Lin28调节小鼠iPS细胞向诱导性PGCs分化及机制探讨

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.

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是体细胞重编程的四个重要调控因子之一，又是原始生殖细胞PGCs分化的调控因子，同时又具有癌基因功能，是内质网相关蛋白翻译的总抑制剂，对肿瘤发生发展起着重要作用。干细胞和肿瘤细胞二者之间存在相似性，因此以LIN28A、MSI1和LAMP1为切入点，进行干细胞和肿瘤细胞的平行研究。发现小鼠iPS细胞IP14D-1向PGCs分化过程中，Lin28对Blimp1具有正向调节作用，Msi1并没有协同Lin28对Blimp1进行调节，但却促进Lin28对let-7g的表达抑制。即Msi1协同Lin28增强了对let-7g的抑制作用，但并没有加强Lin28对Blimp1的正向调控作用。LIN28A对膀胱癌细胞和胚胎干细胞的调控方面具有相似性，LIN28A和MSI1分别敲减或者联合敲减均不影响LAMP1的mRNA水平，但对LAMP1蛋白起抑制作用，MSI1没有协同LIN28A调控LAMP1，LAMP1对癌细胞迁移起促进作用。总之，对干细胞和癌细胞同时开展了LIN28的相关研究，并发现二者的确存在相似性，这对深入理解干细胞的干性和癌细胞恶性转化之间的联系，具有重要的借鉴意义，并为探讨其机制提供新思路。