SIRT1基因shRNA慢病毒载体的构建及其对乳腺癌细胞增殖的影响  

Construction of recombinant lentiviral vector of shRNA targeting SIRT1 gene and its effects on the proliferation of breast cancer cell

作　　者：陶颖[1] 陈可[1] 宋春丽[1] 任吉华[1] 陈娟[1] 

机构地区：[1]重庆医科大学感染性疾病分子生物学教育部重点实验室,重庆400016

出　　处：《重庆医科大学学报》2013年第5期474-477,共4页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:81201282)

摘　　要：目的:探讨慢病毒介导沉默信息调节因子1(silent information regulator 1,SIRT1)基因的shRNA对乳腺癌细胞MCF-7增殖和凋亡的影响。方法:设计、合成2对针对SIRT1基因shRNA的寡核苷酸序列,与pLentilox3.7-GFP载体连接产生重组慢病毒质粒pshSIRT1-1、pshSIRT1-2,同时构建不针对任何基因的shRNA阴性对照pshCont。将表达shRNA的载体plentilox3.7与pLP1、pLP2和pLP/VSVG 3种质粒共转染293FT细胞,包装产生慢病毒并测定病毒滴度。将慢病毒感染人乳腺癌细胞MCF-7,行real time-PCR和Western blot验证SIRT1基因的沉默效果,台盼蓝排斥实验和流式细胞仪分别检测其对MCF-7细胞增殖和凋亡的影响。结果:成功构建靶向SIRT1基因shRNA慢病毒载体并获得了相应的慢病毒。2个靶向SIRT1的shRNA均能有效抑制SIRT1基因的表达(P=0.000 2),并使MCF-7细胞增殖明显减慢,凋亡明显增加(P=0.006 0)。结论:靶向SIRT1基因RNA干扰能显著抑制MCF-7细胞的生长并促进其凋亡。Objective：To construct the lentiviral vector expressing shRNA targeting silent information regulator 1 （SIRT1） gene and to investigate its effects on proliferation and apoptosis of breast cancer cell MCF-7. Methods：Two shRNA sequences targeting SIRT1 gene were designed and synthesized and were connected to pLentilox3.7-green fluorescent protein（GFP） vector to reconstruct lentivi- ral plasmids pshSIRTl-1 and pshSIRT1-2.Meanwhile,shRNA negative control pshCont targeting no gene was constructed. Recombi- nant lentiviral vector expressing shRNA together with pLP1 ,pLP2 and pLP/VSVG were co-tranfected into 293FT cells and lentivirus was produced. The titer of virus was tested according to the expression level of GFP in 293FT cells. MCF-7 cells were infected with recombinant lentivirus and the silence efficiency was measured by real-time PCR and Western blot. Proliferation and apoptosis of MCF-7 cells were determined by trypan blue exclusive assay and flow cytometry respectively. Results ： Lentiviral vectors containing shRNAs targeting SIRT1 gene were successfully established and corresponding lentiviruses were acquired. Real-time PCR and West- ern blot analysis showed that these two shRNA targeting SIRT1 gene significantly decreased SIRT1 mRNA （P=0.000 2） and protein levels. SIRTI silencing resulted in marked decrease of proliferation and increase of apoptosis in MCF-7 cells（P=0.006 0）. Conclu- sions ： Lentivirus-mediated RNA interference of SIRT1 can significantly inhibit MCF-7 cell proliferation and induce its apoptosis.

关 键 词：慢病毒 沉默信息调节因子1(SIRT1) 乳腺癌细胞 RNA干扰 

分 类 号：R322.66[医药卫生—人体解剖和组织胚胎学] R394-33[医药卫生—基础医学]