polo样激酶-1干扰质粒构建及对人肝癌SMMC-7721细胞株凋亡的影响  

Construction of polo-like kinase-1 siRNA-expressing plasmid and its effects on human hepatocellular carcinoma cell line SMMC-7721

作　　者：倪侃[1] 陈佳慧[1] 贾乃昕[1] 吴龙祥[1] 常仁安[1] 陈钟[2] 

机构地区：[1]江苏南通大学附属医院普外科,226001 [2]江苏南通大学附属医院肝胆外科,226001

出　　处：《中华实验外科杂志》2012年第10期1914-1916,共3页Chinese Journal of Experimental Surgery

基　　金：江苏省医学重点人才基金资助项目（RC2007086）;江苏省高校自然科学基金资助项目（09KJD320004）;南通市社会发展基金资助项目（S2009031）

摘　　要：目的构建polo样激酶-1（PLK1）基因小于扰RNA（siRNA），观察其对肝癌细胞凋亡的影响。方法设计合成3个PLK1siRNA（siRNAl、siRNA2、siRNA3）干扰序列，构建干扰载体，转染人肝癌细胞株SMMC-7721，观察48h后转染效率，采用实时定量逆转录-聚合酶链反应（RT—PCR）筛选下调PLK1mRNA效果最好的siRNA；然后选择以该siRNA转染48h的SMMC-7721细胞，分别以实时定量RT－PCR和Westernblot法检测空白组、对照组及转染组细胞中PLK1基因和蛋白表达；流式细胞仪检测细胞周期及细胞凋亡的改变。结果肝癌细胞株SMMC-7721中存在PLK1蛋白的过表达。应用靶向PLK1的siRNA转染SMMC-7721细胞48h后，PLK1mRNA相对水平siRNA2组分别较无关对照组和空白对照组下降了74％和78％，而PLK1蛋白的表达siRNA2组分别较无关对照组和空白对照组下降了83％和88％，抑制率达88％；siRNA2组中出现大量凋亡细胞，与对照组比较差异有统计学意义（P〈0．05）。结论PLK1基因在肝癌细胞增殖中具有重要的调控作用。PLK1-siRNA转染可明显抑制癌细胞增殖，诱导凋亡是其重要机制之一。Objective To study effects of polo-like kinase-1 （PLK1） small interfering RNA （siRNA） on proliferation of human hepatocellular carcinoma （HCC） cells. Methods Three PLK1 siRNAs （siRNA1, siRNA2, siRNA3） were constructed and use to transfect SMMC-7721 cells. Transfection effi- ciency at 48 h was observed. Real-time reverse transcription-polymerase chain reaction （RT-PCR） was used to find the most effective siRNA which was then used to transfect SMMC-7721 cells. RT-PCR and Western blotting were used to detect PLK1 expression in SMMC-7721 cells which were divided into different groups. Flow cytometry （FCM） was used to detect apoptosis rate and phase distribution of cell cycles. Re- suits PLK1 protein was overexpressed in SMMC-7721 cells. SMMC-7721 cells transfected with low doses of siRNAs targeted against PLK1 had greatly decreased levels of PLK1 mRNA and protein. Compared to blank control and negative control 48 h after transfectian, siRNA2 reduced PLK1 mRNA by 74% and 78%, and PLK1 protein by 83% and 88%, respectively. Apoptosis rate was increased remarkably and the phenotypes of apoptosis could be seen in transfected cells 48 h after transfection. Conclusion PLK1 may play an important role in proliferation of HCC cells. PLK1 siRNA transfection can inhibit proliferation of HCC ceils through apoptosis induction.

关 键 词：癌 肝细胞 polo样激酶-1 小干扰RNA 

分 类 号：R735.7[医药卫生—肿瘤]