miR-328-5p靶向SPON2介导FAK/Src信号调控胃癌细胞迁移和侵袭的机制研究

MicroRNA-328-5p inhibits migration and invasion of gastric cancer cells through targeting SPON2 and mediating FAK/Src signaling

目的探讨miR-328-5p对胃癌细胞迁移和侵袭的作用及其分子机制。方法将30只小鼠按照随机数字表法分为胃癌组和正常组,每组15只,采用N-甲基-N-亚硝基脲(MNU)化学诱导法构建小鼠胃癌模型,采用RT-PCR法检测两组小鼠胃组织中miR-328-5p、脊椎蛋白2(SPON2)相对表达量;Western blot法检测两组小鼠胃组织中酪氨酸蛋白激酶(Src)、磷酸化Src(p-Src)、局部黏着斑激酶(FAK)、磷酸化FAK(p-FAK)蛋白表达水平。将胃癌细胞系SGC7901分为miR-328-5p组、miR-con组、NC组共3组,采用RT-PCR法检测3组细胞miR-328-5p、波形蛋白(Vimentin)、基质金属蛋白酶-9(MMP-9)相对表达量;采用Transwell小室实验检测3组细胞迁移和侵袭数。采用荧光素酶报告基因实验检测胃癌细胞miR-328-5p、miR-con+SPON2基因野生型(WT)和突变型(MUT)的荧光素酶相对活力值。结果与正常组比较,胃癌组胃组织中miR-328-5p相对表达量明显降低,SPON2相对表达量明显升高(均P<0.05);与正常组比较,胃癌组胃组织中p-FAK、p-Src蛋白表达水平均明显升高(均P<0.05)。与NC、miR-con组比较,miR-328-5p组miR-328-5p相对表达量明显升高,同时Vimentin、MMP-9相对表达量均明显降低(均P<0.05);与NC、miR-con组比较,miR-328-5p组细胞迁移数和侵袭数均明显降低(均P<0.05)。与miR-328-5p+SPON2 WT组比较,miR-con+SPON2 WT组、miR-con+SPON2 MUT组、miR-328-5p+SPON2 MUT组荧光素酶相对活力值均明显为高(均P<0.05)。结论miR-328-5p在胃癌组织中呈低表达,过表达miR-328-5p可抑制胃癌细胞的迁移和侵袭能力;miR-328-5p直接靶向SPON2基因3'UTR序列,调控其转录表达,并抑制其下游FAK/Src信号活化及其下游Vimentin、MMP-9的表达。

Objective To explore the effect of microRNA-328-5p(miR-328-5p)on migration and invasion of gastric cancer cells and its molecular mechanism.Methods The mouse gastric cancer model was induced by N-methyl-Nnitrosourea(MNU)method,and the relative expression of miR-328-5p and Spondylin 2(SPON2)in mouse gastric cancer tissue was detected.The expression levels of local focal adhesion kinase(FAK)/Src kinase(Src)and its protein phosphorylation were detected by Western blotting.The overexpression of miR-328-5p in gastric cancer SGC7901 cells was detected by fluorescence quantitative PCR(RT-PCR).The mRNA expression of migration related markers vimentin(Vimentin),matrix metalloproteinase 9(MMP9)was detected by RT-PCR.Cell migration and invasion ability were determined by Transwell assay.Target scan 7.2 software analysis and prediction of miR-328-5p target gene SPON2 were performed.The luciferase reporter gene experiment was used to detect the relative luciferase activity of miR-328-5p,miR-con gastric cancer cell SPON2 gene wild type(WT)and mutant type(MUT).Results The level of miR-328-5p in gastric cancer tissue of mice bearing gastric cancer was significantly lower than that in normal group(both P<0.05),while the level of SPON2 in gastric cancer group was significantly higher than that in normal group(both P<0.05).Western blotting showed that FAK/Src phosphorylation level in gastric tissue of mice bearing gastric cancer was significantly increased.RT-PCR results showed that the expression of Vimentin and MMP-9 in the overexpression miR-328-5p group was significantly lower than that in the NC and miR-con groups(all P<0.05),the number of cell migration and invasion in the miR-328-5p overexpression group was significantly lower than that in NC,miR-con group(all P<0.05).Targetscan 7.2 software predicts that miR-328-5p directly targets the 3'UTR sequence of SPON2 gene,and the results of luciferase reporter gene showed mutations of SPON2 and miR-328-5p after binding sites,the relative activity of luciferase in the miR-328-5p-SPON2 MUT group was significantly lower than that in the miR-con-SPON2 WT group(all P<0.05).Conclusion The expression of miR-328-5p is down-regulated in gastric cancer,and over-expressed of miR-328-5p can inhibit the migration and invasion of gastric cancer cells.It is found that miR-328-5p directly targets the 3'UTR sequence of the SPON2 gene,regulates its transcriptional expression,and inhibits its downstream FAK/Src signal activation and downstream Vimentin,Expression of MMP-9.