RNA干扰MAGEA3对人肝癌细胞凋亡的影响  

Effects of MAGEA3 inhibition by shRNA on apoptosis in human hepatocellular cancer cells

作　　者：李文瑜[1] 郭爱林[1] 杨素清[1] 胡少为[2] 文剑明[2] 

机构地区：[1]广东省人民医院肿瘤中心医学研究中心,广东广州510080 [2]中山大学医学院病理教研室,广东广州510080

出　　处：《中国病理生理杂志》2006年第12期2372-2376,共5页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30100218)

摘　　要：目的:探讨小发夹RNA(shRNA)载体介导抑制人肝癌细胞中黑色素瘤相关抗原A3(MAGEA3)的表达和对肝癌细胞凋亡的影响。方法:构建pS ilencer-MAGEA3短发夹状siRNA真核基因表达载体,采用脂质体介导的方法将其转染到人肝癌细胞(m el-ed1)中,用W estern印迹法、RT-PCR检测MAGEA3基因在肝癌细胞中的表达;通过原位末端标记TUNEL法、DNA片段化及Annexin V/PI双标记法检测,观察其对肝癌细胞凋亡的影响。结果:成功构建pS ilencer-MAGEA3短发夹状siRNA真核基因表达载体,转染特异性小发夹RNA质粒表达载体的MEL-ED1细胞中MAGEA3基因表达显著抑制(90%)。与对照组比较,pS ilencer-MAGEA3转染组肝癌细胞培养48 h后,DNA片段化检测出“DNA ladder”、TUNEL法显示细胞典型凋亡特征,Annexin V/PI双标检测转染组细胞凋亡率为21.41%±1.98%,明显高于pS ilencer-neo空载体转染组和未转染组(P<0.01)。结论:MAGEA3小发夹RNA质粒载体可显著抑制MEL-ED1细胞中的MAGEA3基因的表达,促进肝癌细胞的凋亡,MAGEA3基因具有抑制凋亡的作用。该研究为应用RNA i进行肿瘤的基因治疗提供了实验依据。AIM ： To investigate the inhibitory effeet of veetor - based RNA interferenee （ RNAi ） on the expression of melanoma assoeiated antigen A3 （MAGEA3） protein in hepatoeellular eareinoma eells and on apotposis of hepatoeellular eareinoma eells. METHODS： A veetor for transeribing speeifie small hairpin RNA （shRNA） targeting MAGEA3 gene was eonstrueted ,introduced into hepatoeellular eareinoma MEL - EDI eells by Lipofeetamine 2000. The MAGEA3 protein and mRNA expression levels of MEL -EDI eells were deteeted by Western blotting and RT - PCR, respeetively. The eell apoptosis was studied by DNA fragmentation, eleetron mieroseopy ,TUNEL assay, and annexin V/PI staining. RESULTS： The veetor of RNA interferenee was sueeessfully eonstrueted and MAGEA3 expression was desereased signifieantly in MEL - EDI eells. After the shRNA expression veetor was transfeeted into the MEL - EDI eells, the expression of MAGEA3 gene was inhibited signifieantly （ by 90% ）. DNA fragmentation,eleetron mieroseopy and TUNEL assay showed elassie apoptosis eharaeters in the MEL - EDI eells transfeeted with pSileneer - MAGEA3 plasmid with an apoptosis rate of 21.41% ± 1.98%, significantly higher than those in the negative control group transfeeted with pSilencer - neo and in the non - transfeeted group （ both P 〈0. 01 ）. CONCLUSION： The speeifie small hairpin RNA targeting MAGEA3 mRNA ean inhibit the expression of MAGEA3 and eause apoptosis of hepatoeellular eareinoma eells, whieh suggests inhibitory effeet of MAGEA3 on apoptosis in cancer and provides an experimental basis for treating human tumors with RNAi.

关 键 词：RNA干扰 黑色素瘤相关抗原A3 肝肿瘤 细胞凋亡 

分 类 号：R737.31[医药卫生—肿瘤]