MiR-9-5p对宫颈鳞癌细胞迁移和侵袭能力及上皮间质转化的影响

Effects of miR-9-5p on the migration,invasion and epithelial-mesenchymal transition process in cervical squamous cell carcinoma

目的:宫颈鳞癌是女性生殖系统中最常见的恶性肿瘤,本研究探讨微RNA(microRNA,miR)-9-5p对宫颈鳞癌细胞迁移、侵袭能力及上皮间质转化(epithelial-mesenchymal transition,EMT)进程的影响。方法:利用生物信息学网站预测能与E-钙黏蛋白(E-cadherin,E-cad)靶向结合的miRs;利用癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库,从部分宫颈鳞癌组织和正常宫颈组织中分离并提取有显著差异表达的miRs;采用将野生型E-cad的3'-非编码区(untranslated regions,UTR)(E-cad-WT 3'-UTR)或突变体E-cad的3'-UTR(E-cad-MUT 3'-UTR)分别与miR-9-5p模拟物(mimic)对照、miR-9-5p mimic共转染人胚肾细胞293T;采用双萤光素酶报告实验检测miR-9-5p与E-cad的靶向结合关系。采用real-time PCR检测人正常宫颈上皮细胞株H8与宫颈鳞癌细胞株(C33A、siha、caski、Me180)中的miR-9-5p表达;实验分为空白对照组、过表达对照组(mimic-NC组)、miR-9-5p过表达组(miR-9-5p mimic组)、抑制剂对照组(inhibitor-NC组)、miR-9-5p抑制组(miR-9-5p inhibitor组),采用划痕实验检测各组细胞迁移能力变化,利用Transwell侵袭实验分析各组细胞侵袭能力变化,利用real-time PCR技术和蛋白质印迹法分别检测各组细胞中的Ecad、波形蛋白mRNA和蛋白的表达变化。结果:MiR-9-5p与E-cad有靶向结合关系;与细胞株H8相比,miR-9-5p在宫颈癌细胞株中均呈高表达(均P<0.05);在miR-9-5p mimic组中,E-cad-MUT的3'-UTR荧光素酶活性比E-cad-WT增加(P<0.05);与空白对照组相比,miR-9-5p mimic组的E-cad蛋白质及mRNA表达均降低(均P<0.05),而miR-9-5p inhibitor组的E-cad蛋白质及mRNA表达均增加(均P<0.05);与正常宫颈细胞H8相比,miR-9-5p在宫颈鳞癌细胞株中均呈高表达(均P<0.05);与mimic-NC组相比,miR-9-5p mimic组中划痕愈合距离、侵袭至膜下面的caski、Me180细胞数以及波形蛋白的mRNA、蛋白质表达均增加(均P<0.05),而E-cad的mRNA、蛋白质表达均减少(均P<0.05);与inhibitor-NC组相比,miR-9-5p inhibitor组中划痕愈合距离、侵袭至膜下面的caski、Me180细胞数以及波形蛋白的mRNA、蛋白质表达均减少(均P<0.05),而E-cad mRNA、蛋白质表达均增加(均P<0.05)。结论:MiR-9-5p在宫颈鳞癌中呈高表达,能提高癌细胞的迁移、侵袭能力,加快EMT进程。

Objective:Cervical squamous cell carcinoma is the most common cancer in female reproductive system.This study aims to explore the effect of microRNA-9-5p(miR-9-5p)on the migration,invasion,and epithelial-mesenchymal transition(EMT)process of cervical squamous cells.Methods:Bioinformatics were used to predict the miRNAs that could bind to E-cadherin(E-cad).The Cancer Genome Atlas(TCGA)database was used to analyze and extract significantly differentially expressed miRNAs from part of cervical squamous cell carcinoma tissues and normal cervical tissues,and miR-9-5p was selected as the main research target.The translated regions(UTR)of wild-type E-cad(E-cad-WT 3'-UTR)or the 3'-UTR of mutant E-cad(E-Cad-MUT 3'-UTR)was transfected with miR-9-5p mimic normal control(NC),and miR-9-5p mimic was co-transfected human embryonic kidney cells(293T).The relationship between miR-9-5p and E-cad was detected by double luciferase assay.The expression of miR-9-5p in normal cervical epithelial cell lines(H8)and cervical squamous cell lines(C33A,siha,caski and Me180)were detected by quantitative real-time PCR.Then,the experiments were divided into groups as follows:a block control group,an overexpression control group(mimic-NC group),a miR-95p overexpression group(mimic group),an inhibitory expression control group(inhibitor-NC group),and a miR-9-5p inhibitory expression group(inhibitor group).The changes of migration ability were detected by scratch assay.Transwell invasion assay was used to analyze the changes of invasion ability,and the mRNA and protein changes of E-cad and vimentin were detected by quantitative real-time PCR and Western blotting.Results:MiR-9-5p had a targeting binding relationship with E-cad.Compared with the normal cervical tissue H8 cell line,the miR-9-5p was highly expressed in cervical cancer cell lines(C33A,siha,caski and Me180)(all P<0.05).The luciferase activity of E-cad-MUT was increased compared with that of E-cad-WT in miR-9-5p mimic cells(P<0.05).Compared with the blank control group,the protein and mRNA expressions of E-cad were decreased in the miR-9-5p mimic group(both P<0.05),which were increased in the miR-9-5p inhibitor group(both P<0.05).Compared with H8 cell line,the miR-9-5p was highly expressed in the cervical squamous cell lines(all P<0.05).Compared with the mimic-NC group,the distance of wound healing,the number of caski and Me180 cells invaded below the membrane,and the mRNA and protein expressions of vimentin were all increased in the miR-9-5p mimic group(all P<0.05),while the mRNA and protein of E-cad were decreased(both P<0.05).Compared with the inhibitor-NC group,the distance of wound healing,the number of caski and Me180 cells invading the membrane,and the mRNA and protein expressions of vimentin were decreased in the miR-9-5p inhibitor group(all P<0.05),but the mRNA and protein expressions of E-cad were increased(both P<0.05).Conclusion:The miR-9-5p is highly expressed in cervical squamous cell carcinoma,which can increase the migration and invasion ability,and promote the EMT process of cancer cells.