应用CRISPR/Cas9系统下调长链非编码RNA HOTAIR

doi:10.13560/j.cnki.biotech.bull.1985.2017-0394

摘要/Abstract

摘要： 近来CRISPR/Cas9系统作为一种成熟的基因编辑工具,被广泛应用。然而由于长链非编码RNA特殊的结构,该系统针对其研究的报道并不多见。选取与肿瘤生成、发展密切相关的长链非编码RNA HOTAIR为研究对象,通过构建Cas9核酸酶稳定遗传表达细胞株,建立pgRNA文库,对其进行基因敲除。qRT-PCR结果显示,pgRNA/Cas9系统靶向HOTAIR基因可使其表达下调60%。MTT增殖检测及细胞划痕实验结果表明：此系统介导的HOTAIR下调可明显抑制HeLa细胞的增殖与迁移。同时,肿瘤抑制因子p21、p53、pRB和DLC1转录水平的变化也暗示,HOTAIR对细胞功能的影响与其调控相关肿瘤抑制因子之间存在紧密联系。因此,利用Cas9技术抑制HOTAIR的表达对全面了解其功能具有重要意义。

关键词: CRISPR/Cas9, pgRNA, 长链非编码RNA, HOTAIR, 增殖, 迁移, 肿瘤抑制因子

Abstract: Recently,as a mature gene editing technique,the CRISPR/Cas9 system has been widely applied. However,it is rarely used in the studies of long non-coding RNAs（lncRNAs）,owe to their specific secondary structure. The lncRNA HOTAIR was called oncogenic lncRNA overexpressing in the vast majority of cancer types,regulating various biological processes such as cell growth,apoptosis,proliferation and metastasis. Our study performed that HOTAIR was knocked out using the Cas9 system based on the stably genetic cell lines HeLa-cas9 and a pgRNA library was built. The qRT-PCR analysis suggested that the RNA expression of HOTAIR decreased by 60% with pgRNA/Cas9. MTT proliferation detection and cell scratch test revealed that HeLa cell proliferation and migration were significantly inhibited after the knockout of HOTAIR. Meanwhile,results indicated that it was associated between HOTAIR and some tumor suppressors by measuring the RNA level of p21,p53,pRB and DLC1 after the knockout of HOTAIR. Thus,it provided a novel insight to comprehensively understand the function of HOTAIR in cancer cells.

Key words: CRISPR/Cas9, pgRNA, LncRNA, HOTAIR, proliferation, migration, tumor suppressor

刘宏博, 郑鹏, 印泽, 张同存. 应用CRISPR/Cas9系统下调长链非编码RNA HOTAIR[J]. 生物技术通报, 2017, 33(11): 180-187.

LIU Hong-bo, ZHENG Peng, YIN Ze, ZHANG Tong-cun. Knockdown of Long Non-coding RNA HOTAIR by CRISPR/Cas9 System[J]. Biotechnology Bulletin, 2017, 33(11): 180-187.

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