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

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. Key words: Hepatocellular carcinoma; Human monopolar spindle-one-binder 2; Clustered regularly interspaced short palindromic repeats/Cas9; Migration