TOX3基因RNAi慢病毒载体的构建及对乳腺癌ZR-75-1细胞增殖的影响

doi:10.13560/j.cnki.biotech.bull.1985.2018-0868

生物技术通报 ›› 2019, Vol. 35 ›› Issue (7): 141-147.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0868

TOX3基因RNAi慢病毒载体的构建及对乳腺癌ZR-75-1细胞增殖的影响

韩翠翠^1,2, 刘立琨³, 王玉春¹, 杨莹¹, 刘吉成³, 周忠光²

1. 齐齐哈尔医学院药学院,齐齐哈尔 161006;
2. 黑龙江中医药大学基础医学院,哈尔滨 150040;
3. 齐齐哈尔医学院医药科学研究院,齐齐哈尔 161006

收稿日期:2018-10-10 出版日期:2019-07-26 发布日期:2019-07-29
作者简介:韩翠翠,女,博士,教授,博士生导师,研究方向：肿瘤药理学,E-mail：hancuicui-1234@163.com
基金资助:
齐齐哈尔医学院院内博士专项科研基金（QY2016B-19）,齐齐哈尔市科学技术局社会发展攻关项目（SFGG-201549）,国家自然科学基金面上项目（81573660）

Construction of TOX3 Gene Lentiviral RNA Interference Vector and Effect on Proliferation of Human Breast Cancer Cells ZR-75-1

HAN Cui-cui^1,2, LIU Li-kun³, WANG Yu-chun¹, YANG Ying¹, LIU Ji-cheng³, ZHOU Zhong-guang²

1. College of Pharmacy,Qiqihar Medical University,Qiqihar 161006;
2. Basic Medical College,Heilongjiang University of Chinese Medicine,Harbin 150040;
3. Research Center of Medical Science,Qiqihar Medical University,Qiqihar 161006

Received:2018-10-10 Published:2019-07-26 Online:2019-07-29

摘要/Abstract

摘要： 旨在构建TOX3基因RNAi慢病毒载体并观察其对人乳腺癌ZR-75-1细胞增殖能力的影响。针对TOX3基因设计干扰靶序列,构建载体,测序正确后进行慢病毒包装及滴度测定。转染ZR-75-1细胞,荧光显微镜下观察表达GFP的细胞数目,实时荧光定量PCR及Western blot实验验证转染后ZR-75-1细胞中TOX3 mRNA和蛋白的表达。MTT及平板单克隆实验检测TOX3对ZR-75-1细胞增殖能力的影响。结果显示,各组载体序列正确,病毒滴度均>2×10⁸TU/mL,表达GFP细胞数目均可达95%以上。各干扰组TOX3 mRNA及蛋白表达水平均降低,其中TOX3-shRNA-3组干扰效果最佳,转染后ZR-75-1细胞的增殖和单克隆形成能力下降。成功构建TOX3基因的RNAi慢病毒载体,沉默TOX3后ZR-75-1细胞的增殖能力下降。

关键词: TOX3基因, 慢病毒载体, RNA干扰, ZR-75-1细胞

Abstract: This work aims to construct TOX3 gene lentiviral RNA interference（RNAi）vector and observe its effect on the proliferation of human breast cancer ZR-75-1cells. First,designing interference sequence targeting the TOX3,constructing vectors and then packaging lentiviral vectors and measuring the virus titer after sequence identification were conducted. Then the recombinant lentiviral vector was transfected into human ZR-75-1cells,the number of cells expressing GFP was observed under fluorescence microscope,and then the expression of TOX3 in the transfected ZR-75-1cells was detected by real time-PCR and Western blot. Finally,the effect of TOX3 on ZR-75-1 cells proliferation was measured by MTT assay and colony formation experiment. The results showed that the vector sequence of each group was confirmed correctly by gene sequencing,the virus titer was > 2×10⁸TU/mL,and over 95% cells expressed GFP under the fluorescence microscope after being transfected,the expression of TOX3 mRNA and protein significantly reduced in the interference groups,particularly the interfere effect of TOX3-shRNA-3 group was the best,the proliferation and monoclonal ability of ZR-75-1cells decreased after the transfection. In sum,the study successfully constructed TOX3 gene lentiviral RNA interference vector,and the proliferation of ZR-75-1cells decreased after TOX3 was silenced.

Key words: TOX3 gene, lentiviralvector, RNA interference, ZR-75-1 cells

韩翠翠, 刘立琨, 王玉春, 杨莹, 刘吉成, 周忠光. TOX3基因RNAi慢病毒载体的构建及对乳腺癌ZR-75-1细胞增殖的影响[J]. 生物技术通报, 2019, 35(7): 141-147.

HAN Cui-cui, LIU Li-kun, WANG Yu-chun, YANG Ying, LIU Ji-cheng, ZHOU Zhong-guang. Construction of TOX3 Gene Lentiviral RNA Interference Vector and Effect on Proliferation of Human Breast Cancer Cells ZR-75-1[J]. Biotechnology Bulletin, 2019, 35(7): 141-147.

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TOX3基因RNAi慢病毒载体的构建及对乳腺癌ZR-75-1细胞增殖的影响

Construction of TOX3 Gene Lentiviral RNA Interference Vector and Effect on Proliferation of Human Breast Cancer Cells ZR-75-1

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