Effect of prmt3 knockout on A549 cell line using CRISPR/Cas9
10.7644/j.issn.1674-9960.2018.02.009
- VernacularTitle:CRISPR/Cas9介导的prmt3基因敲除对A549细胞的影响研究
- Author:
Yue ZHU
1
;
Chang-Min PENG
;
Ya-Li CHEN
;
Xiao-Ming YANG
;
Hua-Dong PEI
Author Information
1. 安徽医科大学研究生院
- Keywords:
PRMT3;
CRISPR/Cas9;
non-small cell lung cancer;
cell proliferation
- From:
Military Medical Sciences
2018;42(2):119-123,157
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct plasmids for knock-out of protein arginine methyltransferase 3 (prmt3) gene using CRISPR/Cas9 gene editing method and examine the effect of prmt3 knockout on the proliferation of human non-small cell lung cancer(NSCLC)A549 cells.Methods Synthesized sgRNA oligos targeting prmt3 gene were cloned into LentiCRISPR vector and positive constructs confirmed by sequencing later .After infection with the packaged virus , A549 cells were screened with puromycin , and then the single clones were isolated .The protein level of PRMT3 in individual cell clones was analyzed with Western blot . Biological assay of clone formation , wound healing , flow cytometry assay and mass spectrometry ( MS) analysis were used to compare cellular proliferation behavior changes between control cells and cells with prmt3 gene knockout .Results The LentiCRISPR plasmids targeting prmt3 gene were confirmed by sequencing , and the PRMT3 protein level was significantly decreased in PRMT 3 KO cells compared with control cells .Depletion of PRMT3 promoted cell proliferation and led to cell cycle arrest at G 2/M phase, but had no influence on cell migration .Besides, some PRMT3 substrate candidates were identified with mass spectrum assays .Conclusion A549 cells with prmt3 gene knockout based on CRISPR/Cas9 are successfully established .PRMT3 can regulate cell cycle and proliferation .
