HuR和 miR-890竞争调控ARDS关键始动因子HMGB1表达的机制研究

The acute respiratory distress syndrome (ARDS) is a common clinical critical disease which is lack of effective therapies. Several clinical disorders including sever infection, major trauma and etc. can increase HMGB1, the key initiation factor of ARDS, release heavily and then activate the cytokine storm. However, the regulatory mechanism of HMGB1 is still unknown. Based on our previous researches, we found that microRNA expression profiles significantly changed in the peripheral blood of patients who were suffering from ARDS caused by different factors. Further bioinformatics predictions and pre-experiments showed that miR-890 and HuR known as an RNA-binding protein could both bind to HMGB1 mRNA and regulate its expression, but yet the precise mechanism and effects on ARDS still remained to be studied. Considering literatures and preliminary experiment results, we propose that HuR and miR-890 compete for the binding site on HMGB1 mRNA to determine the expression level of HMGB1, and then influence the occurrence of ARDS. In the subject, we would like to use PCR, adenovirus transfection, RNA interference, immunofluorescence and other means on cell lines and gene knockout mice models to illustrate the competitive regulatory mechanism of HuR and miR-890 on the expression of HMGB1. We anticipate that it may provide a general target of prevention and treatment for ARDS caused by different inducements.

ARDS是常见临床危重症，治疗手段缺乏。严重感染和创伤等多种诱因均导致ARDS关键始动因子HMGB1大量释放，形成炎症风暴，而HMGB1表达调控机制仍不明确。申请人前期研究发现，不同诱因导致ARDS患者外周血中microRNA谱发生显著变化。进一步生物信息学预测及预实验表明，miR-890及RNA结合蛋白HuR均可与HMGB1的mRNA结合，导致其表达变化，但具体作用机制及其对ARDS的影响有待探索。综合文献及预实验结果，我们认为：HuR与miR-890两者竞争HMGB1 mRNA结合位点，从而决定HMGB1表达水平，影响ARDS发生。本课题拟通过细胞和基因敲除小鼠模型，采用PCR、腺病毒转染、RNA干扰和免疫荧光等手段，从分子、细胞以及动物水平等多方面研究阐明HuR和miR-890在HMGB1表达中的调控作用及其内在机制。本研究将为临床防治多种诱因导致的ARDS提供通用治疗靶点。

ARDS是常见临床危重症，病死率高，治疗手段缺乏。严重感染和创伤等多种诱因均导致ARDS关键始动因子HMGB1大量释放，形成炎症风暴，而HMGB1表达调控机制仍不明确。在本研究中，我们发现不同诱因导致ARDS患者外周血中microRNA谱发生显著变化。我们利用肺微血管内皮细胞模型，过表达或抑制HuR后，再用LPS刺激细胞，观察炎症反应变化。研究发现在外界刺激时，HuR由细胞核转移到细胞质。此时HuR通过与HMGB1 mRNA 3’UTR区域结合稳定HMGB1的mRNA，进而导致HMGB1的过度表达，影响ARDS发生。我们构建了选择性血管内皮细胞HuR基因敲除小鼠。利用基因敲除小鼠和对照小鼠构建了ARDS模型。研究发现基因敲除小鼠肺微血管内皮细胞上HMGB1表达和释放减少，炎症反应减轻，减弱ARDS发生发展。我们接下来在肺微血管内皮细胞模型中转染miR-890 类似物，再用LPS刺激细胞，研究发现miR-890通过与HMGB1 mRNA结合抑制HMGB1表达，减弱炎症反应，进一步减弱ARDS的发生发展。最终，我们在肺微血管内皮细胞模型中同时抑制HuR并转染miR-890 mimics，LPS刺激细胞后没有发现HuR与miR-890两者竞争改变HMGB1表达水平。此外，我们还发现miR-574–5p也可以在体外和体内调控HMGB1的表达。由此我们得出课题研究结论：HuR和miR-890在调控ARDS关键促炎因子HMGB1的表达中分别起重要作用，从而影响ARDS的发生发展，两者未发现有竞争关系。miR-574–5p对lps诱导的炎症反应提供负反馈，缓解ARDS。本研究为深入阐明ARDS病理生理过程提供了新的证据，为临床防治感染导致的 ARDS 提供治疗靶点。