反义人DNMT1基因对乳腺癌细胞MDA-MB-435s增殖和细胞周期的影响  

Effect of DNMT1 antisense RNA on proliferation and cell cycle of human breast cancer cell MDA-MB-435s

作　　者：孙荣斌[1] 鲁雅洁[2] 魏钦俊[2] 

机构地区：[1]南京医科大学药学院,南京210029 [2]南京医科大学基础医学院生物技术系

出　　处：《山西医科大学学报》2010年第3期205-209,240,共6页Journal of Shanxi Medical University

基　　金：南京医科大学校基金资助项目(NY04003)

摘　　要：目的构建表达人DNMT1基因(DNA methyltransferase 1,DNMT1)反义RNA的真核表达质粒,观察其对乳腺癌细胞MDA-MB-435s增殖的影响。方法应用DNA重组技术,将人DNMT1基因的cDNA片段克隆至pcDNA3.1(-)载体中,构建DNMT1反义RNA真核表达质粒,测序验证,将其转染入人乳腺癌细胞系中,利用荧光定量PCR法检测转染前后细胞DN-MT1基因mRNA表达水平的变化;Western blot检测转染后DNMT1蛋白表达变化;应用MTT法检测细胞生长状况,绘制细胞生长曲线;流式细胞术分析细胞周期的变化。结果成功构建出能够表达DNMT1反义RNA的真核表达质粒。转染该质粒后,与未转染组和转染正义质粒组相比,MDA-MB-435s细胞中DNMT1基因mRNA和DNMT1蛋白表达量均显著下降;细胞生长变慢;细胞周期分析可见S期明显减少,而G1/G0期细胞显著增加。结论反义DNMT1能够特异性下调该基因的表达,并抑制乳腺癌细胞的增殖,为乳腺癌基因治疗提供了新的靶标。Objective To construct the antisense RNA expression vector targeting DNMT1 gene, and to investigate its effect on cell cycle, proliferation and apoptosis of breast cancer cell line MDA-MB-435s. Methods cDNA of DNMTI gene was inserted into pcD- NA3.1 （ - ） vector to construct antisense RNA recombinant plasmids by recombinant DNA technology. After transfection into breast cancer MDA-MB-435s cells,the mRNA expression levels of DNMT1 gene and protein were detected by real-time quantitative PCR and Results The recombinant plasmid p-anti-DNMT1 was successfully constructed. After transfection of p-anti-DNMT1 ,the DNMT1 mR- NA and protein Western blot. The cell cycle was analyzed by flow cytometry. MTT assay was used to detect the status of cell growth, ex- pression levels were significantly down-regulated in MDA-MB-435s cells. The proliferation of MDA-MB-435s cells was markedly inhibi- ted after transfected with p-anti-DNMTl. The S phase MDA-MB-435s cells obviously reduced while G1/G0 phase cells significantly in- creased. Conclusion P-anti-DNMT1 can efficiently and specifically down-regulate the expression of DNMT1 gene in MDA-MB-435s cells and inhibit the proliferation of breast cancer, which may provide a new target for gene therapy of human breast cancer. K

关 键 词：DNMT1基因 反义RNA 乳腺癌 MDA-MB-435S 

分 类 号：R737.9[医药卫生—肿瘤]