Table6_MicroRNA-155 mediates multiple gene regulations pertinent to the role of human adipose-derived mesenchymal stem cells in skin regeneration.XLSX

Introduction: The role of Adipose-derived mesenchymal stem cells (AD-MSCs) in skin wound healing remains to be fully characterized. This study aims to evaluate the regenerative potential of autologous AD-MSCs in a non-healing porcine wound model, in addition to elucidate key miRNA-mediated epigenetic regulations that underlie the regenerative potential of AD-MSCs in wounds.
Methods: The regenerative potential of autologous AD-MSCs was evaluated in porcine model using histopathology and spatial frequency domain imaging. Then, the correlations between miRNAs and proteins of AD-MSCs were evaluated using an integration analysis in primary human AD-MSCs in comparison to primary human keratinocytes. Transfection study of AD-MSCs was conducted to validate the bioinformatics data.
Results: Autologous porcine AD-MSCs improved wound epithelialization and skin properties in comparison to control wounds. We identified 26 proteins upregulated in human AD-MSCs, including growth and angiogenic factors, chemokines and inflammatory cytokines. Pathway enrichment analysis highlighted cell signalling-associated pathways and immunomodulatory pathways. miRNA-target modelling revealed regulations related to genes encoding for 16 upregulated proteins. miR-155-5p was predicted to regulate Fibroblast growth factor 2 and 7, C-C motif chemokine ligand 2 and Vascular cell adhesion molecule 1. Transfecting human AD-MSCs cell line with anti-miR-155 showed transient gene silencing of the four proteins at 24 h post-transfection.
Discussion: This study proposes a positive miR-155-mediated gene regulation of key factors involved in wound healing. The study represents a promising approach for miRNA-based and cell-free regenerative treatment for difficult-to-heal wounds. The therapeutic potential of miR-155 and its identified targets should be further explored in-vivo.

引言：脂肪间充质干细胞（Adipose-derived mesenchymal stem cells, AD-MSCs）在皮肤伤口愈合中的作用尚未完全阐明。本研究旨在评估自体AD-MSCs在难愈性猪伤口模型中的再生潜能，同时阐明介导AD-MSCs伤口再生潜能的关键微小RNA（microRNA, miRNA）依赖型表观遗传调控机制。
方法：本研究采用组织病理学及空间频域成像（spatial frequency domain imaging）技术，在猪模型中评估自体AD-MSCs的再生潜能。随后，通过对原代人AD-MSCs与原代人角质形成细胞（keratinocytes）进行整合分析，探究AD-MSCs中miRNA与蛋白的表达关联。此外，通过AD-MSCs转染实验验证生物信息学分析结果。
结果：与对照组伤口相比，自体猪AD-MSCs可促进伤口上皮形成及皮肤屏障功能修复。本研究鉴定出26种在人AD-MSCs中上调表达的蛋白，包括生长及血管生成因子、趋化因子与炎性细胞因子。通路富集分析显示，显著富集的通路涉及细胞信号通路与免疫调节通路。miRNA靶标预测模型显示，16种上调蛋白的编码基因存在miRNA介导的调控关系。其中，miR-155-5p被预测可调控成纤维细胞生长因子2（Fibroblast growth factor 2, FGF2）、成纤维细胞生长因子7（Fibroblast growth factor 7, FGF7）、C-C基序趋化因子配体2（C-C motif chemokine ligand 2, CCL2）及血管细胞黏附分子1（Vascular cell adhesion molecule 1, VCAM1）的表达。向人AD-MSCs细胞系转染抗miR-155寡核苷酸后，转染24小时即可观察到上述4种蛋白的瞬时基因沉默效应。
讨论：本研究揭示了miR-155介导的伤口愈合关键因子正向调控通路。本研究为难愈性伤口的miRNA靶向及无细胞再生治疗提供了极具前景的研究思路。未来可在体内实验中进一步探究miR-155及其鉴定出的靶标的治疗潜力。