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目的探索microRNA-31(miR-31)的高表达对AGS人胃癌细胞生物学行为及其成纤维细胞核受体(LRH-1)蛋白表达的影响,并探索miR-31调控胃癌发生和发展的机理。方法将AGS细胞分为3组,分别给予转染miR-31(MT组)、转染miR-31阴性对照序列即空脂质体(NC组)及滴加PBS溶液(BC组)处理。分别采用细胞增殖和细胞毒性试剂盒(CCK-8试剂盒)、流式细胞仪和Transwell实验检测3组细胞的增殖、凋亡、细胞周期和细胞迁移情况。采用Western blot法检测3组细胞中LRH-1蛋白的表达水平;采用双荧光素酶报告系统检测miR-31的作用位点。结果 CCK-8实验结果显示,转染4 d后,MT组、NC组及BC组细胞A450值的均值分别为1.31、2.26和2.14,MT组较低(P<0.01);流式细胞仪检测结果表明,MT组、NC组及BC组细胞凋亡率的均值分别为39.5%、9.3%和10.0%,G1+S期细胞比例分别为92.54%、73.23%及74.58%,MT组的该2个指标均较高(P<0.05);Transwell实验结果表明,MT组的迁移细胞数低于NC组及BC组(P<0.05)。Western blot结果表明,MT组细胞LRH-1蛋白的表达较BC组和NC组下调(P<0.01)。结论上调miR-31的表达对AGS细胞的增殖具有明显的抑制作用,且可促进其凋亡并降低细胞的迁移能力;miR-31可使LRH-1蛋白的表达下调,推测LRH-1基因可能是miR-31参与胃癌发生和发展的靶标之一。
Objective To explore the effect of microRNA-31 (miR-31) overexpression on the biological behavior of AGS human gastric cancer cells and the expression of LRH-1 protein and to explore the mechanism of miR-31 in regulating the occurrence and development of gastric cancer . Methods AGS cells were divided into three groups and transfected into miR-31 (MT group) respectively. The transfected miR-31 negative control group was treated with empty liposome (NC group) and PBS solution (BC group). The proliferation, apoptosis, cell cycle and cell migration of the three groups were detected by using cell proliferation and cytotoxicity kit (CCK-8 kit), flow cytometry and Transwell assay respectively. The expression of LRH-1 protein in three groups of cells was detected by Western blot. The dual luciferase reporter system was used to detect the site of miR-31. Results The results of CCK-8 assay showed that the average A450 values of MT, NC and BC were 1.31, 2.26 and 2.14, respectively, and were lower in MT group (P <0.01) after 4 d of transfection. The results of flow cytometry The results showed that the average apoptotic rates of MT group, NC group and BC group were 39.5%, 9.3% and 10.0%, respectively. The percentage of cells in G1 + S phase were 92.54%, 73.23% and 74.58% The results of Transwell assay showed that the number of migrating cells in MT group was lower than that in NC group and BC group (P <0.05). Western blot results showed that the expression of LRH-1 protein in MT group was lower than that in BC group and NC group (P <0.01). Conclusion Upregulation of miR-31 can significantly inhibit the proliferation of AGS cells and promote its apoptosis and decrease the migration ability of cells. MiR-31 can down-regulate the expression of LRH-1 protein and speculate that LRH-1 may be MiR-31 is involved in the occurrence and development of gastric cancer.