miR-375在人胚胎干细胞定向分化为beta-细胞过程中的作用及对其成熟的影响

miR-375 is highly evolutionary conserved miRNA which has been cloned from a mouse insulinoma pancreatic β-cell line. MiR-375 is expressed in islet β-cells as well as in non-β-cells of the pancreas, and in the pituitary gland. In human and mouse pancreatic islets, miR-375 appears to be the miRNA with the most robust expression. During pancreas organogenesis, miR-375 exhibits increased expression ocurring together with augmented insulin transcript expression and β-cell proliferation. The contribution of miR-375 in pancreas genesis has first been revealed in zebrafish.Using gain and loss function approach,the authors demonstrated that miR-375 is essential for the formation of the insulin-secreting β-cells. More rencently, mice lacking miR-375(375KO) have been generated. Using this model, Poy et al. demonstrated that deletion of miR-375 infulences not only β-cell mass but also α-cell mass by regulating a cluster of gene controlling cellular growth and proliferation.To sum up, the studies on miR-375 indicate that this miRNA exerts mulitiple and distinct effects, and is involved not only in pancreas development by regulating β-cell and α-cell mass,but also in the safeguard of β-cell physiology. Human embryonic stem cells (hESCs) with the capacity of self-renewal and multilineage differentiation are promosing sources for generation of Pancreatic β-cells for cell replacement therapy in diabetes. Researches showed miR-375 highly expressed in definitive endoderm and decreased at later stages during the process to diferentiating hESCs to β-cells. The insulin producing cells can be generated by only overexpression of miR-375 in hESCs and induced puripotent stem cells (iPSCs). To further figure out the function of miR-375 on formation and maturation of hESCs derived β-cells, we are going to build miR-375 tet-off/on system in INSGFP/w hES cell line. The modified cell line will be used to differentiate to β-cells and miR-375 will be induced at different stage of differentiation by adding doxycyline in the medium. At the end of differentiation, β-cells will be analyzed by flow cytometry and immunofluorescent staining. Furthermore, the obtained INS+GFP+ cells by flow cytometry will be reaggreated to form spheres and induced by doxycylcine to overexpress miR-375. After reculture for 7,15,30 and 45 days. The cells will be used for electrophysiology and insulin secretion analysis. This project will provide a new way to further mature hESC derived β-cells and help scientists to further understend the function of miR-375 in pancreas development.

miR-375是人和小鼠胰岛中表达量最高的一个miRNA,在胰腺的发生过程中,其表达量随着胰岛素转录表达和β-细胞的增殖而升高。人胚胎干细胞和诱导多能干细胞中过表达miR-375,在不添加任何生长因子的情况下,可以形成胰岛素分泌细胞，表明miR-375是潜在治疗糖尿病的重要microRNA。为了深入研究miR-375对胚胎干细胞分化为胰岛细胞过程中的作用，我们将在人INSGFP/w胚胎干细胞的基础上构建miR-375四环素调控系统，通过研究在分化不同阶段过表达miR-375对胰岛素分泌细胞形成的影响，确定其在人类胰岛细胞发生和发育过程中的作用，为去除多激素共表达细胞，获得更多成熟仅表达胰岛素并具有分泌能力的β-细胞提供新思路;通过研究在分化获得的β-细胞中维持miR-375表达水平，对促进β-细胞的增殖成熟和胰岛素分泌能力的影响，为临床上糖尿病的诊断和治疗提供理论依据。

研究表明，miR-375不但在胰腺发育过程中调节β-细胞和 α-细胞的数量，而且在保障β-细胞的功能上都起着重要作用。为了确定miR-375在胚胎干细胞向胰岛素分泌细胞分化中的作用，本研究构建了具有miR-375四环素调控系统（Tet-off/on）的人胚胎干细胞系，优化人胚胎干细胞向胰岛素分化方案，对分化不同时期的hESCs进行脂质体介导的miR-375 mimic转染，发现在分化早期过表达miR-375会提高Foxa2和Cxcr4的相对表达量，在分化中期过表达miR-375会提高Ngn3和Pdx1的相对表达量，在分化末期过表达miR-375会提高Insulin的相对表达量同时降低Glucagon和Somatostatin的相对表达量。并且瞬时过表达miR-375可促进胰岛素的合成和分泌。因此，在hESCs向IPCs分化过程中过表达miR-375可提高胰岛β细胞发育成熟相关基因的表达水平，降低分化末期细胞多激素共表达程度，促进β细胞成熟和胰岛素分泌。