Construction And Growth Of SPATA20^-/- Triple Negative Breast Cancer Cell Lines By Dual SgRNA/Cas9

Objectives: The purpose of this study was to explore the expression level of Spermatogenesis associated 20(SPATA20)in breast cancer cells MDA-MB-468 and its effects on the proliferation,migration and invasion of breast cancer cells.To explore whether SPATA20 can be a new target for targeted therapy of breast cancer.Methods:1.Use small guide RNA(sg RNA)to design a website online and obtain sg RNA sequences targeting the human SPATA20 gene.2.The sg RNA screened from the web sites were synthesized in vitro and the knockout efficiency of sg RNA was verified by in vitro enzyme digestion system.3.SPATA20 knockout vector was constructed,lentivirus was packaged,and human embryonic kidney cells HEK293 T were infected.The feasibility of the knockout scheme and identification scheme was determined through monoclonal selection and PCR identification.4.SPATA20 was knocked out in MDA-MB-468 breast cancer cells using a stable double sg RNA knockout protocol and the single clone of the knockout cell line was screened.5.Protein western blotting was used to detect the expression level of SPATA20 protein in MDA-MB-468 breast cancer cells with SPATA20 protein knockout andthe control group.6.The effect of SPATA20 knockdown on the proliferation of breast cancer cell MDA-MB-468 was detected by cell proliferation assay.7.The effect of SPATA20 knockdown on the migration and invasion of breast cancer MDA-MB-468 cells was detected by Transwell assay.8.To explore the potential mechanism of SPATA20 affecting the proliferation,migration and invasion of breast cancer cell MDA-MB-468 using differential transcription profiling technology.Results: 1.Efficient sg RNA sequences sg RNA #98 and sgRNA#258 were obtained.2.The double sg RNA lentivirus transfection system and the identification system of single clone of knockout cell line were established,and the human embryonic kidney cell HEK293 T with SPATA20 knockout was successfully constructed.3.SPATA20 was successfully knocked out in breast cancer cell MDAMB-468.4.The proliferation,migration and invasion ability of MDA-MB-468 breast cancer cells with SPATA20 knocked out were significantly reduced compared with the control group.5.After differential expression gene(DEGs)analysis,14 significantly up-regulated genes(KRT17,IFIT1,IFIT2,CCL5,OASL,INA,DSTN,IFIT3,GINS1,MAL2,ACSS1,DPYSL3,PYGB,PCDH10)were screened out in SPATA20 knockdown breast cancer cells and one significantly down-regulated gene(SCGB3A2).6.Differentially expressed genes(DEGs)were enriched.GO enrichment analysis showed that they were mainly enriched in histone modification,regulation of DNA damage stress response,peptidyl-lysine modification,autophagy regulation,Golgi vesicles transport,etc.The results of KEGG enrichment analysis showed that the main enrichment factors were cell cycle,ubiquitin-mediated proteolysis,cell senescence,adhesion plaques,and axon orientation.Conclusions: 1.Knockout of SPATA20 inhibits the proliferation,migration and invasion of MDA-MB-468 breast cancer cells,suggesting that SPATA20 may promote the occurrence and development of breast cancer.2.The enrichment of differentially expressed genes related to SPATA20 in the regulation of DNA damage stress response suggests that SPATA20 may be involved in the regulation of DNA damage stress response of breast cancer cells.3.The related differentially expressed genes of SPATA20 are enriched in the cell cycle,suggesting that SPATA20 may play a role in promoting the migration and invasion of breast cancer cells.