Blocking the MIR155HG/miR-155 axis reduces CTGF-induced inflammatory cytokine production and α-SMA expression via upregulating AZGP1 in hypertrophic scar fibroblasts

Yize Li; Yujie Xiao; Yongfeng Han; Huayu Zhu; Juntao Han; Hongtao Wang

doi:10.1016/j.cellsig.2024.111202

Blocking the MIR155HG/miR-155 axis reduces CTGF-induced inflammatory cytokine production and α-SMA expression via upregulating AZGP1 in hypertrophic scar fibroblasts

Cell Signal. 2024 May 9:120:111202. doi: 10.1016/j.cellsig.2024.111202. Online ahead of print.

Authors

Yize Li¹, Yujie Xiao², Yongfeng Han³, Huayu Zhu⁴, Juntao Han⁵, Hongtao Wang⁶

Affiliations

¹ Department of Clinical Oncology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, China.
² Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, China.
³ Department of Ultrasound, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, China.
⁴ Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, China. Electronic address: huayuzhu@fmmu.edu.cn.
⁵ Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, China. Electronic address: hanjt@fmmu.edu.cn.
⁶ Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, China. Electronic address: wanght@fmmu.edu.cn.

PMID: 38729323
DOI: 10.1016/j.cellsig.2024.111202

Abstract

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.

Keywords: Cytokine production; DNA methylation; Hypertrophic scar; MIR155HG; miR-155; α-SMA.