LncRNA ICR regulates endothelium inflammation through feedback activation of NF-?B signaling

The responsive and abundant lncRNAs to pro-inflammatory factors in human ECs were screened and validated by RNA sequencing and qPCR. ICAM1-related non-coding RNA (ICR) was identified as the most potential candidate. In vivo data confirmed ICR was upregulated in atherosclerotic plaque. Knock down and overexpression experiments showed that ICR plays an essential role for EC adhesion and migration as well as for the expression of its adjacent ICAM1 gene. Mechanistically, we unexpectedly found in quiescent ECs, ICR regulates mRNA stabilization of ICAM1 through a RNA duplex formation mechanism while in activated ECs, ICR modulated ICAM1 transcription via a NF-?B-dependent manner. RNA-seq and PCR analysis demonstrated a vast number of pro-inflammatory genes downstream of NF-?B were regulated by ICR. Further, CHIP assay showed p65 directly binds to ICR promoter and facilitate the transcription. Interestingly, we found p65 phosphorylation and degradation was in turn regulated by ICR, forming a feedback activation of NF-?B signaling. Finally, we screened siRNA of mouse ICR and delivered the shRNA adenovirus by intravenous injection in a high fat diet induced ApoE-/- atherosclerosis model, it was found that both aortic root stenosis and plaque area could be markedly inhibited by suppressing ICR. Overall design: silenced ICR with or without TNF-a treatment performed RNA-seq in endothelial cells

本研究通过RNA测序（RNA-seq）与实时定量聚合酶链反应（qPCR），筛选并验证了人类内皮细胞（endothelial cells，ECs）中响应促炎因子且高表达的长链非编码RNA（long non-coding RNAs，lncRNAs）。与细胞间黏附分子1（intercellular adhesion molecule 1，ICAM1）相关的非编码RNA（ICR）被鉴定为最具潜力的候选靶点。体内实验数据证实，ICR在动脉粥样硬化斑块中呈现上调表达。基因敲低与过表达实验结果显示，ICR在内皮细胞的黏附、迁移过程以及邻近基因ICAM1的表达调控中发挥关键作用。从机制层面而言，我们意外发现：在静息内皮细胞中，ICR通过RNA双链形成机制调控ICAM1的mRNA稳定性；而在活化内皮细胞中，ICR则通过核因子κB（nuclear factor kappa-light-chain-enhancer of activated B cells，NF-κB）依赖的途径调控ICAM1的转录。RNA测序与聚合酶链反应分析表明，NF-κB下游的大量促炎基因均受ICR调控。进一步的染色质免疫沉淀（chromatin immunoprecipitation，ChIP）实验证实，p65可直接结合ICR的启动子并促进其转录。值得注意的是，我们还发现p65的磷酸化与降解过程反过来亦受ICR调控，由此形成了NF-κB信号通路的反馈激活环。最后，我们筛选了小鼠ICR的小干扰RNA（small interfering RNA，siRNA），并通过静脉注射将靶向ICR的短发夹RNA（short hairpin RNA，shRNA）腺病毒递送至高脂饮食诱导的载脂蛋白E基因敲除（Apolipoprotein E knockout，ApoE-/-）动脉粥样硬化模型小鼠体内，结果显示，抑制ICR表达可显著减轻主动脉根部狭窄程度并缩小斑块面积。实验整体设计：在内皮细胞中，分别在肿瘤坏死因子α（tumor necrosis factor α，TNF-α）处理与未处理的条件下沉默ICR后开展RNA测序。