摘要
目的:探讨miR-30a在非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞中的表达情况及miR-30a在肺癌细胞迁移和侵袭过程中的作用及其机制。方法:采用qRT-PCR检测miR-30a在不同NSCLC细胞株中的表达情况;采用脂质体2000转染miR-30a mimics和E盒结合锌指蛋白2(E-box binding zinc finger protein 2,ZEB2)siRNA;通过qRT-PCR检验miR-30a mimics和ZEB2 siRNA的转染效率及转染后ZEB2 mRNA的表达水平变化;Western blot检测转染miR-30a mimics和ZEB2 siRNA后ZEB2蛋白表达情况;采用双荧光素酶报告实验验证miR-30a和ZEB2的相互作用机制;划痕实验和Transwell小室侵袭实验检测上调miR-30a和干扰ZEB2对A549细胞迁移和侵袭能力的影响。结果:实验结果显示,与正常人支气管上皮细胞相比,在不同NSCLC细胞株中miR-30a的表达呈不同程度下调;NSCLC细胞转染miR-30a mimics和ZEB2 siRNA后均获得满意的转染效果:miR-30a mimics和ZEB2 siRNA明显降低了NSCLC细胞中ZEB2的mRNA和蛋白的表达水平,组间差异具有统计学意义。双荧光素酶报告实验验证miR-30a对ZEB23'UTR具有直接调控作用。细胞迁移实验和侵袭实验结果显示,与Blank组和NC组相比,miR-30a过表达组和ZEB2基因沉默组的A549细胞迁移和侵袭能力均明显降低,说明miR-30a mimics和ZEB2 siRNA均对A549细胞细胞迁移和侵袭有抑制作用。结论:上调miR-30a的表达水平可以负调控ZEB2转录后表达水平,通过抑制上皮-间质转化(epithelial-mesenchymal transition,EMT)的进程来抑制肺癌细胞的转移和侵袭。
Objective:To investigate the exression of miR-30a on lung cancer,and the effect and mechanism of miR-30a in lung cancer cell invasion and migration.Methods:The expression of miR-30a in different NSCLC cell lines was detected by qRT-PCR.miR-30a mimics and ZEB2 siRNA were transfected with liposome 2000.The transfection efficiency of miR-30a mimics and ZEB2 siRNA and the expression level of ZEB2 mRNA were tested by qRT-PCR.Western blot analysed ZEB2 protein expression after transfection of qRT-PCR.The interaction mechanism between miR-30a and ZEB2 was verified by double luciferase assay.The effects of upregulation of miR-30a and interference of ZEB2 on migration and invasion ability of A549 cells were detected by scratch test and Transwell invasion test.Results:Compared with normal bronchial epithelial cells,the expression of miR-30a in NSCLC cell line was down-regulated on different degrees.NSCLC cells had satisfactory transfection with miR-30a mimics and ZEB2 siRNA.miR-30a mimics and ZEB2 siRNA significantly reduced the expression levels of ZEB2 mRNA and protein in NSCLC cells(P<0.05).The dual luciferase assay confirmed that miR-30a had a direct regulatory effect on ZEB23'UTR.Cell migration and invasion experiments showed that compared with the Blank group and NC group,the ability of migration and invasion in A549 cell were significantly decreased in the miR-30a overexpression group and ZEB2 gene silencing group,suggesting that miR-30a mimics and ZEB2 siRNA could inhibit cell migration and invasion of NSCLC cells.Conclusion:These results suggest that upregulation of miR-30a can negatively regulate the post-transcriptional expression of ZEB2 and inhibit the metastasis and invasion of lung cancer cells by inhibiting the process of EMT.
作者
李智慧
姚菲菲
王富霞
LI Zhihui;YAO Feifei;WANG Fuxia(Department of Respiratory and Critical Care Medicine,People's Hospital of Henan University of Traditional Chinese Medicine,Zhengzhou People's Hospital,Henan Zhengzhou 450000,China)
出处
《现代肿瘤医学》
CAS
北大核心
2021年第11期1840-1846,共7页
Journal of Modern Oncology
基金
河南省自然科学基金资助项目(编号:H201723154)。
