miR-154-3p抑制非小细胞肺癌细胞转移的研究

The inhibitory effect of miR-154-3p on metastasis of non-small cell lung cancer cell lines

目的 :检测微RNA(micro RNA,mi R)-154-3p在非小细胞肺癌(nonsmall cell lung cancer,NSCLC)组织和细胞系中的表达情况,并探讨其在NSCLC细胞转移中可能的作用机制。方法:采用实时荧光定量PCR(real-time l uorogenic quantitative-PCR,RFQ-PCR)法检测NSCLC组织及其癌旁组织中、发生转移和未发生转移的NSCLC组织中、NSCLC细胞系(A549、H292、H1975、95D和SPC-A-1)和人胚肺细胞(WI38)中mi R-154-3p的表达水平。采用脂质体法将mi R-154-3p-模拟物(mimics)转入A549和H292细胞,以mi R-154-3p-阴性对照(mi R-154-3p-negative control,mi R-154-3p-NC)作为阴性对照;分别采用划痕实验和Transwell小室法检测mi R-154-3p对A549和H292细胞迁移和侵袭能力的影响;蛋白质印迹法检测mi R-154-3p过表达后对上皮-间质转化(epithelial-mesenchymal transition,EMT)标志蛋白上皮细胞钙黏蛋白(E-cadherin)、紧密连接蛋白-1(zonula occludens-1,ZO-1)、神经钙黏蛋白(N-cadherin)和波形蛋白(vimentin)表达的影响。利用生物信息学软件预测mi R-154-3p的潜在靶基因,采用双荧光素酶报告基因系统和蛋白质印迹法进一步验证预测结果。结果 :mi R-154-3p在NSCLC组织(P<0.001)、发生转移的NSCLC组织(P<0.05)和NSCLC细胞系(P<0.01)中的表达水平均低于相应的对照组。过表达mi R-154-3p后,A549和H292细胞的迁移和侵袭能力减弱(P值均<0.05);E-cadherin和ZO-1蛋白的表达水平上调(P值均<0.05);A549细胞中vimentin蛋白的表达水平下调(P<0.01),H292细胞中N-cadherin蛋白的表达水平下调(P<0.05)。双荧光素酶报告基因和蛋白质印迹法检测结果提示,mi R-154-3p能负调控BMI1蛋白的表达。结论 :mi R-154-3p在NSCLC中呈低表达,其过表达可以抑制NSCLC细胞的迁移和侵袭能力,阻碍NSCLC细胞发生EMT,BMI1可能为其靶基因之一。

Objective：To investigate the expression of microRNA-1 54-3p（miR-1 54-3p） in non-small cell lung cancer（NSCLC） tissues and cell lines,and explore the role of miR-1 54-3p in metastasis process of NSCLC.Methods：The expression levels of miR-1 54-3p in NSCLC tissues and the corresponding paracarcinoma tissues,metastatic and non-metastatic NSCLC tissues,NSCLC cell lines（A549,H292,H1975,95 D and SPC-A-1） and human embryo lung WI38 cells were detected by realtime fluorogenic quantitative-PCR（RFQ-PCR）.The miR-1 54-3p-negative control（miR-1 54-3p-NC,as a negative control） or miR-1 54-3p-mimics was transiently transfected into A549 and H292 cells by liposome.The effects of miR-1 54-3p on migration and invasion abilities of NSCLC A549 and H292 cells were determined by wound healing assay and Transwell invasion assay.The expressions of epithelial-mesenchymal transition（EMT）-related proteins E-cadherin,zonula occludens-1（ZO-1）（epithelial marker）,N-cadherin and vimentin（mesenchymal marker）induced by miR-1 54-3p overexpression were detected by Western blotting.The bioinformatic softwares were used to predict the potential targets of miR-1 54-3p,and the interested target genes were further validated by Dual-Luciferase Reporter Assay and Western blotting.Results：The expression levels of miR-1 54-3p were significantly lower in NSCLC tissues（P 0.001）,metastatic NSCLC tissues（P 0.05） and NSCLC cell lines（P 0.01） than those in their corresponding controls.Overexpression of miR-1 54-3p attenuated the migration and invasion abilities of both A549 and H292 cells（P 0.05）.The expressions of E-cadherin and ZO-1 proteins were up-regulated in A549 and H292 cells（all P 0.05）,whereas the vimentin expression in A549 cells and N-cadherin expression in H292 cells were down-regulated（both P 0.05）.Dual-Luciferase Reporter Assay and Western blotting results revealed that miR-1 54-3p could negatively regulate the expression of BMI1.Conclusion：MiR-154-3p is lowly expressed in NSCLC tissues and cell lines.The overexpression of miR-1 54-3p can inhibit the migration and invasion abilities of NSCLC cells and block the EMT progression.BMI1 may be one target gene of miR-1 54-3p.