miR-34a稳定表达株构建及其对宫颈癌Caski细胞功能影响的观察  

Construction of miR-34astable expression strain in cervical cancer Caski cell and preliminary study of its function

作　　者：汤文凡[1] 佐满珍[1] 刘朝奇[2] 查锦芬[1] 汤超[2] 

机构地区：[1]三峡大学人民医院妇产科,湖北宜昌443000 [2]三峡大学医学院,湖北宜昌443002

出　　处：《中华肿瘤防治杂志》2013年第19期1503-1507,共5页Chinese Journal of Cancer Prevention and Treatment

基　　金：三峡大学硕士学位论文培优基金(2012PY048)

摘　　要：目的:构建人miR-34a的真核表达载体,并研究其对宫颈癌细胞株Caski细胞活性的影响。方法:以人正常组织基因组DNA为模板,用PCR法扩增得到miR-34a的前体序列,构建miR-34a真核表达载体pEGFP-C1-miR34a。同时用重组载体pEGFP-C1-miR34a转染宫颈癌细胞株Caski细胞,筛选miR-34a稳定表达细胞系作为实验组,pEGFP-C1转染组为对照组(Control),用RT-PCR及Real time RT-RCR法鉴定miR-34a在克隆细胞中的表达,并用MTT、FCM分析miR-34a的生物学特性,RT-PCR研究检测其靶基因表达改变。结果:成功构建了人miR-34a真核表达载体pEGFP-C1-miR34a,并在宫颈癌Caski细胞中上调miR-34a的表达,实验组miR-34a的表达相对于对照组升高约3.13倍;MTT提示,实验组miR-34a能够显著的抑制Caski细胞增殖,差异有统计学意义,P=0.018;实验组G0/G1期百分率为77.7%,与对照组的50%比较,差异有统计学意义,P=0.004;而实验组S期百分率为20.3%,对照组为35.4%,两组比较差异有统计学意义,P=0.027;实验组细胞miR-34a靶基因细胞周期基因CDK4、CDK6、Cyclin E2和E2F1mRNA的表达下调,与对照组比较差异有统计学意义,P=0.002。结论:成功构建人miR-34a的真核表达载体,并能显著抑制宫颈癌细胞株Caski细胞活性,为进一步研究miR-34a在宫颈癌中的功能及基因调控机制奠定了实验基础。OBJECTIVE：To construct an eukaryotic expression vector of miR-34aand explore its influence on the cell viability in cervical cancer cell lines Caski cells.METHODS：The miR-34aprecursor sequences were amplificated by PCR with human genomic DNA as template.The eukaryotic expression vector of pEGFP-C1-miR34awas constructed.The cervical cancer cell lines Caski cells were transfected with the vector pEGFP-C1-miR34ain experimental group and Caski cells were transfected with pEGFP-C1vector as control group.RT-PCR and Real-time RCR were used to identify miR-34aeukaryotic expression in clone cells.The biological characteristics of the miR-34ain caski cells were studied by MTT,FCM and RT-PCR methods,the expression levels of cell cycle gene CDK4,CDK6,Cyclin E2,E2F1 mRNA were tested by RT-PCR.RESULTS：The eukaryotic expression vector of miR-34apEGFP-C1-miR-34awas successfully constructed and it could be expressed in cervical cancer cell lines Caski cells.Expression of miR-34ain the experimental group was3.13times higher than that of the control group;MTT suggested that miR-34acan significantly inhibit the proliferation of Caski cells,and there was statistical significance（P=0.018）;cell cycle of G0/G1in the experimental group was77.7%,while the control group was 50%,and the difference was statistically significant（P=0.004）.The percentage of S phase cells in experimental group was 20.3%,compared with the control group（35.4%）the difference was statistically significant（P=0.027）;The downregulated expression of the miR-34atarget genes of cell cycle genes CDK4,CDK6,Cyclin E2,E2F1mRNA in experiment group had statistically significant difference compared with the control group（P=0.002）.CONCLUSION：The eukaryotic expression vector of miR-34acan be effectively expressed in cervical cancer cell lines Caski cells,inhibit the cell proliferation and downregulate the mRNA expression of cell cycle genes CDK4,CDK6,Cyclin E2,E2F1,which has laid the experimental foundation for further studying the function of miR-34ain

关 键 词：宫颈肿瘤 微小RNAS MIR-34A 表达载体 基因表达 细胞周期 细胞增殖 

分 类 号：R737.33[医药卫生—肿瘤]