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

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.

Keywords: CCAT1; Chemoresistance; Epithelial ovarian carcinoma; LncRNA; Survivin; miR-454.

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.