miR-375、miR-92b靶向NLK对非小细胞肺癌细胞生物学功能及上皮间质转化的影响

Effects of miR-375 and miR-92b targeting Nemo-like kinase on biological function and epithelial-mesenchymal transformation of non-small cell lung cancer cells

目的探讨miR-375、miR-92b靶向Nemo样激酶(Nemo-like kinase,NLK)对非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞增殖、迁移、侵袭及上皮间质转化(epithelial-mesenchymal transition,EMT)的影响。方法以人NSCLC细胞A549为实验细胞,分为7组:正常对照组、miR-375阴性对照组、miR-92b阴性对照组、miR-375+miR92b阴性对照组、miR-375抑制组、miR-92b抑制组和miR-375+miR-92b抑制组。采用双荧光素酶报告法观察miR-375、miR-92b与NLK之间的靶向关系。培养72 h时采用实时荧光定量PCR(qRT-PCR)法检测miR-375、miR-92b、NLK mRNA表达,CCK-8法检测细胞增殖(OD值),划痕实验法(wound healing)检测细胞迁移,Transwell实验法检测各组细胞侵袭,蛋白免疫印迹法(Western blot)检测EMT标记蛋白(E-cadherin、vimentin)的表达差异。结果miR-375、miR-92b与NLK存在靶向结合位点且在A549细胞中存在靶向关系。细胞培养72 h,miR-375抑制组、miR-92b抑制组、miR-375+miR-92b抑制组与相应的阴性对照组和正常对照组比较,NLK mRNA表达、OD值、迁移距离、侵袭细胞数降低(均P<0.05)。miR-375阴性对照组、miR-92b阴性对照组、miR-375+miR-92b阴性对照组与正常对照组比较,E-cadherin、vimentin表达差异不具有统计学意义(P>0.05);与相应的阴性对照组和正常对照组比较,miR-375抑制组、miR-92b抑制组、miR-375+miR-92b抑制组E-cadherin蛋白表达均增高,vimentin蛋白表达均降低,差异具有统计学意义(P<0.05)。结论miR-375、miR-92b靶向NLK对NSCLC的生物学功能和EMT具有抑制作用,NLK表达调控可能是NSCLC的预后生物标志物和治疗靶点。

Objective To investigate the effects of miR-375 and miR-92b targeting Nemo-like kinase(NLK)on the proliferation,migration,invasion and epithelial-mesenchymal transition(EMT)of non-small cell lung cancer(NSCLC)cells.Methods Human NSCLC A549 cells were divided into 7 groups:normal control group,miR-375 negative control group,miR-92b negative control group,miR-375+miR92b negative control group,miR-375 inhibitor group,miR-92b inhibitor group,and miR-375+miR92b inhibitor group.The targeting relationships between miR-375,miR-92b and NLK were analyzed using a dual luciferase reporter assay.After culture for 72 h,the expression levels of miR-375,miR-92b and NLK mRNA were detected by real-time fluorescence quantitative PCR(qRT-PCR),the cell proliferation(OD value)was detected by CCK-8 assay,the cell migration was observed by wound healing assay,the cell invasion was observed by Transwell assay,and the expression levels of EMT-related proteins(E-cadherin and vimentin)were detected by Western blot.Results The miR-375,miR-92b and NLK had targeted binding sites and targeting relationships in A549 cells.Compared with the corresponding negative control groups and normal control group,NLK mRNA expression,OD value,the migration distance and the number of invasive cells were reduced in miR-375 inhibitor group,miR-92b inhibitor group and miR-375+miR-92b inhibitor group after cell culture for 72 h(all P<0.05).E-cadherin and vimentin expression showed no statistically significant difference between miR-375 negative control group,miR-92b negative control group,miR-375+miR92b negative control group and normal control group(P>0.05).Compared with the corresponding negative control groups and normal control group,E-cadherin protein expression was increased while vimentin protein expression was decreased in miR-375 inhibitor group,miR-92b inhibitor group and miR-375+miR-92b inhibitor group(all P<0.05).Conclusion The miR-375 and miR-92b targeting NLK can inhibit the biological function and EMT of NSCLC cells.The regulation of NLK expression may be a prognostic biomarker and therapeutic target of NSCLC.