摘要
以高表达Polo样激酶1(Polo-likekinase1,PLK1)的宫颈癌细胞系HeLa细胞为模型,观察针对PLK1基因的短发夹状RNA(shorthairpinRNA,shRNA)对其凋亡和增殖的影响.设计并合成了针对PLK1的shRNA,将其导入构建的携带增强型绿色荧光蛋白(EGFP)的RNAi(RNAinterference)表达载体中.通过RT-PCR和Western印迹分别检测HeLa细胞PLK1基因和蛋白水平的表达,以流式细胞仪和PI-Hochest双染法测细胞凋亡,MTT法检测细胞的增殖水平.成功构建了携带EGFP的RNAi表达载体pEGFP-H1.转染shRNA后,HeLa细胞PLK1的表达降低至30%.与对照组和空载体转染组相比,shRNA转染组的HeLa细胞凋亡率明显增加,其增殖活性则明显降低.本课题构建的RNAi表达载体便于观察靶基因的转染情况,且不影响H1启动子的体内转录.PLK1的基因沉默能明显增加HeLa细胞的凋亡,抑制该细胞的增殖,有可能为未来肿瘤的治疗找到新的靶点和有效途径.
HeLa cell, one kind of Cervix carcinoma cell hairpin RNA on PLK1 gene with high expression of PLK1. line, was used to investigate the effect of short Short hairpin RNA targeting at Polo-like kinase 1 gene was designed and synthesized, which was tmnsfected into RNAi expression vector with EGFP. PLK1 expression in HeLa cells was detected by RT-PCR and Western blotting respectively. Cells apoptosis were measured by flow cytometry, and proliferation was detected via MTT assay, pEGFP-H1, an RNAi expression vector, was successfully constructed. Transfected with shRNA, PLK1 expression in HeLa cells was decreased to 30%. Compared with control group and mock-transfected group, apoptosis of HeLa cells transfected with shRNA was remarkably increased（ P 〈 0.05）, but their proliferation activity was reduced（ P 〈 0.05）. The RNAi expression vector constructed was more conveniently to detect the genes transfection and did not affect its transcription. PLK1 gene silence can obviously increase apoptosis of HeLa cells and inhibit their proliferation, which may be the new target and efficient way for tumor therapy in the future.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2006年第12期991-995,共5页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金资助项目(No.30240022)资助课题~~
