TNF-α通过特定microRNA抑制喉黏膜间充质干细胞免疫调控作用的机制研究

Chronic laryngitis, which is the most common disease in respiratory tract, is harmful for local and systemic health. Our research group have identified a laryngeal mesenchymal stem cells (LMSCs), which play important role in laryngeal mucosa maintenance, repair and regeneration. Moreover, LMSCs are potent to regulate local inflammatory events. During chronic laryngitis, the immune-regulatory property of MSCs is inhibited by TNF-α, which might be a key factor to aggravate inflammation. But the molecular mechanism remains unknown. Our further research revealed that the inhibition of MSCs immune-regulatory property is inreversible. We also found that microRNA significantly regulated MSCs biological behavior through epigenetic pathway. Taken together, we hypothesized that TNF-α lead to dysfunction of MSCs through regulating miRNAs. In this study, we plan to perform miRNA micro-array screening between MSCs derived from normal and inflammatory mucosa; find out the key miRNAs; confirm the function of selected miRNAs and their targeting genes using strategies such as miRNAs mimics/inhibitor, and lentivirus overexpression/knockdown vector; determine the downstream regulatory pathway of TNF-α using CHIP and western blot analysis. The study will be helpful for us to understand undermining relationship between inflammation micro-environment and MSCs, to determine the mechanism of specific miRNAs regulation to MSCs, and to provide basic experimental evidence for novel strategy of inflammatory diseases.

慢性喉炎是发病率最高的呼吸道疾病之一，严重危害人体健康，其发病分子机制尚不清楚。本项目组前期研究发现间充质干细胞（MSCs）在喉黏膜组织的维持、修复及再生中发挥重要作用，并对局部炎症具有强大的调节功能，而TNF-α导致MSCs免疫调控功能下降，是炎症反应加剧的关键原因。我们发现慢性炎症组织来源MSCs的免疫调控能力受到不可逆的抑制，而miRNA介导的表观遗传调控对MSCs功能至关重要，据此推测炎症微环境通过表观遗传调控导致MSCs免疫功能异常。依此本项目拟对正常与炎症喉黏膜组织来源MSCs进行miRNA表达谱高通量筛选，明确关键miRNA，利用合成miRNA模拟物/抑制物、慢病毒过表达/干扰载体转染等手段研究其对于MSCs免疫调节功能的影响及靶基因，通过CHIP等技术明确TNF-α作用靶点及下游通路。从而探索miRNA表观遗传修饰对炎症MSCs的调控机制、探寻基于miRNA的炎症治疗策略。

慢性喉炎是发病率最高的呼吸道疾病之一，严重危害呼吸道及全身健康。本项目组前期研究发现间充质干细胞（MSCs）在喉黏膜组织的维持、修复及再生中发挥重要作用，并且对于局部炎症反应具有强大的调节功能，而慢性炎症中TNF-α导致MSCs免疫调控功能下降，是炎症反应加剧的关键原因，但机制不明。我们进一步研究证实，慢性炎症组织来源MSCs的免疫调控能力受到不可逆的抑制，同时发现miRNAs介导的表观遗传调控对MSCs生物学行为至关重要，据此推测TNF-α通过miRNAs途径导致MSCs的功能异常。本项目通过对正常与炎症组织来源MSCs进行miRNA高通量筛选，筛查直接受TNF-α调控并在炎症微环境中差异表达的miRNAs，并利用生物信息学手段进行相依miRNAs的功能预测出11个差异miRNA。从中，我们选择了let-7a进行深入研究。利用合成miRNAs模拟物/抑制物进行上调和下调let-7a表达，我们发现let-7a能够调控MSCs的免疫调节能力。进一步机制研究证实let-7a能否通过直接结合3’UTR区负调控Fas及FasL，而Fas/FasL在MSCs介导的T细胞凋亡中发挥关键作用。TNF-α可以促进let=7a的表达，从而抑制Fas/FasL系统，最终导致MSCs免疫调节能力的异常。上述研究为深入认识炎症微环境和MSCs之间的密切关系、探索特定表观遗传修饰对MSCs的调控机制、探寻炎症疾病防治新的途径提供实验依据。