PLZF Up-regulatiion CXCR4 To Promote Dairy Goat Male Germline Stem Cells Proliferation Via Targeting MiR-146a

Differentiation and self-renewal of SSCs is critical for spermatogenesis.The differentiation and self-renewal of SSCs is a strictly regulated event, making the study of this significant for spermatogenesis. It has been reported that PLZF, CXCR4, and miR-146 a are all associated with SSC self-renewal. In this study, we isolated and purified gSSCs to the interaction of PLZF, miR-146 a, and CXCR4. Our results demonstrated that PLZF promoted CXCR4 expression through inhibiting transcription of mi R-146 a, and this event would enhance ERK1/2 phosphorylation, thereby facilitating the self-renewal and proliferation of gSSCs.1. The expression pattern of PLZF, miR-146 a and CXCR4 in dairy goat testisAnalyzed by qRT-PCR, we found the expression pattern of PLZF and CXCR4 in 3, 6, 9, 12, 18 mouth old dairy goat testis was basically the same, but the transcription of miR-146 a was contrary. Meanwhile we identified the expression and location of CXCR4 in testis by immunofluorescent staining, it show that the mRNA and protein level of CXCR4 expression pattern was similar, the CXCR4 and PLZF was co-expression in the gSSCs2. The interaction between PLZF, miR-146 a and CXCR4.We analyzed the expression of CXCR4 and miR-146 a by qRT-PCR and Western Blot in gSSCs was overexpressed or inhibited PLZF. The result show that overexpressed PLZF promoted the expression of CXCR4 but decreased the transcription of miR-146 a, but it was contrary in inhibiting PLZF expression. According to the luciferase reporter assay, we found the PLZF was binding to the promoter region of miR-146 a and suppressed transcription. Overexpression miR-146 a in gSSCs can decrease the expression of CXCR4 analyzed by qRT-PCR and Western. Unexpectedly, miR-146 a can not bind to the 3’UTR region of CXCR4 according to the dual-luciferase reporter assay.3. The effect of PLZF, miR-146 a and CXCR4 on gSSCs self-renew and proliferationWe identified the phosphorylation level of ERK1/2, the result showed that increased expression of PLZF and CXCR4 can promote the phosphorylation of ERK1/2, but decreased PLZF expression was contrary. Used the FCM to analyze the cell cycle, we found that PLZF and CXCR4 can enhance the proliferation of gSSCs.