miR-365通过靶向FoxO1抑制胃癌细胞的增殖

miR-365 Inhibits Gastric Cancer Cell Proliferation by Targeting FoxO1

为了探究miR-365对胃癌细胞活力、增殖的影响及作用机制,使用RT-qPCR技术检测胃癌细胞(AGS和MGC80-3)与正常胃上皮细胞(GES-1)中miR-365的内源性表达水平及临床样本中胃癌组织和癌旁组织中miR-365的内源性表达水平;使用CCK-8检测细胞增殖活力;使用RT-qPCR及Western-blot检测周期蛋白依赖性激酶4(cyclin-dependent kinase 4,CDK4)、CDK6、周期蛋白D1(cyclinD1)和FoxO1的mRNA及蛋白质水平;采用荧光素酶报告实验验证miR-365与FoxO13´-UTR的结合能力。实验结果显示,与正常胃上皮细胞相比,两种胃癌细胞内miR-365的表达下调;转染miR-365模拟物后,胃癌细胞的增殖活力下降,cyclinD1、CDK4、CDK6及FoxO1的mRNA和蛋白质水平均下调;miR-365可结合FoxO1的3´-UTR,进而抑制FoxO1蛋白的表达;与癌旁组织比较,癌组织中miR-365的表达降低。以上结果表明,miR-365可通过靶向结合FoxO1的3´-UTR抑制FoxO1蛋白表达,进而抑制胃癌细胞的增殖。

In order to explore the effect of miR-365 on the viability and proliferation of gastric cancer cells and its mechanism of action, RT-qPCR technology was used to detect the endogenous expression levels of miR-365 in gastric cancer cells(AGS and MGC80-3) and normal gastric epithelial cells(GES-1). At the same time, the endogenous expression levels of miR-365 in gastric cancer and adjacent tissues in clinical samples were detected. CCK-8 was used to detect cell proliferation, RT-qPCR and Western-blot were used to detect the mRNA and protein levels of cyclin-dependent kinase 4(CDK4), CDK6, cyclinD1 and FoxO1, and the luciferase reporter experiment was used to verify the binding ability of miR-365 and FoxO1 3′-UTR. The experimental results showed that, compared with normal gastric epithelial cells, the two gastric cancer cells showed down-regulated expression of miR-365. After miR-365 mimics were transfected, the proliferation activity of gastric cancer cells was decreased, and the mRNA and protein levels of cyclinD1, CDK4, CDK6 and FoxO1 were down-regulated. miR-365 could bind to the 3′-UTR of FoxO1, inhibiting the expression of FoxO1 protein. Compared with adjacent tissues, cancer tissues showed reduced expression of miR-365. The above results indicate that miR-365 could inhibit FoxO1 protein expression by targeting the 3′-UTR of FoxO1,thereby inhibiting the proliferation of gastric cancer cells.