基础研究(Basic Research)

尾型同源盒转录因子2 shRNA慢病毒表达载体的构建及其转染对结肠癌细胞生长的影响

Published at: 2016年第25卷第4期

王训凯 1 , 郑见宝 1 , 孙学军 1 , 王孝珑 1 , 余钧辉 1 , 李孝斌 1
1 西安交通大学第一附属医院 普通外科,陕西 西安 710061
通讯作者 学军 孙 Email: sunxy@mail.xjtu.edu.cn
DOI: 10.3978/j.issn.1005-6947.10.3978/j.issn.1005-6947.2016.04.011
基金:
国家自然科学基金资助项目 81101874;81172362
陕西省科学技术研究发展计划资助项目 2011-K12-19
陕西省科技统筹创新工程计划资助项目 2013KTCQ03-08

摘要

目的:构建尾型同源盒转录因子2(CDX2)shRNA慢病毒表达载体,并观察下调CDX2基因其对结肠癌细胞生长的影响。方法:根据CDX2 mRNA序列设计shRNA,并合成shRNA的互补序列,通过连接酶链接到GV248载体上,用测序方法鉴定阳性克隆后,将构建好的慢病毒表达载体与慢病毒包装载体用Lpofectamine 2000共转染293T细胞,产生慢病毒颗粒并用稀释法进行滴度测定。将CDX2 shRNA慢病毒表达载体转染人结肠癌细胞SW480、HT29后,分别用qRT-PCR和Western blot检测CDX2 mRNA及蛋白水平,CCK8实验及克隆形成实验检测细胞增殖能力。结果:DNA测序鉴定证实,CDX2 shRNA表达片段正确插入GV248载体中,包装后的病毒滴度为1×109 TU/mL;SW480、HT29细胞转染CDX2-shRNA慢病毒表达载体后,CDX2 mRNA及蛋白表达水平均明显降低(均P<0.05),但细胞的增殖与克隆形成能力无明显改变(均P>0.05)。结论:成功构建shRNA慢病毒表达载体,其转染结肠癌细胞后能有效地抑制CDX2基因的表达;下调CDX2基因对人结肠癌细胞的增殖无明显影响。


Construction of lentiviral vectors expressing shRNA against caudal-related homeobox 2 gene and effect of their transfection on growth of colon cancer cells

Abstract

Objective: To construct recombinant lentiviral vectors expressing the shRNA against caudal-related homeobox 2 (CDX2) gene, and observe the effect of down-regulation of CDX2 gene expression on growth ability of colon cancer cells. Methods: According to CDX2 mRNA sequence, the shRNA sequences were designed and their complementary sequences were synthesized, which were then inserted into the GV248 vectors by using ligase. Positive clones were verified by DNA sequencing. Lentiviral particles were produced by cotransfection of lentiviral expression vectors with lentiviral packaging plasmids into 293T cells by lipofectamine2000, and then the viral titer was determined by serial dilution. Human colon cancer SW480 and HT29 cells were transfected with the CDX2-shRNA expressing lentiviral vectors, and then the CDX2 mRNA and protein expression were determined by qRT-PCR and Western blot analysis, and the proliferative ability was examined by CCK 8 assay and colony formation assay, respectively. Results: The results of DNA sequencing showed that CDX2-shRNA expression segments were correctly inserted into GV248 vectors, and viral titer was 1×109 TU/mL after packaging. In both SW480 and HT29 cells after transfection of CDX2-shRNA expressing lentiviral vectors, the mRNA and protein expression levels of CDX2 mRNA and protein were significantly down-regulated (all P<0.05), but no significant change was found in cell proliferation and colony forming ability (all P>0.05). Conclusion: CDX2-shRNA expressing lentiviral vectors are successfully constructed and their transfection can effectively inhibit CDX2 gene expression. Down-regulation of CDX2 gene expression exerts no obvious effect on growth of colon cancer cells.


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引用

引用本文: 训凯 王, 见宝 郑, 学军 孙, 孝珑 王, 钧辉 余, 孝斌 李. 尾型同源盒转录因子2 shRNA慢病毒表达载体的构建及其转染对结肠癌细胞生长的影响[J]. 中国普通外科杂志, 2016, 25(4): 529-534.
Cite this article as: WANG Xunkai, ZHENG Jianbao, SUN Xuejun, WANG Xiaolong, YU Junhui, LI Xiaobin . Construction of lentiviral vectors expressing shRNA against caudal-related homeobox 2 gene and effect of their transfection on growth of colon cancer cells[J]. Chin J Gen Surg, 2016, 25(4): 529-534.