N-Ras基因表达沉默与二羟环氧苯并芘转化的人支气管上皮细胞生长的关系  

Relationship between N-Ras silencing and growth of BPDE-transformed 16HBE cells

作　　者：周兰兰[1,2] 蒋义国[1] 谭爱军[2] 沈月兰[1] 刘林华[1] 杨巧媛[1] 

机构地区：[1]广州医学院化学致癌研究所与预防医学系,广东广州510182 [2]珠海市疾病预防控制中心

出　　处：《毒理学杂志》2009年第2期92-95,共4页Journal of Toxicology

基　　金：国家自然科学基金资助项目(30571546;30771780);教育部留学回国人员科研启动基金资助项目(2007-24);广东省自然科学基金资助项目(07117550);广东省高校自然科学重点项目(06Z021)

摘　　要：目的构建N-Ras基因小发卡结构RNA(shRNA)干扰真核表达质粒载体,并初步观察其对二羟环氧苯并芘(BPDE)转化的人支气管上皮细胞(16HBE-T)生长的影响。方法根据GenBank提供的N-Ras cDNA序列,设计并合成shRNA寡核苷酸片段,与含U6启动子的pGPU6/GFP/Neo质粒定向连接,构建4个真核表达载体,并经限制性内切酶酶切和DNA测序进行鉴定。转染16HBE-T细胞48 h后,采用逆转录-聚合酶链反应(RT-PCR)和Western blot检测重组质粒对N-Ras基因表达的影响;用噻唑蓝法(MTT)观察重组质粒对细胞生长的抑制作用。结果构建了4个N-Ras shRNA真核表达载体,经限制性内切酶酶切和DNA测序证实与设计完全一致。构建的4个表达载体pGPU6/GFP/Neo-NRas-566、pGPU6/GFP/Neo-NRas-305、pGPU6/GFP/Neo-NRas-613和pGPU6/GFP/Neo-NRas-791分别转染16HBE-T细胞48 h后,RT-PCR显示N-Ras基因mRNA表达水平依次下调95%、70%、92%和60%;Western blot显示蛋白表达依次降低92%、45%、58%和34%。MTT发现16HBE-T细胞的生长受到明显抑制,且与N-Ras基因表达水平正相关。结论成功构建了N-Ras基因特异性shRNA真核细胞表达载体,有效沉默了N-Ras在16HBE-T细胞中的表达,抑制了恶性转化细胞的生长。Objective To construct eukaryotic expression vector of small hairpin RNA （shRNA） of N-Ras and investigate the effect of recombinant plasmid on suppressing growth of malignantly transformed human bronchial epithelial cell induced by anti-benzo（a） pyrene-trans-7, 8-dihydrodiol-9, 10-epoxide （BPDE） （16HBE-T）. Methods Four shRNAs of N-Ras gene were designed and synthesized, and inserted into plasmid pGPU6/GFP/Neo including U6 promoter. The recombinant expression vectors were identified by restriction map and sequence analysis, and transfected into 16HBE-T cells. After 48h of transfection, RT-PCR and Western blot were conducted to assess the efficency of N-Ras silencing; and MTI？ was used to observe the proliferation of 16HBE-T cells. Results The four recombinant plasmids were verified by restriction map and sequence analysis. The sequences completely coincided with the designs. After 48h of transfection with pGPU6/GFP/Neo-NRas-566, pGPU6/GFP/Neo-NRas-305, pGPU6/GFP/Neo-NRas-613 and pGPU6/GFP/ Neo-NRas-791 recombinant plasmid, RT-PCR showed that the N-Ras mRNA expression was decreased by 95%, 70%, 92% and 60%, respectively, and Western blot exhibited that its protein expression was decreased by 92%, 45%, 58% and 34%, correspondingly. MTT demonstrated the growth of 16HBE-T transfected cells was suppressed significantly, and its proliferation ability correlated with N-Ras expression level. Conclusion The shRNA eukaryotic expression vector against N-Ras gene is successfully constructed. It effectively silences the expression of N-Ras in 16HBE-T ceils and suppresses the cell growth.

关 键 词：二羟环氧苯并芘 RNA干扰 小发夹RNA N-RAS 

分 类 号：R994.6[医药卫生—毒理学]