摘要
目的采用常间回文重复序列丛集(CRISPR)/CRISPR相关蛋白9(Cas9)技术,构建RNA甲基化转移酶(METTL)3与METTL14的基因同时敲除的人膀胱癌细胞株T24细胞,为职业性肿瘤的靶向基因干预提供新的工具与手段。方法设计并合成靶向METTL3和METTL14的小向导RNA(sgRNA)寡核苷酸序列,构建至Lenti-multiCRISPR载体;采用慢病毒系统将诱导型质粒Lenti-i Cas9-neo转染至T24细胞,以流式细胞术分选阳性克隆,将重组质粒Lenti-multi-METTL3-METTL14转染至阳性分选细胞并筛选,以免疫印迹法检测T24-Lenti-multi-CRISPR空载体细胞(对照组)、METTL3-METTL14双基因敲除T24细胞株T24-KO-METTL3-METTL14(双基因敲除组)的METTL3和METTL14蛋白的相对表达量。结果目的 sgRNA寡核苷酸双链成功插入酶切后的Lenti-multi-CRISPR质粒载体,测序正确;诱导型质粒Lenti-i Cas9-neo成功转染至T24细胞中,经流式分选获得大量阳性细胞;经强力霉素诱导后,获得METTL3-METTL14双基因敲除的稳定细胞株。强力霉素处理96 h后,双基因敲除组细胞的METTL3、METTL14蛋白相对表达量较对照组细胞分别下降52.08%和64.04%(P<0.01)。结论首次获得METTL3-METTL14双基因稳定敲除的T24细胞,为人膀胱癌细胞的候选基因干预靶点及膀胱癌的防治机制研究奠定基础。
Objective To construct a cell model with simultaneous knockout of RNA methyltransferase-like （METTL）3 and METTL14 using clustered regularly interspaced palindromic repeats （CRISPR）/CRISPR-associated protein 9 （Cas9） system in human bladder cancer T24 cells, in order to provide new tools and means for further studying gene intervention in occupational cancers. Methods The small guide RNA （sgRNA） oligonucleotide sequences targeting METTL3 and METTL14 were designed, synthesized and constructed into the Lenti-multi-CRISPR vector. The Lenti-iCas9-neo inducible plasmids were transfected into T24 cells by lentivirus system, and the positive cells were sorted by flow cytometry. Finally, the recombinant plasmid Lenti-muhi-METTL3-METTL14 was transfected into positively sorted cells and screened to obtain stable cell lines. T24-Lenti-multi-CRISPR cells and T24-KO-METTL3-METTLI4 were used as control group and multi- gene knockout group. The Western blot was used to examine relative expression of METTL3 and METTL14 protein in the two groups. Results The targeting sgRNA was successfully inserted into the Lenti-muhi-CRISPR plasmid and confirmed by sequencing. The flow cytometry was used to obtain a large amount of the positive cells. The stable cell line inducted of doxycycline with simultaneous knockout of METTL3 and METTL14 gene was obtained. The relative expression of METTL3 and METTL14 protein decreased respectively by 52.08% and 64.04% after 96 hours of doxycycline treatment in the multi-gene knockout group compared with the control group （P 〈 0. 01 ）. Conclusion The stable T24 bladder cancer cell with simuhaneous double gene knockout of METTL3 and METTL14 was obtained for the first time, which will be helpful for providing a foundation for mechanistic study for prevention and treatment of candidate tumor targets and bladder cancer.
出处
《中国职业医学》
CAS
北大核心
2017年第6期664-670,共7页
China Occupational Medicine
基金
国家自然科学基金(81473014
81472999
81772699)
广东省自然科学基金重点项目(2015A030311038)
广东省"百千万工程"青年拔尖人才(87316004)
