The long non-coding RNA ANRIL promotes proliferation and cell cycle progression and inhibits apoptosis and senescence in epithelial ovarian cancer

Abstract

Antisense non-coding RNA in the INK4 locus (ANRIL) has been implicated in a variety of cancers. In the present study, we evaluated ANRIL expression in epithelial ovarian cancer (EOC) and defined its clinical implications and biological functions. ANRIL was overexpressed in EOC tissues relative to normal controls. Overexpression correlated with advanced International Federation of Gynecologists and Obstetricians stage and high histological grade. Multivariate analysis indicated that ANRIL is an independent prognostic factor for overall survival in EOC. Gain- and loss-of-function experiments demonstrated that ANRIL promotes EOC cell proliferation both in vitro and in vivo. The proliferative effect was linked to the promotion of cell cycle progression and inhibition of apoptosis and senescence. Down-regulation of P15INK4B and up-regulation of Bcl-2 by ANRIL may partially explain ANRIL-induced EOC cell proliferation. This study is the first to establish that ANRIL promotes EOC progression and is a potential prognostic biomarker.

Keywords: ANRIL; apoptosis; cell cycle; proliferation; senescence.

Figure 1. Relative ANRIL expression levels and their association with poor prognosis in EOC

(A) Relative ANRIL expression levels in EOC and normal ovarian tissues. (B) Kaplan-Meier analysis of OS was performed based on ANRIL expression levels.

Figure 2. ANRIL knockdown inhibits the proliferation of A2780 and OVCA433 cells

(A) Relative ANRIL expression levels in EOC cell lines (A2780, Hey, SKOV3, OVCA429, OVCA433, and OVCAR3). (B) Relative ANRIL expression levels in A2780 and OVCA433 cells transfected with si-NC or ANRIL siRNAs. *P < 0.05. (C) MTT assays were performed to evaluate the proliferation of A2780-KD and OVCA433-KD cells compared to controls. The data represent the mean ± standard deviation (SD) of three independent experiments. Bars denote the SD. *P < 0.05. (D) Representative colony formation assay results for A2780-KD and OVCA433-KD cells and corresponding controls. *P < 0.05.

Figure 3. ANRIL knockdown inhibits cell cycle progression and promotes apoptosis and senescence in A2780 and OVCA433 cells

(A) Cell cycle analysis was performed using flow cytometry. The bar graph on the right presents the percentage of cells in the G0-G1, S, or G2-M phases of the cell cycle. Representative histograms are presented on the left. The results shown are representative of three independent experiments. *P < 0.05. (B) Apoptosis was assessed using flow cytometry. The bar graph on the right presents the percentage of apoptotic cells. Representative quadrant figures are presented on the left. The results shown are representative of three independent experiments. *P < 0.05. (C) Cells were stained with the senescence marker β-galactosidase. The blue staining around the nucleus in A2780-KD and OVCA433-KD cells indicates cellular senescence. *P < 0.05.

Figure 4. Overexpression of ANRIL promotes EOC cell proliferation and cell cycle progression, and inhibits apoptosis and senescence in OVCA429 cells

(A) Relative ANRIL expression levels in OVCA429-OE cells and control cells. *P < 0.05. (B) MTT assays were performed to evaluate the proliferation of OVCA429-OE and OVCA429-Vector cells. *P < 0.05. (C) Representative colony formation assay results for OVCA429-OE and OVCA429-Vector cells. *P < 0.05. (D) Cell cycle analysis was performed using flow cytometry in OVCA429-OE and OVCA429-Vector cells. The bar graph on the right presents the percentage of cells in the G0–G1, S, or G2-M phases of the cell cycle. *P < 0.05. (E) Apoptosis was assessed using flow cytometry. The bar graph on the right presents the percentage of apoptotic cells. *P < 0.05. (F) OVCA429-OE and OVCA429-Vector cells were stained with β-galactosidase. The blue staining around the nucleus indicates cellular senescence. *P < 0.05.

Figure 5. Knockdown and overexpression of ANRIL alters P15^INK4B and Bcl-2 expression

(A) Western blots showing that ANRIL knockdown increases P15^INK4B and decreases Bcl-2 levels in A2780 and OVCA433 cells. (B) Western blots showing that overexpression of ANRIL decreases P15^INK4B and increases Bcl-2 protein levels in OVCA429 cells. (C) Increased P15^INK4B and decreased Bcl-2 mRNA levels were detected by qRT-PCR in A2780-KD and OVCA433-KD cells while decreased P15^INK4B and increased Bcl-2 mRNA levels were detected in OVCA429-OE cells.

Figure 6. ANRIL knockdown inhibits A2780 cell proliferation in vivo

(A) Growth curves of tumor xenografts. The volumes of tumors originating from A2780-KD1 and A2780-KD2 cells were substantially smaller than those originating from A2780-NC cells, and the size difference between the two groups increased over time (six mice per group; *P < 0.05). (B) The weights of the A2780-NC tumors were significantly greater than those of the A2780-KD1 and A2780-KD2 tumors. *P < 0.05. (C) Representative images of tumors in nude mice after subcutaneous injection of A2780-NC, A2780-KD1, and A2780-KD2 cells. (D) Immunohistochemical staining showing that tumors originating from A2780-KD1 and A2780-KD2 cells had increased P15^INK4B and decreased ki67 and Bcl-2 levels compared to those originating from A2780-NC cells.