Ku70基因稳定敲除HeLa细胞株的建立及其生物学功能研究

Establishment and Biological Function of Ku70 Gene Stably Knockout HeLa Cell Lines

目的:利用CRISPR/Cas9技术构建人Ku70基因稳定敲除的HeLa细胞株,并检测其生物学功能。方法:设计并构建向导RNA载体p Cas-g RNA和同源重组供体DNA载体p Back Zero-T-Ku70,2种重组质粒共转染HeLa细胞,加入潮霉素B进行抗性筛选,通过基因组PCR和Western印迹检验Ku70基因是否被敲除;进而,选择Ku70稳定敲除的细胞株,分别采用CCK-8和Transwell实验检测细胞增殖和迁移能力;此外,提取细胞总RNA,反转录成c DNA后用荧光定量PCR仪检测5种miRNA(hsa-miR-649、hsa-miR-544a、hsa-miR-562、hsa-miR-548a、hsa-miR-492)的表达水平。结果:g RNA表达质粒p Cas-g RNA和DNA供体质粒p Back Zero-T-Ku70构建成功;2种重组质粒共转染HeLa细胞,基因组PCR扩增出特异的基因重组DNA片段,Western印迹结果显示Ku70蛋白已基本无表达。细胞增殖和迁移实验显示敲除Ku70基因的HeLa细胞增殖和迁移能力均有所减弱。q RT-PCR结果显示,敲除Ku70基因致hsa-miR-649、hsa-miR-544a和hsa-miR-562水平有所升高,而hsa-miR-548a和hsa-miR-492水平未有明显变化。结论:获得Ku70基因稳定敲除的细胞株;Ku70蛋白可能参与了HeLa细胞增殖和迁移过程;其还可能调节部分miRNA的表达。

Objective:To knock out Ku70 gene of HeLa cells by CRISPR/Cas9 technology and explore the biological functions of Ku70 knockout cell lines.Methods:The guide RNA expressing vector pCas-gRNA and homologous recombination donor DNA vector pBackZero-T-Ku70 were designed and obtained.After co-transfection of the two plasmids,cells were screened with hygromycin B for obtaining Ku70 gene knockout cells.Genomic PCR and Western blot assays were used to identify Ku70 gene knockout cells.The proliferation and migration of Ku70 knockout cell lines were assayed.In order to detect the levels of five miRNAs(hsa-miR-649,hsa-miR-544a,hsamiR-562,hsa-miR-548a and hsa-miR-492),total RNA of Ku70 gene knockout cell lines was extracted and reversely transcribed to cDNA.qPCR assay was used to quantify the expression of these miRNAs in the cDNA preparation.Results:We constructed the plasmids pCas-gRNAs and pBackZero-T-Ku70 successfully,and after cotransfection and screening,we obtained Ku70 gene knockout HeLa cell lines.The results of cell proliferation and migration assays showed that after knockout of Ku70 gene,the proliferation and migration of HeLa cells were attenuated.qPCR results indicated that among the five kinds of miRNAs we chose,the levels of three miRNAs(hsamiR-649,hsa-miR-544a,hsa-miR-562)were increased in Ku70 knockout cells than those in the wide type cells,while the level of hsa-miR-548a and hsa-miR-492 had no obvious changes.Conclusion:Ku70 gene knockout HeLa was obtained by using CRISPR/Cas9 technology.Ku70 protein appears to have effects on proliferation and migration of Hela cells.Moreover,it appeared that Ku70 protein could regulate expression of some miRNAs.