增殖细胞核抗原siRNA序列的设计、合成与筛选  

Design,preparation and screening of siRNA of proliferating cell nuclear antigen

作　　者：蒋建伟[1] 史金桃[1] 吴智慧[1] 曾慧兰[2] 严玉霞[1] 陈涛[1] 丁文婷[1] 

机构地区：[1]暨南大学医学院生化教研室,广东广州510632 [2]暨南大学附属第一医院血液科,广东广州510632

出　　处：《中国病理生理杂志》2008年第3期484-489,共6页Chinese Journal of Pathophysiology

基　　金：广州市科技计划资助项目(No.2002J1-C0361);广东省医学科研基金资助项目(No.A2006352)

摘　　要：目的:设计并筛选能有效抑制人肝癌SMMC-7721细胞、人结肠癌Caco2细胞、人胃癌SCG-7901细胞增殖的增殖细胞核抗原(PCNA)siRNA。方法:总结以往提出的siRNA设计原则的基础上,设计4条PCNAsiRNA,体外转录合成PCNAsiRNA;并作用于3种肿瘤细胞,WST-8法检测细胞增殖活性,筛选能有效抑制肿瘤细胞增殖的siRNA序列;RT-PCR检测细胞PCNA mRNA水平;免疫细胞化学方法检测PCNA蛋白表达水平。结果:4条PCNA siRNA中,有3条序列(No.2、No.4、No.3)的siRNA能有效抑制3种肿瘤细胞的增殖;其中No.2、No.4作用效果最优,以50 nmol/L浓度作用48 h,对3种肿瘤细胞的增殖抑制率均可达到62%以上,No.2、No.4 PCNAsiRNA均能在mRNA和蛋白水平显著下调PCNA的表达。结论:PCNA siRNA能在体外有效抑制SMMC-7721细胞、Caco2细胞、SCG-7901细胞的增殖,其适宜作用浓度为50 nmol/L,适宜作用时间为48 h。AIM： To design, prepare and screen out functional small interfering RNA （siRNA） for specifically silencing proliferating cell nuclear antigen （PCNA） gene expression and effectively inhibiting cell proliferation on human hepatoma cell line SMMC -7721, human gastric carcinoma cell line SGC -7901 and human colorectal carcinoma cell line Caco2. METHODS： PCNA siRNA was designed based on previous studies about design guidelines and synthesized in vitro by T7 Mega short script reaction kit according to the manufacturer＇ s instructions. After purification, the integrities of siR- NA were identified through 19% denaturing polyacrylamide gel electrophoresis, and the concentrations of the generated siR- NA were measured by testing the absorbance at 260 nm using a spectrophotometer. Four synthesized double - strand siRNA were transfected into three types of carcinoma cell lines by Lipofectamine^TM 2000 reagent, respectively. WST -8 assay was employed to examine the proliferative inhibitions of these three cell lines. The subsequent alterations on PCNA mRNA and protein levels were determined by semi -quantitative reverse transcription- polymerase chain reaction （RT -PCR） and immunocytochemistry, respectively. RESULTS： 3 sequences, No. 2, No. 3 and No. 4 PCNA siRNA showed effective inhibitions on tumor cells among the 4 candidate siRNA, and a single dose of 50 nmol/L of No. 2, No. 4 PCNA siRNA showed the most effective inhibitory rates as more than 62% at 48 h after transfection. 50 nmol/L of No. 2 and No. 4 PCNA siRNA transfection caused 72% decrease of PCNA mRNA level and almost completely loss of the protein in human Caco2 cells. CONCLUSION： No. 2 and No. 4 PCNA siRNA have been screened out in this study, which show the capability to effectively down - regulated PCNA expression and significantly inhibit the proliferation of carcinoma cells. The optimal concentration is 50 nmol/L and satisfactory effects are achieved 48 h after transfection in vitro.

关 键 词：增殖细胞核抗原 SIRNA 结直肠肿瘤 肝肿瘤 胃肿瘤 

分 类 号：R73-362[医药卫生—肿瘤]