Introduction

Fibroblast abnormalities are crucial causes of skin fibrosis, including systemic sclerosis (SSc) and keloids. However, their mechanisms, including underlying microRNA regulatory mechanisms, remain elusive.

Objectives

This study aimed to evaluate the roles, mechanisms, and therapeutic potential of miR-3606-3p in regulating multiple fibroblast abnormalities.

Methods

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.

Results

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.

Conclusions

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.

中文翻译：

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miR-3606-3p 通过整合抑制整合素/FAK、p-AKT/p-ERK 和 TGF-β 信号级联反应来缓解皮肤纤维化

 介绍

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，它们的机制，包括潜在的 microRNA 调节机制，仍然难以捉摸。

 目标

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。

 方法

在皮肤组织和原代成纤维细胞中评估 miR-3606-3p 水平。采用 RNA-seq 和荧光素酶测定法鉴定 miR-3606-3p 靶标。胶原收缩、蛋白质印迹、体内成像和实时细胞分析用于评估成纤维细胞异常。在小鼠中评估 miR-3606-3p 的治疗潜力。

 结果

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） 中的降低，并与疾病严重程度呈负相关。从机制上讲，miR-3606-3p 靶向整合素 αV （ITGAV） 的 3'-非翻译区 （3'-UTR） 、GRB2 相关结合蛋白 1 （GAB1） 和转化生长因子 β 受体 2 （TGFBR2），这三个靶点在皮肤纤维化中均增加。同时，miR-3606-3p 通过抑制 ITGAV/整合素/FAK、GAB1/p-AKT/p-ERK 和 TGFBR2/p-SMAD2/3 信号传导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 信号转导单向激活 p-AKT/p-ERK 和 p-SMAD2/3 通路。敲低 GAB1 和 TGFRB2 降低了 ITGAV 诱导的 p-AKT/p-ERK 和 p-SMAD2/3 活性。miR-3606-3p 、 si-ITGAV 、 si-GAB1 和 si-TGFBR2 对纤维化发生和迁移表现出显著抑制作用。炎症主要受 si-ITGAV 和 si-GAB1 抑制，而增殖主要受 si-TGFBR2 抑制。此外，miR-3606-3p 显着减轻携带瘢痕疙瘩小鼠的皮肤纤维化。

 结论

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发生、迁移、炎症和增殖。从机制上讲，miR-3606-3p 通过靶向 ITGAV 、 GAB1 和 TGFBR2 的 3′-UTR 来抑制它们。ITGAV-、GAB1- 和 TGFBR2 激活的整合素/AKT/ERK/SMAD2/3 信号诱导成纤维细胞异常。在体内，miR-3606-3p 抑制小鼠皮肤纤维化。因此，miR-3606-3p 的多靶点、多表型调节特性表明其在临床治疗中的潜在效用。