Regulation of miR-375 on hepatocyte nuclear factor 1βduring differentiation of human embryonicstem cells into insulin producing cells

目的 探讨miR-375对人胚胎干细胞(hESC)定向分化为胰岛素分泌细胞(IPCs)的调控作用及其潜在机制.方法 体外诱导hESC定向分化为IPCs,分析miR-375与其预测靶基因肝细胞核因子1β(HNF-1β)之间表达水平的动态变化关系;以miR-375过表达的慢病毒载体感染分化第二阶段细胞,实验分为空白对照组、阴性对照(GFP)组及miR-375过表达组;应用实时定量PCR和(或) Western blotting法检测各组分化细胞中miR-375、HNF-1β、胰岛细胞分化或功能相关的关键基因的表达.两组间比较用t检验,多组间比较用单因素方差分析和Q检验.结果 hESC分化五个阶段中miR-375表达水平呈先高后低的变化趋势,相对表达量分别为100%、(472.25 ± 33.53)%、(768.00 ± 25.65)%、(54.25±5.74)%、(30.75±5.70)% (F=1137.57,P<0.001).HNF-1β表达水平在分化第三阶段开始明显上升,在分化第四阶段达到高峰[分别为(279.50 ± 21.30)%、(645.00 ± 64.55)%,F=224.86,P<0.001],变化趋势与1/miR-375的变化趋势相似.慢病毒感染后,miR-375过表达组的HNF-1βmRNA表达水平与GFP组、空白对照组无显著差异(F=1.467,P>0.05),而miR-375过表达组的HNF-1β蛋白表达水平则下降至GFP组的(38.75±9.22)% (F=60.69,P<0.001).与GFP组相比,miR-375过表达组分化第四阶段细胞的胰十二指肠同源盒因子1(Pdx-1)表达水平显著升高,分化第五阶段细胞(IPCs)的Nkx6.1、配对盒因子4(Pax-4)及胰岛素的表达水平则显著降低(F=105.19~484.05,均P<0.001).结论 miR-375对hESC向IPCs定向分化具有重要的调控作用,该作用可能是通过影响转录因子HNF-1β表达而实现的.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-1βand 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<0.001). The expression of HNF-1βincreased from stage 3 ((279.50 ± 21.30)%), and peaked at stage 4 ((645.00 ± 64.55)%,F=224.86, P<0.001). The dynamic level of HNF-1βwas similar as that of 1/miR-375. After the lentivirus infection, the mRNA level of HNF-1βin miR-375-overexpression group was not statistically different from that of both the GFP group and control group (F=1.467, P>0.28), whereas the protein level of HNF-1βdecreased 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.国家自然科学基金资助项目; 教育部博士点新教师基金资助项目中文核心期刊要目总览(PKU)中国科技核心期刊(ISTIC)中国科学引文数据库(CSCD)12730-734