miR-3195对人喉癌Hep-2细胞增殖的抑制作用及其机制

Inhibitory effect of miR-3195 on the proliferation of human laryngeal carcinoma Hep-2 cells and its mechanism

目的:探讨miR-3195对喉癌Hep2细胞增殖的影响及其分子机制。方法:选取2008年1月至2012年8月期间在南华大学教学医院郴州市第一人民医院耳鼻喉科收治的29例喉癌患者的喉癌组织及其相应的癌旁组织标本,采用qPCR检测miR-3195在喉癌和癌旁组织中的表达;构建miR-3195在喉癌Hep-2细胞中稳定高表达的细胞株,采用MTT法观察miR-3195稳定高表达组和对照组的增殖情况;建立裸鼠移植瘤模型,观察miR-3195稳定高表达的人喉癌细胞株Hep-2在裸鼠体内增殖的情况。利用生物信息学预测miR-3195的靶基因,构建TBX13'UTR的荧光素酶载体,双荧光素酶检测系统检测其荧光素酶活性;Western blotting检测稳定高表达miR-3195组和对照组细胞株的TBX1蛋白表达水平。结果:miR-3195在喉癌组织中的表达明显低于癌旁组织(P<0.01);miR-3195上调可抑制Hep-2细胞的增殖(P<0.01),且可明显抑制裸鼠移植瘤瘤体的生长(P<0.05);双荧光素酶报告基因检测结果表明miR-3195可能与TBX1靶向结合(P<0.05),同时Western blotting法验证了miR-3195能够抑制TBX1蛋白的表达(P<0.05)。结论:miR-3195对Hep2细胞有明显的增殖抑制作用,其分子机制可能与负性调控TBX1的表达有关。

Objective:To investigate the effect of miR-3195 on the proliferation of laryngeal carcinoma Hep-2 cells and its molecular mechanism.Methods:From January 2008 to August 2012,the laryngeal cancer tissues and their corresponding paracancerous tissues from 29 patients with laryngeal cancer who were admitted to the Department of Otorhinolaryngology,Chenzhou First People's Hospital Affiliated to teaching hospital of University of South China were selected for this study.qPCR was used to detect the expression of miR-3195 in laryngeal carcinoma and the paracancerous tissues;Hep-2 cell line with stable and high expression of miR-3195 was constructed.The proliferation of miR-3195 over-expressed Hep-2 cells and the control cells was observed by MTT method.A nude mouse xenograft model was established to observe the proliferation of miR-3195 overexpressed Hep-2 cells in nude mice.Bioinformatics tools were used to predict the target gene of miR-3195;the luciferase vector of TBX13'UTR was constructed,and its luciferase activity was examined with dual luciferase detection system;Western blotting was used to detect the TBX1 protein expression in miR-3195 over-expressed cells and control cells.Results:The expression of miR-3195 in laryngeal carcinoma tissues was significantly lower than that in paracancerous tissues(P<0.01);miR-3195 up-regulation could inhibit the proliferation of Hep-2 cells(P<0.01)and significantly inhibit the growth of transplanted tumors in nude mice(P<0.05);The results of the Dual luciferase reporter gene assay indicated that miR-3195 might targetedly bind to TBX1(P<0.05),and Western blotting proved that miR-3195 could inhibit the expression of TBX1 protein(P<0.05).Conclusion:miR-3195 has a significant inhibitory effect on the proliferation of Hep-2 cells,and its molecular mechanism may be related to the negative regulation of TBX1 expression.