Screening Of Key Targets In Response To TGF-β1in Mouse GC-1Spg Cell Line

Male infertility has been a worldwide medical challenge and is closely related to abnormal spermatogenesis. TGF-β1(transforming growth factor β, TGF-β), one of the TGF-β superfamily members, plays different roles at different stages of testicular development and involves in the regulation of spermatogenesis. Therefore, it will better interpret the mechanism of spermatogenesis to find and identify key molecules involved in TGF-β1signaling pathway during germ cell development. Based on these, the mouse spermatogonial epithelial cells GC-1spg was used as experimental material with exogenous TGF-β1stimulation, and then the following three aspects were carried out:By high-throughput microRNA(miRNA) microarray analysis and quantitative PCR, twenty-four miRNA molecules regulated by TGF-β1were screened and then10miRNAs with high expression abundance were selected to verify the results of miRNA microarray. Through bioinformatic analysis and quantitative PCR technology, we initially verified that Hbpl may be the target gene of miR-5112, and Celsr2may be the target gene of miR-199a-3p, and Nr6al may be the target gene of miR-196a.Through high-throughput2-DE (two-dimensional gel electrophoresi s,2-DE) and MALDI-TOF-TOF-MS,eight differentially expressed protei ns regulated by TGF-β1were identified in GC-1spg cells. They are Gelsolin(Gsn),Vinculin(Vcl),Ezrin(Ezr),NDKB(Nme2),PPIA(Ppia),Cofilin-1(Cf11),TERA(Vcp)and IF5A1(Eif5a),and their expression were down-re gulated with TGF-β1treatment.Then the GEO analysis and t-test were performed and found that the target genes of some miRNAs-Nr6a1,Celsr2,Hbp1and the screened proteins-NDKB,COF1,PPIA were differentially expressed in teratozoos permia and seminoma, suggesting that they may involve in spermatoge nesis process.It is noteworthy that further study showed that the down-regulated protein NDKB could be a direct target of the up-regulated miR-199a-3p; and the down-regulated protein PPIA could be the direct target of the up-regulated miR-196a and miR-149. Their expression show an opposite changes in response to TGF-β1. The observation may suggest that these three miRNAs may play the role in spermatogenesis via inhibiting the expression of NDKB, PPIA and other proteins respectively.In conclusion, we screened and verified10differentially expressed miRNA and8differentially expressed proteins in GC-1spg cell lines and found that TGF-β1could inhibit the target NDKB or PPIA via inducing up-regulation of miR-199a-3p,miR-196a and miR-149,and thus participate in spermatogenesis.