Li Jie, Li Yan, Wang Yunchuan, He Xiang, Wang Jing, Cai Weixia, Jia Yanhui, Xiao Dan, Zhang Jian, Zhao Ming, Shen Kuo, Li Zichao, Jia Wenbin, Wang Kejia, Zhang Yue, Su Linlin, Zhu Huayu, Hu Dahai
Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, 710032, China.
Burns Trauma. 2021 Nov 26;9:tkab038. doi: 10.1093/burnst/tkab038. eCollection 2021.
MicroRNA-101 (miR-101) is a tumor suppressor microRNA (miRNA) and its loss is associated with the occurrence and progression of various diseases. However, the biological function and target of miR-101 in the pathogenesis of hypertrophic scars (HS) remains unknown.
We harvested HS and paired normal skin (NS) tissue samples from patients and cultured their fibroblasts (HSF and NSF, respectively). We used quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), fluorescence hybridization (FISH), enzyme-linked immunosorbent assays (ELISA) and Western blot analyses to measure mRNA levels and protein expression of miR-101, enhancer of zeste homolog 2 (EZH2), collagen 1 and 3 (Col1 and Col3) and α-smooth muscle actin (α-SMA) in different conditions. We also used RNA sequencing to evaluate the relevant signaling pathways and bioinformatics analysis and dual-luciferase reporter assays to predict miR-101 targets. We utilized a bleomycin-induced fibrosis mouse model in which we injected miR-101 mimics to evaluate collagen deposition .
We found low expression of miR-101 in HS and HSF compared to NS and NSF. Overexpressing miR-101 decreased Col1, Col3 and α-SMA expression in HSF. We detected high expression of EZH2 in HS and HSF. Knockdown of EZH2 decreased Col1, Col3 and α-SMA in HSF. Mechanistically, miR-101 targeted the 3'-untranslated region (3'UTR) of EZH2, as indicated by the decreased expression of EZH2. Overexpressing EZH2 rescued miR-101-induced collagen repression. MiR-101 mimics effectively suppressed collagen deposition in the bleomycin-induced fibrosis mouse model.
Our data reveal that miR-101 targets EZH2 in HS collagen production, providing new insight into the pathological mechanisms underlying HS formation.
微小RNA-101（miR-101）是一种肿瘤抑制性微小RNA（miRNA），其缺失与多种疾病的发生和发展相关。然而，miR-101在增生性瘢痕（HS）发病机制中的生物学功能和靶点仍不清楚。
我们从患者身上采集了HS和配对的正常皮肤（NS）组织样本，并分别培养其成纤维细胞（分别为HSF和NSF）。我们使用定量逆转录聚合酶链反应（qRT-PCR）、荧光杂交（FISH）、酶联免疫吸附测定（ELISA）和蛋白质印迹分析来测量不同条件下miR-101、zeste同源物2增强子（EZH2）、胶原蛋白1和3（Col1和Col3）以及α-平滑肌肌动蛋白（α-SMA）的mRNA水平和蛋白质表达。我们还使用RNA测序来评估相关信号通路，并通过生物信息学分析和双荧光素酶报告基因测定来预测miR-101的靶点。我们利用博来霉素诱导的纤维化小鼠模型，在其中注射miR-101模拟物以评估胶原蛋白沉积。
我们发现与NS和NSF相比，HS和HSF中miR-101表达较低。过表达miR-101可降低HSF中Col1、Col3和α-SMA的表达。我们检测到HS和HSF中EZH2表达较高。敲低EZH2可降低HSF中Col1、Col3和α-SMA的表达。机制上，miR-101靶向EZH2的3'-非翻译区（3'UTR），这表现为EZH2表达降低。过表达EZH2可挽救miR-101诱导的胶原蛋白抑制。miR-101模拟物在博来霉素诱导的纤维化小鼠模型中有效抑制了胶原蛋白沉积。
我们的数据表明，miR-101在HS胶原蛋白产生过程中靶向EZH2，为HS形成的病理机制提供了新的见解。
