表观遗传调控TLR4信号诱导动脉粥样硬化单核细胞异常活化的机制研究

It has been demonstrated that overexpression of TLR4 leads to monocyte activation and the pathogenesis of atherosclerosis. However, the molecular mechanism of its overexpression has not been fully elucidated. Aberrant DNA methylation and histone acetylation of TLR4 have been reported in inflammatory diseases, suggesting that epigenetic modification plays an important role in the regulation of TLR4 gene expression. The present study was to investigate the epigenetic modification of TLR4 in monocytes isolated from the peripheral blood and whether the effects of LDL on monocyte activation are mediated by epigenetic modification of TLR4. In our previous study, we found that the expression of transcriptional factor RFX1 was downregulated in CD14+ monocytes isolated from patients with coronary arteriosclerosis disease. And the bioinformatics prediction suggests that there are binding sites for RFX1 in the promoter regions of TLR4. RFX1 has been reported to regulate gene transcription by recruitment DNA methyltransferase and histonedeacetylase. Therefore, we further explore whether RFX1 is the key modulator of TLR4 epigenetic modification and its role in the development of atherosclerosis. This project will contribute to clarify the epigenetic mechanisms of atherosclerosis and to provide new ideas and targets for the treatment of atherosclerosis.

目前研究已证实TLR4过度表达是导致单核细胞活化及动脉粥样硬化（AS）发病的重要机制，然而其过度表达的分子机制尚未阐明。多种炎症性疾病中TLR4的DNA甲基化与组蛋白乙酰化水平均发生改变，提示表观遗传学修饰在调控TLR4基因表达中起重要作用，本实验旨在探讨表观遗传学对AS单核细胞TLR4表达的调控及多种AS危险因素LDL对单核细胞的作用是否与诱导TLR4异常表观遗传学修饰有关；在此基础上，我们前期工作发现发现冠心病患者CD14+单核细胞转录因子RFX1表达降低，生物信息学预测TLR4基因转录起始位点上游存在RFX1结合位点，已有研究报道，RFX1可以通过招募DNA甲基转移酶及组蛋白去乙酰化酶，调控基因转录。因此，我们进一步探讨了RFX1对TLR4调控的表观遗传学机制及其对LDL等AS危险因素的介导作用。本项目将有助于进步一步阐明AS发病的表观遗传学机制，为治疗AS提供新的思路和靶点。

TLR4过度表达导致的单核细胞活化在动脉粥样硬化疾病的发生发展中起关键作用。然而，TLR4过度表达的机制尚不清楚。已有研究证明在某些炎症性疾病中TLR4启动子区DNA甲基化和组蛋白乙酰化水平发生改变。然而，在动脉粥样硬化患者单核细胞中TLR4的过度表达是否与其启动子区表观遗传修饰异常有关尚未阐明。本研究通过亚硫酸盐测序和ChIP实验检测发现冠心病患者CD14+单核细胞中TLR4启动子区甲基化水平较正常人低，而组蛋白乙酰化水平较正常人高。并进一步通过ChIP实验证实冠心病患者CD14+单核细胞中TLR4启动子区DNMT1和HDAC1的富集水平高于正常人。我们前期工作发现冠心病患者CD14+单核细胞转录因子RFX1表达降低，同时利用生物信息学预测发现TLR4基因转录起始位点上游存在RFX1结合位点，因此我们探讨了TLR4的异常表观遗传修饰是否与RFX1的表达异常有关。我们首先通过荧光素酶报告基因和ChIP实验进一步证实TLR4启动子区存在RFX1结合位点。然后我们分离健康志愿者外周血CD14+单核细胞，分别建立了RFX1低表达以及过表达的细胞模型，检测了RFX1对TLR4启动子DNA甲基化和组蛋白乙酰化水平的影响。我们发现，RFX1低表达会导致TLR4启动子区DNA甲基化水平降低、组蛋白H3和H4乙酰化水平升高及TLR4启动子区RFX1对DNMT1、HDAC1和SUV39H1的招募水平降低，RFX1高表达会导致TLR4启动子区DNA甲基化水平升高、组蛋白H3和H4乙酰化水平降低及TLR4启动子区RFX1对DNMT1、HDAC1和SUV39H1的招募水平升高。以上研究揭示了RFX1介导的TLR4异常表观修饰水平所致的TLR4过度表达是导致动脉粥样硬化患者单核细胞活化的重要机制之一，该研究结果表明RFX1可能是动脉粥样硬化及其相关疾病治疗的有效新靶点。