DGCR8调控人成体干细胞稳态和衰老的分子机制研究

Aging, characterized by a progressive degeneration of physiological integrity, is caused by the loss of homeostasis or aging of adult stem cells. Increasing evidence indicated that non-coding RNA plays a vital role in regulating intracellular homeostasis especially during cellular senescence. In addition, DGCR8 as a key component of microprocessor has been shown to be involved into the processing of non-coding RNAs including miRNA, snoRNA and lncRNA, as well as the posttranscriptional regulation and the alternative splicing of mRNAs. Our pilot study found that the levels of DGCR8 expression decreased in aging human mesenchymal stem cells. However, the study to explore the mechanism of DGCR8 in regulating cellular homeostasis and senescence is rare. Therefore, based on two types of human adult stem cells including mesenchymal stem cells and neuronal stem cells, which are directly differentiated from the engineered DGCR8-Knock out human embryonic stem cells, this proposed project aims to systematically study on the molecular mechanism of how DGCR8 is involved in aging process with quantitative proteomics, RNAseq, miRNAseq, LncRNA-microarray, etc. The present research will result in a better understanding of DGCR8-related regulatory networks during the aging of human adult stem cells, and provide molecular cues underlying human aging and aging-related diseases.

成体干细胞稳态失衡和衰老目前被认为是引发组织器官退行和机体衰老的重要诱因。非编码RNA被认为是细胞稳态调控和细胞衰老的重要调节子，而DGCR8广泛参与非编码RNA合成， mRNA可变剪接和转录后调控等重要进程。我们最近发现DGCR8表达与人成体干细胞衰老显著相关，但目前鲜见DGCR8参与调控人成体干细胞衰老的分子机制研究。本项目拟利用基因组编辑技术建立DGCR8基因敲除的人胚胎干细胞系，并将其定向分化为间充质干细胞和神经干细胞两种成体干细胞，进而结合基因组、转录组、表观遗传组和系统生物学等技术进行组学测序和系统分析，以研究DGCR8参与调节人成体干细胞衰老的分子机理。本项目研究将有助于理解人成体干细胞衰老和稳态失衡的新型分子调控网络，为延缓衰老和干预衰老相关疾病提供可能的线索和靶点。

本研究首次发现了miRNA合成通路关键因子DGCR8通过稳定异染色质抑制人间充质干细胞衰老的新型生物学功能，为延缓器官衰老、防治衰老相关疾病提供新型潜在干预靶标。DGCR8作为经典miRNA合成通路中的关键蛋白，广泛参与非编码RNA合成、mRNA可变剪接和转录后调控等重要生物学过程，但其在干细胞衰老中的调控作用尚不明确。在本项研究中，我们利用CRISPR/Cas9介导的基因编辑技术和干细胞定向诱导分化技术，获得DGCR8功能缺陷型人间充质干细胞，并观察到这些细胞出现加速衰老的表型。进一步研究发现，DGCR8可与核膜蛋白Lamin B1以及异染色质蛋白KAP1和HP1形成复合物，共同参与维持异染色质结构的稳定性，从而保持人间充质干细胞的年轻态。此外，在复制性衰老、生理性衰老以及早衰症加速衰老的人间充质干细胞中，DGCR8的表达水平均发生下调，而过表达野生型的DGCR8或miRNA加工活性缺失的DGCR8突变体均可延缓人间充质干细胞的衰老。更重要的是，基于DGCR8过表达的基因治疗可有效抑制小鼠关节组织衰老，促进关节软骨再生，缓解损伤性骨关节炎及衰老性骨关节炎的病理表型。本研究首次确立了DGCR8通过稳定异染色质结构调节人间充质干细胞的稳态和衰老的新功能，并揭示了以DGCR8为靶标的骨关节炎基因治疗新路径，相关研究结果证明通过基因疗法导入“干细胞去衰老因子”治疗骨关节炎的可行性，具有潜在的临床应用价值。