Department of Clinical Oncology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, China.
Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, China.
Cell Signal. 2024 Aug;120:111202. doi: 10.1016/j.cellsig.2024.111202. Epub 2024 May 9.
Hypertrophic scarring (HS) is a pathological condition characterized by excessive fibrosis and inflammation, resulting in excessive extracellular matrix formation in the skin. MIR155HG, a long non-coding RNA, is abnormally upregulated in fibrotic tissues; however, its underlying mechanism is poorly understood. Using single-cell sequencing data, we analyzed connective tissue growth factor (CTGF) expression in various cell types in HS and normal skin tissues and MIR155HG expression in clinical samples. To investigate the mechanism of fibrosis, an in vitro model using CTGF-treated hypertrophic scar fibroblasts (HSFBs) was established and qRT-PCR, western blotting and ELISA assays were performed to investigate the expression of interleukin (IL)-1β, IL-6, and mesenchymal markers α-smooth muscle actin (α-SMA). CTGF stimulates MIR155HG level through phosphorylated STAT3 binding to the MIR155HG promoter. We analyzed the methylation of MIR155HG, assessed the levels of miR-155-5p/-3p in CTGF-treated HSFBs and identified differentially expressed genes among HS and NS samples using the Gene Expression Omnibus RNA sequencing data. The binding between miR-155-5p/-3p and AZGP1 was confirmed using a dual-luciferase assay and inflammatory cytokine production and α-SMA expression were investigated in rescue experiments. The findings revealed that CTGF elevated inflammatory cytokine production, α-SMA and MIR155HG expression in HSFBs. MIR155HG is upregulated in HS tissues due to low DNA methylation. Mechanistically, miR-155-5p/-3p was directly bound to MIR155HG 3'UTR. MIR155HG silencing inhibited cytokine production and α-SMA expression by repressing the generation of miR-155-5p/-3p in CTGF-treated HSFBs. Bioinformatics analysis and luciferase reporter assays revealed that miR-155-5p/-3p targets AZGP1. In addition, transfection with plasmids carrying AZGP1 cDNA significantly inhibited the signaling activity of miR-155-5p/-3 p-overexpressing HSFBs. Our findings highlight the importance of the MIR155HG/miR-155/AZGP1 axis in regulating cytokine production and α-SMA in HS.
增生性瘢痕（HS）是一种以过度纤维化和炎症为特征的病理状态，导致皮肤中细胞外基质的过度形成。长链非编码 RNA MIR155HG 在纤维化组织中异常上调，但其潜在机制尚不清楚。我们使用单细胞测序数据分析了 HS 和正常皮肤组织中结缔组织生长因子（CTGF）在各种细胞类型中的表达以及临床样本中 MIR155HG 的表达。为了研究纤维化的机制，我们建立了一个使用 CTGF 处理的增生性瘢痕成纤维细胞（HSFBs）的体外模型，并通过 qRT-PCR、western blot 和 ELISA 检测试剂盒检测白细胞介素（IL）-1β、IL-6 和间充质标志物α-平滑肌肌动蛋白（α-SMA）的表达。CTGF 通过磷酸化 STAT3 与 MIR155HG 启动子结合来刺激 MIR155HG 水平。我们分析了 MIR155HG 的甲基化，评估了 CTGF 处理的 HSFBs 中 miR-155-5p/-3p 的水平，并使用基因表达综合组 RNA 测序数据确定了 HS 和 NS 样本之间差异表达的基因。使用双荧光素酶报告基因检测证实了 miR-155-5p/-3p 与 AZGP1 之间的结合，并在挽救实验中研究了炎症细胞因子的产生和α-SMA 的表达。研究结果表明，CTGF 可提高 HSFBs 中的炎症细胞因子产生、α-SMA 和 MIR155HG 的表达。HS 组织中 MIR155HG 的上调是由于 DNA 低甲基化所致。在机制上，miR-155-5p/-3p 直接与 MIR155HG 3'UTR 结合。在 CTGF 处理的 HSFBs 中沉默 MIR155HG 可通过抑制 miR-155-5p/-3p 的产生来抑制细胞因子产生和α-SMA 的表达。生物信息学分析和荧光素酶报告基因检测表明，miR-155-5p/-3p 靶向 AZGP1。此外，转染携带 AZGP1 cDNA 的质粒可显著抑制 miR-155-5p/-3p 过表达 HSFBs 的信号活性。我们的研究结果强调了 MIR155HG/miR-155/AZGP1 轴在调节 HS 中细胞因子产生和α-SMA 中的重要性。
