靶向SMC1A基因慢病毒介导的RNA干扰对奥沙利铂治疗敏感性的影响  

The study of RNA interfering targeted to SMC1A mediated by lentivirus increases the sensitivity of colorectal cancer cell lines to oxaliplatin

作　　者：冯婉婷[1] 喻晓娟[2] 李进[2] 史玉叶[2] 曹维克[2] 邓之奎[2] 何敬东[2] 

机构地区：[1]南京医科大学,在读硕士研究生210029 [2]南京医科大学附属淮安第一人民医院肿瘤内科,江苏淮安223300

出　　处：《临床肿瘤学杂志》2012年第5期398-402,共5页Chinese Clinical Oncology

基　　金：江苏省卫生厅医学科技发展基金会临床肿瘤学科研基金资助项目(P200950);江苏省卫生厅面上资助项目(H200652)

摘　　要：目的探讨沉默结直肠癌细胞株染色体结构维持-1A(SMC1A)基因对奥沙利铂(L-OHP)敏感性的影响。方法构建SMC1A基因的小分子RNA干扰表达质粒。与慢病毒包装质粒一起转染239T细胞制备pGCSIL-GFP/SMC1A-siRNA慢病毒载体,并将重组质粒转染结直肠癌SW480、HT-29细胞株。RT-PCR、Western blotting法检测SMC1A mRNA和蛋白的表达情况。CCK-8法检测不同浓度L-OHP对细胞的抑制作用。结果 pGCSIL-GFP/SMC1A-siRNA慢病毒载体构建成功,RT-PCR产物为343bp。RT-PCR显示重组质粒转染的SW480、HT-29细胞SMC1A mRNA表达量明显低于其对应的未转染细胞(0.13±0.02 vs.1.02±0.06,0.182±0.019 vs.1.114±0.032;P<0.05);Western blotting显示干扰后的细胞SMC1A蛋白表达受到明显抑制;不同浓度的L-OHP处理细胞48h后,CCK-8显示转染SMC1A-siRNA细胞生长抑制率较未转染细胞高,差异具有统计学意义(P<0.05)。结论沉默结直肠癌细胞株SMC1A基因对奥沙利铂的敏感性显著增加,为临床进一步提高结直肠癌的疗效提供了研究基础。Objective To investigate the inhibitory effects of SMC1A siRNA mediated by lentivirus combined with oxaliplatin （L-OHP） on human colorectal cancer cells. Methods The lentiviral vector of small RNA interference for structure maintenance of chromosome 1A （SMC1A） was constructed for experiment. The siRNA plasmids were transfected together into 293T cells with lentiviral helper plasmid pHelper 1.0 and pHelper 2.0 to generate the respective lentiviruses using Lipofectamine 2000. The recombinant was transfected into SW480 and HT-29 cell lines. The siRNA silenced the expression of SMC1A was detected by RT-PCR and Western blotting. CCK-8 test was used to examine the inhibition rates of cell growth when each group was treated at different concentration of L- OHP. Results The lentiviral vector of small RNA interference for SMCIA was successfully constructed （343bp）. RT-PCR showed that the expression level of SMC1A mRNA in SW480, HT-29 cell lines were lower than negative group（0. 13 ±0.02 vs. 1.02±0.06, 0. 182±0. 019 vs. 1. 114 ± 0. 032; P 〈 0. 05 ）. Western blotting demonstrated that the expressions of SMC1A mRNA and protein in SW480 and HT-29 cell lines with recombinant were significantly lower than those in SW480 and HT-29 cell lines without recombinant （ P 〈 0.05 ）. CCK-8 assay showed that the percentage of inhibition rate in SW480 and HT-29 cell lines transfected with pGCSIL-GFP/ SMC1A-siRNA was higher than those of SW480 and HT-29 cell lines without recombinant after treatment of L-OHP at different concentrations for 48h （ P 〈 0.05 ）. Conclusion SMC1A may increase the Chemosensitivity to L-OHP and make a foundation for the study of treatment of colorectal cancer.

关 键 词：RNA干扰 慢病毒 染色体结构维持-1A基因 奥沙利铂 

分 类 号：R735.3[医药卫生—肿瘤]