miR-127-3p is an epigenetic activator of myofibroblast senescence situated within the miRNA enriched Dlk1-Dio3 imprinted domain on mouse chromosome 12 [miRNA array]

During wound healing, fibroblasts differentiate into non-proliferative contractile myofibroblasts, contribute to skin repair, and eventually undergo apoptosis or become senescent. MicroRNAs (miR) are posttranscriptional regulators of gene expression networks that control cell fate and survival and may also regulate senescence. Here we determined regulated miRs in myofibroblasts isolated from wounds and analyzed their role in senescent myofibroblast formation. Transcriptome profiling showed that a 200 kbp region of the Dlk1-Dio3 imprinted domain on mouse chromosome 12 encodes for most of the upregulated miRs in the entire genome of mouse myofibroblasts. Among those, miR-127-3p induced a myofibroblast-like phenotype associated with a block in proliferation. Molecular analysis revealed that miR-127-3p induced a prolonged cell-cycle arrest with unique molecular features of senescence, including the activation of the senescence-associated ß-galactosidase, increase in p21 levels, inhibition of lamin B1, proliferation factors, and the production of senescence-associated inflammatory and extracellular matrix -remodeling components. Hence, miR-127-3p emerges as an epigenetic activator regulating the transition from repair to remodeling during skin wound healing, but may also induce age-related defects, pathological scarring and fibrosis, all linked to myofibroblast senescence. Non-hematopoietic (CD45-)/non-endothelial (CD31-) cell populations with low expression of SMA-GFP from dermis (SMA-GFP+) and with high expression from wounds 7 days post injury (SMA-GFP+++) were isolated by cell sorting from transgenic SMA-GFP mice. Total RNA of was isolated with TRIZOL (Life Technologies) and RNA quality was confirmed using micro capillary electrophoresis (2100 Bioanalyzer, Agilent). ~50ng of RNA was amplified, labeled and hybridized to Sureprint G3 human GE 8x60K or whole genome mRNA microarray according to the manufacturer’s specifications (Low input Quick Amp Labeling kit, Agilent). The arrays were scanned (Agilent G2595C scanner), data extracted and processed to create a generic gene level experiments from two technologies using the Genespring 14.5 software (Agilent).

在皮肤伤口愈合进程中，成纤维细胞（fibroblasts）会分化为丧失增殖能力的收缩型肌成纤维细胞（myofibroblasts），参与皮肤修复过程，并最终发生凋亡或进入衰老状态。微小RNA（microRNAs, miR）是基因表达网络的转录后调控因子，可调控细胞命运与存活，同时也可能参与衰老过程的调控。本研究对从伤口组织中分离的肌成纤维细胞内的差异表达miR进行了鉴定，并分析其在衰老型肌成纤维细胞形成过程中的作用。转录组分析显示，小鼠12号染色体上Dlk1-Dio3印记结构域（Dlk1-Dio3 imprinted domain）中一段200 kbp的区域，编码了小鼠肌成纤维细胞全基因组中绝大多数上调表达的miR。在这些差异表达的miR中，miR-127-3p可诱导出与增殖阻滞相关的肌成纤维细胞样表型。分子机制分析表明，miR-127-3p可诱导出具有衰老特有分子特征的长期细胞周期阻滞，具体包括激活衰老相关β-半乳糖苷酶（senescence-associated ß-galactosidase）、上调p21蛋白水平、抑制核纤层蛋白B1（lamin B1）与增殖因子的表达，以及产生衰老相关炎症因子与细胞外基质（extracellular matrix）重塑相关组分。综上，miR-127-3p作为表观遗传激活因子，可调控皮肤伤口愈合过程中从修复到重塑的转变，同时也可能诱导与肌成纤维细胞衰老相关的年龄相关性缺陷、病理性瘢痕形成与纤维化。本研究通过细胞分选技术，从转基因平滑肌肌动蛋白-绿色荧光蛋白（SMA-GFP）小鼠中分离得到两类细胞群：一类来源于真皮组织、SMA-GFP表达量较低的非造血（CD45阴性）/非内皮（CD31阴性）细胞；另一类来源于伤后7天伤口组织、SMA-GFP表达量较高（SMA-GFP+++）的非造血/非内皮细胞。使用TRIZOL试剂（Life Technologies公司）提取总RNA，并通过微毛细管电泳技术（安捷伦2100生物分析仪，Agilent 2100 Bioanalyzer）验证RNA质量。按照试剂盒说明书（安捷伦低起始量快速荧光标记试剂盒，Agilent Low input Quick Amp Labeling kit），对约50ng RNA进行扩增、标记，并与Sureprint G3人全基因组表达8x60K芯片或全基因组mRNA微阵列进行杂交。使用安捷伦G2595C扫描仪对芯片进行扫描，通过安捷伦Genespring 14.5软件对原始数据进行提取与处理，整合得到来自两种技术平台的标准化基因水平实验数据。