The Proliferation And Apoptosis Effects Of Knock-out BLM Gene On Human Triple-negative Breast Cancer MDA-MB-231 Cells By CRISPR/Cas9 Technology

Objective:To knock out BLM gene in human breast cancer MDA-MB-231 cell line by using CRISPR/Cas9 genome engineering technology.And to further explore the effect of knock-out BLM gene on human breast cancer MDA-MB-231 cell.Methods:1.According to CRISPR/Cas9 technology targets design principles,two pairs of the small guide RNA(gRNA)target in 3 exons of BLM were designed,and the recombinant PX459 V2-gRNA plasmid was constructed by the PX459 V2 vectors.2.The MDA-MB-231 cells transfected with the recombinant plasmid were selected by puromycin to screen the positive clone cells.The targeting efficiency was detectd by Cruiser~TMM endonuclease,the high targeting efficiency positive clone was cultured,further the BLM knock-out MDA-MB-231 cell was detectd by western blot.Off-target effects was detected by Cruiser~TMM endonuclease.3.The proliferation and apoptosis of the BLM knock-out breast cancer cell line was detected by MTT,Hoechst 33258,FCM method.Results:1.Two gRNA target sites were designed on exon3,and the recombinant plasmid was successfully constructed.2.The targeting efficiency of gRNA2 was higher than other by Cruiser~TMM endonuclease and A strain of BLM protein significantly decreased cell lines was obtained and the BLM-KO cells is not off-target by Cruiser~TMM endonuclease.3.the cell proliferation rate of the BLM knock-out breast cancer cell line was inhibited by MTT method and the cells apoptosis was induced by Hoechst 33258 and FCM method.Conclusion:The BLM knock-out breast cancer MDA-MB-231 cell line had been successfully constructed by using CRISPR/Cas9 system,and the proliferation rate of the BLM knock-out breast cancer cell line was inhibited and the apoptosis was induced,which lays the foundation for further study of the mechanism and founction of BLM in breast cancer.