miR-409-3b通过下调表皮生长因子蛋白7抑制胃癌侵袭和转移的分子机制

mi R-409-3b inhibits invasion and metastasis of gastric cancer by downregulating EGFL7 protein expression

目的:观察mi R-409-3b对胃癌细胞侵袭转移的影响,探讨mi R-409-3b通过下调表皮生长因子蛋白7(epidermal growth factor like domain protein 7,EGFL7)调节胃癌侵袭和转移的具体机制.方法:通过基因芯片技术筛选出胃癌及癌旁组织差异性表达的微小RNA(micro RNA m i R N A);采用生物学信息学技术预测E G F L7调控相关m i R N A,通过对比上述结果,筛选出mi R-409-3b;进一步以mi R-4093b慢病毒及mi R-409-3b mimics过表达mi R-409-3b后,用Western blot检测胃癌细胞中的EGFL7的表达变化;采用Transwell侵袭实验及划痕实验检测mi R-409-3b慢病毒感染后细胞体外侵袭能力的变化;q RT-PCR检测80例胃癌患者癌组织和癌旁组织中mi R-409-3b的表达差异,并分析mi R-409-3b的表达与胃癌患者临床病理之间的关系.结果:基因芯片及生物信息学预测发现m i R-409-3b在胃癌组织中低表达,同时又可能调控E G F L7,双荧光素酶实验证实m i R-409-3b可以结合在E G F L7上,m i R-409-3b慢病毒及mi R-409-3b mimics过表达m i R-409-3b都能在蛋白水平下调E G F L7;m i R-409-3b家族3条m i R N A的q RT-P C R结果表明癌旁组织相对含量高于胃癌组织;Tr a n s w e l l侵袭实验结果表明:m i R-409-3b与感染空载慢病毒相比感染能显著降低胃癌细胞穿的能力.体外划痕实验的结果表明,经LV-mi R-409-3b感染空载慢病毒比BGC-823细胞的迁移能力明显升高.进一步统计结果表明,mi R-409-3b与淋巴结转移有关(P<0.05),mi R-409-3b的C/P值在无远处转移的病例组织比具有远处转移的病例组织中明显升高(P<0.05).结论:mi R-409-3b可以在转录后水平调控EGFL7的表达,从而抑制胃癌细胞的侵袭和转移.

AIM： To investigate the inhibitory effect of miR-409-3b on the invasion and migration of gastric adenocarcinoma and the underlying molecular mechanisms. METHODS： Micro RNA array was used to screen mi RNAs which were significantly differentially expressed between gastric cancer and paracancerous tissue. Bioinformatics was employed to predict the mi RNAs related to the regulation of epidermal growth factor-like domain-containing protein 7（EGFL7）. Gastric cancer cells were infected with lentiviral vectors or transfected with mi R-409-3b mimics to overexpress mi R-409-3b. Western blot was used to detect the changes of EGFL7 in gastric cancer cells. Transwell invasion assay was accomplished to analyze cell invasion ability. Quantitative real-time polymerase chain reaction（q RT-PCR） was adopted to detect the expression of mi R-409-3b in 80 pairs of gastric cancer tissues and adjacent noncancerous tissues. The relationship between clinical pathological data and EGFL7 expression was analyzed. RESULTS： MicroR NA array and bioinformatics prediction results supported that mi R-409-3b was lowly expressed in gastric carcinoma, a n d r e g u l a t e d E G F L 7. D u a l l u c i f e r a s e assay confirmed that mi R-409-3b binds to E G F L 7. W e s t e r n b l o t a n a l y s i s s u g g e s t e d that EGFL7 was downregulated after mi R-409-3b overexpression. q RT-PCR suggested that the relative expression of mi R-409-3b in gastric cancer tissues was lower than that in the adjacent noncancerous tissues. Transwell invasion experiments showed that mi R-409-3b infection could significantly reduce the invasion ability of gastric cancer cells in vitro. The migration ability of mi R-409-3b LV-cells was significantly higher than that of BGC-823 cells. The expression of mi R-409-3b was closely related with lymph node metastasis（P〈0.05）. The expression ratio of mi R-409-3b in gastric cancer tissues to adjacent noncancerous tissues was significantly lower in tissues collected from patients with distant metastasis than in those from patients without distant metastasis（P〈0.05）. CONCLUSION： mi R-409-3b regulates EGFL7 expression at the post-transcriptional level, and then suppresses the invasion and metastasis of gastric adenocarcinoma.