PLK1基因RNAi慢病毒载体的构建及对食管鳞癌细胞侵袭转移的影响  

Lentiviral-mediated RNA interference targeting the PLK1 gene inhibits invasion and metastasis of esophageal squamous cell carcinoma cells

作　　者：于文静[1] 张宝刚[2] 陈丽梅[1] 王守训[3] 冯卫国[1] 杜长青[1] 刘顺梅[1] 赵春玲[1] 

机构地区：[1]潍坊医学院细胞生物学教研室,山东省潍坊市261053 [2]潍坊医学院病理学教研室,山东省潍坊市261053 [3]潍坊医学院生物化学教研室,山东省潍坊市261053

出　　处：《世界华人消化杂志》2013年第22期2128-2135,共8页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;Nos.30901779;81072608;山东省自然科学基金资助项目;No.ZR2010HM065;山东省高等学校科技计划基金资助项目;No.J10LC20~~

摘　　要：目的:研究慢病毒介导的PLK1(Polo-like kinase1)基因RNAi对食管鳞癌细胞侵袭转移的影响.方法:利用RT-PCR和Western blot检测不同食管鳞癌细胞中PLK1的表达.根据人PLK1mRNA序列设计干扰片段,Western blot检测干扰效率;利用划痕愈合实验及Transwell实验研究靶向PLK1的RNAi对食管鳞癌细胞体外侵袭转移能力的影响.将有效干扰序列构建至慢病毒干扰载体pGLV/H1/GFP+Puro中,测序鉴定.重组慢病毒载体与包装质粒共转染293T细胞包装病毒,制备的重组病毒感染食管鳞癌细胞,利用荧光定量PCR和Western blot检测干扰效率;利用裸鼠肺转移模型实验研究慢病毒介导的PLK1基因RNAi对食管鳞癌细胞在体内侵袭转移能力的影响.结果:筛选出一种高表达PLK1的食管鳞癌细胞株TE-8用于实验研究;筛选出有效的干扰片段,并成功构建靶向干扰PLK1基因的慢病毒载体,制备重组病毒颗粒用于感染食管鳞癌细胞;靶向PLK1的RNAi在体内外均能明显抑制食管鳞癌细胞的侵袭与转移.结论:靶向干扰PLK1基因的重组慢病毒能够抑制食管鳞癌细胞的侵袭与转移,PLK1基因影响着食管鳞癌的恶性进展.AIM：To investigate the inhibitory effect of lentiviral-mediated RNA interference targeting the PLK1 gene on invasion and metastasis of esophageal squamous cell carcinoma（ESCC） cells.METHODS：RT-PCR and Western blot were used to detect the expression of PLK1 mRNA and protein in different ESCC cells.Based on the mRNA sequence of human PLK1 gene,interference fragments were designed,and interference efficiency was detected by Western blot.The in vitro effect of PLK1 siRNA on migration and invasion of ESCC cells was assessed by woundhealing assay and Matrigel chemoinvasion assay.The most efficient interference fragment was cloned into the lentiviral vector pGLV/H1/GFP＋Puro and sequenced.The resulting recombinant lentiviral vector and packaging plasmids were transfected into 293T cells,and packaged virus particles were used to infect ESCC cells.Interference efficiency was assessed using ？ uorescence quantitative PCR and Western blot.The in vivo effect of recombinant lentiviral vector on invasion and metastasis of ESCC cells was studied using a nude mouse model of pulmonary metastasis.RESULTS：The ESCC cell line TE-8 overexpressed PLK1,and the most efficient PLK1 siRNA could obviously inhibit migration and invasion of TE-8 cells in vitro.The lentiviral vector for RNA interference targeting the PLK1 gene was successfully constructed.The prepared recombinant virus particles could infect TE-8 cells and signif icantly inhibit the metastasis of ESCC cells in vivo.CONCLUSION：Lentiviral-mediated RNA interference targeting PLK1 could obviously inhibit invasion and metastasis of ESCC cells.PLK1 may promote the malignant development of ESCC.

关 键 词：PLK1 侵袭 转移 慢病毒载体 RNAI 食管鳞癌 

分 类 号：R735.1[医药卫生—肿瘤]