Yonsei Med J.  2019 Dec;60(12):1146-1156. 10.3349/ymj.2019.60.12.1146.

MiR-338-3p Enhances Ovarian Cancer Cell Sensitivity to Cisplatin by Downregulating WNT2B

• Niu Q
• Liu Z
• Gao J
• Wang Q

• KMID: 2463793
• DOI: http://doi.org/10.3349/ymj.2019.60.12.1146

Abstract

PURPOSE
Chemoresistance is a concern in ovarian cancer patients, in whom survival remains. MicroRNA, a novel class of small RNAs, have frequently been found to be dysregulated in human malignancies and to act as negative regulators of gene expression. This study aimed to explore the function of miR-338-3p in cisplatin resistance in ovarian cancer and potential molecular mechanisms thereof.
MATERIALS AND METHODS
The expression levels of miR-338-3p and WNT2B in ovarian cancer tissues and cells were estimated by real-time quantitative polymerase chain reaction (RT-qPCR). In addition, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT), transwell, and flow cytometry assays were used to assess biological role of miR-338-3p in vitro. Western blot assay was conducted to measure protein expression of WNT2B, epithelial-mesenchymal transition (EMT)-related proteins, and apoptosis-related proteins. The relationship between miR-338-3p and WNT2B was confirmed by dual-luciferase reporter. Finally, a xenograft tumor model was developed to explore the effects of overexpression of miR-338-3p on tumor growth in ovarian cancer in vivo.
RESULTS
MiR-338-3p was downregulated in cisplatin resistant ovarian cancer tissues and cells. Mechanistically, high expression of miR-338-3p enhanced cell sensitivity to cisplatin by inhibiting proliferation, motility, and EMT and by promoting apoptosis via targeting WNT2B expression in vitro. Furthermore, overexpression of miR-338-3p increased cisplatin sensitivity among ovarian cancer in an in vivo xenograft tumor model.
CONCLUSION
MiR-338-3p enhances the sensitivity of ovarian cancer cells to cisplatin by downregulating WNT2B.

Keyword

WNT2B; miR-338-3p; ovarian cancer; chemoresistance

MeSH Terms

Apoptosis
Blotting, Western
Cisplatin*
Epithelial-Mesenchymal Transition
Flow Cytometry
Gene Expression
Heterografts
Humans
In Vitro Techniques
MicroRNAs
Ovarian Neoplasms*
Polymerase Chain Reaction
RNA
Cisplatin
MicroRNAs
RNA

Figure

• 

  Fig. 1 Expression of miR-338-3p in ovarian cancer tissues and cell clines. (A) Real-time quantitative polymerase chain reaction (RT-qPCR) assay was performed to examine the expression of miR-338-3p in 54 ovarian cancer tissues (divided into two groups according to resistance to cisplatin) and adjacent normal tissues. (B) The expression levels of miR-338-3p in human normal ovarian cancer cells (IOSE-80) and ovarian cancer cells (A2780 and SKOV3), as well as cisplatin resistant ovarian cancer cells (A2780/DDP and SKOV3/DDP), were assessed by RT-qPCR. (C) The overall survival curve of ovarian cancer patients was analyzed with Kaplan-Meier analysis. *p<0.05; **p<0.01; ***p<0.001.

• 

  Fig. 2 Increased expression of miR-338-3p enhances sensitivity to cisplatin among ovarian cancer cells. A2780/DDP and SKOV3/DDP cells were transfected with miR-NC or miR-338-3p. (A) The relative expression levels of miR-338-3p in transfected cisplatin-resistant ovarian cancer cells was evaluated via real-time quantitative polymerase chain reaction (RT-qPCR). (B–E) IC50 values of cisplatin, as well as cell viability in A2780/DDP and SKOV3/DDP cells, were measured by MTT assay. (F) Apoptotic rate was assessed by flow cytometry in A2780/DDP and SKOV3/DDP cells after transfection. (G and H) Protein levels of cleaved-caspase-3, Bcl-2, and Bax were determined in A2780/DDP and SKOV3/DDP cells by Western blot. (I and J) Cell migration and invasion were evaluated in transfected A2780/DDP and SKOV3/DDP by transwell assay (×100, stained with 0.1% crystal violet). (K and L) Western blot assay was used to assess the expression levels of epithelial-mesenchymal transition-related proteins (E-cadherin, N-cadherin and Vimentin). *p<0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

• 

  Fig. 3 WNT2B is a target of miR-338-3p. (A) The binding sites of miR-338-3p on the 3′UTR of WNT2B was predicted with bioinformatics and its mutant are shown. (B and C) Luciferase activity was estimated in A2780/DDP and SKOV3/DDP co-transfected with WNT2B 3′UTR-WT/MUT luciferase reporters and miR-338-3p/miR-NC. (D–F) The protein and mRNA levels of WNT2B were measured in treatment-responsive patient tissues or cells (A2780 and SKOV3) and treatment-resistant patients tissues and cells (A2780/DDP and SKOV3/DDP), as well as matched normal controls, utilizing real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot assay. (G) The relationship between miR-338-3p and WNT2B was analyzed. (H–K) Gain-of-function experiments were performed to detect mRNA and protein expression levels of WNT2B in A2780/DDP and SKOV3/DDP cells transfected with miR-NC, miR-338-3p, miR-338-3p+pcDNAN or miR-338-3p+WNT2B. *p<0.05; ***p<0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

• 

  Fig. 4 WNT2B knockdown weakens resistance to cisplatin in ovarian cancer cells. A2780/DDP and SKOV3/DDP cells were transfected with sh-NC or sh-WNT2B. (A and B) real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot assays were carried out to evaluate expression levels of WNT2B in transfected ovarian cancer cells. (C–F) MTT analysis was utilized to assess IC50 values for cisplatin and cell viability in ovarian cancer cells post-transfection. (G) Apoptosis of transfected ovarian cancer cells was determined by flow cytometry. (H and I) Levels of apoptosis-related proteins (cleaved-caspase-3, Bcl-2, and Bax) were measured with Western blot. (J and K) Transwell assay was performed to detect the motility of ovarian cancer cells. (L and M) Western blot was used to assess the expression levels of E-cadherin, N-cadherin, and Vimentin. *p<0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

• 

  Fig. 5 Upregulation of miR-338-3p increases cisplatin sensitivity in resistant ovarian cancer cells by negatively regulating WNT2B. A2780/DDP and SKOV3/DDP were transfected with miR-NC, miR-338-3p, miR-338-3p+pcDNA, or miR-338-3p+WNT2B. (A and B) IC50 values for cisplatin was measured by MTT assay in transfected A2780/DDP and SKOV3/DDP cells treated with various doses of cisplatin for 48 h. (C and D) MTT analysis was used to measure the viability of transfected A2780/DDP and SKOV3/DDP cells. (E) Apoptosis rates of 2780/DDP and SKOV3/DDP cells were detected by flow cytometry assay. (F and G) Western blot assay was conducted to test the protein expression levels of apoptosis-related proteins in transfected cells. (H and I) The migration and invasion capabilities in transfected ovarian cancer cells were measured by transwell assay. (J and K) The protein expression levels of epithelial-mesenchymal transition-related in transfected A2780/DDP and SKOV3/DDP cells were detected by Western blot assay. *p<0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

• 

  Fig. 6 Elevated expression of miR-338-3p suppresses tumor growth and enhances cisplatin sensitivity by affecting WNT2B expression in vivo. (A and B) Growth curves for tumor volume and weight were measured in xenograft tumors from nude mice treated with PBS or cisplatin (5.0 mg/kg, once every three days). (C and D) RT-qPCR and Western blot assay were performed to measure the expression levels of miR-338-3p and WNT2B in resected tumor tissues, respectively. *p<0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Reference

1. Torre LA, Trabert B, DeSantis CE, Miller KD, Samimi G, Runowicz CD, et al. Ovarian cancer statistics, 2018. CA Cancer J Clin. 2018; 68:284–296.

2. Ozols RF, Bundy BN, Greer BE, Fowler JM, Clarke-Pearson D, Burger RA, et al. Phase III trial of carboplatin and paclitaxel compared with cisplatin and paclitaxel in patients with optimally resected stage III ovarian cancer: a Gynecologic Oncology Group study. J Clin Oncol. 2003; 21:3194–3200.

3. Agarwal R, Kaye SB. Ovarian cancer: strategies for overcoming resistance to chemotherapy. Nat Rev Cancer. 2003; 3:502–516.

4. Mantia-Smaldone GM, Edwards RP, Vlad AM. Targeted treatment of recurrent platinum-resistant ovarian cancer: current and emerging therapies. Cancer Manag Res. 2011; 3:25–38.

5. Stordal B, Pavlakis N, Davey R. A systematic review of platinum and taxane resistance from bench to clinic: an inverse relationship. Cancer Treat Rev. 2007; 33:688–703.

6. Steg AD, Bevis KS, Katre AA, Ziebarth A, Dobbin ZC, Alvarez RD, et al. Stem cell pathways contribute to clinical chemoresistance in ovarian cancer. Clin Cancer Res. 2012; 18:869–881.

7. Ricci F, Bernasconi S, Perego P, Ganzinelli M, Russo G, Bono F, et al. Ovarian carcinoma tumor-initiating cells have a mesenchymal phenotype. Cell Cycle. 2012; 11:1966–1976.

8. Yonemura Y, Kawamura T, Bandou E, Tsukiyama G, Endou Y, Miura M. The natural history of free cancer cells in the peritoneal cavity. Recent Results Cancer Res. 2007; 169:11–23.

9. Chaffer CL, Weinberg RA. A perspective on cancer cell metastasis. Science. 2011; 331:1559–1564.

10. Singh A, Settleman J. EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene. 2010; 29:4741–4751.

11. Haslehurst AM, Koti M, Dharsee M, Nuin P, Evans K, Geraci J, et al. EMT transcription factors snail and slug directly contribute to cisplatin resistance in ovarian cancer. BMC Cancer. 2012; 12:91.

12. Ha M, Kim VN. Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol. 2014; 15:509–524.

13. Sorrentino A, Liu CG, Addario A, Peschle C, Scambia G, Ferlini C. Role of microRNAs in drug-resistant ovarian cancer cells. Gynecol Oncol. 2008; 111:478–486.

14. Sui GQ, Fei D, Guo F, Zhen X, Luo Q, Yin S, et al. MicroRNA-338-3p inhibits thyroid cancer progression through targeting AKT3. Am J Cancer Res. 2017; 7:1177–1187.

15. Cao R, Shao J, Hu Y, Wang L, Li Z, Sun G, et al. microRNA-338-3p inhibits proliferation, migration, invasion, and EMT in osteosarcoma cells by targeting activator of 90 kDa heat shock protein ATPase homolog 1. Cancer Cell Int. 2018; 18:49.

16. Sun F, Yu M, Yu J, Liu Z, Zhou X, Liu Y, et al. miR-338-3p functions as a tumor suppressor in gastric cancer by targeting PTP1B. Cell Death Dis. 2018; 9:522.

17. Liu X, Wen J, Wang H, Wang Y. Long non-coding RNA LINC00460 promotes epithelial ovarian cancer progression by regulating microRNA-338-3p. Biomed Pharmacother. 2018; 108:1022–1028.

18. Li Z, Hu S, Wang J, Cai J, Xiao L, Yu L, et al. MiR-27a modulates MDR1/P-glycoprotein expression by targeting HIPK2 in human ovarian cancer cells. Gynecol Oncol. 2010; 119:125–130.

19. Eoh KJ, Lee SH, Kim HJ, Lee JY, Kim S, Kim SW, et al. MicroRNA-630 inhibitor sensitizes chemoresistant ovarian cancer to chemotherapy by enhancing apoptosis. Biochem Biophys Res Commun. 2018; 497:513–520.

20. Zhang P, Zhu J, Zheng Y, Zhang H, Sun H, Gao S. miRNA-574-3p inhibits metastasis and chemoresistance of epithelial ovarian cancer (EOC) by negatively regulating epidermal growth factor receptor (EGFR). Am J Transl Res. 2019; 11:4151–4165.

21. Li X, Chen W, Jin Y, Xue R, Su J, Mu Z, et al. miR-142-5p enhances cisplatin-induced apoptosis in ovarian cancer cells by targeting multiple anti-apoptotic genes. Biochem Pharmacol. 2019; 161:98–112.

22. Boyerinas B, Park SM, Murmann AE, Gwin K, Montag AG, Zillhardt M, et al. Let-7 modulates acquired resistance of ovarian cancer to Taxanes via IMP-1-mediated stabilization of multidrug resistance 1. Int J Cancer. 2012; 130:1787–1797.

23. Zhang Y, Shi B, Chen J, Hu L, Zhao C. MiR-338-3p targets pyruvate kinase M2 and affects cell proliferation and metabolism of ovarian cancer. Am J Transl Res. 2016; 8:3266–3273.

24. Jiang YC, Ma JX. The role of MiR-324-3p in polycystic ovary syndrome (PCOS) via targeting WNT2B. Eur Rev Med Pharmacol Sci. 2018; 22:3286–3293.

25. Li G, Wang Y, Liu Y, Su Z, Liu C, Ren S, et al. miR-185-3p regulates nasopharyngeal carcinoma radioresistance by targeting WNT2B in vitro. Cancer Sci. 2014; 105:1560–1568.

26. Zhang Y, Dun Y, Zhou S, Huang XH. LncRNA HOXD-AS1 promotes epithelial ovarian cancer cells proliferation and invasion by targeting miR-133a-3p and activating Wnt/β-catenin signaling pathway. Biomed Pharmacother. 2017; 96:1216–1221.

27. Arend RC, Londoño-Joshi AI, Straughn JM Jr, Buchsbaum DJ. The Wnt/β-catenin pathway in ovarian cancer: a review. Gynecol Oncol. 2013; 131:772–779.

28. Du B, Shim JS. Targeting epithelial–mesenchymal transition (EMT) to overcome drug resistance in cancer. Molecules. 2016; 21:E965.

29. Vleminckx K, Vakaet L Jr, Mareel M, Fiers W, van Roy F. Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role. Cell. 1991; 66:107–119.