摘要
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.
作者
赵振兴
赵振山
李海洋
杨永辉
郝孟辉
代岱
ZHAO Zhen-xing;ZHAO Zhen-shan;LI Hai-yang;YANG Yong-hui;HAO Meng-hui;DAI Dai(Department of Cardiothoracic Surgery,Kailuan General Hospital,Tangshan,Hebei 063000;Department of Oncology,Kailuan General Hospital,Tangshan,Hebei 063000,China)
出处
《热带医学杂志》
CAS
2019年第5期599-604,670,共7页
Journal of Tropical Medicine