Research On The Function And Mechanism Of UTF1 Gene In Regulating The Proliferation Of Human Spermatogonial Stem Cells

Objective To explore the role of UTF1 gene in regulating human spermatogonial stem cell proliferation and its mechanism.Methods In this study,human SSCs were amplified by an in vitro culture system,and then the SSCs cultured in vitro were transfected with lentivirus.UTF1 gene expression in spermatogonial stem cell lines was up-regulated and down-regulated by lentiviral vectors.UTF1 gene expression was detected by Western blot and fluorescent quantitative PCR,CCK8 cell viability and cell proliferation assay and EDU cell cycle assay were used to detect the viability and proliferation of spermatogonial stem cells in each group.Finally,RNA-seq sequencing analysis was used to determine the important signal pathways of UTF1 gene regulating SSCs proliferation,and Western blot and RT-PCR techniques were used to verify the differentially expressed genes screened.Results In this study,we isolated CD90 + / CD49 f + SSC in adult testis tissues by immunomagnetic bead method.Through in vitro culture,we observed that SSCs rapidly proliferated into colonies and expressed undifferentiated SSCs markers such as CD49 f,CD90,UTF1,and PLZF.The positive expression rate was >95%,and the positive rate of CD117 of differentiated SSCs markers was <1%.After interference of UTF1 gene expression,it was found that SSCs in the UTF1 gene overexpression group proliferated actively,SSCs in the UTF1 gene suppression group were inhibited from proliferating,and cell cycle tests revealed that human SSCs showed G1 phase arrest after UTF1 gene expression was down-regulated.A total of 26734 genes were identified by RNA-seq,of which 2651 genes were up-regulated and 2810 genes were down-regulated.KEGG and GSEA analysis showed that a large number of gene expressions in the Cell cycle pathway changed significantly.Western blot and RT-PCR techniques further confirmed this result.Conclusion UTF1 is a key regulatory gene for human SSCs proliferation by regulating cell cycle(HSA04110)pathway gene expression and then regulating human SSCs proliferation.