Long non-coding RNA MIR503HG controls endothelial-to-mesenchymal transition. Long non-coding RNA MIR503HG controls endothelial-to-mesenchymal transition

Endothelial-to-mesenchymal transition (EndMT) is a dynamic transformation process that has a functional impact upon pathological vascular remodelling. The molecular mechanisms that govern EndMT remain largely unknown. By induction of EndMT in human primary endothelial cells (EC), using a combination of transforming growth factor-β2 (TGF-b2) and interleukin-1b (IL-1β), we identified the dramatic loss of the lncRNA MIR503HG, as a common signature across multiple primary EC types. Targeted depletion of MIR503HG spontaneously induced EndMT. Overexpression of MIR503HG repressed EndMT despite TGF-β2 and IL-1β co-stimulation. RNA-seq was carried out to identify the changes in gene expression induced by MIR503HG overexpression. We showed that over 25% of the EndMT-transcriptome signature was inhibited upon MIR503HG overexpression. Crucially, phenotypic changes induced by MIR503HG were independent of the functional regulation of miR-503 and miR-424, both harbored within the MIR503HG locus. Collectively, we identify the lncRNA MIR503HG as an essential regulator of EndMT. Overall design: To analyse the effect of MIR503HG overexpression during EndMT, we performed RNA-Seq on control and EndMT (TGFβ2 and IL1β) HUVEC cells overexpressing MIR503HG isoform 2 (ENST00000440570.5) using lentiviral transfection. Untransfected cells and cells transfected with an empty vector were used as controls. Each condition was carried out in triplicates.

内皮细胞向间充质转化（Endothelial-to-mesenchymal transition, EndMT）是一类动态转化过程，对病理性血管重塑具有功能性影响。目前调控EndMT的分子机制大多仍未明确。本研究通过联合使用转化生长因子-β2（transforming growth factor-β2, TGF-β2）与白细胞介素-1β（interleukin-1β, IL-1β），在人原代内皮细胞（endothelial cells, EC）中诱导EndMT，发现长链非编码RNA（long non-coding RNA, lncRNA）MIR503HG的显著下调是多种原代内皮细胞共有的表达特征。靶向敲低MIR503HG可自发诱导EndMT；即便同时受到TGF-β2与IL-1β的共刺激，过表达MIR503HG仍可抑制EndMT。为明确MIR503HG过表达所诱导的基因表达变化，我们开展了RNA测序（RNA-seq）分析，结果显示MIR503HG过表达可抑制超过25%的EndMT相关转录组特征。尤为关键的是，MIR503HG所介导的表型变化并不依赖于位于其基因座内的miR-503与miR-424的功能调控。综上，本研究证实长链非编码RNA MIR503HG是EndMT的关键调控因子。
整体实验设计：为探究MIR503HG过表达在内皮细胞向间充质转化过程中的调控效应，我们通过慢病毒转染技术，将MIR503HG亚型2（ENST00000440570.5）分别导入对照组人脐静脉内皮细胞（human umbilical vein endothelial cell, HUVEC）以及经TGF-β2与IL-1β诱导发生EndMT的HUVEC中，随后对上述细胞开展RNA测序分析。实验设置未转染细胞与转染空载体细胞作为对照，所有实验组与对照组均设置3次生物学重复。