FUT8基因RNAi慢病毒载体的构建及对MCF-7细胞增殖的影响

doi:10.13560/j.cnki.biotech.bull.1985.2015.05.035

生物技术通报 ›› 2015, Vol. 31 ›› Issue (5): 231-236.doi: 10.13560/j.cnki.biotech.bull.1985.2015.05.035

• 研究报告 • 上一篇

FUT8基因RNAi慢病毒载体的构建及对MCF-7细胞增殖的影响

温宪春¹, 韩翠翠¹, 赵月生², 于海涛¹, 李成冲¹, 岳丽玲¹

（1.齐齐哈尔医学院医药科学研究中心，齐齐哈尔 161006；2.齐齐哈尔医学院附属第三医院乳腺外科，齐齐哈尔 161006）

收稿日期:2014-08-25 出版日期:2015-05-18 发布日期:2015-05-18
作者简介:温宪春，男，硕士，研究方向：肿瘤药理学；E-mail：wenxianchun@sina.com
基金资助:
国家自然科学基金项目（81202084），教育部科学技术研究重点项目（212047）

Construction of FUT8 Gene Lentiviral RNA Interference Vector and Regulation on Proliferation of Human Breast Cancer Cells MCF-7

Wen Xianchun¹, Han Cuicui¹, Zhao Yuesheng², Yu Haitao¹, Li Chengchong¹, Yue Liling¹

（1. Research Center of Medical Science，Qiqihar Medical College，Qiqihar 161006；2. Breast Surgery，the Third Affiliated Hospital of Qiqihaer Medical College，Qiqihar 161006）

Received:2014-08-25 Published:2015-05-18 Online:2015-05-18

摘要/Abstract

摘要： 旨在构建FUT8基因RNA干扰（RNAi）慢病毒载体并观察其对人乳腺癌细胞MCF-7增殖的影响。针对FUT8基因设计3组短发夹RNA序列，退火合成双链DNA，通过连接线性化的pGC-LV-GFP载体，构建miRNA慢病毒载体质粒，并将其转化至感受态细胞DH5α；测序验证正确后进行FUT8基因慢病毒载体的包装及病毒滴度测定，将获得的重组慢病毒pGC-shFUT8转染MCF-7细胞，利用Real time-PCR、Western blot分别验证转染后MCF-7细胞中FUT8 mRNA及蛋白的表达，MTT法及克隆形成实验检测shFUT8对MCF-7细胞增殖能力的影响。测序证实成功构建针对FUT8基因的RNAi慢病毒载体；慢病毒载体经293T细胞包装成功，测定病毒悬液滴度＞5×10⁸TU/mL；荧光显微镜下观察各转染组细胞GFP的表达，转染效率达90%以上；Real-time PCR、Western blot结果显示干扰组FUT8的mRNA及蛋白表达水平较对照组显著降低，其中pGC-shFUT8-2序列对FUT8基因的干扰效率可达80%，干扰效果最佳，FUT8沉默后MCF-7细胞增殖能力下降。

关键词: FUT8基因, 慢病毒载体, RNA干扰, MCF-7细胞

Abstract: This study aimed to construct the lentiviral RNA interference（RNAi）vector of human FUT8 gene and determine its effect on proliferation of human breast cancer cells MCF-7. Designing three short hairpin RNA（shRNA）sequence targeting the FUT8 gene, and then synthesizing the complementary DNA chains containing the target sequence, miRNA lentiviral vector plasmid with linear pGC-LV-GFP carriers were constructed and then transformed to DH5α cells. After identified by sequencing, then packaging lentiviral vectors and measuring the virus titer were done. The recombinant lentiviral vector pGC-shFUT8 was transfected into human MCF-7 cells, and then the expression of FUT8 in transfected MCF-7 cells was detected by real time-PCR and Western Blotting. The effect of shFUT8 on MCL-7 cell proliferation was measured by MTT assay and colony formation experiment. Gene sequencing confirmed the successful construction of RNAi lentiviral vector targeting the FUT8 gene. The lentiviral vectors were packed successfully by 293T cells and the virus titer was more than 5×10⁸TU/mL. Expressed GFP in transfected cells were observed under the fluorescence microscope, and the transfection efficiency was over 90%. Real-time PCR and Western Blotting analysis indicated that the expression levels of FUT8 mRNA and protein in the transfected group significantly reduced when comparing with the negative control group. Particularly the pGC-shFUT8-2 sequence could interfere 80 % of FUT8 gene expression. The proliferation of MCF-7 decreased after FUT8 was inactive.

Key words: FUT8 gene, Lentiviral vector, RNA interference, MCF-7cells

温宪春, 韩翠翠, 赵月生, 于海涛, 李成冲, 岳丽玲. FUT8基因RNAi慢病毒载体的构建及对MCF-7细胞增殖的影响[J]. 生物技术通报, 2015, 31(5): 231-236.

Wen Xianchun, Han Cuicui, Zhao Yuesheng, Yu Haitao, Li Chengchong, Yue Liling. Construction of FUT8 Gene Lentiviral RNA Interference Vector and Regulation on Proliferation of Human Breast Cancer Cells MCF-7[J]. Biotechnology Bulletin, 2015, 31(5): 231-236.

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FUT8基因RNAi慢病毒载体的构建及对MCF-7细胞增殖的影响

Construction of FUT8 Gene Lentiviral RNA Interference Vector and Regulation on Proliferation of Human Breast Cancer Cells MCF-7

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