linc00152/miR-103/p21细胞周期信号轴在人胚胎干细胞造血分化过程中的机制研究

The exploration of hematopoietic differentiation of human embryonic stem cell (hESC) is the hotspot. Hematopoietic differentiation of hESC is the dynamic transformation between the self-renewal and differentiation of stem cell. Cell cycle not only regulates proliferation in self-renewal process, but also the differentiation of stem cell. Long non-coding RNA (LncRNA) has been reported in the regulation of ESC function, which the competitive binding of miRNA to regulate target genes is the main mechanism. Our previous report indicated differentially encoded genes in hESC hematopoietic differentiation were majorly enriched in cell cycle, indicated the critical roles in dynamic regulation. Among the non-coding genes, linc00152 levels were consistently up-regulated. The preliminary data indicated linc00152 significantly promoted ESC hematopoiesis in vitro, and upregulated p21 to block hESC into G0/G1 phase in cell cycle. Besides, competitively binding between linc00152 and miR-103 was observed in hESC. Therefore, we speculated that linc00152 regulates hematopoietic differentiation of hESC via cell cycle, by the miR-103/p21 signal pathway. In the current project, RIP, pull-down, and luciferase technology will be applied to confirm the linc00152/miR-103/p21 pathway. Additionally, our hypothesis about hESC hematopoietic differentiation by regulating cell cycle will be verified by the directional hematopoietic differentiation experiment.

探索人胚胎干细胞（hESC）造血分化调控机制是当前研究热点。hESC造血过程是干细胞自我更新与分化的动态转变。而细胞周期调控自我更新的同时，还可能参与细胞分化。已有报道发现长链非编码(LncRNA)参与hESC生物学功能调控，其主要机制是与miRNA竞争性结合调控靶基因。我们前期对hESC造血分化过程中编码差异基因进行富集分析，发现细胞周期是最重要的生物学过程。而在非编码基因中，发现linc00152水平持续上调。体外实验进一步证实linc00152可以促进hESC造血，并调控p21使其阻滞于G0/G1期。此外，linc00152还可能与miR-103存在竞争性结合。因此，我们猜测linc00152通过下游miR-103/p21信号轴调控细胞周期影响hESC造血分化。本项目拟利用RIP、Pull-down、荧光素酶技术等验证该调控假设，以丰富hESC造血分化的机制理论。

利用人胚胎干细胞(hESC)多向分化能力在体外进行组织重建是当前临床转化医学的热点内容。近年来大量实验数据证实lncRNA可以在多个层面参与基因表达调节。该项目探索了长链非编码RNA-Linc00152在hESC造血分化过程中的作用及机制。本研究前期对hESC造血分化过程中编码差异基因进行富集分析,发现在非编码基因中,linc00152水平持续上调。本项目围绕Linc00152在人胚胎干细胞向造血干细胞分化发育中的功能及机制研究形成总体框架，利用基因编辑技术获取敲除（或诱导）linc00152表达的hPSC亚系，通过免疫荧光、Q-PCR、和WB均证实linc00152可以抑制ESC干性标记表达。流式细胞仪、Q-PCR、和WB证实linc00152阻断细胞周期。免疫荧光、Q-PCR、和体外分化实验表明linc00152促进中胚层发育，加速造血形成。核质分离实验证实在ESC中，linc00152主要定位于细胞质。Q-PCR检测发现干预linc00152可负向影响miR-103表达。瞬时转染miR-103 mimics，反向检测linc00152变化情况，进一步证实miR-103可能是linc00152作用miRNA。本项研究通过高通量基因芯片检测，筛选得到的linc00152进行功能研究证实定位于ESC胞质中的linc00152可抑制干性特征，促进造血分化。具体表现为诱导中胚层发育，加速造血形成，而抑制血管内皮分化。对阐明血细胞发育过程中的分子机制、进一步推动ESC的临床转化应用具有重要意义。