缺氧诱导的miRNA-210靶向HIF-1α促进胃癌发展机制研究

Effects of HIF-1αunder hypoxic condition on SGC7901 and its mechanism

目的研究低氧条件下微小RNA-210(miRNA-210)靶向低氧诱导因子1α(HIF-1α)对人胃癌细胞系SGC-7901的影响,并探讨其机制。方法体外培养人胃癌细胞系SGC-7901,分别经0、100、150、200、300μmol/L氯化钴(CoCl2)作用3 h后,继续培养48 h,CCK-8法检测细胞增殖情况,Transwell实验检测细胞迁移能力;培养密度至80%~90%时,实时荧光定量PCR(qRT-PCR)检测肺组织中miR-210水平变化,蛋白免疫印记(WB)检测HIF-1α、增殖细胞核抗原(PCNA)、胰岛素样生长因子ⅡmRNA结合蛋白3(IMP3)、β-连环蛋白(β-catenin)蛋白变化情况。200μmol/L CoCl2条件下转染antagomic miR-210,WB检测HIF-1α蛋白变化情况。结果与0μmol/L CoCl2组比较,100μmol/L CoCl2组OD450、迁移细胞数量、miR-210、PCNA、β-catenin表达升高(P<0.05),150、200、300μmol/L CoCl2组OD450、迁移细胞数量、miR-210、HIF-1α、PCNA、IMP3、β-catenin表达升高(P<0.05)。与100μmol/L CoCl2组比较,150μmol/L CoCl2组OD450、迁移细胞数量、miR-210、HIF-1α、IMP3、β-catenin表达升高(P<0.05),200、300μmol/L CoCl2组OD450、迁移细胞数量、miR-210、HIF-1α、PCNA、IMP3、β-catenin表达升高(P<0.05)。与150μmol/L CoCl2组比较,200μmol/L CoCl2组OD450、迁移细胞数量、miR-210、HIF-1α、PCNA、IMP3表达升高(P<0.05),300μmol/L CoCl2组OD450、迁移细胞数量、miR-210、HIF-1α、PCNA、IMP3、β-catenin表达升高(P<0.05)。与200μmol/L CoCl2组比较,300μmol/L CoCl2组OD450、miR-210、β-catenin表达升高(P<0.05)。Targetscan预测结果显示,HIF-1α基因3’UTR区与miR-210有结合位点,低氧条件下,200μmol/L CoCl2组和200μmol/L CoCl2+antagomic NC组差异无统计学意义(P>0.05),200μmol/L CoCl2+antagomic组细胞中HIF-1α表达水平降低(P<0.05)。结论缺氧诱导miR-210、HIF-1α上调表达,促进人胃癌细胞系SGC-7901细胞的增殖和迁移。

Objective To investigate the effects of HIF-1αunder hypoxic condition on human gastric cancer cell line SGC-7901(SGC7901),and to explore its action mechanism.Methods Human gastric cancer cell line SGC7901 was cultured in vitro.The cells were cultured for 48h after being treated with 0,100,150,200 and 300μmol/L cobalt chloride(CoCl2)for 3h.then the cell proliferation was detected by CCK8 assay.Transwell assay was used to detect cell migration.Real time fluorescence quantitative PCR(qRTPCR)was used to detect the level of miR210 in lung tissue when the culture density was 80%~90%.Furthermore,Western blot(WB)was used to detect the changes of HIF-1α,PCNA,IMP3 andβcatenin.In addition,anagomic miR210 was transfected with 200μmol/L CoCl2 and the change of HIF-1αprotein was detected by WB.Results As compared with those in 0μmol/L CoCl2 group,the expression levels of OD450,the number of migrating cells,miR210,PCNA andβcatenin in 100μmol/L CoCl2 group and the espressions of OD450,the number of migrating cells,miR210,HIF-1α,PCNA,IMP3 andβcatenin in 150,200 and 300μmol/L CoCl2 groups were significantly increased(P<0.05).As compared with those in 100μmol/L CoCl2 group,the expression levels of OD450,the number of migrating cells,miR210,HIF-1α,IMP3 andβ-catenin in 150μmol/L CoCl2 group and the expression levels of OD450,miR210 andβcatenin in 200,300μmol/L CoCl2 group were significantly increased(P<0.05).As compared with those in 150μmol/L CoCl2 group,the expression levels of OD450,the number of migrating cells,miR210,HIF-1α,PCNA and IMP3 in 200μmol/L CoCl2 group and the expression levels of OD450,the number of migrating cells,miR210,HIF-1α,PCNA and IMP3 in 300μmol/L CoCl2 group were significantly increased(P<0.05).As compared with those in 200μmol/L CoCl2 group,the expression levels of OD450,miR210 and HIF-1αin 300μmol/L CoCl2 group were significantly inceased(P<0.05).Moreover,Targetscan prediction showed that there was a binding site between the 3’UTR region of HIF-1αgene and miR210 and there was no significant difference between 200μmol/L CoCl2 group and 200μmol/L CoCl2+antagomic NC group under hypoxic conditions(P>0.05).In addition the expression levels of HIF-1αof the cells in 200μmol/L CoCl2+antagomicgroup were significantly decreased(P<0.05).Conclusion Hypoxia induces the up-regulations of the expressions of miR210 and HIF-1α,and promotes the proliferation and migration of human gastric cancer cell line SGC7901.