单极纺锤体1结合蛋白2基因敲除对人肝癌SMMC-7721细胞迁移的影响

Construction of monopolar spindle-one-binder 2 knock out hepatocellular carcinoma cell line SMMC-7721 and the effects of expression on Migration

目的运用规律成簇的间隔短回文重复(CRISPR)/Cas9基因编辑技术构建敲除人单极纺锤体1结合蛋白2 (hMOB2)的肝癌SMMC-7721细胞,观察hMOB2基因敲除对其迁移的影响.方法首先将靶向hMOB2基因的导向RNA (sgRNA)的CRISPR序列克隆到慢病毒载体lentiCRISPRv2中,并将制备的慢病毒颗粒感染肝癌SMMC-7721细胞,然后使用嘌呤霉素筛选并挑取单克隆进行培养,用蛋白质印迹法(Western blot)方法检测hMOB2基因敲除效果,利用Transwell、细胞划痕迁移实验检测敲除hMOB2基因对SMMC-7721细胞迁移的影响,运用GraphPad Prism 8.0统计软件分析,计量资料以均值±标准差(Mean±SD)表示,组间比较采用t检验.结果成功构建了靶向hMOB2基因的慢病毒介导的CRISPR-Cas9编辑/敲除系统,筛选、建立了hMOB2基因稳定敲除的SMMC-7721细胞株,Transwell迁移实验显示,与对照组比较,敲除组中挑选的单克隆sgR-1细胞的迁移数为(224.3±6.3)个,sgR-2细胞的迁移数为(166.3±6.5)个,明显高于对照组[(114.3±10.5)个,t=16.680、9.714,P<0.01],细胞划痕实验结果显示,与对照组比较,sgR-1相对愈合面积为1.61±0.03(t =24.610,P<0.01),sgR-2为1.26 ±0.04(t=8.081,P<0.01).结论敲除hMOB2基因可促进人肝癌SMMC-7721细胞迁移.

Objective To construct hepatocellular carcinoma cell line SMMC-7721 with knock out of human monopolar spindle-one-binder 2 (hMOB2) gene using lentivirus-mediated clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR-Cas9) editing/knockout system and to investigate the effect of its expression on migration. Methods Synthesized CRISPR sequences specific targeting hMOB2 gene (sgRNA) were cloned into the lentiviral vector lentiCRISPRv2, and the lentiviruses were produced. SMMC-7721 cells were infected with indicated lentiviruses and screened by using puromycin, and stably transduced monoclone cell culture was selected. Western blotting analysis was used to detect the knockout efficiency of hMOB2, Transwell cell migration and wound healing assay were also performed to evalute the effect of hMOB2 knockout on the migration of SMMC-7721 cells. Using GraphPad 8.0 statistical software analysis, the measurement data were expressed as mean±standard deviation (Mean±SD), and t-test was used for comparison between groups. Results Lentivirus-mediated CRISPR-Cas9 editing/knockout system for targeting hMOB2 was successfully constructed, and the SMMC-7721 cell line with stable knockout of hMOB2 was screened and established. Data from Transwell cell migration assay showed that the number of migrating cells in sgR-1 and sgR-2 group were 224.3±6.3 and 166.3±6.5, significantly higher than that in control group (114.3±10.5, t1=16.680, t2=9.714, P<0.01). Wound healing assay demonstrated that the relative healing area of sgR-1 was 1.61±0.03 (t=24.610, P<0.01), and sgR-2 was 1.26±0.04 (t=8.081, P<0.01) comparing to the control group. Conclusion The migration of the SMMC-7721 cell line with stable knockout of hMOB2 gene was promoted by lentivirus-mediated CRISPR-Cas9 editing/knockout system.