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Nucleolin Promotes Cisplatin Resistance in Cervical Cancer by the YB1-MDR1 Pathway

Nucleolin Promotes Cisplatin Resistance in Cervical Cancer by the YB1-MDR1 Pathway Hindawi Journal of Oncology Volume 2021, Article ID 9992218, 11 pages https://doi.org/10.1155/2021/9992218 Research Article Nucleolin Promotes Cisplatin Resistance in Cervical Cancer by the YB1-MDR1 Pathway Jing Ke , Chunming Gu , Heyan Zhang , Yang Liu , Wenhao Zhang , Huiling Rao , Shan Li , and Fuyun Wu School of Basic Medical Sciences, Hubei University of Medicine, Shiyan 442000, China Correspondence should be addressed to Shan Li; lishanhbmu@126.com and Fuyun Wu; wufuyun@hbmu.edu.cn Received 23 March 2021; Accepted 10 April 2021; Published 22 April 2021 Academic Editor: Prasanna Kumar Santhekadur Copyright © 2021 Jing Ke et al. -is 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. Purpose. Cervical cancer is the fourth most common cancer in women worldwide and is the main cause of cancer-related deaths in women. Cisplatin (DDP) is one of the major chemotherapeutic drugs for cervical cancer patients. But, drug resistance limits the effectiveness of cancer therapy. Nucleolin (NCL) is a nucleocytoplasmic multifunctional protein involved in the development of cancer. It has been reported that NCL may be a potential target for modulation of drug resistance. However, the precise molecular mechanisms are poorly understood. Materials and Methods. Human cervical cancer Hela cells and their cisplatin-resistant cell line Hela/DDP were used in this study. -e protein level of NCL in cervical cancer cells was measured by western blot analysis. Hela cells and Hela/DDP cells were transfected with NCL overexpression plasmid or NCL siRNA separately. MTT and EdU assay were performed to evaluate the cell viability and sensitivity to cisplatin. -e drug efflux function of MDR1 protein was assessed by intracellular rhodamine-123 accumulation assay.-e promoter activity of MDR1 was assessed by using a dual-luciferase reporter assay. Results. We found that the protein level of NCL was elevated in Hela/DDP cells. Overexpression of NCL increased cervical cancer cell proliferation and attenuated the sensitivity to cisplatin. Overexpression of NCL increased Multidrug resistance (MDR1) gene expression and drug efflux. Our results demonstrated that NCL was highly related with cisplatin resistance in cervical cancer. NCL played an important role in MDR1 gene transcription through regulation of the transcription factor YB1. Conclusion. Our findings revealed the novel role of NCL in cisplatin-resistant cervical cancer and NCL may be a potential therapeutic target for chemoresistance. cells and promotes the proliferation, invasion, and migration 1. Introduction of tumor cells through its action on different cellular Cervical cancer is the fourth most common cancer in women pathways [9, 10]. In recent years, it was found that the worldwide and the second most common cancer in women expression of NCL was significantly increased in etoposide- living in less-developed regions [1]. Cisplatin-based che- and mitoxantrone-resistant breast cancer cells [11]. Recent motherapy is an important treatment option for cervical reports have demonstrated that high NCL expression pro- motes drug resistance in acute lymphoblastic leukemia cancer, but resistance to cisplatin often results in chemo- therapy failure [2, 3]. As so far, the mechanism of cisplatin [12].-ese results indicate that NCL may be involved in drug resistance in cervical cancer is not clear. NCL is a multi- resistance of tumor cells, but whether NCL is involved in functional phosphoprotein involved in ribosome assembly, cisplatin resistance in cervical cancer has not been reported. rRNA maturation, mRNA stability, and so on [4, 5]. It is Here, we explored the relationship between NCL and cis- mainly located in the nucleolus, but also found in the nu- platin resistance in cervical cancer. We found a new cleoplasm, cytoplasm, and cell membrane [6]. Many studies mechanism of cisplatin resistance mediated by NCL via have shown that NCL plays an important role in tumor stimulating YB1-induced MDR1 transcription. Our results development [7, 8]. NCL is upregulated in a variety of tumor indicated that NCL may be a potential drug-resistant target. 2 Journal of Oncology with siNCL were seeded into a 96-well plate for overnight. 2. Materials and Methods After 36 h, cells were treated with different concentrations of 2.1. Plasmids. -e human NCL gene was amplified by PCR cisplatin for 24 h. MTT was added to each well and incubated from the cDNA of Hela cells and cloned into the pcDNA4/ ° for 4 h at 37 C, and then, 100 μl dimethyl sulfoxide (DMSO) TO vector. To construct MDR1-luciferase reporter plasmid, was added to each well with plate shaking for 30 minutes. the nt −201 to nt +43 fragment of the MDR1 promoter was Absorbance was read at 540 nm using a 96-well plate reader. inserted into the pGL3-basic vector at the KpnI and HindIII For EdU assay, Hela cells transfected with NCL vector or sites. Y-Box mutated MDR1-luciferase reporter plasmid was Hela/DDP cells transfected with siRNA were seeded in 24- generated by overlap extension PCR using the primer. well plates. After treated with DDP for 24 h, cell proliferation was detected using the EdU Cell Proliferation Assay Kit Forward: 5′-GGTGAGGCTGATCAACTGGGCAG- (KeyGEN, China). -e EdU positive cells were determined by GAAC-3′ fluorescence microscopy and calculated from five different Reverse: 5′-GTTCCTGCCC AGTTGATCAG fields. Each experiment was repeated at least three times. CCTCACC-3′ 2.6. Quantitative Real-Time PCR Analysis. Total cellular 2.2. Cell Culture and Transfection. -e Hela cells were RNA was extracted using Trizol (Invitrogen) according to the cultured in Dulbecco’s modified Eagle’s medium (DMEM, manufacturer’s instructions. First-strand cDNA was gener- Invitrogen) supplemented with 10% fetal bovine serum ated with the HiScript 1 Strand cDNA Synthesis Kit (vazyme, (Hyclone), 50 U/mL penicillin, and 50 μg/mL streptomycin China).Quantitative real-time PCR was conducted using a (Invitrogen) at 37 C with 5% CO , and cisplatin-resistant HiScript one-step qRT-PCR SYBR Green Kit (vazyme, China) cervical cancer cells Hela/DDP were treated with DDP at and performed on the Bio-Rad CFX96 TM Real-time system. 0.5 µg/ml in the complete medium to maintain their resis- Gene expression in each sample was normalized to GAPDH tant phenotypes. Hela cells were transfected with pcDNA4/ expression. Primer sequences were as follows: NCL, 5′-G TO-NCL plasmid using lipofectamine 3000 (Invitrogen) ATCACCTAATGCCAGAAGCCAGC-3′; 5′-CAAAGCCG based on the manufacturer’s instructions. After 48 h, cells CCTCTGCCTCCACCAC-3′; MDR1, 5′-TGACTACCAGG were selected with zeocin (200 μg/ml) for one week, and CTCGCCAATGAT-3′; 5′-TGTGCCACCAAGTAGGCTCA then, the cells were used to assay proliferation. AA-3′; GAPDH, 5′-GGAGTCAACGGATTTGGT-3′; 5′-GT GATGGGATTTCCATTGAT-3′. 2.3. Western Blotting. -e whole cell lysates were extracted using RIPA buffer supplemented with protease inhibitors, and 2.7. Intracellular Rhodamine-123 Accumulation Assay. the concentration of isolated protein was determined using Hela cells transfected with NCL overexpression or empty the BCA protein assay. Samples were resolved by SDS-PAGE vector were plated at 1 × 10 /well in 12-well plates and in- and transferred to PVDF membranes. Membranes were cubated for 24 h, cells were treated with or without DDP blocked in 5% nonfat dry milk in TBST for 1 h at room (2.5 μg/mL) for 12 h, and then, Rh123 was added at a final temperature and then incubated with the primary antibodies ° concentration of 10 μg/ml and incubated with cells at 37 C overnight at 4 C, followed by Horseradish-Peroxidase-linked for 30 min. Cells were harvested and resuspended with cold secondary antibodies for 1 h at room temperature. Protein PBS. -e fluorescence intensity of the cells was measured at signals were detected by using the ECL western blotting 488/575 nm using the FACS Calibur flow cytometer (Becton substrate. -e following antibodies were used in immuno- Dickinson, USA). blotting: rabbit monoclonal antibodies against NCL, YB1, MDR1, and GAPDH purchased from Proteintech Group. 2.8. Luciferase Reporter Assay. -e pGL3-MDR1-promoter- luc plasmid or Y-Box mutated MDR1-luc plasmid and NCL 2.4. siRNA Transfection. To knockdown NCL expression, the overexpression plasmid or empty vector were cotransfected double-stranded small interfering RNA against human NCL into Hela cells. After 24 h, cells were treated with cisplatin for (target sequence: 5′-UUUCUCAAACGAAGUAAGCUU- 24 h. Luciferase activity was measured using the Dual-Lu- dTdT-3′) and nonspecific siRNA (target sequence: 5′-UU- ciferase Reporter Assay System (Promega). CUCCGAACGUGUCACGU-3′) (purchased from GenePharma Company Ltd.) were used for transient transfection. Hela/ 2.9. Statistical Analysis. Data analysis was performed using DDP cells were plated into a 6-well plate. After 24 h, cells GraphPad Prism 5. All results are expressed as means± S. E. M. were transfected with 5 μg NCL siRNA or control siRNA Differences between the two groups were determined using with Lipofectamine 3000 reagent according to the manu- Student’s t-test. P< 0.05 was considered statistically significant. facturer’s instructions. Gene silencing was assessed after 36 h by western blotting. 3. Results 2.5. Cell Proliferation Assay. Cell proliferation was assessed 3.1. NCL Is Involved in Cisplatin-Resistant Cervical Cancer by both MTT and EdU assays. For MTT assay, 3000 Hela cells Cells. To determine whether NCL was associated with cis- transfected with NCL vector or Hela/DDP cells transfected platin resistance in cervical cancer, the expression of NCL Journal of Oncology 3 3.4. NCL Regulates Transcription Factor YB1 Expression. was detected by western blotting in Hela cells and cisplatin- resistant cervical cancer cells Hela/DDP. As shown in Transcriptional activation is generally accepted to be the principle mechanism for upregulating MDR1 gene expres- Figures 1(a) and 1(b), NCL was expressed more abundant in Hela/DDP cells than in Hela cells. We then test the effect of sion. Many transcription factors play a role in the regulation overexpression and knockdown of NCL on the sensitivity to of MDR1 gene expression. YB1 (Y-Box binding protein 1) cisplatin of cervical cancer cells. NCL overexpression vector has been demonstrated to be involved in multidrug resis- (NCL) or empty vector (NC) were transfected into Hela tance and regulation of downstream gene MDR1 cells. Western blot results showed that the expression level (Figure 4(a)).So, we tested whether NCL may affect the of NCL was remarkably elevated in cells transfected with expression of YB1. Results showed a significant increase of NCL overexpressing vector compared with the control YB1 after NCL overexpression (Figures 4(b) and (Figure 1(c)), and MTT assay result indicated that over- 4(c)).Similarly, NCL knockdown dramatically attenuated the expression of YB1 (Figures 4(d) and 4(e)). expression of NCL increased cervical cancer cell prolifera- tion and attenuated the sensitivity to cisplatin (Figure 1(d)). Consistently, siRNA knockdown of NCL inhibited cell 3.5. NCL Regulates the Promoter Activity of MDR1 in an proliferation and enhanced the sensitivity of Hela/DDP cells YB1-Dependent Manner. To further address the mecha- to cisplatin (Figures 1(e) and 1(f)). We then performed the nisms that control MDR1 gene expression by NCL, firstly, EdU assay, in which Hela/DDP cells transfected with siRNA we determined whether NCL modulated the activity of the were treated with or without DDP, then exposed to EdU MDR1 promoter. We constructed the MDR1 promoter/ (10 μM) for 12 h, and visualized under a fluorescent mi- luciferase reporter plasmid and Y-Box mutated MDR1-luc croscope. As expected, knockdown of NCL attenuated the plasmid (Figure 5(a)), then they were cotransfected with cell proliferation and enhanced the sensitivity of cisplatin NCL overexpression vector or empty vector into Hela cells as (Figures 1(g) and 1(h)).-ese results suggested that NCL was described in Section 2. After treatment with or without DDP, involved in the cisplatin resistance to cervical cancer. luciferase activities were measured. As shown in Figure 5(b), MDR1 promoter activity was increased by about 2-fold after NCL overexpression compared with the control, and lu- 3.2. NCL Regulates MDR1 Expression. Mechanisms of ciferase activity was markedly increased by about 4-fold multidrug resistance in cancer are very complicated. One of following DDP treatment, while no change was observed in the most important mechanisms responsible for multidrug the NC group. As expected, NCL failed to activate luciferase resistance is the overexpression of multidrug resistance activity in transfectants containing Y-Box mutated MDR1- protein 1 (MDR1), also known as P-glycoprotein 1 (P-gp) or luciferase vector. In contrast, MDR1 promoter activity was ATP-binding cassette subfamily B member 1 (ABCB1), dramatically rEduced in NCL-silenced cells. Moreover lu- which acts as an efflux pump for a wide spectrum of anti- ciferase activity was at a much lower level when cotrans- cancer drugs. So, MDR1 proteins may be involved in the fected with Y-Box mutated MDR1-luciferase vector mechanism of cisplatin resistance mediated by NCL. To test (Figure 5(c)). -ese results suggest that NCL regulates the this possibility, we detected the effect of overexpression and expression of MDR1 by controlling its promoter activity in a knockdown of NCL on MDR1 protein expression level. NCL YB1-dependent manner. overexpression plasmid or vector control was transfected in the Hela cells, and MDR1 and NCL were detected by western blotting. As shown in Figures 2(a) and 2(b), the MDR1 3.6. NCL Induces Cisplatin Resistance in Cervical Cancer Cells protein level was increased in NCL expressing cells, com- by the YB1-MDR1 Pathway. To further test whether NCL pared with that of cells expressing empty vector control. induces cisplatin resistance in cervical cancer cells by the Conversely, a significantly decreased MDR1 level was ob- YB1-MDR1 pathway, we also performed the EdU assay. As served after NCL knockdown cells when compared with the shown in Figure 6(a), NCL overexpression promoted cell control (Figures 2(c) and 2(d)). Furthermore, we measured proliferation and attenuated the sensitivity of cells to DDP the mRNA level of MDR1 in the Hela cells transfected with compared with the NC control. However, a significant NCL siRNA by qRT-PCR. Compared with the siNC group, decrease in cell proliferation and increase in the sensitivity to the mRNA level of MDR1 was markedly decreased DDP was observed after YB1 knockdown in NCL over- (Figure 2(e)). expression cells. 4. Discussion 3.3. NCL Overexpression Increases Drug Efflux. It has been reported that MDR1 functions as a drug efflux pump and Chemotherapy resistance is one of the main obstacles to extrudes multiple anticancer drugs contributing to multi- successful clinical cancer therapy, and drug resistance can drug resistance in many human cancers. So, we investigated be obtained by different mechanisms, including drug efflux, whether overexpression of NCL affected the function of drug apoptosis suppression, enhancing DNA repair, altering efflux. Rhodamine-123 accumulation assay was carried out drug metabolism and persistence of cancer stem cells by flow cytometry. As shown in Figure 3, the fluorescence (CSCs), and epithelial-mesenchymal transition (EMT) intensity of rhodamine-123 was lower in NCL over- [13–15]. Cisplatin is the major chemotherapeutic drug for expression cells than control cells. cervical cancer, but cisplatin resistance has been a common 4 Journal of Oncology 0.8 0.6 NCL 0.4 GAPDH 0.2 0.0 Hela cells Hela/DDP cells (a) (b) 0.6 NCL 0.4 GAPDH 0.2 0.0 5 10 15 20 25 DDP concentration (μM) NC NCL (c) (d) 0.4 0.3 NCL 0.2 GAPDH 0.1 0.0 5 10 15 20 25 DDP concentration (μM) siNC siNCL (e) (f) Figure 1: Continued. NC Hela siNC Hela/DDP siNCL NCL cells Protein relative expression Inhibition rate Inhibition rate Journal of Oncology 5 250 ∗∗∗ ∗∗∗ siNC siNCL siNC + DDP siNCL + DDP (g) (h) Figure 1: NCL is involved in cisplatin-resistant cervical cancer cells. (a) Western blot analysis and (b) quantification of NCL expression in cervical cancer, Hela cell, and cisplatin-resistant cervical cancer cell Hela/DDP. GAPDH was used as a loading control. P< 0.05, compared with Hela cells. (c) -e NCL expression level was detected by western blot in Hela cells transfected with NCL overexpression vector or empty vector. (d) NCL overexpression cells were treated with graded concentrations of DDP; then, cellular viability was assessed by MTT assay. (e) -e same as that in (c), but Hela/DDP cells were transfected with NCL siRNA or control siRNA. (f) Same as that in (d), but with NCL knockdown cells. -e data in the curves represent mean ± SD from three independent experiments. (g, h) Edu proliferation assay analysis of ∗∗∗ the effect of NCL knockdown on the growth of Hela/DDP cells treated with DDP. P< 0.001, compared with control cells. NC NCL NC NCL 0.8 DDP – – + + 0.6 MDR1 0.4 ∗∗ NCL ∗∗∗ 0.2 GAPDH 0.0 NCL MDR1 (a) (b) siNC siNCL siNC siNCL ∗∗∗ 0.8 DDP – – + + ∗∗ MDR1 0.6 NCL 0.4 GAPDH 0.2 0.0 NCL MDR1 (c) (d) Figure 2: Continued. Relative protein levels Relative protein levels Edu positive cell numbers siNC NC siNCL NCL siNC siNC + DDP NC + DDP siNCL siNCL + DDP NCL + DDP siNC + DDP siNC NC siNCL + DDP siNCL NCL siNC + DDP NC + DDP siNCL + DDP NCL + DDP 6 Journal of Oncology 2.5 2.0 1.5 1.0 0.5 0.0 NCL MDR1 siNC siNCL (e) Figure 2: NCL regulates MDR1 expression. (a) Western blot analysis and (b) quantification of MDR1 expression in NCL overexpression Hela cells with or without DDP treatment. (c, d) -e same as that in (a) and (b), but with NCL knockdown cells. (e) MDR1 mRNA expression in NCL knockdown cells was determined by qRT-PCR, and the mRNA levels were normalized to GAPDH. NC + Rh123 NCL + Rh123 0 0 0 50 100 0 50 100 5 5 FSC-A (×10 ) FSC-A (×10 ) NC + DDP + Rh123 NCL + DDP + Rh123 0 0 0 50 100 0 50 100 5 5 FSC-A (×10 ) FSC-A (×10 ) (a) Figure 3: Continued. Count Count Relative mRNA levels Count Count Journal of Oncology 7 NC NCL NC + NCL + DDP DDP (b) Figure 3: Rhodamine-123 (Rh123) efflux assay measuring MDR1 activity. (a) Flow cytometry analysis of intracellular accumulation of Rh123 in NCL overexpression cells with or without DDP treatment. (b) Mean fluorescence intensity of intracellular accumulation of Rh123. Results are mean± SD of three independent experiments. P< 0.05. YB1 binding site NC NCL NC NCL DDP – – + + CTGATTGGCTG YB1 –82 –72 +1 MDR1 NCL GAPDH (a) (b) 2.0 siNC siNCL siNC siNCL DDP –– + + ∗∗ 1.5 ∗∗ YB1 NCL 1.0 GAPDH 0.5 0.0 NCL YB1 (c) (d) Figure 4: Continued. Relative protein levels NC NCL NC + DDP NCL + DDP Relative fluorescence intensity NC NCL NC + DDP NCL + DDP 8 Journal of Oncology ∗∗∗ 1.5 ∗∗ 1.0 0.5 0.0 NCL YB1 (e) Figure 4: NCL regulates YB1 expression. (a) Schematic representation of human MDR1 gene promoter that contains a binding site for YB1. (b) Western blot analysis and (c) quantification of YB1 expression in NCL overexpression Hela cells with or without DDP treatment. (d, e) -e same as that in (b) and (c), but in the NCL knockdown cells. ∗∗∗ MDR1 promoter Luciferase ∗∗∗ YB1 binding site –201 ......CTGATTGGCTG....... +43 WT MDR1-Pro-Luci –201 ......CTGATCAACTG....... +43 Mut-MDR1-Pro-Luci ∗∗∗ ∗∗∗ 3.0 2.0 1.0 0.0 Wt-MDR1-promoter Mut-MDR1-promoter (a) (b) Figure 5: Continued. Relative protein levels siNC siNCL siNC + DDP siNCL + DDP Relative luciferase activity siNC siNCL NC siNC + DDP NCL siNCL + DDP NC + DDP NCL + DDP NC NCL NC + DDP NCL + DDP Journal of Oncology 9 ∗∗∗ ∗∗ ∗∗∗ 2.0 ∗∗∗ 1.5 1.0 0.5 0.0 Wt-MDR1-promoter Mut-MDR1-promoter (c) Figure 5: NCL regulates the promoter activity of MDR1 in a YB1-dependent manner. (a) Structures of the MDR1 promoter/luciferase reporter plasmid and Y-Box mutated MDR1-luc plasmid. (b) Functional analysis of MDR1 promoter activity by luciferase assays: NCL overexpression plasmid or empty vector were cotransfected with the wildtype or mutant MDR1 promoter reporter vector into Hela cells and then treated with or without DDP. After that, the cells were lysed and the luciferase activity was measured. Data are mean± SD, N � 3. (c) -e same as that in (b), but the Hela/DDP cells were cotransfected with siRNA and MDR1 promoter/luciferase reporter plasmid. ∗∗∗ ∗∗∗ NC NCL NC NCL NC + NCL + DDP DDP NC + DDP NCL + DDP (a) (b) Figure 6: Continued. Relative luciferase activity siNC siNCL siNC + DDP siNCL + DDP siNC Edu positive cell numbers siNCL siNC + DDP siNCL + DDP 10 Journal of Oncology ∗∗∗ ∗∗∗ NCL + siNC NCL + siYB1 siNC siYB1 siNC + siYB1 + DDP DDP NCL + siNC + DDP NCL + siYB1 + DDP (c) (d) Figure 6: NCL induces cisplatin resistance in cervical cancer cells by the YB1-MDR1 pathway. (a) Edu proliferation assay analysis of the effect of NCL overexpression on the growth of Hela cells treated with DDP. (b) Quantification of Edu positive cell number. (c, d) -e same as that in (a) and (b), but NCL overexpression cells transfected with YB1 siRNA or control siRNA. -e results presented are representative of three independent experiments. and serious problem in the treatment of cervical cancer. NCL contributed to the drug resistance by regulation the expression and function of MDR1. Transcriptional acti- Several reports have been published showing that over- expression of MDR1 gene enhancing drug efflux is asso- vation is the principle mechanism for upregulating MDR1 ciated with multidrug resistance in cervical cancer [16–18]. gene expression [24]. YB1, as a major transcription factor NCL, as a multifunctional protein with oncogenic prop- for the MDR1 gene, has been reported to regulate the erties involved in many key cellular processes, has attracted expression of MDR1 and mediated multidrug resistance in attention as a potential therapeutic target [19, 20]. NCL was a variety of tumors [25–27]. Here, we provided evidence able to traffic from the nucleus to the cytoplasm and cell that overexpression of NCL led to a markedly increased surface, acting as a surface receptor for a variety of ligands level of YB1 expression. Moreover, the results of luciferase implicated in tumorigenesis and angiogenesis. AS1411, an reporter assay showed that NCL could modulate MDR1 promoter activity in a YB1-dependent manner (Figures 4 NCL-targeted DNA aptamer, has antiproliferative activity against a wide range of cancer cells [21–23]. It was reported and 5). Furthermore, we demonstrated that NCL over- expression in cervical cancer cells led to cisplatin resistance that the NCL gene was upregulated in etoposide- and mitoxantrone-resistant breast cancer cells and associated depending on YB1 (Figure 6). Taken together, NCL is with the drug resistance in acute lymphoblastic leukemia. closely associated with drug resistance, so NCL inhibitor or However, the role of NCL in cisplatin resistance in cervical the combination of NCL and MDR1 inhibitors may provide cancer is not clearly understood. In this study, we found a potential therapeutic option for the treatment of mul- that NCL was overexpressed in cisplatin-resistant cervical tipdrug resistance in cervical cancer. cancer cells and NCL expression was closely related to cisplatin sensitivity in cervical cancer cells. Overexpression 5. Conclusions of NCL significantly attenuated the sensitivity of cisplatin resistance in Hela cells, while NCL knockdown inhibited We found a new mechanism of cisplatin resistance me- cell proliferation and reversed the cisplatin-resistant in diated by NCL in cervical cancer. NCL contributed to the Hela/DDP cell lines (Figure 1).Furthermore, we investi- drug resistance by regulation the expression and function gated the relationship between NCL and the expression of MDR1 in a YB1-dependent manner. Our results indi- level of MDR1 protein because one of the best characterized cated that NCL may be a potential drug-resistant target, drug resistance mechanisms is overexpression of MDR1 and blocking its function may be a potential strategy to which acts as a drug efflux and reduces the drug accu- enhance the treatment efficacy in cisplatin-resistant cer- mulation. We observed that knockdown of NCL expression vical cancer. with siRNA dramatically decreased MDR1 expression levels in protein and mRNA levels. Conversely, a signifi- Data Availability cantly increased MDR1 level and intracellular accumula- tion of Rhodamine-123 were observed in NCL -e data that support the findings of this study are available overexpression cells (Figures 2 and 3).It suggested that from the corresponding author upon reasonable request. Edu positive cell numbers Journal of Oncology 11 [13] B. Mansoori, A. Mohammadi, S. Davudian, S. Shirjang, and Conflicts of Interest B. Baradaran, “-e different mechanisms of cancer drug resistance: a brief review,” Advanced Pharmaceutical Bulletin, -e authors report no conflicts of interest in this work. vol. 7, no. 3, pp. 339–348, 2017. [14] L. Galluzzi, L. Senovilla, I. Vitale et al., “Molecular mecha- Authors’ Contributions nisms of cisplatin resistance,” Oncogene, vol. 31, no. 15, pp. 1869–1883, 2012. Jing Ke and Chunming Gu contributed equally to this work. [15] S. N. Aleksakhina, A. Kashyap, and E. N. Imyanitov, “Mechanisms of acquired tumor drug resistance,” Biochimica Acknowledgments et Biophysica Acta (BBA)—Reviews on Cancer, vol. 1872, no. 2, Article ID 188310, 2019. -is work was supported by the National Natural Science [16] Z. Chen, K. Ling, Y. Zhu, L. Deng, Y. Li, and Z. 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Ono, “Oncogenic nucleolin-AKT pathway,” Nature Communications, vol. 9, Y-box binding protein-1 as an effective therapeutic target in no. 1, p. 4113, 2018. drug-resistant cancer,” Cancer Science, vol. 110, no. 5, [11] Z. Fu and C. Fenselau, “Proteomic evidence for roles for pp. 1536–1543, 2019. nucleolin and poly[ADP-ribosyl] transferase in drug resis- tance,” Journal of Proteome Research, vol. 4, no. 5, pp. 1583–1591, 2005. [12] J. Hu, Y. Chen, Z. Wu et al., “Targeting nucleolin for reversal of chemotherapy resistance in acute lymphoblastic leukemia,” Blood, vol. 134, no. S1, p. 5058, 2019. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Oncology Hindawi Publishing Corporation

Nucleolin Promotes Cisplatin Resistance in Cervical Cancer by the YB1-MDR1 Pathway

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Copyright © 2021 Jing Ke 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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10.1155/2021/9992218
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Abstract

Hindawi Journal of Oncology Volume 2021, Article ID 9992218, 11 pages https://doi.org/10.1155/2021/9992218 Research Article Nucleolin Promotes Cisplatin Resistance in Cervical Cancer by the YB1-MDR1 Pathway Jing Ke , Chunming Gu , Heyan Zhang , Yang Liu , Wenhao Zhang , Huiling Rao , Shan Li , and Fuyun Wu School of Basic Medical Sciences, Hubei University of Medicine, Shiyan 442000, China Correspondence should be addressed to Shan Li; lishanhbmu@126.com and Fuyun Wu; wufuyun@hbmu.edu.cn Received 23 March 2021; Accepted 10 April 2021; Published 22 April 2021 Academic Editor: Prasanna Kumar Santhekadur Copyright © 2021 Jing Ke et al. -is 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. Purpose. Cervical cancer is the fourth most common cancer in women worldwide and is the main cause of cancer-related deaths in women. Cisplatin (DDP) is one of the major chemotherapeutic drugs for cervical cancer patients. But, drug resistance limits the effectiveness of cancer therapy. Nucleolin (NCL) is a nucleocytoplasmic multifunctional protein involved in the development of cancer. It has been reported that NCL may be a potential target for modulation of drug resistance. However, the precise molecular mechanisms are poorly understood. Materials and Methods. Human cervical cancer Hela cells and their cisplatin-resistant cell line Hela/DDP were used in this study. -e protein level of NCL in cervical cancer cells was measured by western blot analysis. Hela cells and Hela/DDP cells were transfected with NCL overexpression plasmid or NCL siRNA separately. MTT and EdU assay were performed to evaluate the cell viability and sensitivity to cisplatin. -e drug efflux function of MDR1 protein was assessed by intracellular rhodamine-123 accumulation assay.-e promoter activity of MDR1 was assessed by using a dual-luciferase reporter assay. Results. We found that the protein level of NCL was elevated in Hela/DDP cells. Overexpression of NCL increased cervical cancer cell proliferation and attenuated the sensitivity to cisplatin. Overexpression of NCL increased Multidrug resistance (MDR1) gene expression and drug efflux. Our results demonstrated that NCL was highly related with cisplatin resistance in cervical cancer. NCL played an important role in MDR1 gene transcription through regulation of the transcription factor YB1. Conclusion. Our findings revealed the novel role of NCL in cisplatin-resistant cervical cancer and NCL may be a potential therapeutic target for chemoresistance. cells and promotes the proliferation, invasion, and migration 1. Introduction of tumor cells through its action on different cellular Cervical cancer is the fourth most common cancer in women pathways [9, 10]. In recent years, it was found that the worldwide and the second most common cancer in women expression of NCL was significantly increased in etoposide- living in less-developed regions [1]. Cisplatin-based che- and mitoxantrone-resistant breast cancer cells [11]. Recent motherapy is an important treatment option for cervical reports have demonstrated that high NCL expression pro- motes drug resistance in acute lymphoblastic leukemia cancer, but resistance to cisplatin often results in chemo- therapy failure [2, 3]. As so far, the mechanism of cisplatin [12].-ese results indicate that NCL may be involved in drug resistance in cervical cancer is not clear. NCL is a multi- resistance of tumor cells, but whether NCL is involved in functional phosphoprotein involved in ribosome assembly, cisplatin resistance in cervical cancer has not been reported. rRNA maturation, mRNA stability, and so on [4, 5]. It is Here, we explored the relationship between NCL and cis- mainly located in the nucleolus, but also found in the nu- platin resistance in cervical cancer. We found a new cleoplasm, cytoplasm, and cell membrane [6]. Many studies mechanism of cisplatin resistance mediated by NCL via have shown that NCL plays an important role in tumor stimulating YB1-induced MDR1 transcription. Our results development [7, 8]. NCL is upregulated in a variety of tumor indicated that NCL may be a potential drug-resistant target. 2 Journal of Oncology with siNCL were seeded into a 96-well plate for overnight. 2. Materials and Methods After 36 h, cells were treated with different concentrations of 2.1. Plasmids. -e human NCL gene was amplified by PCR cisplatin for 24 h. MTT was added to each well and incubated from the cDNA of Hela cells and cloned into the pcDNA4/ ° for 4 h at 37 C, and then, 100 μl dimethyl sulfoxide (DMSO) TO vector. To construct MDR1-luciferase reporter plasmid, was added to each well with plate shaking for 30 minutes. the nt −201 to nt +43 fragment of the MDR1 promoter was Absorbance was read at 540 nm using a 96-well plate reader. inserted into the pGL3-basic vector at the KpnI and HindIII For EdU assay, Hela cells transfected with NCL vector or sites. Y-Box mutated MDR1-luciferase reporter plasmid was Hela/DDP cells transfected with siRNA were seeded in 24- generated by overlap extension PCR using the primer. well plates. After treated with DDP for 24 h, cell proliferation was detected using the EdU Cell Proliferation Assay Kit Forward: 5′-GGTGAGGCTGATCAACTGGGCAG- (KeyGEN, China). -e EdU positive cells were determined by GAAC-3′ fluorescence microscopy and calculated from five different Reverse: 5′-GTTCCTGCCC AGTTGATCAG fields. Each experiment was repeated at least three times. CCTCACC-3′ 2.6. Quantitative Real-Time PCR Analysis. Total cellular 2.2. Cell Culture and Transfection. -e Hela cells were RNA was extracted using Trizol (Invitrogen) according to the cultured in Dulbecco’s modified Eagle’s medium (DMEM, manufacturer’s instructions. First-strand cDNA was gener- Invitrogen) supplemented with 10% fetal bovine serum ated with the HiScript 1 Strand cDNA Synthesis Kit (vazyme, (Hyclone), 50 U/mL penicillin, and 50 μg/mL streptomycin China).Quantitative real-time PCR was conducted using a (Invitrogen) at 37 C with 5% CO , and cisplatin-resistant HiScript one-step qRT-PCR SYBR Green Kit (vazyme, China) cervical cancer cells Hela/DDP were treated with DDP at and performed on the Bio-Rad CFX96 TM Real-time system. 0.5 µg/ml in the complete medium to maintain their resis- Gene expression in each sample was normalized to GAPDH tant phenotypes. Hela cells were transfected with pcDNA4/ expression. Primer sequences were as follows: NCL, 5′-G TO-NCL plasmid using lipofectamine 3000 (Invitrogen) ATCACCTAATGCCAGAAGCCAGC-3′; 5′-CAAAGCCG based on the manufacturer’s instructions. After 48 h, cells CCTCTGCCTCCACCAC-3′; MDR1, 5′-TGACTACCAGG were selected with zeocin (200 μg/ml) for one week, and CTCGCCAATGAT-3′; 5′-TGTGCCACCAAGTAGGCTCA then, the cells were used to assay proliferation. AA-3′; GAPDH, 5′-GGAGTCAACGGATTTGGT-3′; 5′-GT GATGGGATTTCCATTGAT-3′. 2.3. Western Blotting. -e whole cell lysates were extracted using RIPA buffer supplemented with protease inhibitors, and 2.7. Intracellular Rhodamine-123 Accumulation Assay. the concentration of isolated protein was determined using Hela cells transfected with NCL overexpression or empty the BCA protein assay. Samples were resolved by SDS-PAGE vector were plated at 1 × 10 /well in 12-well plates and in- and transferred to PVDF membranes. Membranes were cubated for 24 h, cells were treated with or without DDP blocked in 5% nonfat dry milk in TBST for 1 h at room (2.5 μg/mL) for 12 h, and then, Rh123 was added at a final temperature and then incubated with the primary antibodies ° concentration of 10 μg/ml and incubated with cells at 37 C overnight at 4 C, followed by Horseradish-Peroxidase-linked for 30 min. Cells were harvested and resuspended with cold secondary antibodies for 1 h at room temperature. Protein PBS. -e fluorescence intensity of the cells was measured at signals were detected by using the ECL western blotting 488/575 nm using the FACS Calibur flow cytometer (Becton substrate. -e following antibodies were used in immuno- Dickinson, USA). blotting: rabbit monoclonal antibodies against NCL, YB1, MDR1, and GAPDH purchased from Proteintech Group. 2.8. Luciferase Reporter Assay. -e pGL3-MDR1-promoter- luc plasmid or Y-Box mutated MDR1-luc plasmid and NCL 2.4. siRNA Transfection. To knockdown NCL expression, the overexpression plasmid or empty vector were cotransfected double-stranded small interfering RNA against human NCL into Hela cells. After 24 h, cells were treated with cisplatin for (target sequence: 5′-UUUCUCAAACGAAGUAAGCUU- 24 h. Luciferase activity was measured using the Dual-Lu- dTdT-3′) and nonspecific siRNA (target sequence: 5′-UU- ciferase Reporter Assay System (Promega). CUCCGAACGUGUCACGU-3′) (purchased from GenePharma Company Ltd.) were used for transient transfection. Hela/ 2.9. Statistical Analysis. Data analysis was performed using DDP cells were plated into a 6-well plate. After 24 h, cells GraphPad Prism 5. All results are expressed as means± S. E. M. were transfected with 5 μg NCL siRNA or control siRNA Differences between the two groups were determined using with Lipofectamine 3000 reagent according to the manu- Student’s t-test. P< 0.05 was considered statistically significant. facturer’s instructions. Gene silencing was assessed after 36 h by western blotting. 3. Results 2.5. Cell Proliferation Assay. Cell proliferation was assessed 3.1. NCL Is Involved in Cisplatin-Resistant Cervical Cancer by both MTT and EdU assays. For MTT assay, 3000 Hela cells Cells. To determine whether NCL was associated with cis- transfected with NCL vector or Hela/DDP cells transfected platin resistance in cervical cancer, the expression of NCL Journal of Oncology 3 3.4. NCL Regulates Transcription Factor YB1 Expression. was detected by western blotting in Hela cells and cisplatin- resistant cervical cancer cells Hela/DDP. As shown in Transcriptional activation is generally accepted to be the principle mechanism for upregulating MDR1 gene expres- Figures 1(a) and 1(b), NCL was expressed more abundant in Hela/DDP cells than in Hela cells. We then test the effect of sion. Many transcription factors play a role in the regulation overexpression and knockdown of NCL on the sensitivity to of MDR1 gene expression. YB1 (Y-Box binding protein 1) cisplatin of cervical cancer cells. NCL overexpression vector has been demonstrated to be involved in multidrug resis- (NCL) or empty vector (NC) were transfected into Hela tance and regulation of downstream gene MDR1 cells. Western blot results showed that the expression level (Figure 4(a)).So, we tested whether NCL may affect the of NCL was remarkably elevated in cells transfected with expression of YB1. Results showed a significant increase of NCL overexpressing vector compared with the control YB1 after NCL overexpression (Figures 4(b) and (Figure 1(c)), and MTT assay result indicated that over- 4(c)).Similarly, NCL knockdown dramatically attenuated the expression of YB1 (Figures 4(d) and 4(e)). expression of NCL increased cervical cancer cell prolifera- tion and attenuated the sensitivity to cisplatin (Figure 1(d)). Consistently, siRNA knockdown of NCL inhibited cell 3.5. NCL Regulates the Promoter Activity of MDR1 in an proliferation and enhanced the sensitivity of Hela/DDP cells YB1-Dependent Manner. To further address the mecha- to cisplatin (Figures 1(e) and 1(f)). We then performed the nisms that control MDR1 gene expression by NCL, firstly, EdU assay, in which Hela/DDP cells transfected with siRNA we determined whether NCL modulated the activity of the were treated with or without DDP, then exposed to EdU MDR1 promoter. We constructed the MDR1 promoter/ (10 μM) for 12 h, and visualized under a fluorescent mi- luciferase reporter plasmid and Y-Box mutated MDR1-luc croscope. As expected, knockdown of NCL attenuated the plasmid (Figure 5(a)), then they were cotransfected with cell proliferation and enhanced the sensitivity of cisplatin NCL overexpression vector or empty vector into Hela cells as (Figures 1(g) and 1(h)).-ese results suggested that NCL was described in Section 2. After treatment with or without DDP, involved in the cisplatin resistance to cervical cancer. luciferase activities were measured. As shown in Figure 5(b), MDR1 promoter activity was increased by about 2-fold after NCL overexpression compared with the control, and lu- 3.2. NCL Regulates MDR1 Expression. Mechanisms of ciferase activity was markedly increased by about 4-fold multidrug resistance in cancer are very complicated. One of following DDP treatment, while no change was observed in the most important mechanisms responsible for multidrug the NC group. As expected, NCL failed to activate luciferase resistance is the overexpression of multidrug resistance activity in transfectants containing Y-Box mutated MDR1- protein 1 (MDR1), also known as P-glycoprotein 1 (P-gp) or luciferase vector. In contrast, MDR1 promoter activity was ATP-binding cassette subfamily B member 1 (ABCB1), dramatically rEduced in NCL-silenced cells. Moreover lu- which acts as an efflux pump for a wide spectrum of anti- ciferase activity was at a much lower level when cotrans- cancer drugs. So, MDR1 proteins may be involved in the fected with Y-Box mutated MDR1-luciferase vector mechanism of cisplatin resistance mediated by NCL. To test (Figure 5(c)). -ese results suggest that NCL regulates the this possibility, we detected the effect of overexpression and expression of MDR1 by controlling its promoter activity in a knockdown of NCL on MDR1 protein expression level. NCL YB1-dependent manner. overexpression plasmid or vector control was transfected in the Hela cells, and MDR1 and NCL were detected by western blotting. As shown in Figures 2(a) and 2(b), the MDR1 3.6. NCL Induces Cisplatin Resistance in Cervical Cancer Cells protein level was increased in NCL expressing cells, com- by the YB1-MDR1 Pathway. To further test whether NCL pared with that of cells expressing empty vector control. induces cisplatin resistance in cervical cancer cells by the Conversely, a significantly decreased MDR1 level was ob- YB1-MDR1 pathway, we also performed the EdU assay. As served after NCL knockdown cells when compared with the shown in Figure 6(a), NCL overexpression promoted cell control (Figures 2(c) and 2(d)). Furthermore, we measured proliferation and attenuated the sensitivity of cells to DDP the mRNA level of MDR1 in the Hela cells transfected with compared with the NC control. However, a significant NCL siRNA by qRT-PCR. Compared with the siNC group, decrease in cell proliferation and increase in the sensitivity to the mRNA level of MDR1 was markedly decreased DDP was observed after YB1 knockdown in NCL over- (Figure 2(e)). expression cells. 4. Discussion 3.3. NCL Overexpression Increases Drug Efflux. It has been reported that MDR1 functions as a drug efflux pump and Chemotherapy resistance is one of the main obstacles to extrudes multiple anticancer drugs contributing to multi- successful clinical cancer therapy, and drug resistance can drug resistance in many human cancers. So, we investigated be obtained by different mechanisms, including drug efflux, whether overexpression of NCL affected the function of drug apoptosis suppression, enhancing DNA repair, altering efflux. Rhodamine-123 accumulation assay was carried out drug metabolism and persistence of cancer stem cells by flow cytometry. As shown in Figure 3, the fluorescence (CSCs), and epithelial-mesenchymal transition (EMT) intensity of rhodamine-123 was lower in NCL over- [13–15]. Cisplatin is the major chemotherapeutic drug for expression cells than control cells. cervical cancer, but cisplatin resistance has been a common 4 Journal of Oncology 0.8 0.6 NCL 0.4 GAPDH 0.2 0.0 Hela cells Hela/DDP cells (a) (b) 0.6 NCL 0.4 GAPDH 0.2 0.0 5 10 15 20 25 DDP concentration (μM) NC NCL (c) (d) 0.4 0.3 NCL 0.2 GAPDH 0.1 0.0 5 10 15 20 25 DDP concentration (μM) siNC siNCL (e) (f) Figure 1: Continued. NC Hela siNC Hela/DDP siNCL NCL cells Protein relative expression Inhibition rate Inhibition rate Journal of Oncology 5 250 ∗∗∗ ∗∗∗ siNC siNCL siNC + DDP siNCL + DDP (g) (h) Figure 1: NCL is involved in cisplatin-resistant cervical cancer cells. (a) Western blot analysis and (b) quantification of NCL expression in cervical cancer, Hela cell, and cisplatin-resistant cervical cancer cell Hela/DDP. GAPDH was used as a loading control. P< 0.05, compared with Hela cells. (c) -e NCL expression level was detected by western blot in Hela cells transfected with NCL overexpression vector or empty vector. (d) NCL overexpression cells were treated with graded concentrations of DDP; then, cellular viability was assessed by MTT assay. (e) -e same as that in (c), but Hela/DDP cells were transfected with NCL siRNA or control siRNA. (f) Same as that in (d), but with NCL knockdown cells. -e data in the curves represent mean ± SD from three independent experiments. (g, h) Edu proliferation assay analysis of ∗∗∗ the effect of NCL knockdown on the growth of Hela/DDP cells treated with DDP. P< 0.001, compared with control cells. NC NCL NC NCL 0.8 DDP – – + + 0.6 MDR1 0.4 ∗∗ NCL ∗∗∗ 0.2 GAPDH 0.0 NCL MDR1 (a) (b) siNC siNCL siNC siNCL ∗∗∗ 0.8 DDP – – + + ∗∗ MDR1 0.6 NCL 0.4 GAPDH 0.2 0.0 NCL MDR1 (c) (d) Figure 2: Continued. Relative protein levels Relative protein levels Edu positive cell numbers siNC NC siNCL NCL siNC siNC + DDP NC + DDP siNCL siNCL + DDP NCL + DDP siNC + DDP siNC NC siNCL + DDP siNCL NCL siNC + DDP NC + DDP siNCL + DDP NCL + DDP 6 Journal of Oncology 2.5 2.0 1.5 1.0 0.5 0.0 NCL MDR1 siNC siNCL (e) Figure 2: NCL regulates MDR1 expression. (a) Western blot analysis and (b) quantification of MDR1 expression in NCL overexpression Hela cells with or without DDP treatment. (c, d) -e same as that in (a) and (b), but with NCL knockdown cells. (e) MDR1 mRNA expression in NCL knockdown cells was determined by qRT-PCR, and the mRNA levels were normalized to GAPDH. NC + Rh123 NCL + Rh123 0 0 0 50 100 0 50 100 5 5 FSC-A (×10 ) FSC-A (×10 ) NC + DDP + Rh123 NCL + DDP + Rh123 0 0 0 50 100 0 50 100 5 5 FSC-A (×10 ) FSC-A (×10 ) (a) Figure 3: Continued. Count Count Relative mRNA levels Count Count Journal of Oncology 7 NC NCL NC + NCL + DDP DDP (b) Figure 3: Rhodamine-123 (Rh123) efflux assay measuring MDR1 activity. (a) Flow cytometry analysis of intracellular accumulation of Rh123 in NCL overexpression cells with or without DDP treatment. (b) Mean fluorescence intensity of intracellular accumulation of Rh123. Results are mean± SD of three independent experiments. P< 0.05. YB1 binding site NC NCL NC NCL DDP – – + + CTGATTGGCTG YB1 –82 –72 +1 MDR1 NCL GAPDH (a) (b) 2.0 siNC siNCL siNC siNCL DDP –– + + ∗∗ 1.5 ∗∗ YB1 NCL 1.0 GAPDH 0.5 0.0 NCL YB1 (c) (d) Figure 4: Continued. Relative protein levels NC NCL NC + DDP NCL + DDP Relative fluorescence intensity NC NCL NC + DDP NCL + DDP 8 Journal of Oncology ∗∗∗ 1.5 ∗∗ 1.0 0.5 0.0 NCL YB1 (e) Figure 4: NCL regulates YB1 expression. (a) Schematic representation of human MDR1 gene promoter that contains a binding site for YB1. (b) Western blot analysis and (c) quantification of YB1 expression in NCL overexpression Hela cells with or without DDP treatment. (d, e) -e same as that in (b) and (c), but in the NCL knockdown cells. ∗∗∗ MDR1 promoter Luciferase ∗∗∗ YB1 binding site –201 ......CTGATTGGCTG....... +43 WT MDR1-Pro-Luci –201 ......CTGATCAACTG....... +43 Mut-MDR1-Pro-Luci ∗∗∗ ∗∗∗ 3.0 2.0 1.0 0.0 Wt-MDR1-promoter Mut-MDR1-promoter (a) (b) Figure 5: Continued. Relative protein levels siNC siNCL siNC + DDP siNCL + DDP Relative luciferase activity siNC siNCL NC siNC + DDP NCL siNCL + DDP NC + DDP NCL + DDP NC NCL NC + DDP NCL + DDP Journal of Oncology 9 ∗∗∗ ∗∗ ∗∗∗ 2.0 ∗∗∗ 1.5 1.0 0.5 0.0 Wt-MDR1-promoter Mut-MDR1-promoter (c) Figure 5: NCL regulates the promoter activity of MDR1 in a YB1-dependent manner. (a) Structures of the MDR1 promoter/luciferase reporter plasmid and Y-Box mutated MDR1-luc plasmid. (b) Functional analysis of MDR1 promoter activity by luciferase assays: NCL overexpression plasmid or empty vector were cotransfected with the wildtype or mutant MDR1 promoter reporter vector into Hela cells and then treated with or without DDP. After that, the cells were lysed and the luciferase activity was measured. Data are mean± SD, N � 3. (c) -e same as that in (b), but the Hela/DDP cells were cotransfected with siRNA and MDR1 promoter/luciferase reporter plasmid. ∗∗∗ ∗∗∗ NC NCL NC NCL NC + NCL + DDP DDP NC + DDP NCL + DDP (a) (b) Figure 6: Continued. Relative luciferase activity siNC siNCL siNC + DDP siNCL + DDP siNC Edu positive cell numbers siNCL siNC + DDP siNCL + DDP 10 Journal of Oncology ∗∗∗ ∗∗∗ NCL + siNC NCL + siYB1 siNC siYB1 siNC + siYB1 + DDP DDP NCL + siNC + DDP NCL + siYB1 + DDP (c) (d) Figure 6: NCL induces cisplatin resistance in cervical cancer cells by the YB1-MDR1 pathway. (a) Edu proliferation assay analysis of the effect of NCL overexpression on the growth of Hela cells treated with DDP. (b) Quantification of Edu positive cell number. (c, d) -e same as that in (a) and (b), but NCL overexpression cells transfected with YB1 siRNA or control siRNA. -e results presented are representative of three independent experiments. and serious problem in the treatment of cervical cancer. NCL contributed to the drug resistance by regulation the expression and function of MDR1. Transcriptional acti- Several reports have been published showing that over- expression of MDR1 gene enhancing drug efflux is asso- vation is the principle mechanism for upregulating MDR1 ciated with multidrug resistance in cervical cancer [16–18]. gene expression [24]. YB1, as a major transcription factor NCL, as a multifunctional protein with oncogenic prop- for the MDR1 gene, has been reported to regulate the erties involved in many key cellular processes, has attracted expression of MDR1 and mediated multidrug resistance in attention as a potential therapeutic target [19, 20]. NCL was a variety of tumors [25–27]. Here, we provided evidence able to traffic from the nucleus to the cytoplasm and cell that overexpression of NCL led to a markedly increased surface, acting as a surface receptor for a variety of ligands level of YB1 expression. Moreover, the results of luciferase implicated in tumorigenesis and angiogenesis. AS1411, an reporter assay showed that NCL could modulate MDR1 promoter activity in a YB1-dependent manner (Figures 4 NCL-targeted DNA aptamer, has antiproliferative activity against a wide range of cancer cells [21–23]. It was reported and 5). Furthermore, we demonstrated that NCL over- expression in cervical cancer cells led to cisplatin resistance that the NCL gene was upregulated in etoposide- and mitoxantrone-resistant breast cancer cells and associated depending on YB1 (Figure 6). Taken together, NCL is with the drug resistance in acute lymphoblastic leukemia. closely associated with drug resistance, so NCL inhibitor or However, the role of NCL in cisplatin resistance in cervical the combination of NCL and MDR1 inhibitors may provide cancer is not clearly understood. In this study, we found a potential therapeutic option for the treatment of mul- that NCL was overexpressed in cisplatin-resistant cervical tipdrug resistance in cervical cancer. cancer cells and NCL expression was closely related to cisplatin sensitivity in cervical cancer cells. Overexpression 5. Conclusions of NCL significantly attenuated the sensitivity of cisplatin resistance in Hela cells, while NCL knockdown inhibited We found a new mechanism of cisplatin resistance me- cell proliferation and reversed the cisplatin-resistant in diated by NCL in cervical cancer. NCL contributed to the Hela/DDP cell lines (Figure 1).Furthermore, we investi- drug resistance by regulation the expression and function gated the relationship between NCL and the expression of MDR1 in a YB1-dependent manner. Our results indi- level of MDR1 protein because one of the best characterized cated that NCL may be a potential drug-resistant target, drug resistance mechanisms is overexpression of MDR1 and blocking its function may be a potential strategy to which acts as a drug efflux and reduces the drug accu- enhance the treatment efficacy in cisplatin-resistant cer- mulation. We observed that knockdown of NCL expression vical cancer. with siRNA dramatically decreased MDR1 expression levels in protein and mRNA levels. Conversely, a signifi- Data Availability cantly increased MDR1 level and intracellular accumula- tion of Rhodamine-123 were observed in NCL -e data that support the findings of this study are available overexpression cells (Figures 2 and 3).It suggested that from the corresponding author upon reasonable request. Edu positive cell numbers Journal of Oncology 11 [13] B. Mansoori, A. Mohammadi, S. Davudian, S. Shirjang, and Conflicts of Interest B. Baradaran, “-e different mechanisms of cancer drug resistance: a brief review,” Advanced Pharmaceutical Bulletin, -e authors report no conflicts of interest in this work. vol. 7, no. 3, pp. 339–348, 2017. [14] L. Galluzzi, L. Senovilla, I. Vitale et al., “Molecular mecha- Authors’ Contributions nisms of cisplatin resistance,” Oncogene, vol. 31, no. 15, pp. 1869–1883, 2012. Jing Ke and Chunming Gu contributed equally to this work. [15] S. N. Aleksakhina, A. Kashyap, and E. N. Imyanitov, “Mechanisms of acquired tumor drug resistance,” Biochimica Acknowledgments et Biophysica Acta (BBA)—Reviews on Cancer, vol. 1872, no. 2, Article ID 188310, 2019. -is work was supported by the National Natural Science [16] Z. Chen, K. Ling, Y. Zhu, L. Deng, Y. Li, and Z. 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Published: Apr 22, 2021

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