miR-27a促进胃癌细胞AGS增殖与侵袭及其机制探讨

miR-27a promotes the proliferation and invasion of gastric cancer cells AGS and its mechanism

目的:探究miR-27a对胃癌细胞AGS增殖与侵袭能力的影响,并进一步探讨能否靶向调控盐诱导激酶1(salt induce kinase 1,SIK1)和F框/WD_40域蛋白7(F-box and WD_40 repeat domain protein 7,FBXW7)。方法:利用TCGA数据库分析胃癌组织与邻近正常胃组织之间miR-27a表达水平的差异;利用脂质体Lipofectamine^(TM)2000将miR-27a mimics和miR-27a NC转入AGS细胞中,实时定量聚合酶链反应(Real-time PCR)检测miR-27a的表达,CCK-8实验检测细胞增殖活力,细胞平板克隆形成实验检测细胞克隆能力,Transwell实验检测细胞侵袭能力。生物信息学预测miR-27a的靶基因,通过蛋白印迹实验验证miR-27a对SIK1的调控作用;通过双荧光酶素报告基因、Real-time PCR和蛋白印迹实验验证miR-27a对FBXW7的调控作用。利用TCGA数据库分析胃癌组织与邻近正常胃组织之间FBXW7 mRNA表达水平的差异,分析FBXW7 mRNA表达水平与胃癌患者5年生存率的关系。结果:TCGA数据库分析表明miR-27a在胃癌组织中的表达高于邻近正常胃组织。Real-time PCR表明miR-27a在模拟物组中的表达较对照组高,细胞功能实验表明过表达miR-27a能促进胃癌细胞的增殖与侵袭能力;TargetScan预测SIK1为miR-27a的潜在靶基因,蛋白印迹实验表明SIK1不能被miR-27a靶向负性调控。TargetScan预测FBXW7为miR-27a的潜在靶基因,双荧光素酶报告基因结果表明miR-27a可抑制FBXW73’UTR双荧光素酶活性,突变其结合位点后抑制作用消失。Real-time PCR和蛋白印迹实验表明过表达miR-27a后FBXW7的蛋白与mRNA表达均明显下调。TCGA数据库分析表明胃癌组织中FBXW7 mRNA的表达较邻近正常胃组织明显下降,FBXW7 mRNA表达低的组胃癌患者5年生存率更低。结论:过表达miR-27a能增强胃癌细胞AGS的增殖与侵袭能力,可能是通过负性调控FBXW7发挥的促瘤作用。在胃癌细胞AGS中,SIK1不能被miR-27a负性调控,可能被FBXW7靶向介导泛素化降解。

Objective:To observe the effect of microRNA(miR)-27a on the proliferation and invasion abilities of gastric cancer cells AGS,and to further explore whether it can regulate salt induce kinase 1(SIK1)and F-box and WD_40 repeat domain protein 7(FBXW7).Methods:The TCGA database was used to analyze the expression of miR-27a in the gastric cancer tissues and normal tissues.Lipofectamine^(TM) 2000 was used to transfer miR-27a mimics and miR-27a NC into AGS cells.Real-time PCR was used to detect the expression of miR-27a,and CCK-8 assay was used to detect cell proliferation.The clone formation assay was used to detect cell cloning ability,and the Transwell assay was used to detect cell invasion ability.Bioinformatics was used to predict the target genes of miR-27a in gastric cancer cells.Western blot assay was used to verify whether SIK1 was regulated by miR-27a.Dual-luciferase reporter gene analysis,Real-time PCR and Western blot assays were performed to confirm whether FBXW7 was downstream target gene of miR-27a.The TCGA database was used to analyze the expression of FBXW7 mRNA in the gastric cancer tissues and normal tissues.Relationship between 5-year survival rate and expression level of FBXW7 mRNA was revealed by analyzing the TCGA database.Results:The expression level of miR-27a in gastric cancer tissues was higher than that in the normal tissues.The expression level of miR-27a in the miR-27a mimics group was higher than that in the miR-27a NC group.Cell functional assay suggested that the proliferation and invasion abilities of gastric cancer cells were enhanced significantly when miR-27a was overxpressed.SIK1 and FBXW7 were predicted as the potential target genes of miR-27a in TargetScan.Western blot revealed that the expression of SIK1 cannot be down-regulated by miR-27a.The dual-luciferase reporter gene result showed that overexpression of miR-27a can inhibit the FBXW73'UTR dual-luciferase activity,and the suppressive effect disappeared once the binding site was mutated.The mRNA expression and protein expression of FBXW7 in the miR-27a mimics group were significantly lower than those in the miR-27a NC group.The analysis of TCGA database showed that FBXW7 mRNA level was lower in gastric cancer tissues than that in normal tissues and the 5-year survival rate was lower in the low-level group of FBXW7 mRNA.Conclusion:Overexpression of miR-27a can promote the proliferation and invasion abilities of AGS cells.miR-27a exert tumor-promoting effect probably by negatively regulating the expression of FBXW7.SIK1 could not be down-regulated by miR-27a and it may be degenerated in ubiquitin pathway mediating by FBXW7.