反义RNA技术对抑制人肝癌细胞SMMC-7721中POLD1基因表达的研究  

Inhibition of POLD1gene by RNA antisense in the human liver cancer cell line SMMC-7721

作　　者：吴琼[1] 黄文涛[1] 黄怡[1] 徐恒[2] 

机构地区：[1]广西医科大学病理学教研室,广西南宁530021 [2]广西医科大学医学实验中心,广西南宁530021

出　　处：《中华肿瘤防治杂志》2013年第4期241-244,共4页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(30950028)

摘　　要：目的:应用反义RNA技术抑制人肝癌细胞SMMC-7721中POLD1基因的表达,探索采用该技术干预肝癌的可行性。方法:制备人POLD1基因的反义RNA表达质粒,同时建立空白对照组与阴性对照组,由此将实验分为阴性对照组(转染空载体的SMMC-7721细胞)、空白对照组(肝癌细胞SMMC-7721)和实验组(转染人POLD1基因反义RNA表达质粒的肝癌SMMC-7721细胞)3组。MTT实验分析细胞增殖变化,并在转染SMMC-7721细胞48h后,实时荧光定量PCR和蛋白质印迹法检测POLD1基因的表达。结果:MTT实验显示,与空白对照和阴性对照组相比,转染了反义RNA的实验组细胞增殖受到明显抑制。在转染后24～72h,实验组吸光度24h为0.306 7±0.015 4,48h为0.459 2±0.033 2,72h为0.567 1±0.061 1,与空白和阴性对照组相比,P值均<0.05。实时荧光定量PCR实验显示,实验组POLD1的mRNA表达明显下调为0.142 5±0.020 5,阴性对照组为1.017±0.188,空白对照组为1.00±0.00(F=26.5,P<0.05),说明POLD1基因表达受抑制。蛋白质印迹实验显示,POLD1基因蛋白水平(p125)变化趋势与mRNA相同,均为下调,其中实验组为0.222 3±0.009 7,阴性对照组为0.237 9±0.005 9,空白对照组为0.235 4±0.003 4(F=1 365.754,P值均<0.05),说明反义RNA可抑制p125蛋白表达,结论与基因水平趋势一致。结论:针对POLD1基因设计的反义RNA体外抑制了肝癌细胞SMMC-7721的增殖,这与POLD1在基因和蛋白水平的表达下调有关。OBJECTIVE：To inhibit the expression of POLD1 gene by antisense RNA in the cells of human liver canc- er SMMC-7721,and explore the feasibility of intervention for liver cancer. METHODS： Expression plasmid with human POLD1 antisense RNA was constructed. The blank control group and negative control group were established in the same time. SMMC-7721 cells were divided into three groups：blank control group （untransfected cells） , negative control group （cells transfected with blank vector）, and experimental group （cells transfected with antisense RNA）. MTT assay, RT-PCR and western blot were used to analyze the data. RESULTS： MTT assay showed that cell proliferation was signifi- cantly lower in transfected group. Compared with negative control group and blank control group, the A490 value of exper- imental group was 0. 306 7±0. 015 4 in 24 h,0. 459 25=0. 033 2 in 48 h,0. 567 1±0. 061 1 in 72 h afer transfacting （P〈 0.05）. The mRNA expression of POLD1 gene was down-regulated by real-time RT-PCR,which were 0. 142 52=0. 020 5 in experimental group,1. 017 2= 0. 188 in negative control group and 1.00 2= 0. 00 in blank control group （F= 26. 5, P〈 0.05）. It meant antisense RNA could inhibit the gene expression. The protein expression of POLD1 gene was down-regu- lating by Western Blot,which were 0. 237 9-4-0. 005 9 in negative control group,0. 222 3~±. 009 7 in experimental group and 0. 235 4 5= 0. 003 4 in blank control group （F= 136 5. 754,P〈0.05）. Similar results were also obtained for the protein expression levels of p125. It means antisense RNA can inhibit the protein expression. CONCLUSION：Antisense RNA could inhibit the proliferation of human liver cancer cell SMMC-7721 possibly by down-regulating POLD1 expression.

关 键 词：肝肿瘤 POLD1 反义RNA 基因表达 

分 类 号：R735.7[医药卫生—肿瘤]