A Study On The Construction Of Stable CACYBP/SIP Gene Knockout MCF-7 Strain And The Influences On Its Phenotype

Object Using gene editing technology(crispr/cas9)building CACYBP/SIP gene knockout of a single clone of human breast cancer cell line mcf-7,to investigate the effect of CACYBP/SIP gene deletion on phenotype of mcf-7 cells.In order to clear the possible role and mechanism of CACYBP/SIP in breast cancer.Methods Target CACYBP/SIP sgRNA gene was deigned and the plasmid of Lenti-CAS9-sgRNA-puro was constructed.Then the plasmid was transfere d into MCF-7 cells by electroporation.Through screening of puromycin resistance,detection of sgRNA activity by Cruiser?,and sequencing of CACYBP/SIP gene knockout cells,we had gained the stable gene defect cell line.Next,we detected the CACYBP/SIP gene mRNA expression by Real time PCR and CACYBP/SIP protein expression by Western-blot to verify these cells.To investigate these cells phenotypic change,cell proliferation was detected by Brdu and MTT kit,cell viability was detected by plate colony forming assay,and cell apoptosis was detected by flow cytometry.Statistical analysis was performed by using independent student T test.Results By constructing crispr/cas9 gene knockout technology,we succeeded with the breast cancer cell line MCF-7 CACYBP/SIP knockout monoclonal strains.Real time PCR and Western blot results showed that the expression of CACYBP/SIP mRNA was zero and the CACYBP/SIP protein was not expressed.Further on phenotype detection of CACYBP/SIP deficient MCF-7 cell lines,MTT results showed that the knockout cell proliferation ability decreased significantly(p<0.05);Brdu results showed that the knockout strain of cell proliferation decreased significantly(p<0.05);experimental results show that the clone deletion mutant clone formation the number was significantly decreased(p<0.05);the results of apoptosis showed that knockout strains significantly increased the percentage of apoptosis(p<0.05).Conclusion This study successfully constructed CACYBP/SIP gene knockout vector,and obtained MCF-7 breast cancer cell line CACYBP/SIP gene kknockout monoclonal strains,the phenotype analysis showed that cell proliferation and colony formation ability decreased,the apoptosis rate increased,suggesting that CACYBP/SIP gene may play an active role in promoting cancer in breast cancer.