Let-7d表达质粒的构建及其对卵巢癌细胞高迁移率A2蛋白及ras蛋白表达的抑制作用  

Construction of let-7d expression vector and its inhibitory effect on HMGA2 and ras expression in human ovarian cancer cells in vitro

作　　者：叶海燕[1] 陈建国[1] 黄小穗[2] 郭爱林[2] 郝佩佩[3] 

机构地区：[1]广东省人民医院//广东省医学科学院妇科,广东广州510080 [2]广东省人民医院//广东省医学科学院医学研究中心,广东广州510080 [3]南方医科大学生物技术学院生物技术系暨分子免疫学研究所,广东广州510515

出　　处：《南方医科大学学报》2012年第12期1752-1757,共6页Journal of Southern Medical University

基　　金：广东省科技计划项目(2008B060600037);广东省医学科研基金(A2008004);广东省自然科学基金(0151008004000033)

摘　　要：目的构建let-7d真核表达载体,研究let-7d对卵巢癌细胞生物学行为及蛋白表达的影响。方法根据let-7d序列设计合成microRNA片段,定向克隆到pcDNATM6.2GW/EmGFPmiR真核表达载体上,运用基因转染技术将其导入卵巢癌IGROV1细胞中,使let-7d基因过表达,以实时荧光定量PCR验证let-7d及高迁移率A2(HMGA2)基因的表达,并用Western blotting检测相关蛋白HMGA2的差异表达。以MTT绘制细胞生长曲线,流式细胞术检测细胞凋亡率,分析let-7d对IGROV1细胞增殖生长的影响。结果成功构建针对靶向let-7d的表达质粒,将表达质粒转染人卵巢癌IGROV1细胞后,明显下调HMGA2的表达水平,而下调ras蛋白的作用较弱,没有前者明显。细胞表型研究显示,抑制HMGA2表达可能改变该细胞的恶性表型,表现在细胞增殖能力明显下降,自发凋亡率明显增加等。结论 Let-7d通过对HMGA2的调控,在改变卵巢癌IGROV1细胞恶性表型方面可能起重要作用。Objective To elucidate the role of let-7d in regulating the biological behavior of ovarian cancer cells and their expressions of HMGA2 and ras proteins. Methods The pre-let-7d sequence was synthesized and inserted into pcDNA6.2GW/ EmGFPmiR and transfected into ovarian cancer IGROV1 cells to cause pre-let-7d overexpression. Real-time quantitative RT-PCR was employed to examine the expression levels of let-7d miRNA and HMGA2 mRNA, and Western blotting was performed to detect the expressions of HMGA2 and ras protein in the transfected cells. The effect of pcDNA6.2GW-let-7d transfection on IGROV1 cell proliferation was determined using MTT assay and the cell apoptosis rate was measured using flow cytometry. Results The eukaryotic expression vector containing the target gene let-7d was successfully constructed and transfected into IGROV1 cells. The transfected cells showed a marked reduction of HMGA2 expression but a less obvious down-regulation of ras expression. Transfection with pcDNA6.2GW-let-7d to suppress the expression of HMGA2 caused alterations of the phenotype of IGROV1 cells shown by a reduced proliferative activity and increased cell apoptosis. Conclusion Let-7d plays an important role in altering the malignant cell phenotype of ovarian cancer IGROV1 cells by regulating the expression of HMGA2.

关 键 词：LET-7 卵巢癌 高迁移率蛋白A2 RAS 

分 类 号：R737.31[医药卫生—肿瘤]