环状RNAcircANRIL调控LPS-TLRs-NF-kβ信号通路在细胞增殖和缺血性卒中易感性中的作用

CircRNA associates with a series of complex diseases. In our previous study, we found SNPs in 9p21 region may confers to IS suscepbility in which Long non-coding RNA ANRIL plays an important role. But the exact mechanism remains unclear, According to the latest research, we suppose that circANRIL formed by exon 5-7 contribute to the progression of atherosclerosis and ischemic stroke through modulating LPS-TLRs-NF-kβ signaling pathway,which has not been reported.This project aims to observe circANRIL expression of different rs4977574 genotypes in peripheral blood of patients with ischemic stroke, then generates cell line stably expresses circANRIL of rs4977574 risk genotype and protective genotype, another knockout cell lines that deletes exon 5-20 by Capser cas9 -mediated gene editing will also established, cell apoptosis and proliferation will be detected after circANRIL expression upregulated by tranfection CMV-circANRIL vector and inhibited by siRNA.LPS induced NF-kβ,IL2/IL6 expression changes and cell apoptosis and proliferation will detected after circANRIL overexpressed and inhibited by siRNA,circANRIL and IL2/IL6 expression and cell apoptosis and proliferation will also be observed when NF-kβ is inhibited by siP65, CHIP assay will preform to explore the possible binding site that circANRIL cooperatively activate IL2 and IL6 expression upon LPS treatment. This project will enhance understanding of the etiology of IS and provides potential therapeutic target for treatment of IS.

环状RNA与多种复杂疾病相关。我们在前期研究中发现9p21区域的单核苷酸多态性与缺血性卒中有关，非编码基因ANRIL在其中发挥了重要作用，机制未明。据最新研究，我们推测ANRIL外显子5-7以“外显子跳读”模式形成circANRIL介导LPS-TLRs-NF-kb信号通路影响细胞增殖参与动脉粥样硬化及缺血性卒中的发生，相关研究未见报道。本项目拟观察9p21各基因型缺血性卒中患者体内circANRIL表达，建立稳定表达circANRIL的rs4977574突变型及linANRIL表达沉默细胞株，LPS诱导,分别检测circANRIL及NF-kβ在不同干预下LPS-TLR-NF-kβ-circANRIL-IL2/IL6通路各因子表达及细胞凋亡与增殖情况，寻找该通路与circANRIL作用的靶因子，以阐明circANRIL调控该通路在细胞增殖及缺血性卒中当中的作用机制，为诊疗提供新的思路和靶点。

环状RNA circANRIL与缺血性卒中的风险相关，但其生物学功能及作用机制并不明确。本研究首先利用ANRIL的环状转录体之一建立稳定过表达、敲减circANRIL的HEK93细胞株，并在两组细胞株中进一步抑制线性ANRIL及P65功能，在LPS诱导前后, 观察各组细胞中LPS-NF-kβ-circANRIL-IL2/IL6通路重要因子表达及细胞凋亡与增殖情况，结果发现circANRIL表达水平与细胞增殖尤其是凋亡均有关，circANRIL低表达细胞株中凋亡细胞比例明显增高，且伴有p65和IL-2表达水平明显下调，同时P65核转位水平下降，IL-6表达水平增高，LPS刺激并未改变变化趋势,提示circANRIL敲减后NF-kβ信号通路可能被抑制，IL-2表达下调提示可能存在MAPK级联信号通路的参与。进一步对circANRIL过表达细胞进行差异基因分析发现上调基因富集于MAPK信号通路，也证实其可能影响circANRIL生物学功能，另外, circANRIL并非通过海绵mircoRNA发挥生物学功能，WDR3、COL4A基因表达在circANRIL的生物学作用中可能发挥一定作用，与神经系统发育和细胞凋亡相关的差异表达基因SYTL3和SLC22A3的部分单倍体型与缺血性卒中风险有关。总之，本研究证实了circANRIL在抑制细胞凋亡中的作用并发现了2个相关细胞因子,2条信号通路及4个主要基因。但需要进一步利用细胞株和动物实验及人群样本研究证实。挖掘MAPK相关信号通路在circANRIL生物学功能实现中的意义，进一步探讨WDR3和COL4A与circANRIL作用的联系可作为下一步研究的方向；SYTL3作为与神经修复有关的基因，与缺血性卒中后神经细胞修复的关系也值得进一步研究。本研究为寻找以上信号通路与circANRIL作用的靶因子，进一步以阐明circANRIL调控MAPK和NF-Kβ通路在细胞增殖、凋亡及缺血性卒中当中的作用机制，为诊疗提供新的思路和靶点提供了理论基础。

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