Regulation of miR-375 on hepatocyte nuclear factor 1β during differentiation of human embryonic stem cells into insulin producing cells

Abstract

Objective To investigate the effect and underlying mechanism of miR-375 on regulating differentiation of human embryonic stem (hES) cells into insulin producing cells (IPCs). Methods hES cells were induced into IPCs in vitro. The dynamic expression of miR-375 and hepatocyte nuclear factor 1β (HNF-1β) the predicted target gene, during the differentiation was analyzed. The stage 2-cells during the differentiation infected with miR-375-overexpression lentiviral vector were divided into 3 groups: control group, GFP control group and miR-375-overexpression group. The expressions of miR-375, HNF-1β and the specific markers related to pancreatic islet differentiation or function were determined by realtime PCR and/or Western blotting. Independent sample t test was used to analyze the difference between two group. ANOVA and Q test were used to analyze the difference among multiple groups. Results miR-375 expression in the stage 1-to 5-cells were 100%, (472.25±33.53)%, (768.00±25.65)%, (54.25±5.74)% and (30.75±5.70)%, respectively (F=1137.57, P 0.28), whereas the protein level of HNF-1β decreased to (38.75±9.22)% of the GFP group in the miR-375-overexpression group (F=60.69, P<0.001). miR-375 overexpression upregulated the expression of pancreatic and duodenal homeobox 1 (Pdx-1) in stage 4-cells (F=412.15, P<0.001), and downregulated the expression of the pancreatic islet specific markers, e.g. Nkx6.1, paired box 4 (Pax-4) and insulin in the stage 5-cells (F=105.19-484.05, all P<0.001) when compared with the GFP group. Conclusion miR-375 plays a modulatory role in the IPC differentiation from hES cells. That may be mediated via regulating the expression of the transcription factor HNF-1β. Key words: Pancreatic islet; Human embryonic stem cell; Hepatocyte nuclear factor 1β; miR-375