MiR-544Regulates The Dairy Goat Male Germline Stem Cells’ Self-renewal Through Targeting To PLZF

The initiation of mammal spermatogenesis is depending on the proliferation anddifferentiation of male-germ line stem cells （mGSCs）. mGSCs’ self-renewal ability is thebasis for the Reproduction of mammal. The proliferation and differentiation of mGSCs arethe biological processes that under Precise control and studies on factors that regulatingmGSCs’ self-renewal could be helpful for us to gain deeper insight into the mammalspermatogenesis. Prom-yelocytic leukaemia zinc finger (PLZF) is a zinc finger protein thatcontaining nine carboxyterminal zinc fingers and an amino termina BTB/POZ domain,which suppresses transcription by binding to specific DNA sequences with the help ofrecruiting co-repressors. Previous researches had showed that PLZF could maintain themGSCs’ self-renewal ability in human and mice, and it could cooperate with otherdifferentiation associated regulators, building a network to regulate the mGSCs. miRNAs’study is one of the current biological research focuses. miRNAs play a critical role in manybiological events, including cell proliferation, differentiation and cell apoptosis. Researchesof miRNAs’ role on their targeted gene transcription and translation regulation are criticalfor the studies of mGSCs’ self-renewal.As one of the18miRNAs in14q32miRNA-clusters, miR-544regulates both cells’proliferation and cell cycle of varies cancers, and it could be treated as a diagnose marker ofboth Acute lymphocytic leukemia and neuroglioma.We focus on the function of PLZF on mGSCs’ self-renewal. We predicted theprospective miRNAs that targeting to PLZF by using the online Bioinformaticsdatabase-Targetscan. The dual-luciferase reconstructed vector ofpsiCHCEKTM-2-PLZF-3’UTR or psiCHCEKTM-2-Mut-PLZF-3’UTR was co-transfectedwith miR-544mimics (miR-544m), miR-544inhibitor(miR-544i) and scrambledoligonucleotides (NC) into mGSCs. We detected expression of genes that related withmGSCs’ self-renewal and differentiation after miRNA transfection, and we found thatmiR-544could promote the differentiation of mGSCs by targeting to PLZF.1. The regulation of PLZF on mGSCs’ self renewalWe detected the PLZF expression in dairy goat testis of30dpp,6M and10M by usingthe immunohistochemistry method, and we found that the highest expression level of PLZF in dairy goat testicular tissue was at6M, following by30dpp,10M. The whole PLZFmRNA sequence of dairy goat was obtained by In silicon cloning, and the recombinantvector (pPLZF-IRES2-EGFP) was constructed. We transfected the PLZF over-expressionvector in mGSCs and then detected the expression of genes that related with mGSCs’self-renewal and differentiation by QRT-PCR and Immunofluorescence methods. We foundthat PLZF could maintain the expression of GFRa1、 VASA、 OCT4and the cells’proliferation ability(higher BRDU expression than control group). On the contrary, thegenes’ expression of GFRa1、VASA、OCT4were lost and proliferation ability declinesignificantly in control group. All the results varied that the PLZF could resist the cells’differentiation which causing by High concentrations of serum, in other words, the role ofPLZF on mGSCs’ self renewal was verified.2. Screening and confirmation of prospective miRNAs that target to PLZFWe got the4prospective miRNAs: miR-544、miR-370、miR-874、miR-378byusing the Targetscan Bioinformative Software, and then miR-544and miR-378picked upby PLZF expression detection after miRNAs transfection in mGSCs. Finally, miR-544wasidentified as the most promising one by dual-luciferase experiment after co-transfectionpsiCHCEKTM-2-PLZF-3’UTR and miR-544or miR-378in mGSCs; the result of miR-544targeting to PLZF directly was confirmed by Western blot and the dual-luciferase afterco-transfection psiCHCEKTM-2-PLZF-3’UTR or psiCHCEKTM-2-Mut-PLZF-3’UTR withmiR-544m and NC.3. The function of miR-544on mGSCs’ self renewalWe detected the morphology changing and apoptosis cells’ rate of mGSCs after miR-544mand miR-544i transfection. The expression of genes that related with mGSCs’ self-renewaland differentiation were detected by QRT-PCR, Western blot and Flow cytometry methods.We find that miR-544over-expression could make mGSCs’ volume become larger andappear more apoptosis cells, and it could inhibit the expression of self-renewal relativegenes like PLZF、CXCR4and GFRa1, while promote the mGSCs’ differentiation relatedgenes KIT, DAZL. The results above show that miR-544could promote the proliferation ofmGSCs by targeting to PLZF.