miRNA调控细胞程序性坏死的分子机制及其功能研究

Necroptosis is a form of programmed cell death that critically depends on receptor-interacting protein 1 (RIP1)，receptor-interacting protein 3 (RIP3) and mixed lineage kinase domain-like (MLKL) activation. Necroptosis is involved in many pathological diseases. By now, the negative regulatory mechanism of necroptosis is still scarce. microRNAs (miRNAs) is well-known to inhibit target genes by promoting target mRNAs for cleavage or translational repression. In this project, we have identified multiple miRNAs that negatively regulate necroptosis, including the miR-324-5p and miR-148/152 family, with a highly efficient necroptosis inducing system. We found that miR-324-5p directly targeted MLKL to inhibit necroptosis, whereas the miR-148/152 family did not directly target a known necroptotic regulatory protein. Moreover, we observed that the expression of miR-324-5p was regulated by the IFN signaling pathway. On this basis, we will investigate the mechanism of interferon regulating microRNA-324-5p, and elucidate the mechanism through which miR-324-5p regulates necroptosis in pathological conditions. Furthermore, we will identify the target protein of miR-148/152 family and uncover its action in necroptosis. We aim to fully understand the molecular mechanisms of these miRNAs in the regulation of necroptosis and their roles in the necroptosis-associated diseases.

细胞程序性坏死是由RIP1/RIP3/MLKL介导的程序性死亡方式，参与许多重要疾病的病理过程。目前，细胞程序性坏死的负调控机制知之甚少。miRNA是一类能负调控靶基因表达的非编码RNA。本项目的初期研究已鉴定出多个负调控细胞程序性坏死的miRNAs，包括miR-324-5p和miR-148/152家族。进一步的靶基因分析发现miR-324-5p直接靶向MLKL从而抑制程序性坏死，而miR-148/152家族并不直接靶向已知的程序性坏死调控蛋白。我们还发现miR-324-5p的表达受到IFN信号通路调控。在此基础上，我们将深入研究干扰素调控miRNA-324-5p的机理，阐明miR-324-5p在病原体感染等病理情况下调控程序性坏死的机制，并揭示miR-148/152负调控程序性坏死的靶蛋白及其作用机理，充分阐述这些miRNAs调控细胞程序性坏死的机制及其在程序性坏死相关疾病中的调控作用。

甲型流感病毒感染（IAV）可以导致患者高烧、头痛、全身性肌肉酸痛等一系列症状，并可能对例如老年人和有潜在健康问题的的人群构成危险。世界卫生组织估计每年全球季节性流感病毒的重症病例达300万例以上，死亡人数为29万到65万人。因此，预防和控制流感是关乎人类健康的重要课题。近年来的研究表明，IAV感染可引发感染细胞发生细胞程序性坏死，导致组织损伤，加重流感症状。而细胞程序性坏死是由RIPK1/RIPK3/MLKL介导的程序性死亡方式，参与许多重要疾病的病理过程，包括清除被病原体感染的细胞等。了解IAV和细胞程序性坏死之间的关系可以深入了解流感发病机制，并在发展新的治疗策略方面起到重要作用。但是目前，细胞程序性坏死的负调控机制知之甚少。而miRNA是一类能够负调控靶基因表达的非编码RNA，参与了各种生理学病理学的过程。因此，我们构建了miRNA筛选文库，从而鉴定出多个负调控细胞程序性坏死的miRNAs，包括miR-324-5p。MiR-324-5p通过特异性靶向3'UTR以一种不依赖于种子区域的方式下调MLKL的表达。在干扰素（IFNs）的作用下，miR-324-5p通过JAK/STAT信号通路被下调，从而解除了MLKL mRNA的转录后抑制，提高了MLKL的表达进一步促进细胞程序性坏死激活。在甲型流感病毒（IAV）感染的人原代巨噬细胞后，导致IFNs的表达被激活从而下调miR-324-5p。通过miR-324-5p的过表达可以减弱IAV依赖的细胞程序性坏死并增强病毒复制，而当细胞缺失miR-324-5p时，细胞程序性坏死被增强并病毒复制受到抑制。因此，我们的实验证明miR-324-5p通过调控MLKL表达负向调控细胞程序性坏死，而IFNs下调miR-324-5p促进细胞的坏死从而更好的激活宿主抗病毒防御。这对甲型型流感病毒药物的开发具有一定的借鉴意义。