The Research Of The Mechanism Of The Long Non-coding RNA NEAT1 In Ovarian Cancer Occurrence And Migration

Objective:To research the expression of long non-coding RNA NEAT1 in ovarian cancer tissue, correlations of NEAT1 expression with clinicopathological features in patients with ovarian cancer, to explore the influence of NEAT1 the biological functions of ovarian cancer, to evaluate the influence of NEAT1 in patients with ovarian cancer.Methods:1. We tested the expression of NEAT1 for 75 cases of ovarian cancer and 20 cases of ovarian cyst by qRT-PCR; 2. We tested the expression level of NEAT1 in five ovarian cancer cell lines (SKOV3, C200, COC1, HP-8910 and OVCAR3) by qRT-PCR and chosen the two higher expression of NEAT1 from them;3. SKOV3 and OVCAR3 cells were transfected with siRNA; 4. Cell proliferation measured by Cell Counting Kit (CCK-8); Evaluation of cell cycle and apoptosis by flow cytometry; Cell migration and invasion was evaluated by using Transwell chambers; 5.Measured the expression of cell apoptosis-associated proteins (BCL-2、Bax、Caspase3、Caspase9) after NEAT1 siRNA transfected cells by qRT-PCR and Western Blot.6.Detected expression of cell invasion-related proteins (Snail、MMP2、MMP9、TGF-β1) after NEAT 1 siRNA transfected cells by qRT-PCR and Western Blot in ovarian cancer.Results:1. NEAT1 was differently expressed in primary epithelial ovarian cancer tissues than noncancerous cyst tissues by qRT-PCR. Furthermore, higher expression of NEAT1 was associated with more advanced tumor stage and bigger tumor size (P=0.0110, P=0.0107).2. The expression level measured by qRT-PCR of NEAT1 in SKOV3 and OVCAR3 is higher than cancer cell lines C200, COC1, HP-8910.3. A notably reduced expression of NEAT1 was observed in SKOV3 and OVCAR3 cells transfected with NEAT1 siRNA.The transfection efficiency was 92.7%,89.01%.4. NEAT1 siRNA inhibits cell proliferation in SKOV3 and OVCAR3 cells measured by CCK-8 assay (P<0.01).5. NEAT1 silencing affects the cell cycle distribution of ovarian cancer cells. Compared with NC cells, cell population in G0/G1 phase was increased 15.4% and 20.8% in SKOV3 and OVCAR3 transfected with NEAT1 siRNA, respectively (P<0.01).6. NEAT1 siRNA induces cell apoptosis in SKOV3 and OVCAR3 cells(P<0.001).7. NEAT1 silencing reduces the migration and invasive capacity of SKOV3 and OVCAR3 cells(P<0.05).8.Silencing of NEAT1 expression significantly reduced Bcl-2 expression(P<0.01), but remarkably increased expression of Bax, Caspase 9 and Caspase 3(P<0.001).9. NEAT1 knockdown significantly downregulted the expression of MMP-2, MMP-9, Snaill and TGF-β1.Conclusion:1. NEAT1 was overexpression in ovarian cancer tissues and higher expression of NEAT1 was associated with more advanced tumor stage and bigger tumor size.2. Down-regulation of NEAT1 by siRNA transfection in two ovarian cancer cell lines, SKOV3 and OVCAR3 resulted in a significant decrease of cell proliferation via inducing cell cycle arrest and cell apoptosis.We found that suppressing of NEAT1 expression notably repressed the migration and invasive ability of both ovarian cancer cell lines.3.NEAT1 siRNA treatment notably affected the expression of cell apoptosis-associated proteins (Bcl-2, Bax, Caspase 3 and Caspase 9) and cell invasion-related proteins (MMP-2, MMP-9, Snaill and TGF-β1). We suggested that NEAT1 may target these proteins and involved in migration and invasiono of ovarian cancer.4.NEAT1 may act as an oncogene on ovarian cancer and may become a durg target of ovarian cancer therapy.