摘要
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、mutYES1-3′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-205mimics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。
Objective To detect the expression levels of the miR-205 in lung cancer tissue and A549 cells and its targeted gene YES1 using qRT-PCR and dual fluorescence protein repoter assay system,and to explore the possible mechanism of miR-205 to inhibit the proliferation of lung cancer A549 cells.Methods The expression levels of miR-205 in 10 cases 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 A549 cells after transfected＆amp;nbsp;by miR-205 mimics 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 A549 cells,the extracted protein was detected with fluorescence spectrophotometer.Results Compared with adjacent normal lung tissue,the expression levels of miR-205 in lung cancer tissue and A549 cells were decreased （P〈0.05 ）;the proliferation rate of A549 cells in miR-205 mimics group was lower than that in control mimics group （P〈0.05）. The fluorescence protein expression level in YES1-3′UTR and miR-205 mimics 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 A549 cells in highly expressed YES1 group was higher than that in cell control group （P〈0.05）.Conclusion MiR-205 may inhibit the proliferation of A549 cells through regulating of the expression of YES1 directly.miR-205 and YES1 are potential therapeutic targets for the biological treatment of tumor.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2014年第3期493-498,共6页
Journal of Jilin University：Medicine Edition
基金
国家自然科学基金青年基金资助课题(81201281/H1904)
河北省科技厅自然科学基金项目资助课题(H2013209180
C2012401037
H2013209040)
河北省唐山市科技计划项目资助课题(12140209A-4)
关键词
miR-205
YES1
肺肿瘤
细胞增殖
miR-205
v-yes-1 yamaguchi sarcoma viral oncogene homolog 1
lung neoplasms
cell proliferation
作者简介
程远（1989-），男，河北省唐山市人，在读医学硕士，主要从事肺癌分子学机制的研究。
[通信作者]刘志勇（Tel：0315—3721567，E-mail：macromicro@126．com）；
崔和勤（Tel：0315—3725918，E-mail：503171553@qq．com）
