Lin28调节小鼠iPS细胞向诱导性PGCs分化及机制探讨

Lin28是PGCs分化新调控因子，最新发现RNA结合蛋白Musashi1(Msi1)对Lin28具有协同作用。我们前期结果表明，小鼠iPS细胞IP14D-1和形成的拟胚体(iEBs)具备诱导性原始生殖细胞(induced PGCs, iPGCs)分化潜能，全反式视黄酸(atRA)可能不参与PGCs分化基因Blimp1的调控。因此本项目提出"Lin28为中心，Msi1为辅助，通过抑制let-7miRNA继而影响Blimp1调控小鼠iPS细胞向iPGCs分化"。拟采用IP14D-1和形成iEBs经atRA诱导，根据报告基因和抗体采用FACS方法从iEBs中分离假定iPGCs并鉴定。免疫共沉淀和蛋白定位确定Lin28和Msi1共同参与iPGCs分化，RNAi和超表达后检测Blimp1和let-7表达以及嵌体制备探讨其分化机制。为研究生殖细胞分化、不孕不育以及生殖细胞肿瘤发病机制提供一个新思路。

Lin28 is a novel upstream regulator of PGCs development related to Blimp1. Musashi1(Msi1) is a RNA-binding protein, and Msi1 works in concert with Lin28 to regulate differentiation of mESCs-derived EBs. .In our previous studies, the mouse induced pluripotent stem cells (iPSCs), IP14D-1, and IP14D-1-derived induced embryoid bodies (iEBs) have potency to differentiate into induced primordial germ cells (iPGCs). Meanwhile, Blimp1 expression may be independent of the effect of atRA( all-trans retinoic acid) on PGC differentiation, and atRA may promote the start of a period in which there is a low level of Lin28 expression during PGC differentiation. .This project proposed the hypothesis"Msi1 collaborates Lin28 to regulate the mouse iPSCs differentiating into iPGCs through Blimp1". The iEBs derived from IP14D-1 were treated with all-trans retinoic acid (atRA) for 4d, 7d and 9d. We identifed the reporter gene Oct4-GFP and SSEA1 specific antibody as markers that allow the isolation of putative iPGCs from the 4d, 7d and 9d iEBs. Real-time PCR and Western blot analysis were performed to detect the relative levels of PGC differentiation-associated genes (Lin28, Blimp1, Stra8 and Mvh) and the corresponding proteins, as well as let-7 family microRNA members including let-7b and mir98, respectively. Co-immunoprecipitation(Co-IP), immunofluorescence, RNAi，ectopic expression and chimeras preparation were used to address whether Msi1 is involved in Lin28-mediated regulation of iPS differentiating into iPGCs. To establish the technology system of iPS(IP14D-1)-iPGCs, and to investigate the mechanism of Lin28 serving as a centre factor to regulate the iPGCs specification. In addition, Msi1 cooperates with Lin28 to inhibit let-7 miRNA biogenesis and to direct the downstream factor Blimp1 in the development of iPS(IP14D-1)-iPGCs. These will provide new insights into germ cell formation, development, infertility pathogenesis and cancer therapy.