.54 μM to 22.34 μM in MHCC-97H cells and expression of miR-106-5p in HCC tissues was 1.45-fold from 21.2 μM to 13.23 μM in HCC-LM3 cells (Figure 2 (f)). higher than that in matched adjacent tissues (P � 0.04; 0ese results suggest that miR-106b-5p promotes cell pro- Figure 1(b)), which is consistent with the data from the liferation and metastasis, along with sorafenib resistance in Cancer Genome Atlas (TCGA; Figure 1(c)). Moreover, FISH HCC cells. results showed that miR-106b-5p was significantly increased in HCC tissues compared with matched adjacent tissues and was mainly located in the nucleus (Figure 1(d)) 3.3. miR-106b-5p Directly Targets BTG3. Considering In addition, comparing clinicopathological factors of miRNAs act through inhibition of downstream target genes, high and low miR-106b-5p expression groups (Table 1), we we searched for potential target genes of miR-106b-5p in four found that high miR-106b-5p expression positively corre- databases, including miRanda (https://www.microrna.org), lated with tumor size (χ2 � 4.263; P � 0.039), α-fetoprotein DIANA (https://diana.imis.athena-innovation.gr), TargetS- (AFP) (χ2 � 4.097; P � 0.043), and hepatitis B surface an- can (https://www.targetscan.org), and miRDB (https://www. tigen (HBsAg) (χ2 � 9.996; P � 0.002). Silico analysis of mirdb.org). Among the searching results, we identified BTG3 TCGA revealed that high miR-106b-5p expression is neg- as a potential target of miR-106b-5p. In previous studies, atively associated with the overall survival of HCC patients BTG3 has been identified as a candidate tumor suppressor (Figure 1(e)). To validate the importance of miR-106b-5p in gene that plays an important role in tumor growth and HCC, the expression of miR-106b-5p was next analyzed in a metastasis [19–21]. Our findings are consistent with these panel of human HCC cell lines, and the normal liver cell line findings. Overexpressed miR-106b-5p downregulated BTG3 L02 using qPCR. Similarly, the relative expression levels for mRNA and protein levels in PLC/PRF/5 and MHCC-97L miR-106b-5p in these seven HCC cells were 1.21- to 4.28- cells, while downregulated miR-106b-5p elevated BTG3 levels fold compared to that in L02 cells (Figure 1(f)). 0ese results in HCC-LM3 and MHCC-97H cells (Figures 3(a) and 3(b)). suggest that increased miR-106b-5p is a frequent event in Furthermore, a luciferase reporter assay was performed to test human HCC. the effect of miR-106b-5p on BTG3 in HEK-293T cells expressing wild-type or mutant 3′ UTR BTG3 reporter gene. Twenty-four hours after cotransfection with miR-106b-5p 3.2. miR-106b-5p Promotes HCC Cell Proliferation and Sor- mimic or negative control, the luciferase activities were found afenib Resistance. To gain insight into the biological role of to be inhibited in wild-type 3′ UTR reporter gene, rather than miR-106b-5p in HCC, we performed both inhibition studies the mutant gene by the miR-106b-5p mimic (Figure 3(c)). using miR-106b-5p inhibitor and overexpression studies Moreover, the results from clinical samples revealed that the using miR-106b-5p mimics (Supplementary Figure 1). First, BTG3 expression was significantly downregulated in tumor CCK8 and cell cycle were carried out to evaluate the effect of tissues compared with adjacent nontumor tissues miR-106b-5p on cell proliferation. Overexpression of miR- (Figure 3(d)). 0us, these findings suggest that BTG3 is a 106b-5p significantly promoted cell growth and G1/S cell direct downstream target of miR-106b-5p. cycle transition in PLC/PRF/5 and MHCC-97L cells. In contrast, suppression of miR-106b-5p markedly inhibited cell proliferation and G1/S cell cycle transition in HCC-LM3 3.4. Effects of BTG3 on HCC Cells. To further examine whether BTG3 mediated miR-106b-5p-induced HCC pro- and MHCC-97H cells (Figures 2(a) and 2(b)). Subsequently, wound-healing and Transwell assays were performed to liferation and metastasis, the pEX3-BTG3 plasmid was Journal of Oncology 5 * 6 *** miR−532−3p miR−130b−3p 2 miR−222−3p miR−106b−5p miR−93−5p miR−378d 0 2 miR−424−5p −1 -5 miR−125b−5p miR−10a−5p 0 -10 −2 Tumor Non-tumor miR−195−5p Tumor Non-tumor (a) (b) (c) 100 8 miR-106b-5p 80 DAPI Logrank P-value = 0.0123 0 1000 2000 3000 4000 Merge Days Low miR-106b-5p expression Tumor Non-tumor High miR-106b-5p expression (d) (e) (f) Figure 1: miR-106b-5p is frequently upregulated in HCC tissues and cell lines. (a) A heatmap showing differential expression of miRNAs in 7 paired HCC tissues by miRNA microarray. (b) miR-106b-5p expression in HCC and adjacent para-tumor tissues by qPCR (n � 90). (c) miR-106b-5p expression in HCC (n � 371) and adjacent para-tumor tissues (n � 50) from TCGA database. (d) miR-106b-5p ex- pression in HCC and nontumor tissues by FISH. Scale bar represents 50 μm. (e) Overall survival rate in HCC patients with the low and high miR-106b-5p expression groups. (f) qPCR analysis of miR-106b-5p in HCC cell lines and L02 cells. Data were analyzed using −(ΔΔCT) ∗ ∗∗∗ formula 2 . Data represent mean± SD for (b) and (c). P< 0.05 and P< 0.001. Table 1: Clinical characteristics of selected 75 pairs of HCC and matched nontumor specimens. miR-106b-5p expression Characteristics χ P Low High Male 35 40 Gender 0.224 0.636 Female 6 9 ≤60 17 18 Age (years) 0.210 0.647 >60 24 31 ≤5 cm 24 18 Tumor size 4.263 0.039 >5 cm 17 31 I-II 27 30 Pathological grade 0.206 0.650 III-IV 14 19 I-II 33 34 TNM stage 1.446 0.229 III-IV 8 15 − 13 3 HBsAg 9.996 0.002 + 28 46 ≤200 31 27 AFP 4.097 0.043 >200 10 22 − 29 28 Vascular invasive 1.775 0.183 + 12 21 TNM, tumor-node-metastasis; HBsAg, hepatitis B surface antigen; AFP, α-fetoprotein; χ , chi-square. constructed to overexpress BTG3, along with BTG3-siRNA 106b-5p mimic and consequently resulted in increased cell being employed to inhibit BTG3 in HCC cells. 0e CCK8, proliferation (Figure 4(a)), G1/S transition (Figure 4(d)), and migratory and invasive ability in PLC/PRF/5 and cell cycle, wound-healing, and Transwell assays were then performed to investigate the function of BTG3 in HCC cell MHCC-97L cells (Figures 4(b) and 4(c)). In contrast, the lines. Downregulation of BTG3 simulated the effect of miR- overexpression of BTG3 also had a similar effect of miR- 5T 6T 9T 10T 11T 13T 19T 5P 6P 9P 10P 11P 13P 19P Overall survival (%) miR-106b-5p relative expression miR-106-5p relative exression miR-106b-5p relative expression (1×10 ) L02 PLC/PRF/5 Hep3B MHCC-97L MHCC-97H BEL-7402 HepG2 HCC-LM3 6 Journal of Oncology PLC/PRF/5 MHCC-97L PLC/PRF/5 MHCC-97L 2.0 2.0 80 80 mimic mimic ** ** ** 1.5 NC 1.5 NC * * 60 60 1.0 1.0 40 * 40 0.5 0.5 20 20 0 0 0 0 024 48 72 024 48 72 G0/G1 S G2/M G0/G1 S G2/M Time (h) Time (h) mimic mimic NC NC MHCC-97H HCC-LM3 MHCC-97H HCC-LM3 2.0 2.0 inhibitor inhibitor 80 ** 1.5 1.5 * NC NC *** 60 60 ** 1.0 * 1.0 ** 0.5 0.5 20 ** 0 0 024 48 72 024 48 72 G0/G1 S G2/M G0/G1 S G2/M Time (h) Time (h) inhibitor inhibitor NC NC (a) (b) PLC/PRF/5 MHCC-97L PLC/PRF/5 MHCC-97L mimic NC mimic NC 80 80 *** 60 60 ** 0 h 0 h 40 40 20 20 0 0 72 h 72 h MHCC-97H HCC-LM3 MHCC-97H HCC-LM3 inhibitor NC inhibitor NC 80 80 60 60 *** 0 h 0 h 40 40 20 20 0 0 72 h 72 h (c) MHCC-97L PLC/PRF/5 PLC/PRF/5 MHCC-97L 80 150 ** migration invasion migration invasion 60 ** 0 0 mimic mimic NC NC MHCC-97H HCC-LM3 migration invasion migration invasion MHCC-97H HCC-LM3 300 300 *** ** 200 200 100 100 0 0 inhibitor inhibitor NC NC (d) Figure 2: Continued. NC inhibitor NC mimic Absorbtion Absorbtion (450 nm) (450 nm) Absorbtion Absorbtion NC inhibitor NC mimic (450 nm) (450 nm) Proportion of Proportion of cells (%) cells (%) Cell Number Percent wound Percent wound Cell Number of closure(%) of closure(%) migration migration inhibitor mimic invasion invasion NC NC Proportion of Proportion of cells (%) cells (%) Cell Number Cell Number Percent wound Percent wound of closure(%) of closure(%) migration migration inhibitor mimic invasion invasion NC NC Journal of Oncology 7 PLC/PRF/5 MHCC-97L PLC/PRF/5 mimic NC E-cadherin IC50=18.07 ng/mL IC50=15.75 ng/mL 120 120 1.00 2.69 0.94 2.69 60 60 N-cadherin 0 0 1.00 0.47 1.05 0.51 0 1.0 2.0 0 1.0 2.0 Vimentin log (Sorafenib), ng/mL 1.00 0.48 1.16 0.22 MHCC-97L β-actin mimic NC IC50=23.27 ng/mL IC50=17.44 ng/mL 120 120 60 60 MHCC-97H HCC-LM3 0 0 0 1.0 2.0 0 1.0 2.0 E-cadherin 1.00 0.25 0.85 0.36 log (Sorafenib), ng/mL N-cadherin MHCC-97H 1.00 3.66 1.19 4.02 inhibitor NC Vimentin IC50=22.34 ng/mL IC50=26.54 ng/mL 120 120 1.00 1.28 0.45 2.58 60 60 β-actin 0 0 0 1.0 2.0 0 1.0 2.0 log (Sorafenib), ng/mL HCC-LM3 inhibitor NC IC50=13.23 ng/mL IC50= 21.2 ng/mL 120 120 60 60 0 0 0 1.0 2.0 0 1.0 2.0 log (Sorafenib), ng/mL (e) (f) Figure 2: miR-106b-5p promotes HCC cell proliferation, metastasis, and sorafenib resistance. (a) 0e effect of miR-106b-5p on cell proliferation in HCC cells using CCK8. (b) 0e effect of miR-106b-5p on the cell cycle of HCC cells using flow cytometry. (c) 0e effect of miR-106b-5p on the migration of HCC cells using the wound-healing assay. Scale bar, 100 μm. (d) 0e effect of miR-106b-5p on the invasion of HCC cells using Transwell assay. (e) 0e effect of miR-106b-5p on the sorafenib resistance in HCC cells using CCK-8. Data represent ∗ ∗∗ ∗∗∗ mean± s.d. (n � 3), NC, negative control. P< 0.05, P< 0.01, and P< 0.001 by Student’s t-test. 1.5 2.0 *** ** ** 1.5 1.0 1.0 0.5 0.5 0 0 PLC/PRF/5 MHCC-97L MHCC-97H HCC-LM3 miR-106b-5p mimics miR-106b-5p inhibitor NC NC (a) PLC/PRF/5 MHCC-97L MHCC-97H HCC-LM3 miR-106b-5p mimic +–+– –––– miR-106b-5p inhibitor –––– +–+– NC–+ + – + – + – BTG3 1.00 2.93 0.26 0.60 1.35 0.35 0.74 0.64 GAPDH (b) Figure 3: Continued. BTG3 relative expression inhibitor mimic NC NC inhibitor mimic NC NC BTG3 relative expression Cell viability (%) Cell viability (%) Cell viability (%) Cell viability (%) 8 Journal of Oncology 1T 1N 2T 2N 3T 3N 4T 4N BTG3 1.00 3.61 0.08 1.00 0.69 1.15 0.09 0.45 1.5 GAPDH 1.0 5T 5N 6T 6N 7T 7N 8T 8N BTG3 0.5 1.00 2.29 0.15 0.29 0.17 0.55 0.01 0.10 0.0 GAPDH (c) (d) Figure 3: BTG3 is a direct target of miR-106b-5p. 0e expression of BTG3 in HCC cells after transfected with miR-106b-5p mimic, inhibitor, or its negative control using qPCR (a) and western blotting (b). (c) Sketch of the construction of wild-type or mutant BTG3 3′UTR vectors. (d) Expression of BTG3 in HCC tissues and matched para-tumor tissues from 8 patients. NC, negative control ( P< 0.05). 106b-5p inhibitor and resulted in decreased cell proliferation expression was increased by miR-106b-5p mimic (Figure 4(a)), G1/S transition (Figure 4(d)), migratory and (Figure 5(c)) and siBTG3 (Figure 5(d)). In contrast, miR- invasive ability in HCC-LM3 and MHCC-97H cells 106b-5p inhibitor (Figure 5(c)) and BTG3 overexpression (Figures 4(b) and 4(c)). 0ese findings suggest that miR- (Figure 5(d)) had an opposite effect on G1/S-specific pro- 106b-5p enhances HCC cell proliferation and metastasis, at teins and sorafenib resistance-related proteins in HCC-LM3 and MHCC-97H cells. Finally, we detected above protein least in part by restraining BTG3 expression. levels in the presence of sorafenib in HCC cell lines. We found that compared to the control group, Bcl-xL, cyclin E1, 3.5. miR-106b-5p Induces Cell Proliferation and Sorafenib and CDK2 were decreased, while BTG3 and p27 were in- Resistance through the BTG3/Bcl-xL/p27 Signaling Pathway. creased in all four HCC cell lines (Figures 5(c) and 5(d)). To determine whether miR-106b-5p promotes HCC pro- Together, these results indicate that miR-106b-5p induces liferation and chemoresistance via inhibition of BTG3, cell proliferation and sorafenib resistance through the downstream genes of the BTG3 signaling pathway were also BTG3/Bcl-xL/p27 signaling pathway. analyzed. Based on the results above, we used immuno- fluorescence staining, co-IP, and western blotting to confirm whether miR-106b-5p and BTG3 affected cell cycle regu- 3.6. miR-106b-5p Mediates HCC Proliferation In Vivo. To latory proteins. Recent studies have demonstrated that TOB, verify the phenotype of miR-106b-5p from the in vitro studies, we subcutaneously injected cells treated with miR- one BTG/TOB family member, is negatively associated with Bcl-xL, a cell cycle-related protein [22, 23]. 0us, we hy- 106b-5p, miR-106b-5p sponge, or corresponding controls pothesized that BTG3 might regulate Bcl-xL. into tumor xenografts. After 25 days of injection, the mice To test this, we first detected the two proteins’ localization treated with miR-106b-5p sponge presented a smaller tumor in MHCC-97H and HCC-LM3 cells by immunofluorescence burden than controls by IHC (Figures 6(a) and 6(c)) and staining. BTG3 is mainly colocalized with Bcl-xL in the cy- displayed lower expression for Ki67, proliferating cell nu- toplasm (Figure 5(a)). Furthermore, the reciprocal co-IP clear antigen (PCNA), Bcl-xL, and cyclin E1, along with a showed that BTG3 and Bcl-xL were seen in the cell lysate. higher expression for BTG3 in tumor tissues relative to Both proteins were immunoprecipitated from cell lysate in controls by western blotting (Figures 6(d) and 6(e)). In contrast, the mice injected cells treated with miR-106b-5p MHCC-97H cells (Figure 5(b)), indicating that BTG3 in- teracts with Bcl-xL in HCC cells. had more considerable tumor burden than controls (Figures 6(b) and 6(c)) and displayed higher expression for Moreover, we found that G1/S-specific protein, in- cluding cyclin E1 and CDK2, were increased, while p27 was Ki67, PCNA, Bcl-xL, and cyclin E1, along with a lower decreased by miR-106b-5p mimic (Figure 5(c)) and siBTG3 expression for BTG3 in tumor tissues relative to controls (Figure 5(d)) in PLC/PRF/5 and MHCC-97L cells using (Figures 6(d) and 6(e)). 0ese results demonstrate that miR- western blotting. Subsequently, we analyzed the sorafenib 106b-5p promotes HCC proliferation through the BTG3/ resistance-related protein Bcl-xL. We observed that Bcl-xL Bcl-xL/p27 pathway (Figure 6(f)). BTG3-NC BTG3-WT BTG3-MUT BTG3-NC+miRNA-NC BTG3-WT+miRNA-NC BTG3-MUT+miRNA-NC BTG3-NC+miRNA-mimic BTG3-WT+miRNA-mimic BTG3-MUT+miRNA-mimic Relative Fold Change Journal of Oncology 9 PLC/PRF/5 MHCC-97L 2.0 2.0 1.5 * *** 1.5 ** 1.0 *** 1.0 0.5 0.5 0 0 024 48 72 024 48 72 Time (h) Time (h) siBTG3 siBTG3 NC NC MHCC-97H HCC-LM3 2.0 2.0 1.5 1.5 *** *** *** 1.0 1.0 ** 0.5 0.5 0 0 024 48 72 024 48 72 Time (h) Time (h) pEX3-BTG3 pEX3-BTG3 NC NC (a) PLC/PRF/5 MHCC-97L siBTG3 NC siBTG3 NC MHCC-97L PLC/PRF/5 80 80 ** 60 60 *** 40 40 20 20 0 0 mimic NC mimic NC MHCC-97H HCC-LM3 pEX3-BTG3 NC pEX3-BTG3 NC MHCC-97H HCC-LM3 *** 80 80 60 60 * 40 40 20 20 0 0 mimic NC mimic NC (b) PLC/PRF/5 MHCC-97L PLC/PRF/5 MHCC-97L 150 100 80 80 60 60 PLC/PRF/5 MHCC-97L * * 40 40 migration invasion migration invasion 40 * ** 20 20 0 0 0 0 siBTG3 siBTG3 siBTG3 siBTG3 NC NC NC NC MHCC-97H HCC-LM3 MHCC-97H HCC-LM3 MHCC-97H HCC-LM3 150 150 migration invasion migration invasion 80 80 60 60 100 100 40 40 * * 50 50 * * 20 * *** 0 0 0 pEX3-BTG3 pEX3-BTG3 pEX3-BTG3 pEX3-BTG3 NC NC NC NC (c) (d) Figure 4: BTG3 suppresses HCC cell proliferation and metastasis. (a) CCK8 measured the effect of BTG3 on cell proliferation in HCC cells. 0e wound-healing assay (b) and Transwell assay (c) were performed to analyze the effect of BTG3 on the migration and invasion of HCC cells. (d) Flow cytometric assay was performed to analyze the effect of BTG3 on the cell cycle of HCC cells. NC, negative control. P< 0.05, ∗∗ ∗∗∗ P< 0.01, and P< 0.001. modulate chemoresistance and their therapeutic potentials 4. Discussion remain to be thoroughly elucidated. In this study, we report Several studies, including our previous work, demonstrated that miR-106b-5p promotes cell proliferation, cell cycle, and that the aberrant miRNA expression is related to the the resistance of HCC cells to sorafenib through the BTG3/ pathogenesis of HCC [17, 24–27]. However, miRNAs that Bcl-xL/p27 pathway. We anticipate that these findings, for NC pEX3-BTG3 NC siBTG3 72 h 0 h 72 h 0 h NC pEX3-BTG3 NC siBTG3 Absorbtion Absorbtion (450 nm) (450 nm) 72 h 0 h 72 h 0 h Cell Number Cell Number migration migration invasion invasion Absorbtion Absorbtion (450 nm) (450 nm) Cell Number Cell Number Percent wound Percent wound of closure(%) of closure(%) migration migration invasion invasion Percent wound Percent wound of closure(%) of closure(%) Proportion of cells (%) Proportion of cells (%) G0/G1 G0/G1 S S G2/M G2/M Proportion of cells (%) Proportion of cells (%) G0/G1 G0/G1 S S G2/M G2/M 10 Journal of Oncology DAPI BTG3-myc Bcl-xL Merge IP Input IgG BTG3-myc Bcl-xL MHCC-97H BTG3-myc IP Input IgG Bcl-xL HCC-LM3 BTG3-myc Bcl-xL (a) (b) PLC/PRF/5 MHCC-97L MHCC-97H HCC-LM3 Sorafenib + –– –– + +– – –– + miR-106b-5p mimic + +– +–+ ––– ––– miR-106b-5p inhibitor –––––– +–++ – + NC –– + – + – – – + – + – BTG3 1.00 0.25 0.93 0.05 0.15 0.27 1.00 0.69 0.23 0.42 0.17 0.43 Bcl-xL 1.00 1.92 0.82 1.21 0.82 0.96 1.00 0.63 1.85 1.66 1.32 0.79 P27 1.00 0.24 1.13 0.04 0.04 0.06 1.00 0.43 0.36 0.07 0.04 0.45 CDK2 1.00 3.77 3.95 2.80 1.79 1.20 1.00 2.45 4.23 3.86 3.99 1.54 cyclin E1 1.00 2.57 1.34 0.91 0.25 0.16 1.00 4.37 6.78 7.94 16.85 6.20 Tubulin (c) PLC/PRF/5 MHCC-97L MHCC-97H HCC-LM3 Sorafenib +– – –– + +– – –– + siBTG3 ++ – +–+ ––– ––– pEX3 BTG3 ––– ––– NC –– + –+– BTG3 1.00 0.38 0.68 0.08 0.31 0.33 1.00 0.55 0.15 0.34 0.14 0.48 Bcl-xL 1.00 2.45 0.76 1.35 0.41 0.16 1.00 3.16 7.45 22.58 36.08 4.36 P27 1.00 1.20 1.30 0.10 0.32 0.60 1.00 0.36 0.05 0.33 0.08 0.76 CDK2 1.00 2.29 2.03 0.47 0.20 0.18 1.00 7.75 9.42 8.49 10.84 8.21 cyclin E1 1.00 2.91 2.15 1.15 0.21 0.27 1.00 2.56 2.51 6.46 11.21 2.32 Tubulin (d) Figure 5: miR-106b-5p induces sorafenib resistance of HCC cells through BTG3/Bcl-xL/p27. (a) Immunofluorescence staining analysis showed the colocalization of BTG3 and Bcl-xL in MHCC-97L and HCC-LM3 cells. Scale bar represents 15 μm. (b) 0e co-IP study showing the interaction between BTG3 and Bcl-xL in MHCC-97H cells. IgG was used as a negative control. (c) 0e protein expressions (including BTG3, Bcl-xL, p27, cyclin E1, and CDK2) in HCC cells treated with sorafenib, miR-106b-5p mimic/inhibitor, or sorafenib plus miR-106b-5p mimic/inhibitor for 48 h. (d) Expression of BTG3, Bcl-xL, p27, cyclin E1, and CDK2 was detected after HCC cells incubated with sorafenib, transfected with siBGT3/pEX3-BTG3, or sorafenib plus transfected with siBGT3/pEX3-BTG3 for 48 h NC, negative control. the first time, will provide novel mechanistic insights into miR-106b-5p belongs to the miR-106b/25 cluster. miR-106b-5p-related hepatocarcinogenesis and chemo- Overexpression of the miRNA-106b-25 cluster was identi- fied in HCC tissues and cell lines, as well as in breast and therapeutic resistance. –+– +– – + –+ –+ + Journal of Oncology 11 ** miR-106b-5p Vector *** miR-106b-5p ** MHCC-97L Vector 0 5 1015202530 Time (day ) miR-106b-5p Vector (a) Vector miR-106b-5p sponge *** *** Vector ** MHCC-97H miR-106b-5p sponge 0 5 1015202530 Time (day ) miR-106b-5p sponge Vector (b) MHCC-97L MHCC-97H 2.5 1.5 2.0 1.0 1.5 ** 1.0 0.5 0.5 miR-106b-5p Vector miR-106b-5p Vector sponge (c) MHCC-97L MHCC-97H BTG3 1.00 9.11 4.37 1.49 Bcl-xL 1.00 0.62 0.88 1.30 p27 1.00 2.06 2.69 0.76 CDK2 1.00 0.77 1.50 1.31 Tubulin (d) Figure 6: Continued. miR-106b-5p relative expression miR-106b-5p Vector miR-106b-5p miR-106b-5p relative expression sponge Vector Volume (mm ) Volume (mm ) cyclin-CDK G0 12 Journal of Oncology miR-106-5p MHCC-97L MHCC-97H E-cadherin miR-106b-5p miR-106b-5p Vector Vector sponge BTG3 BTG3 mRNA binding N-cadherin AAA Vimentin Bcl-xL p27 CDK2-r160 Rb-E2F1 E2F1 Sorafenib resistance G1 phase Mitosis phase (e) (f) Figure 6: (a, b) In vivo effect of miR-106b-5p. 0e in vivo effect of miR-106b-5p was evaluated in nude mice, subcutaneously injecting miR- 106b-5p, miR-106b-5p sponge, or control cells into nude mice, n � 4 per group. Tumor volume was periodically measured for each mouse, and tumor growth curves were plotted. (c) Expression of miR-106b-5p from tumor tissues was validated by qPCR. (d) Expression of BTG3, Bcl-xL, p27, and CDK2 from tumor tissue of nude mice was measured by western blotting. (e) Representative H&E, as well as HC staining for Ki67, PCNA, BTG3, Bcl-xL, and cyclin E1 for tumor tissues. Scale bar represents 30 μm. (f) Schematic diagram illustrating regulatory ∗ ∗∗ ∗∗∗ signaling of miR-106b-5p contributing to proliferation, metastases, and sorafenib resistance in HCC. P< 0.05, P< 0.01, and P< 0.001. prostate cancers [28, 29]. Various targets of the cluster have apoptosis, and metastasis in various cancers [21, 31, 32]. been successively confirmed in HCC and other cancers, and Based on the dual-luciferase assay and western blotting, our these targets are involved in tumor growth, apoptosis, and study revealed that BTG3 was a direct downstream target of metastasis. Substantial evidence suggested that clustered miR-106b-5p. In vitro loss- or gain-of-function studies miRNAs cooperatively regulate a similar set of genes be- demonstrated that BTG3 might function as a tumor sup- pressor in HCC. longing to the specific signaling pathways and thus govern biological processes in a coordinated manner. However, in Several studies have demonstrated that the Bcl-2 family influences multiple cellular processes, including cell cycle our study, BTG3 is the target of miR-106b-5p, rather than other members of the miR-106b/25 cluster in HCC (data not [33–35]. Followingly, we showed that Bcl-xL, a member of shown). Given the heterogeneity of HCC, it is possible that the Bcl-2 family, is downstream of BTG3. 0e results of the target of the cluster is dependent on the genetic aber- immunofluorescence staining and co-IP confirm the exis- rations within the tumor. tence of protein-protein interaction between BTG3 and Bcl- Although previous studies reported that miR-106b-5p xL. Since the proteins of the Bcl-2 family are the critical was involved in the development of HCC [30], no biological regulator of cellular processes, abnormalities in its function activity was observed. Herein, our results suggest that have been implicated in many diseases, including HCC [36]. overexpressed miR-106b-5p induces greater tumorigenicity One study suggested that Bcl-xL-overexpressing HCC pa- tients had significantly shorter disease-free survival after and enhances chemotherapeutic resistance, whereas inhibited miR-106b-5p has opposite effects. Identifications surgery [37]. Other studies indicated that the Bcl-2 family proteins were central components of the sorafenib cyto- of direct target genes of miRNAs are critical steps for un- derstanding miRNA-related mechanisms of HCC progres- toxicity in hepatoma cells [38], and Bcl-xL level had been sion. BTG3 is a member of the antiproliferative BTG/Tob (B- connected to HCC growth and sorafenib-resistance [39, 40]. cell translocation gene/transducer of ErbB2) protein family, We then sought to identify whether miR-106b-5p could and identified as a tumor suppressor gene involved in the influence the effects of sorafenib on the viability of HCC suppression of proliferation, cell cycle progression, cells. Sorafenib is an oral multikinase inhibitor that Start or R point G1/S transition S phase cyclin E1 Bcl-xL BTG3 PCNA ki-67 HE Metastasis Journal of Oncology 13 improved the overall survival of HCC patients, but the Conflicts of Interest clinical response to sorafenib is limited due to resistance 0e authors declare that they have no conflicts of interest. [3]. Recent data demonstrated that sorafenib-induced changes in miRNAs [41], while others indicated that several miRNAs were involved in sorafenib resistance in HCC Authors’ Contributions [42, 43]. Of note, it has been suggested that miR-106b-5p participated in drug resistance in multiple cancers [13]. Enkhnaran Bilegsaikhan and Guang-Cong Zhang contrib- However, whether miR-106b-5p is involved in sorafenib uted equally to this work. resistance of HCC is still unknown. Our study revealed that miR-106b-5p expression was decreased by sorafenib (data Acknowledgments not shown), and its inhibitor sensitized HCC to sorafenib in vitro. 0is study was partly supported by the National Natural To better understand the mechanisms underlying this Science Foundation of China (81472673 and 81672720) and effect, cell cycle analysis and western blotting were per- the funding from Shanghai Municipal Population and formed. miR-106b-5p inhibitor blocked G1/S transition; Family Planning Commission (20174Y0151). sorafenib potentiated this effect. Whereas, the miR-106b-5p mimic effect was antagonized by sorafenib. We finally found Supplementary Materials that miR-106b-5p interferes with the expression of BTG3, Bcl-xL, BAX, p27, cyclinE1, and CDK2, possibly hampering Supplementary Figure 1: HCC cells transfected with miR- the effect of sorafenib. It is well established that cell cycle 106b-5p mimics, inhibitors, and corresponding negative progression is a predominant factor promoting tumor cell controls; qPCR analyzed miR-106b-5p levels in indicated proliferation and inducing chemotherapeutic resistance to ∗ ∗∗ cells. NC, negative control. P< 0.05, P< 0.01, and sorafenib. Sorafenib can also suppress several critical cell- ∗∗∗ P< 0.001. Supplementary Figure 2: HCC cells transfected cycle regulators, including cyclin D, CDKs, and E2F1-Rb- with miR-106b-5p mimics, inhibitors, and corresponding cyclin E1 complex, which plays crucial roles in mediating negative controls; qPCR analyzed E-cadherin/N-cadherin/ sorafenib resistance in HCC [44]. In our study, miR-106b-5p vimentin levels in indicated cells. NC, negative control. induces sorafenib resistance of HCC through Bcl-xL and ∗ ∗∗ ∗∗∗ P< 0.05, P< 0.01, and P< 0.001. (Supplementary cyclin E1. 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Journal of Oncology – Hindawi Publishing Corporation
Published: Mar 17, 2022
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