MiR-25-PKCζ-STAT3与AMPK通路的交互调控在增生性瘢痕纤维化中的作用及机制研究

Hypertrophic scar (HS) has always been a difficult problem for clinical treatment. The preliminary study of our group showed that miR-25 inhibits the expression of fibrosis markers in HS fibroblasts (HFb), improves the collagen structure of rabbit ear scar and targets PKCζ; activating the energy-stress molecule AMPK effectively antagonizes HFb fibrosis. STAT3 is a downstream molecule of PKCζ-induced fibrosis. Previous studies have shown that AMPK can inhibit STAT3 activity. Furthermore, we found that PKCζ represses AMPK activity in HFb, suggesting that there may be cross talk between miR-25-PKCζ-STAT3 and AMPK pathway, resulting in amplification of the signal cascade and promoting the development of HS. In current study, the expression of key molecules of miR-25-PKCζ-STAT3 and AMPK pathways in HS and HFb will be firstly determined. The exogenous intervention of these molecules by overexpression/knockdown or activator/inhibitor will be performed to detect changes in the expression of fibrosis-associated proteins and cell cycle distribution and explore the potential mechanisms of interaction between pathways. Finally, further validation is conducted in rabbit ear scar model. Our study aims to elucidate the mechanism of miR-25 antagonizing HS and to enrich the molecular mechanism of HS formation, providing new ideas for the study of HS therapy.

增生性瘢痕（HS）是临床治疗的难题。课题组初步研究表明miR-25抑制HS成纤维细胞（HFb）中纤维化指标的表达，改善兔耳瘢痕胶原结构，且作用靶点为PKCζ；激活能量应激分子AMPK有效拮抗HFb纤维化。STAT3是PKCζ诱导纤维化的下游分子。既往研究表明AMPK能抑制STAT3活性，此外，我们发现PKCζ抑制AMPK活性，提示miR-25-PKCζ-STAT3和AMPK通路间交互调控，致使信号级联放大，促进HS发生发展。本课题拟首先检测miR-25-PKCζ-STAT3和AMPK通路的关键分子在HS及HFb中的表达；通过过表达/敲减、激活剂/抑制剂对这些分子进行外源干预，观察纤维化相关蛋白的表达变化以及细胞周期分布，并探讨通路间交互调控的潜在机制，最终在兔耳瘢痕模型中进一步验证。本研究旨在阐明miR-25拮抗HS的机制，丰富HS形成分子机理，为HS的治疗研究提供新思路。

增生性瘢痕（HS）是创面愈合过程中结缔组织增生修复而引起的皮肤纤维化疾病。表现为肌成纤维细胞的持续活化、增殖和凋亡抵抗，并分泌过量的胶原。PKCζ是非典型蛋白激酶C亚家族的成员，是多种胞外刺激的下游信号分子。我们收集了5对HS组织和邻近的正常皮肤组织 (NS)，并分离其中3对组织的成纤维细胞 (HFs/NFs) 以评估PKCζ与HS的临床相关性。免疫组化和Western blot实验发现PKCζ在HS/HFs中高表达，且其T410位点的磷酸化水平明显升高。进一步研究发现，磷酸化激活的PKCζ通过调控C/EBPβ T235位点磷酸化，促进HFs中胶原蛋白Col I、肌成纤维细胞标志α-SMA以及抗凋亡蛋白Bcl2、BclxL的表达，从而调控HFs纤维化表型、增强凋亡抗性。ChIP-PCR和双荧光素酶实验表明C/EBPβ是调控Col I、α-SMA、Bcl2和BclxL表达的重要转录因子，且其转录活性依赖于T235位点的激活。Hippo通路效应因子YAP是肿瘤和纤维化研究领域的明星分子。我们发现YAP对HFs纤维化表型和凋亡抵制的诱导与其对PKCζ的调控密不可分。免疫共沉淀实验表明，HFs中的内源性YAP与TEAD1结合。ChIP-PCR和双荧光素酶实验表明，YAP辅助TEAD1直接转录激活PKCζ。miR-25-5p属于miR-106b-25基因簇，参与调控肝脏、肺脏等纤维化疾病。miR-25-5p在HS/HFs中表达降低。生物信息学预测PKCζ的3’UTR存在miR-25-5p的结合位点。实验结果显示miR-25-5p mimic能从转录水平抑制PKCζ的表达，且显着抑制野生型PKCζ的荧光素酶报告基因活性。因此，PKCζ是miR-25-5p的靶基因。功能研究发现，miR-25-5p mimic通过抑制PKCζ调控Col I、α-SMA的表达和C/EBPβ的磷酸化水平，并促进细胞凋亡。博来霉素刺激小鼠皮肤，导致真皮层增厚，胶原严重沉积，这是皮肤纤维化的典型病理特征。miR-25-5p激动剂抑制了PKCζ的表达、明显改善了纤维化状态，然而过表达PKCζ则使miR-25-5p激动剂对抗纤维化的效果明显降低。综上，课题初步阐明了miR-25-5p/PKCζ/C/EBPβ拮抗HS纤维化的分子机制，为HS的防治及药物靶点的筛选提供新的切入点。