miR-381靶向 TMEM16A对胃癌细胞增殖和侵袭的影响

Effects of miR-381 on proliferation and invasion of gastric cancer cells by targeting TMEM16A

目的探究miR-381靶向调控跨膜蛋白16A(TMEM16A)对胃癌细胞增殖和侵袭的影响及其机制。方法选取行手术切除的胃癌患者34例,采集其癌组织及癌旁正常组织(距癌组织<3cm);正常培养胃癌细胞AGS、MKN45、SGC7901和正常人胃黏膜细胞GES-1,qRT-PCR检测组织和细胞中miR-381的表达;将miR-381-mimics、TMEM16A-siRNA转染AGS细胞,另设置空白对照组,检测细胞中miR-381和TMEM16AmRNA的表达;应用在线生物信息学预测软件和双荧光素酶实验分析miR-381和TMEM16A的靶向关系;MTT法检测细胞增殖情况,流式细胞仪检测细胞凋亡情况,Transwell小室检测细胞侵袭能力;Westernblot检测细胞中p27基因、含半胱氨酸的天冬氨酸蛋白水解酶(caspase-3、caspase-9)、E钙黏素、波形蛋白、基质金属蛋白酶(MMP-2、MMP-9)的表达。结果与正常组织和正常胃黏膜细胞相比,胃癌组织和细胞中miR-381表达降低(P<0.05),而TMEM16AmRNA表达升高(P<0.05);与空白对照组相比,miR-381-mimics组miR-381表达上升,TMEM16AmRNA表达下降(P<0.05),TMEM16A-siRNA组TMEM16AmRNA表达下降(P<0.05),联合转染组miR-381表达上升(P<0.05),但与miR-381-mimics组比较差异无统计学意义(P>0.05),TMEM16A mRNA表达下降(P<0.05),且低于miR-381-mimics组、TMEM16A-siRNA组(P<0.05);与空白对照组相比,miR-381-mimics组、TMEM16A-siRNA组及联合组细胞增殖明显抑制,细胞凋亡率、p27、caspase-3、caspase-9、E-cadherin的表达增加,细胞侵袭数、Vi-mentin、MMP-2及MMP-9的表达下降(P<0.05)。结论miR-381可负向调控TMEM16A抑制胃癌细胞AGS的增殖和侵袭,并促进其凋亡。

Objective To explore the effects of miR-381 on proliferation and invasion of gastric cancer cells by targeting transmembrane protein 16 A(TMEM16 A) and its mechanism.Methods 34 gastric cancer patients who underwent surgical resection were enrolled. Their cancer tissues and adjacent normal tissues(less than 3 cm away from the cancer) were collected. The gastric cancer cells AGS, MKN45 and SGC7901, and normal human gastric mucosal cells GES-1 were cultured. The expression of miR-381 in tissues and cells was detected by qRT-PCR. The miR-381-mimics and TMEM16 A-siRNA were transfected into AGS cells. The blank control group was set up. The expression of miR-381 and TMEM16 A mRNA in cells was detected. The targeted relationship between miR-381 and TMEM16 A was analyzed by online bioinformatics prediction software and dual-luciferase reporter assay. The cell proliferation was detected by MTT assay. The apoptosis was detected by flow cytometry. The cells invasion ability was detected by Transwell chamber. The expression of p27 gene, cysteine-containing aspartate proteolytic enzyme(caspase-3, caspase-9), E-cadherin, Vimentin and matrix metalloproteinase(MMP-2, MMP-9) in cells was detected by Western blot.Results Compared with normal tissues and normal gastric mucosa cells, the expression of miR-381 in gastric cancer tissues and cells decreased(P<0.05), while expression of TMEM16 A mRNA increased(P<0.05).Compared with blank control group, the expression of miR-381 increased, while the expression of TMEM16 A mRNA decreased in miR-381-mimics group(P<0.05), the expression of TMEM16 A mRNA decreased in TMEM16 A-siRNA group(P<0.05), and the expression of miR-381 increased in combination transfection group(P<0.05). However, there was no significant difference compared with that in miR-381-mimics group(P>0.05). The expression of TMEM16 A mRNA decreased(P<0.05), which was lower than that in miR-381-mimics group and TMEM16 A-siRNA group(P<0.05). Compared with blank control group, cell proliferation in miR-381-mimics group, TMEM16 A-siRNA group and combination group was significantly inhibited, the apoptosis and the expression of p27, caspase-3, caspase-9 and E-cadherin increased, while the number of cell invasion, expression of Vimentin, MMP-2 and MMP-9 decreased(P<0.05).Conclusion MiR-381 can inhibit proliferation and invasion of gastric cancer AGS cells by negatively regulating TMEM16 A, and promote their apoptosis.