外泌体miR-212 促进人iPSCs定向分化为β细胞的分子机制

Type I diabetes mellitus is caused by the autoimmune destruction of β cells in pancreatic islets. While replacing pancreatic β cells would be the ideal solution for this therapeutic problem, but there are two difficult issues for clinical application, including donor source deficiency and long-time using Immunosuppressive drugs, the generation of β cells from stem cells is an attractive alternative. Although development of induced pluripotent stem cells (iPSCs) will cast a new light on above-mentioned problems, but also confront with numerous difficulties, especially formation effect of β cells and its unclear differentiation mechanism. In our previously research, β cells were induced formation from iPSCs using exosome incubation derived from cultured medium of mature human β cells, and illuminated function of exosomal miRNA in β cells formation. We found that exosomal miR-212 play an important role in formation of β cells from iPSCs, however, the mechanism of exosomal miR-212 on promoting formation of β cells remain poorly understood. To elucidate the mechanism of transcriptional regulation during the formation of β cells and to better understand the role of exosomal miR-212 in the differentiation of β cells from iPSCs, in the current study, (1) we will describe the mRNA and protein expression profile in iPSCs after exosomal miR-212 over-expression, and then screen targets of exosomal miR-212 in iPSCs. (2) Potential target genes of exosomal miR-212 were selected in differentially expressed genes, identified its targets using luciferase reporter assay and regulatory relationship of genes. (3) To elucidate the mechanism of its targets and downstream genes during the formation of β cells from iPSCs, RNAi/GRISP-Cas 9 will be used to knock down/out these genes in iPSCs to investigate their effects on β cells formation. Additional studies focusing on exosomal-miRNAs involved in the formation of β cells from iPSCs may advance the development of effective cell transplant therapies for diabetes mellitus.

胰岛移植是治疗I 型糖尿病的希望，面临供体来源和免疫抑制药物长期使用两大难题。iPSCs技术为上述问题带来新的曙光，但受到β细胞分化效率低及分化机制不清晰等瓶颈制约。本项目前期利用β细胞外泌体诱导人iPSCs定向分化为β细胞，并研究外泌体miRNAs发挥的功能，发现外泌体miR-212 在iPSCs定向分化中起重要作用，其可促进iPSCs向β细胞分化，但其调控机制尚未清晰。针对上述问题，本项目重点研究：(1) miR-212在iPSCs定向分化中如何影响mRNA及蛋白表达的变化，筛选miR-212的靶基因；(2)根据靶基因初步筛选结果，明确miR-212的靶基因；(3)针对靶基因及互作下游基因，验证miR-212靶基因及下游基因在iPSCs定向分化的分子功能，最终阐明miR-212促进iPSCs分化的分子机制，为患者自体细胞移植提供技术支持与理论依据。

胰岛移植是治疗I型糖尿病的希望，面临供体来源和免疫抑制药物长期使用两大难题。iPSCs技术为上述问题带来新的曙光，但受到β细胞分化效率低及分化机制不清晰等瓶颈制约。本项发现外泌体miR-212/132在iPSCs程序化分化为β细胞的过程中，抑制靶基因FBW7的表达，FBW7可与内分泌特异基因NGN3直接结合形成泛素降解复合物，使NGN3经泛素化途径降解，外泌体miR-212/132在iPSCs化分化为β细胞的第四阶段通过抑制FBW7表达，稳定细胞中NGN3蛋白表达，促进分化的细胞向内分泌细胞分化。miR-212/132在正常胰岛β细胞中具有维持和增强Insulin的分泌功能，而在不同分化阶段miR-212/132的表达在第四阶段显著增加，表明内源性miR-212/132在该阶段进行转录表达，实验证明，胰岛特异转录因子PDX1可与miR-212/132的启动子结合促进其转录，同时，NGN3可与PDX1互做结合形成复合物促进其转录功能，通过上述研究证明外泌体miR-212/132在iPSCs程序化分化为β细胞中的分子机制。在研究其他干细胞向胰岛β细胞分化过程中，发现Active A在间充质干细胞向β细胞分化的第3阶段，通过增加Smad2、Smad3磷酸化水平，促进Smad4的核表达量，激活TGF-β/Smad信号通路，Smad4入核后启动内分泌特异转录因子NGN3和特异miRNAs(miR-375和miR-26a)的转录表达，促进间充质干细胞向胰岛β细胞分化，在分化过程中，加入TGF-β/Smad信号通路抑制剂SB-431542，反向证明了Active A激活TGF-β/Smad信号通路促进间充质干细胞向胰岛β细胞分化，通过本项目研究，为胰岛细胞移植面临的供体来源不足提供了材料支撑与理论依据。