Effect of long non-coding RNA PVT1 on cell proliferation and migration in melanoma

Abstract

The present study aimed to investigate the potential role of the long non‑coding RNA (lncRNA) Pvt1 oncogene (non‑protein coding) (PVT1) in the progression and metastasis of malignant melanoma, and to reveal its possible molecular mechanisms. The expression of lncRNA PVT1 in melanoma tissues and adjacent normal skin from patients with melanoma, and in the melanoma A‑375 and sk‑mel‑5 cell lines, was analyzed using reverse transcription‑quantitative polymerase chain reaction and western blot analyses. The effects of PVT1 expression on cell proliferation, the cell cycle, cell migration and cell invasion were analyzed using MTT assay, flow cytometry, Transwell and scratch assays, respectively. The interaction between PVT1 and enhancer of zeste homolog 2 (EZH2) in melanoma cells was analyzed using RNA immunoprecipitation (RIP) assay. The effect of PVT1 on microRNA‑200c (miR‑200c) expression was analyzed by chromatin immunoprecipitation (ChIP) assay. PVT1 was highly expressed in the melanoma tissues and cells. Silencing of PVT1 significantly inhibited cell proliferation, migration and invasion, and arrested the cell cycle at the G0/G1 stage. Additionally, PVT1 silencing significantly decreased the cyclin D1 expression in the melanoma cells. The expression of E‑cadherin was significantly increased and the expression of N‑cadherin and vimentin was significantly decreased in the PVT1‑silenced group. The RIP assay found that endogenous PVT1 was highly enriched by EZH2 RIP compared with that of the negative control. The ChIP assay revealed that the expression of miR‑200c was decreased significantly in the PVT1‑silenced group compared with the controls. Overall, the present study demonstrated that the lncRNA PVT1 may contribute to the tumorigenesis and metastasis of melanoma through binding to EZH2 and regulating the expression of miR‑200c. lncRNA PVT1 may serve as a potential target for the therapy of melanoma.

Figure 1

(A) Expression of long non-coding RNA PVT1 in melanoma tissues and (B) cell lines assayed by reverse transcription-quantitative polymerasae chain reaction. ^*P<0.05 and ^**P<0.01 compared with the control/adjacent tissues. PVT1, Pvt1 oncogene (non-protein coding).

Figure 2

Effects of lncRNA PVT1 expression on cell proliferation and the cell cycle. (A) The expression of lncRNA PVT1 in melanoma cells following transfection with si-PVT1. (B) Silenced lncRNA PVT1 significantly inhibited melanoma cell proliferation, as assessed by MTT assay. (C and D) Silenced lncRNA PVT1 significantly decreased the number of melanoma cell colonies, as assessed by clonogenic assay. (E) Silenced lncRNA PVT1 significantly increased the percentage of cells in the G0/G1 stage. Silenced lncRNA PVT1 significantly decreased cyclin D1 expression, but had no significant effects on cyclin E1 and A1 expression in the melanoma cells, as assessed by (F) reverse transcription-quantitative polymerasae chain reaction and (G) western blot analysis. ^*P<0.05 and ^**P<0.01 compared with the control. PVT1, Pvt1 oncogene (non-protein coding); lncRNA, long non-coding RNA; si, small interfering; OD, optical density; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 3

Effects of lncRNA PVT1 suppression on melanoma cell migration and invasion. (A and B) Transwell assays showed that silenced lncRNA PVT1 significantly decreased the number of migrated cells. (C and D) Scratch assay showed that silenced lncRNA PVT1 significantly decreased the number of invading cells. (E) Reverse transcription-quantitative polymerasae chain reaction and (F) western blot analysis showed that silenced lncRNA PVT1 significantly increased E-cadherin expression, and decreased N-cadherin and vimentin expression. ^*P<0.05 and ^**P<0.01 compared with the control. PVT1, Pvt1 oncogene (non-protein coding); lncRNA, long non-coding RNA; si, small interfering; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 4

Effects of lncRNA PVT1 expression on miR-200c expression. (A) The expression of miR-200c in melanoma and adjacent tissues, as detected by RT-qPCR. (B) Silenced lncRNA PVT1 significantly decreased the miR-200c expression in melanoma cells detected by RT-qPCR. ^**P<0.01 compared with the control/adjacent tissue. PVT1, Pvt1 oncogene (non-protein coding); lncRNA, long non-coding RNA; si, small interfering; RT-qPCR, reverse transcription-quantitative polymerasae chain reaction; miR-200, microRNA-200.

Figure 5

Effects of lncRNA PVT1 expression on cell metastasis were mediated by EZH2 and the regulation of miR-200c. (A) RNA immunoprecipitation assay showed that lncRNA PVT1 was significantly enriched in the EZH2 group compared with that in the IgG group. (B) Chromatin immunoprecipitation assay showed that miR-200c expression was significantly decreased in the lncRNA PVT1-silenced group compared with that in the control. (C) The regulatory mechanism of lncRNA PVT1 in melanoma. PVT1, Pvt1 oncogene (non-protein coding); lncRNA, long non-coding RNA; si, small interfering; EZH2, enhancer of zeste homolog 2; miR-200, microRNA-200; IgG, immunoglobulin G; EMT, epithelial-mesenchymal transition ; PRC2, polycomb repressive complex-2.