hsa-miR-302a-3p靶向VEGFA抑制胃癌细胞增殖的机制研究

Mechanism of hsa-miR-302a-3p-targeted VEGFA in the Inhibition of Proliferation of Gastric Cancer Cell

目的探讨过表达hsa-miR-302a-3p下调血管内皮生长因子A(VEGFA)对胃癌细胞SGC-7901存活、运动和上皮间质转化的调节作用。方法采用细胞转染,将hsa-miR-302a-3p mimic转染于miR组细胞,pcVEGFA转染于VEGFA组细胞,同时将两个基因共转染于miR+VEGFA组。采用RT-PCR和Western blot检测转染效率,生物信息学靶向预测和荧光素实验验证两个基因靶向关系,采用CCK-8法检测各组细胞增殖,Transwell检测各组细胞侵袭能力,划痕实验检测各组细胞迁移能力,显微镜下观察细胞的形态是否发生上皮间质转化(EMT),采用Western blot检测各组细胞存活相关蛋白Ki67和Caspase-3、EMT相关蛋白E-cadherin、Vimentin、N-cadherin和Snail以及VEGFA下游靶基因p-P38、p-MAPKAPK和p-Hsp27蛋白表达水平。结果VEGFA是miR-302a-3p的预测靶位点;与对照组相比,miR组细胞数量、侵袭和迁移率均下降(P<0.05),VEGFA组细胞数量、侵袭和迁移率均升高(P<0.05);与VEGFA组相比,miR+VEGFA组细胞数量、侵袭和迁移率均下降(P<0.05),E-cadherin蛋白表达水平上调(P<0.05),Vimentin、N-cadherin和Snail蛋白表达水平下调(P<0.05),p-P38、p-MAPKAPK和p-Hsp27蛋白表达水平下调(P<0.05)。结论 hsa-miR-302a-3p通过抑制VEGFA的表达,抑制胃癌细胞SGC-7901的增殖、侵袭和迁移。

Objective To explore the role mechanism of hsa-miR-302a-3p overexpression in the inhibition of proliferation of gastric cancer cell SGC-7901 by targeted-regulating vascular endothelial growth factor A(VEGFA).Methods The cell transfection was used to transfect hsa-miR-302a-3p mimic into miR mimic group and transfect pcVEGFAinto VEGFA group,and the two genes were co-transfected into miR+VEGFA group.The transfection efficiency was detected by RT-PCR and Western blot.The bioinformatics targeting prediction and fluorescein assay were used to verify the targeting relationship between the two genes.Cell proliferation was detected by CCK-8test,and Transwell assay was used to detect the invasion ability of each group,and scratch assay was used to detect the migration ability of each group.The morphology changes of epithelial-mesenchymal transition(EMT)in cells were observed under microscope.Western blot was used to detect the protein expression levels of survival-related proteins Ki67 and Caspase-3,EMT-related proteins E-cadherin,Vimentin,N-cadherin and Snail and VEGFA downstream target genes p-P38,p-MAPKAPK and p-Hsp27.Results VEGFA was the predicted target site of miR-302a-3p.Compared with control group,the number of cells,the invasion and migration rates were also reduced(P<0.05)in miR mimic group,and the number of cells was increased(P<0.05)as well as the invasion and migration rates in VEGFA group.Compared with VEGFA group,the number of cells,the invasion and migration rates were also decreased(P<0.05)in miR+VEGFA group.The protein expression level of E-cadherin was up-regulated(P<0.05)while the protein expression levels of Vimentin,N-cadherin and Snail were down-regulated(P<0.05),and the protein expression levels of p-P38,p-MAPKAPK and p-Hsp27 were also down-regulated(P <0.05).Conclusion hsa-miR-302a-3p overexpression can inhibit the proliferation and promote apoptosis of gastric cancer cell SGC-7901 by targeting negative regulation of VEGFAexpression.