miR-802通过Fzd5-Creb1-Sox9信号轴调控小鼠胰腺祖细胞增殖及分化OA

Objective To investigate the specific role and potential mechanisms of miR-802 in the maintenance of stemness and the regulation of proliferation in mouse pancreatic progenitor cells(PPCs).Methods A three-dimensional semi-solid culture system based on matrigel and methylcellulose was employed to isolate and culture PPCs from mouse pancreatic ductal tissues.The biological effects of miR-802 were systematically studied using lentivirus-mediated overexpression and knockdown strategies.Immunofluores-cence was used to detect the expression levels of the stemness gene Pdx1 and the proliferation marker Ki67 in PPCs.Flow cytometry was employed to analyze the proportion of CD133-positive cells.Quantitative real-time PCR was used to detect the expressions of stem-ness-,proliferation-,and endocrine differentiation-related genes(Pdx1,Ki67,CD133,Ins1,Ins2,Ngn3,NeuroD1).Western blot was used to detect the expression levels of Fzd5,phosphorylated Creb1(p-Creb1),Creb1,and Sox9 proteins.Results Knockdown of miR-802 significantly enhanced the single-cell clonal colony formation ability of PPCs(P<0.01),increased the colony diameter(P<0.05),promoted the cell proliferation(P<0.001),upregulated the expressions of stemness genes Pdx1 and CD133(P<0.001),and downregulated the expressions of endocrine differentiation genes Ngn3 and NeuroD1(P<0.05).Conversely,the overexpression of miR-802 significantly inhibited single-cell clonal colony formation(P<0.01),reduced the colony diameter(P<0.05),suppressed the cell proliferation(P<0.01),downregulated the expressions of stemness genes Pdx1 and CD133(P<0.01),and upregulated the expressions of endocrine differentiation genes Ins1,Ins2,Ngn3,and NeuroD1(P<0.01).Moreover,the proportion of CD133/Ki67 double-positive cells was significantly increased in the pancreatic tissues of miR-802 knockout mice(P<0.001).Further studies revealed that knock-down of miR-802 upregulated Fzd5 expression(P<0.05),promoted the phosphorylation level of Creb1(P<0.01),and enhanced Sox9 expression(P<0.001).In contrast,the overexpression of miR-802 downregulated Fzd5 expression(P<0.001),inhibited the phosphory-lation level of Creb1(P<0.001),and suppressed Sox9 expression(P<0.001).Conclusion miR-802 regulates the balance of proli-feration and differentiation in PPCs by modulating the Fzd5-Creb1-Sox9 signaling axis,thereby promoting the expression of stemness genes and inhibiting the expression of endocrine differentiation genes in PPCs.