Background 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. Methods 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 in situ 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 in vitro 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 in vivo. Results 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. Conclusions Our data reveal that miR-101 targets EZH2 in HS collagen production, providing new insight into the pathological mechanisms underlying HS formation.

中文翻译：

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miR-101的过表达通过靶向肥厚性瘢痕成纤维细胞中的EZH2抑制胶原蛋白合成

背景 MicroRNA-101（miR-101）是一种抑癌基因microRNA（miRNA），其缺失与多种疾病的发生和发展有关。然而，miR-101在肥厚性瘢痕（HS）发病机制中的生物学功能和靶点仍然未知。方法 我们从患者身上采集 HS 和配对的正常皮肤 (NS) 组织样本，并培养他们的成纤维细胞（分别为 HSF 和 NSF）。我们使用定量逆转录酶聚合酶链反应 (qRT-PCR)、荧光原位杂交 (FISH)、酶联免疫吸附测定 (ELISA) 和蛋白质印迹分析来测量 miR-101、zeste 同源物增强剂的 mRNA 水平和蛋白质表达2 (EZH2)、胶原蛋白 1 和 3 (Col1 和 Col3) 和 α-平滑肌肌动蛋白 (α-SMA) 在不同的体外条件下。我们还使用 RNA 测序来评估相关的信号通路和生物信息学分析以及双荧光素酶报告基因分析来预测 miR-101 靶标。我们利用博来霉素诱导的纤维化小鼠模型，在该模型中我们注射了 miR-101 模拟物来评估体内胶原蛋白的沉积。结果 我们发现 HS 和 HSF 中 miR-101 的表达低于 NS 和 NSF。过表达 miR-101 会降低 HSF 中 Col1、Col3 和 α-SMA 的表达。我们检测到 EZH2 在 HS 和 HSF 中的高表达。EZH2 的敲低降低了 HSF 中的 Col1、Col3 和 α-SMA。机制上，miR-101 靶向 EZH2 的 3'-非翻译区 (3'UTR)，如 EZH2 表达减少所示。过表达 EZH2 挽救了 miR-101 诱导的胶原抑制。MiR-101 模拟物有效抑制博来霉素诱导的纤维化小鼠模型中的胶原蛋白沉积。