miR-205靶定YES1对肺癌细胞A549的增殖抑制作用

目的：利用实时定量RT-PCR技术和双荧光蛋白报告基因分析系统检测miR-205在肺癌组织及A549细胞系中的表达水平以及其直接靶基因YES1，探讨miR-205抑制肺癌细胞A549增殖的可能机制。方法: 实时定量RT-PCR技术检测10例肺癌组织和10例癌旁正常肺组织中miR-205的表达水平；miR-205 mimics和control mimics分别转染A549细胞，利用细胞计数和集落形成实验检测转染后A549细胞的增殖情况。选取表达绿色荧光蛋白的质粒 pcDNA3/EGFP，将YES1 3′UTR的一段特异性序列、YES1 3′UTR（miR-205互补位点）点突变后的序列分别插入该质粒中，构建YES1-3′UTR和mut-YES1-3′UTR的绿色荧光表达质粒。实验分为YES1-3′UTR、YES1-3′UTR与miR-205 mimics、YES1-3′UTR与control mimics、mut-YES1-3′UTR、mut-YES1-3′UTR与miR-205 mimics和mut-YES1-3′UTR与control mimics共6组，均与表达红色荧光蛋白pDsRed2-N1共同转染肺癌细胞系A549，荧光分光光度计进行蛋白定性和定量检测。结果：与癌旁正常肺组织比较，miR-205在肺癌组织及A549细胞中表达水平降低(P<0.05)；miR-205mimics 转染组A549细胞增殖率明显低于转染control mimics对照组（P<0.05）；YES1-3′UTR与miR-205 mimics共转实验组荧光蛋白表达水平低于YES1-3′UTR与control mimics共转染组（P<0.01）；YES1蛋白高表达组A549细胞细胞克隆形成数高于细胞对照组（P<0.05）。结论： miR-205可能通过靶定靶基因YES1抑制了肺癌细胞A549的增殖，提示miR-205和YES1有可能成为肿瘤生物治疗的新靶点。

Inhibitory effect of miR-205 targeted YES1 on proliferation of A549 cells

Objective To detect the expression levels of the miR-205in lung cancer tissue and A549cells and its targeted gene YES1using qRT-PCR and dual fluorescence protein repoter assay system,and to explore the possible mechanism of miR-205to inhibit the proliferation of lung cancer A549cells.Methods The expression levels of miR-205in 10cases of lung cancer tissue and adjacent normal lung tissue were detected with qRT-PCR.The cell growth curve and colony formation assay were used to determine the proliferation rate of A549cells after transfected by miR-205mimics and control mimics.The sequences of YES1 3′UTR(untranslated region)and mutation target sites of YES1 3′UTR were inserted into the plasmid which expressed green fluorescence protein(pcDNA3/EGFP) respectively to construct the green fluorescence protein plasmids of YES1-3′UTR and mut-YES1-3′UTR.There were six groups in the study:YES1-3′UTR,YES1-3′UTR and miR-205 mimics,YES1-3′UTR and control mimics,mut-YES1-3′UTR,mut-YES1-3′UTR and miR-205 mimics,mut-YES1-3′UTR and control mimics; after the plasmids expressed red fluorescent protein(pDsRed2-N1)were cotransfected into A549cells,the extracted protein was detected with fluorescence spectrophotometer.Results Compared with adjacent normal lung tissue,the expression levels of miR-205in lung cancer tissue and A549cells were decreased(P<0.05);the proliferation rate of A549cells in miR-205mimics group was lower than that in control mimics group(P<0.05). The fluorescence protein expression level in YES1-3′UTR and miR-205mimics co-transfected group was lower than that in YES1-3′UTR and control mimics co-transfected group,the difference was statistically significant(P< 0.01).The number of cell colony formation of A549cells in highly expressed YES1group was higher than that in cell control group(P<0.05).Conclusion MiR-205may inhibit the proliferation of A549cells through regulating of the expression of YES1directly.miR-205and YES1are potential therapeutic targets for the biological treatment of tumor.