Chen Yahui, Gong Yiyi, Shi Mengkun, Zhu Haoxing, Tang Yulong, Huang Delin, Wang Wei, Shi Chenyi, Xia Xueyi, Zhang Ying, Liu Jianlan, Huang Jia, Liu Mengguo, Chen Huyan, Ma Yanyun, Wang Ziyu, Wang Lei, Tu Wenzhen, Zhao Yinhuan, Lin Jinran, Jin Li, Distler Jörg Hw, Wu Wenyu, Wang Jiucun, Shi Xiangguang
Department of Dermatology, Huashan Hospital and Human Phenome Institute, Fudan University, Shanghai, China.
Department of Thoracic Surgery, Huashan Hospital & Cancer Metastasis Institute, Fudan University, Shanghai, China.
J Adv Res. 2024 Nov 20. doi: 10.1016/j.jare.2024.11.027.
Fibroblast abnormalities are crucial causes of skin fibrosis, including systemic sclerosis (SSc) and keloids. However, their mechanisms, including underlying microRNA regulatory mechanisms, remain elusive.
This study aimed to evaluate the roles, mechanisms, and therapeutic potential of miR-3606-3p in regulating multiple fibroblast abnormalities.
The miR-3606-3p levels were evaluated in skin tissues and primary fibroblasts. RNA-seq and luciferase assays were employed to identify miR-3606-3p targets. Collagen contraction, western blotting, in vivo imaging, and real-time cellular analysis were used to assess fibroblast abnormalities. The therapeutic potential of miR-3606-3p was evaluated in mice.
MiR-3606-3p decreased in skin tissues (SSc: Fold Change (FC) = - 2.95, P = 0.0101; keloid: FC = - 3.42, P < 0.0001) and primary fibroblasts (SSc: FC = - 12.74, P = 0.0278; keloid: FC = - 2.08, P = 0.0021) from skin fibrosis patients, and negatively correlated with disease severity. Mechanistically, miR-3606-3p targeted the 3'-untranslated regions (3'-UTRs) of Integrin αV (ITGAV), GRB2-associated binding protein 1 (GAB1), and transforming growth factor beta receptor 2 (TGFBR2), all of these three targets increased in skin fibrosis. Simultaneously, miR-3606-3p inhibited fibroblast's fibrogenesis, migration, inflammation, and proliferation by inhibiting ITGAV/integrin/FAK, GAB1/p-AKT/p-ERK, and TGFBR2/p-SMAD2/3 signaling. ITGAV-mediated integrin/FAK signaling unidirectionally activated the p-AKT/p-ERK and p-SMAD2/3 pathways. Knockdown of GAB1 and TGFRB2 reduced ITGAV-induced p-AKT/p-ERK and p-SMAD2/3 activities. MiR-3606-3p, si-ITGAV, si-GAB1, and si-TGFBR2 exhibited significant inhibition of fibrogenesis and migration. Inflammation was primarily inhibited by si-ITGAV and si-GAB1, while proliferation was primarily inhibited by si-TGFBR2. Moreover, miR-3606-3p significantly attenuates skin fibrosis in keloid-bearing mice.
MiR-3606-3p is downregulated in skin fibrosis. Moreover, it negatively correlates with disease severity. Functionally, miR-3606-3p inhibits fibrogenesis, migration, inflammation, and proliferation of fibroblasts. Mechanistically, miR-3606-3p inhibits ITGAV, GAB1, and TGFBR2 by targeting their 3'-UTRs. ITGAV-, GAB1-, and TGFBR2-activated integrin/AKT/ERK/SMAD2/3 signaling induced fibroblast abnormalities. In vivo, miR-3606-3p inhibits skin fibrosis in mice. Therefore, the multi-targeting, multi-phenotypic regulatory properties of miR-3606-3p suggest its potential utility in clinical treatment.
成纤维细胞异常是皮肤纤维化的关键原因，包括系统性硬化症（SSc）和瘢痕疙瘩。然而，其机制，包括潜在的微小RNA调控机制，仍不清楚。
本研究旨在评估miR-3606-3p在调节多种成纤维细胞异常中的作用、机制和治疗潜力。
评估皮肤组织和原代成纤维细胞中miR-3606-3p的水平。采用RNA测序和荧光素酶测定法鉴定miR-3606-3p的靶标。使用胶原收缩、蛋白质印迹、体内成像和实时细胞分析来评估成纤维细胞异常。在小鼠中评估miR-3606-3p的治疗潜力。
miR-3606-3p在皮肤纤维化患者的皮肤组织（SSc：倍数变化（FC）=-2.95，P=0.0101；瘢痕疙瘩：FC=-3.42，P<0.0001）和原代成纤维细胞（SSc：FC=-12.74，P=0.0278；瘢痕疙瘩：FC=-2.08，P=0.0021）中降低，且与疾病严重程度呈负相关。机制上，miR-3606-3p靶向整合素αV（ITGAV）、GRB2相关结合蛋白1（GAB1）和转化生长因子β受体2（TGFBR2）的3'非翻译区（3'-UTR），这三个靶标在皮肤纤维化中均增加。同时，miR-3606-3p通过抑制ITGAV/整合素/黏着斑激酶（FAK）、GAB1/p-AKT/p-ERK和TGFBR2/p-SMAD2/3信号通路来抑制成纤维细胞的纤维化、迁移、炎症和增殖。ITGAV介导的整合素/FAK信号通路单向激活p-AKT/p-ERK和p-SMAD2/3通路。敲低GAB1和TGFRB2可降低ITGAV诱导的p-AKT/p-ERK和p-SMAD2/3活性。miR-3606-3p、si-ITGAV、si-GAB1和si-TGFBR2对纤维化和迁移表现出显著抑制作用。炎症主要被si-ITGAV和si-GAB1抑制，而增殖主要被si-TGFBR2抑制。此外，miR-3606-3p可显著减轻瘢痕疙瘩小鼠的皮肤纤维化。
miR-3606-3p在皮肤纤维化中下调。此外，它与疾病严重程度呈负相关。在功能上，miR-3606-3p抑制成纤维细胞的纤维化、迁移、炎症和增殖。机制上，miR-3606-3p通过靶向其3'-UTR来抑制ITGAV、GAB1和TGFBR2。ITGAV、GAB1和TGFBR2激活的整合素/AKT/ERK/SMAD2/3信号通路诱导成纤维细胞异常。在体内，miR-3606-3p可抑制小鼠皮肤纤维化。因此，miR-3606-3p的多靶点、多表型调控特性表明其在临床治疗中的潜在应用价值。
