Cancer Res Treat.  2020 Jul;52(3):798-814. 10.4143/crt.2019.498.

Long Non-coding RNA CCAT1 Sponges miR-454 to Promote Chemoresistance of Ovarian Cancer Cells to Cisplatin by Regulation of Surviving

Affiliations
  • 1Department of Gynaecology, The 2nd Affiliated Hospital of Harbin Medical University, Harbin, China

Abstract

Purpose
Colon cancer-associated transcript 1 (CCAT1) was identified as an oncogenic long non-coding RNA (lncRNA) in a variety of cancers. However, there was a lack of understanding of the mechanism by which CCAT1 conferred cisplatin (also known as DDP) resistance in ovarian cancer cells.
Materials and Methods
Cell viability of A2780, SKOV3, A2780/DDP, and SKOV3/DDP cells upon cisplatin treatment was monitored by MTT assay. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) detected the expression levels of CCAT1 and miR-454. The effect of sh-CCAT1 on cisplatin response was investigated in xenografts study. Bioinformatic analysis, luciferase reporter assay and qRT-PCR were conducted to validate the direct interaction among CCAT1, miR-454, and survivin. Apoptosis was determined by flow cytometry after dual staining of Annexin-V-FITC/propidium iodide, and the expression of apoptosis-related proteins Bcl-2, Bax and survivin were detected by qRT-PCR and Western blotting. Xenograft study was conducted to monitor in vivo tumor formation.
Results
CCAT1 was highly expressed in cisplatin-resistant ovarian cancer cell line A2780/DDP and SKOV3/DDP. Knockdown of CCAT1 restored sensitivity to cisplatin in vitro and in vivo. Our data revealed that silencing of CCAT1 promoted cisplatin-induced apoptosis via modulating the expression of pro- or anti-apoptotic proteins Bax, Bcl-2, and survivin. CCAT1 directly interacted with miR-454, and miR-454 overexpression potentiated cisplatin-induced apoptosis. Survivin was identified as a functional target of miR-454, restoration of survivin attenuated the effect of miR-454 on cisplatin response. In addition, miR-454 inhibitor or overexpression of survivin was found to abolish sh-CCAT1–induced apoptosis upon cisplatin treatment.
Conclusion
CCAT1/miR-454/survivin axis conferred cisplatin resistance in ovarian cancer cells.

Keyword

Epithelial ovarian carcinoma; Chemoresistance; LncRNA; CCAT1; miR-454; Survivin

Figure

  • Fig. 1. Knockdown of colon cancer-associated transcript 1 (CCAT1) enhanced sensitivity to cisplatin in A2780 and A2780/DDP cells. (A) Cell viability was monitored by MTT assay. A2780 and A2780/DDP cells were treated with different doses of cisplatin (0, 5, 10, 20, 40, and 80 μM) for 24 hours. (B) IC50 of cisplatin in A2780 and A2780/DDP cells. (C) CCAT1 expression in A2780 and A2780/DDP cells was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). lncRNA, long non-coding RNA. (D) CCAT1 expression was determined by qRT-PCR in A2780 and A2780/DDP cells transfected with sh-NC or sh-CCAT1. (E) Cell viability was monitored by MTT assay. A2780 and A2780/DDP cells were transfected with sh-NC or sh-CCAT1, followed by treatment with different doses of cisplatin (0-80 μM) for 24 hours. (F) IC50 of cisplatin in control and CCAT1 knockdown cells. Values are presented as the mean±standard deviation and performed in triplicate. *p < 0.05, **p < 0.01.

  • Fig. 2. Colon cancer-associated transcript 1 (CCAT1) modulated cisplatin sensitivity of ovarian cancer cells via promoting apoptosis. (A) A2780 and A2780/DDP cells were transfected with negative control (NC) or sh-CCAT1 and treated with cisplatin (A2780 for 8 μM and A2780/DDP for 20 μM) for 24 hours. Apoptosis of cells were examined by flow cytometry. PI, propidium iodide. (B, C) Expression of Bcl-2, Bax, and survivin in transfected A2780 and A2780/DDP cells without cisplatin were determined by quantitative reverse transcription polymerase chain reaction (B) and Western blotting (C). GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Values are presented as the mean±standard deviation and performed in triplicate. *p < 0.05, **p < 0.01.

  • Fig. 3. Colon cancer-associated transcript 1 (CCAT1) acted as miR-454 sponge to down-regulate its expression. (A) miR-454 levels in A2780 and A2780/DDP cells were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). (B) Up-regulation of miR-454 by sh-CCAT1 in A2780 and A2780/DDP cells. miR-454 level was determined by qRT-PCR. (C) miR-454 level was determined by qRT-PCR in A2780 and A2780/DDP cells transfected with miR-NC or miR-454 mimics. (D) An illustration of vector and miR-454 binding sequence in CCAT1. A mutation was generated in the CCAT1 sequence in the complementary site for miR-454 binding. (E) A2780 and A2780/DDP cells were co-transfected with CCAT1-WT/CCAT1-MUT and mimics control/miR-454. Luciferase activity was examined by dual luciferase reporter assay. Renilla luciferase activity was used to normalize the activity of firefly luciferase activity. Data were presented as the mean±standard deviation and performed in triplicate. *p < 0.05, **p < 0.01.

  • Fig. 4. Overexpression of miR-454 modulated cisplatin sensitivity of ovarian cancer cells via promoting cell apoptosis. (A) Cell viability was monitored by MTT assay. A2780 and A2780/DDP cells were transfected with mimics control (miR-NC) or miR-454 mimics, followed by treatment with different doses of cisplatin (0-80 μM) for 24 hours. (B) IC50 of cisplatin in miRNC and miR-454 overexpression A2780 and A2780/DDP cells. (C) A2780 and A2780/DDP cells were transfected with miRNC or miR-454 mimics and treated with cisplatin (A2780 for 8 μM and A2780/DDP for 20 μM) for 24 hours. Apoptosis of cells were examined by flow cytometry. PI, propidium iodide. (D) Expression of Bcl-2 and Bax in transfected ovarian cancer cells without cisplatin were determined by Western blotting. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Values are presented as the mean±standard deviation and performed in triplicate. *p < 0.05, **p < 0.01.

  • Fig. 5. Identification of survivin as a direct target of miR-454 in ovarian cancer cells. (A) An illustration of vector and miR-454 binding sequence in survivin 3′-untranslated region (3′-UTR). A mutation was generated in the 3′-UTR of survivin in the complementary site for miR-454 binding. (B) A2780 and A2780/DDP cells were co-transfected with vector/survivin-WT/survivin-MUT and miR-NC/miR-454 mimics. Luciferase activity was examined by dual luciferase reporter assay. Renilla luciferase activity was used to normalize the activity of firefly luciferase activity. (C) Down-regulation of survivin by miR-454. Survivin level was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting. (D) Cells were transfected with pcDNA3.1-survivin vector or negative control (vector) and the expression level of survivin was determined by qRT-PCR and Western blotting. (E) A2780 and A2780/DDP cells were transfected with transfected with miR-NC, miR-454 with or without pcDNA3.1-survivin under cisplatin condition (A2780 for 8 μM and A2780/DDP for 20 μM) for 24 hours. Apoptosis of cells were examined by flow cytometry. PI, propidium iodide. (F) Expression of Bcl-2, Bax, and survivin in transfected ovarian cancer cells without cisplatin were determined by Western blotting. Values are presented as the mean±standard deviation and performed in triplicate. *p < 0.05, **p < 0.01.

  • Fig. 6. Colon cancer-associated transcript 1 (CCAT1)/miR-454/survivin axis regulated cisplatin resistance in A2780 and A2780/DDP cells. (A) Apoptosis of A2780 and A2780/DDP cells transfected with negative control (NC)/sh-CCAT1/sh-CCAT1+miR-454 inhibitor/survivin and treated with cisplatin (A2780 for 8 μM and A2780/DDP for 20 μM) for 24 hours was examined by flow cytometry. For sh-CCAT1+Z-VAD-FMK+DDP group, CCAT1 depleting cells were treated with 20 μM Z-VAD-FMK for 2 hours. PI, propidium iodide. (B) The expression level of Bcl-2, Bax, and survivin in transfected ovarian cancer cells without cisplatin were determined by Western blotting. Values are presented as the mean±standard deviation and performed in triplicate. *p < 0.05, **p < 0.01.

  • Fig. 7. Knockdown of colon cancer-associated transcript 1 (CCAT1) restored cisplatin sensitivity in vivo. (A) A2780 and A2780/DDP cells were transfected with negative control (NC) or sh-CCAT1. These cells were then implanted subcutaneously into the right flank of mice. After 7 days the mice were treated with cisplatin (A2780 for 5 mg/kg and A2780/DDP for 10 mg/kg). The tumor volumes were measured every 5 days. (B) Images of tumor size in different groups of mice on 30 days. (C) The final tumor weight was measured on day 30 following implanted. (D) Survivin protein level in different tumors was determined by western blotting. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Values were presented as the mean±standard deviation and performed in 3 mice. *p < 0.05, **p < 0.01.


Reference

References

1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2017. CA Cancer J Clin. 2017; 67:7–30.
Article
2. Jiang X, Tang H, Chen T. Epidemiology of gynecologic cancers in China. J Gynecol Oncol. 2018; 29:e7.
Article
3. Oronsky B, Ray CM, Spira AI, Trepel JB, Carter CA, Cottrill HM. A brief review of the management of platinum-resistant-platinum-refractory ovarian cancer. Med Oncol. 2017; 34:103.
Article
4. Nikpayam E, Tasharrofi B, Sarrafzadeh S, Ghafouri-Fard S. The role of long non-coding RNAs in ovarian cancer. Iran Biomed J. 2017; 21:3–15.
Article
5. Vergote I, Trope CG, Amant F, Kristensen GB, Ehlen T, Johnson N, et al. Neoadjuvant chemotherapy or primary surgery in stage IIIC or IV ovarian cancer. N Engl J Med. 2010; 363:943–53.
Article
6. Fatica A, Bozzoni I. Long non-coding RNAs: new players in cell differentiation and development. Nat Rev Genet. 2014; 15:7–21.
Article
7. Chen J, Zhang K, Song H, Wang R, Chu X, Chen L. Long noncoding RNA CCAT1 acts as an oncogene and promotes chemoresistance in docetaxel-resistant lung adenocarcinoma cells. Oncotarget. 2016; 7:62474–89.
Article
8. Nissan A, Stojadinovic A, Mitrani-Rosenbaum S, Halle D, Grinbaum R, Roistacher M, et al. Colon cancer associated transcript-1: a novel RNA expressed in malignant and pre-malignant human tissues. Int J Cancer. 2012; 130:1598–606.
Article
9. Deng L, Yang SB, Xu FF, Zhang JH. Long noncoding RNA CCAT1 promotes hepatocellular carcinoma progression by functioning as let-7 sponge. J Exp Clin Cancer Res. 2015; 34:18.
Article
10. Ma MZ, Chu BF, Zhang Y, Weng MZ, Qin YY, Gong W, et al. Long non-coding RNA CCAT1 promotes gallbladder cancer development via negative modulation of miRNA-218-5p. Cell Death Dis. 2015; 6:e1583.
Article
11. Liu SP, Yang JX, Cao DY, Shen K. Identification of differentially expressed long non-coding RNAs in human ovarian cancer cells with different metastatic potentials. Cancer Biol Med. 2013; 10:138–41.
12. Wang Q, Zhang W, Hao S. LncRNA CCAT1 modulates the sensitivity of paclitaxel in nasopharynx cancers cells via miR-181a/CPEB2 axis. Cell Cycle. 2017; 16:795–801.
Article
13. Yang X, Zheng F, Xing H, Gao Q, Wei W, Lu Y, et al. Resistance to chemotherapy-induced apoptosis via decreased caspase-3 activity and overexpression of antiapoptotic proteins in ovarian cancer. J Cancer Res Clin Oncol. 2004; 130:423–8.
Article
14. Salmena L, Poliseno L, Tay Y, Kats L, Pandolfi PP. A ceRNA hypothesis: the Rosetta Stone of a hidden RNA language? Cell. 2011; 146:353–8.
Article
15. Wang S, Huang X, Lee CK, Liu B. Elevated expression of erbB3 confers paclitaxel resistance in erbB2-overexpressing breast cancer cells via upregulation of Survivin. Oncogene. 2010; 29:4225–36.
Article
16. Zhong Y, Gao D, He S, Shuai C, Peng S. Dysregulated expression of long noncoding RNAs in ovarian cancer. Int J Gynecol Cancer. 2016; 26:1564–70.
Article
17. Lai XJ, Cheng HF. LncRNA colon cancer-associated transcript 1 (CCAT1) promotes proliferation and metastasis of ovarian cancer via miR-1290. Eur Rev Med Pharmacol Sci. 2018; 22:322–8.
18. Inoue S, Salah-Eldin AE, Omoteyama K. Apoptosis and anticancer drug resistance. Hum Cell. 2001; 14:211–21.
19. Helm CW, States JC. Enhancing the efficacy of cisplatin in ovarian cancer treatment - could arsenic have a role. J Ovarian Res. 2009; 2:2.
Article
20. Fraser M, Leung B, Jahani-Asl A, Yan X, Thompson WE, Tsang BK. Chemoresistance in human ovarian cancer: the role of apoptotic regulators. Reprod Biol Endocrinol. 2003; 1:66.
21. Hu B, Zhang H, Wang Z, Zhang F, Wei H, Li L. LncRNA CCAT1/miR-130a-3p axis increases cisplatin resistance in non-small-cell lung cancer cell line by targeting SOX4. Cancer Biol Ther. 2017; 18:974–83.
Article
22. Liang HL, Hu AP, Li SL, Xie JP, Ma QZ, Liu JY. MiR-454 prompts cell proliferation of human colorectal cancer cells by repressing CYLD expression. Asian Pac J Cancer Prev. 2015; 16:2397–402.
Article
23. Zhou L, Qu YM, Zhao XM, Yue ZD. Involvement of miR-454 overexpression in the poor prognosis of hepatocellular carcinoma. Eur Rev Med Pharmacol Sci. 2016; 20:825–9.
24. Li Q, Liu J, Meng X, Pang R, Li J. MicroRNA-454 may function as an oncogene via targeting AKT in triple negative breast cancer. J Biol Res (Thessalon). 2017; 24:10.
Article
25. Wang X, Liu B, Wen F, Song Y. MicroRNA-454 inhibits the malignant biological behaviours of gastric cancer cells by directly targeting mitogen-activated protein kinase 1. Oncol Rep. 2018; 39:1494–504.
Article
26. Zhao X, Li X, Zhou L, Ni J, Yan W, Ma R, et al. LncRNA HOXA11-AS drives cisplatin resistance of human LUAD cells via modulating miR-454-3p/Stat3. Cancer Sci. 2018; 109:3068–79.
Article
27. Chen X, Duan N, Zhang C, Zhang W. Survivin and tumorigenesis: molecular mechanisms and therapeutic strategies. J Cancer. 2016; 7:314–23.
Article
28. Xu Y, Wang S, Chan HF, Lu H, Lin Z, He C, et al. Dihydromyricetin induces apoptosis and reverses drug resistance in ovarian cancer cells by p53-mediated downregulation of survivin. Sci Rep. 2017; 7:46060.
Article
29. Chen Q, Zhang H. Smac combined with DDP can inhibit drug resistance of ovarian cancer through regulation of Survivin expression. Cancer Biomark. 2018; 22:1–6.
Article
Full Text Links
  • CRT
Actions
Cited
CITED
export Copy
Close
Share
  • Twitter
  • Facebook
Similar articles
Copyright © 2022 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: koreamed@kamje.or.kr