miR-93-5p对非小细胞肺癌A549细胞增殖和迁移的影响

Effect of miR-93-5p on proliferation and migration of non-small cell lung cancer A549 cells

目的研究miR-93-5p对非小细胞肺癌A549细胞增殖和迁移的影响,并探讨其作用机制。方法体外培养非小细胞肺癌细胞和正常肺细胞,检测细胞中miR-93-5p、P53表达水平,将A549细胞分为miR-93-5p低表达(miR-93-5p inhibitor)组、阴性对照(NC)组和空白对照(CK)组,转染48 h后,CKK-8法检测细胞增殖情况,Transwell实验检测细胞迁移能力,生物信息学预测P53是miR-93-5p的直接靶标,进一步采用双荧光素酶实验进行验证,蛋白免疫印迹(Western Blot)检测miR-93-5p低表达对P53蛋白表达及STAT3信号通路相关蛋白表达的影响。结果与正常肺细胞比较,H1299、HCC827、A549肺癌细胞中miR-93-5p表达水平升高,差异有统计学意义(P<0.05),P53表达水平降低,差异有统计学意义(P<0.05);转染miR-93-5p inhibitor后,A549细胞中miR-93-5p、p-STAT3、JAK、STAT3表达水平降低,差异有统计学意义(P<0.05),P53相对表达量增加,差异有统计学意义(P<0.05);与CK组、NC组相比,A549细胞的增殖、迁移能力均下降,差异有统计学意义(P<0.05);TargetScan数据库预测显示P53是miR-93-5p的直接靶标,双荧光素酶报告基因试验证实二者存在靶向关系。Western Blot结果显示:过表达P53后,与CK组、NC组相比,A549细胞中P53蛋白相对表达量增加,差异有统计学意义(P<0.05),p-STAT3、JAK、STAT3蛋白表达水平均降低,差异有统计学意义(P<0.05)。结论 MiR-93-5p可通过靶向P53调控STAT3信号通路的激活,进而调控肺癌A549细胞的增殖与迁移。

Objective To study the effect of miR-93-5 p on proliferation and migration of non-small cell lung cancer A549 cells,and explore its mechanism.Methods Non-small cell lung cancer cells and normal lung cells were cultured in vitro.The expression levels of miR-93-5 p and P53 were detected.A549 cells were divided into miR-93-5 p low expression(miR-93-5 p inhibitor)group,negative control(NC)group and blank control(CK)group.After 48 hours of transfection,CKK-8 assay was used to detect cell proliferation;transwell assay was used to detect cell migration.Bioinformatics prediction showed that P53 was the direct target of miR-93-5 p,the double luciferase test was used to further verify the results.Western Blot was used to detect the effects of low expression of miR-93-5 p on the expressions of P53 protein and STAT3 signaling pathway-related protein.Results Compared with normal lung cells,the expression of miR-93-5 p in H1299,HCC827,and A549 lung cancer cells increased,the difference was statistically significant(P<0.05),while the expression of P53 decreased,difference was statistically significant(P<0.05).After transfection of miR-93-5 p inhibitor,the expression levels of miR-93-5 p,p-STAT3,JAK,and STAT3 in A549 cells decreased,difference was statistically significant(P<0.05),and the relative expression of P53 increased,difference was statistically significant(P<0.05).Compared with CK group and NC group,the proliferation and migration abilities of A549 cells decreased,difference was statistically significant(P<0.05).TargetScan database prediction showed that P53 was a direct target of miR-93-5 p,and double luciferase reporter gene test confirmed that there was a targeting relationship between them.Western Blot results showed that after overexpression of P53,compared with CK and NC groups,the relative expression of P53 protein in A549 cells increased,difference was statistically significant(P<0.05),while the expression levels of p-STAT3,JAK,and STAT3 decreased,difference was statistically significant(P<0.05).Conclusion MiR-93-5 p could regulate the activation of STAT3 signaling pathway by targeting P53,and then regulate the proliferation and migration of lung cancer A549 cells.