应用载体介导的RNA干扰技术抑制肝癌细胞H-ras^(Val12)的表达  

Inhibition of H-ras^(Val12) Expression by Vector-based RNA Interference in Hepatocellular Carcinoma Cells

作　　者：孟繁杰[1] 曹斌[1] 冯增利[1] 王海刚[1] 马顺茂[1] 赵文增[1] 尹东剑[1] 

机构地区：[1]河北医科大学附属华北石油管理局总医院普外科,河北省任丘市062552

出　　处：《中国全科医学》2013年第9期1000-1003,共4页Chinese General Practice

基　　金：河北省科学技术研究与发展指导计划(072761710);美国中华医学基金会资助项目(06-837)

摘　　要：目的观察载体介导的RNA干扰(RNAi)技术对肝癌细胞突变的H-rasVal12表达的抑制作用。方法将针对突变H-ras的短发夹RNA(shRNA)质粒表达载体转染肝癌细胞株BEL7402和SMMC7721,用免疫印迹(Western blot)法检测H-ras蛋白的表达,用流式细胞仪检测细胞凋亡情况。计量资料比较采用t检验;计数资料比较采用χ2检验。结果 (1)转染前、后SMMC7721的H-ras蛋白表达量〔光密度值分别(1 752±21)和(1 736±28)〕比较,差异无统计学意义(t=0.474,P=0.646);而转染了H1-shRNA的BEL7402的H-ras蛋白表达量(920±11)与转染前(1 221±11)比较,差异有统计学意义(t=20.126,P<0.01);转染了H2-shRNA的BEL7402的H-ras蛋白表达量(872±10)与转染前(1 201±10)比较,差异有统计学意义(t=23.477,P<0.01)。H1-shRNA组和H2-shRNA组H-ras蛋白表达下调了62.8%和63.4%。(2)流式细胞仪检测表明,BEL7402转染H1-shRNA质粒后细胞凋亡率(47.7%)与转染H2-RNA质粒后细胞凋亡率(49.3%)比较,差异无统计学意义(χ2=0.005,P=0.94);但均高于BEL7402转染对照质粒后细胞凋亡率(30.2%),差异有统计学意义(χ2H1=6.810,P<0.01;χ2H2=6.103,P<0.01)。结论载体介导的RNAi技术可有效特异抑制肝癌细胞突变的H-rasVal12表达,对野生型的H-ras表达无影响。Objective To investigate the inhibition of H - ras^vail2 expression by vector - based RNA interference （RNAi） in hepatocellular carcinoma cells. Methods shRNA （ short haipin RNA） for H - ras were synthesized and then insert- ed into pSilencerTM 4. 1 - CMV hygro. Recombinant plasmids were transfected into hepatocellular carcinoma cell line BELT402 and SMMC7721. The expression level of H - ras was detected by Western blot. Apoptosis of cells was analyzed by flow cytome- try. Results The Western blot showed that in BEL7402 cell line transfected with H1 - shRNA and H2 - shRNA the protein level of H - ras were decreased by 62.8% and 63.4% respectively when compared with untransfected group （ P 〈0. 01 ） . The protein level of H - ras in SMMC7721 before and after transfected were not statistically significant [ the optical density values were （ 1 752±21 ） and （1 736±28） respectively]. The protein level of H -ras in BEL7402 before and after transfected with H1 -shRNA were （ 1 221±11 ） and （ 920±11 ） respectively. Differences were statistically significant （ t = 20. 126, P 〈 0. 01 ） . The protein level of H - ras （ 872±10 ） in BEL7402 transfected with H2 - shRNA was significantly lower than that （ 1 201±10 ） in BEL7402 before transfected （ t =23.477, P 〈0.01 ） . The flow eytometry showed that in groups transfected with H1 - shRNA and H2 -shRNA apoptosis rate was 47.7% and 49. 3% respectively, significantly higher than that in group transfected with con- trol plasmid （30. 2% ） （X^2H1= 6. 810, P 〈 0. 01 ; X^2H2 = 6. 103, P 〈 0. 01 ） . Conclusion Conclusion The recombinant plasmid encoding mutant - specific shRNA for H - ras^Val12, can significantly inhibit the expression of H - ras^Val12 without affection of wild - type H -ras in hepatocellular carcinoma cells.

关 键 词：RNA干扰 肝肿瘤 短发夹RNA 基因 抑制 

分 类 号：R735.7[医药卫生—肿瘤]