lncRNA EGFR-AS1靶向miR-577对卵巢癌细胞增殖和凋亡的影响

Effect of lncRNA EGFR-AS1 Targeting miR-577 on the Proliferation and Apoptosis of Ovarian Cancer Cells

目的:研究长链非编码RNA(lncRNA)EGFR-AS1对微小RNA-577(miR-577)的靶向关系及对卵巢癌细胞增殖和凋亡的影响。方法:实时荧光定量PCR(q PCR)检测卵巢癌组织中lncRNA EGFR-AS1和miR-577表达。Lnc Base Predicted v.2预测和双荧光素酶报告实验分析lncRNA EGFR-AS1与miR-577的靶向关系。卵巢癌细胞SKOV3中转染si-EGFR-AS1或miR-577,噻唑蓝(MTT)和流式细胞术分别检测细胞增殖与凋亡,蛋白质印迹法(Western blot)检测细胞周期蛋白D1(Cyclin D1)、p21、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)表达。si-EGFR-AS1和anti-miR-577共转染,观察抑制miR-577表达在干扰lncRNA EGFR-AS1表达诱导的SKOV3细胞增殖和凋亡中的作用。结果:卵巢癌组织中lncRNA EGFR-AS1表达量显著高于癌旁组织,miR-577表达量显著低于癌旁组织(P<0.01)。lncRNA EGFR-AS1与miR-577具有靶向结合位点,转染miR-577的WT-EGFR-AS1细胞荧光素酶相对活性显著低于转染miR-NC组(P<0.01)。干扰lncRNA EGFR-AS1表达明显降低SKOV3细胞24,48,72 h的吸光度(A)值及Cyclin D1、Bcl-2蛋白表达量,显著增加细胞凋亡率及p21、Bax蛋白水平(P<0.05或P<0.01)。miR-577过表达显著降低SKOV3细胞48和72 h的A值及Cyclin D1、Bcl-2蛋白水平,显著提高细胞凋亡率及p21、Bax蛋白表达量(P<0.05)。与si-EGFR-AS1和anti-miR-NC共转染比较,si-EGFR-AS1和anti-miR-577共转染显著减少SKOV3的细胞凋亡率及p21、Bax蛋白表达量,显著提高24,48,72 h的A值及Cyclin D1、Bcl-2蛋白水平(P<0.05)。结论:lncRNA EGFR-AS1在卵巢癌中表达上调,干扰lncRNA EGFR-AS1表达可抑制卵巢癌细胞增殖,并诱导凋亡,其作用机制与靶向调控miR-577表达有关。

Objective: To investigate the targeting relationship of lncRNA EGFR-AS1 to miR-577 and its effects on the proliferation and apoptosis of ovarian cancer cells. Methods: q PCR was used to detect the expressions of lncRNA EGFR-AS1 and miR-577 in ovarian cancer tissues. The Lnc Base Predicted v.2 prediction and dual luciferase reporter assays analyzed the targeting relationship between lncRNA EGFR-AS1 and miR-577. Ovarian cancer cell SKOV3 was transfected with si-EGFR-AS1 or miR-577. MTT and flow cytometry was used to detect the cell proliferation and apoptosis,respectively. Western blot was used to detect Cyclin D1,p21,Bcl-2 and Bax expressions. Si-EGFR-AS1 and anti-miR-577 were co-transfected to observe the inhibition role of miR-577 expression in the proliferation and apoptosis of SKOV3 cells induced by lncRNA EGFR-AS1 expression. Results: The expression of lncRNA EGFR-AS1 in ovarian cancer tissue was significantly higher than that in the adjacent tissues,and the expression of miR-577 was significantly lower than that in the adjacent tissues( P<0.01). lncRNA EGFR-AS1 and miR-577 had targeted binding sites. The relative luciferase activity of WT-EGFR-AS1 cells transfected with miR-577 was significantly lower than that of the miR-NC group( P<0.01). Interfering with lncRNA EGFR-AS1 expression significantly reduced the absorbance( A) value at 24,48 and 72 h,Cyclin D1 and Bcl-2 protein expression of SKOV3 cells,and significantly increased the apoptosis rate and the levels of p21 and Bax proteins( P<0.05 or P<0.01). The overexpression of miR-577 significantly reduced the A value at 48 h and 72 h,Cyclin D1 and Bcl-2 protein levels of SKOV3 cells,and significantly increased the apoptosis rate and the expression of p21 and Bax proteins( P<0.05). Compared with co-transfection with siEGFR-AS1 and anti-miR-NC,co-transfection with si-EGFR-AS1 and anti-miR-577 significantly reduced the apoptosis rate,and the protein expressions of p21 and Bax of SKOV3 cells,significantly increased the A at 24,48 and 72 h,and the protein expressions of Cyclin D1 and Bcl-2( P<0.05). Conclusion: lncRNA EGFR-AS1 is up-regulated in ovarian cancer,and interference with lncRNA EGFR-AS1 expression inhibits the proliferation and induces the apoptosis of ovarian cancer cells,and its mechanism is related to targeted regulation of miR-577 expression.