CRISPR-Cas9 mediated gene knockout in human coronary artery endothelial cells reveals a pro-inflammatory role of TLR2
CRISPR-Cas9 mediated gene knockout in human coronary artery endothelial cells reveals a pro-inflammatory role of TLR2
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CRISPR-Cas9介导的人冠状动脉内皮细胞基因敲除揭示了TLR2的促炎作用
DOI：
10.1002/cbin.10885
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发表时间：
2018
影响因子：
3.9
通讯作者：
Liang Jingyan
中科院分区：
文献类型：
--
作者：
Wang Yingge;Chen Lu;Tian Zheng;Shen Xueyi;Wang Xiaohong;Wu Honghai;Wang Yayi;Zou Jiayu;Liang Jingyan
血管内皮炎性反应促进动脉粥样硬化的发生发展。研究表明，Toll样受体2(TLR2)与血管内皮细胞炎症有关。然而，TLR2对人冠状动脉内皮细胞(HCAECs)炎症反应的影响在很大程度上仍不清楚。在此，我们验证了TLR2激动剂刺激HCAECs后可增强炎症反应的假说。首先，我们利用CRISPR-Cas9技术对HCAEC中的TLR2基因进行了敲除。然后用TLR2激动剂肽聚糖(PGN)处理TLR2-KO和野生型HCAECs。采用实时定量聚合酶链式反应、免疫印迹和酶联免疫吸附试验检测细胞间黏附分子-1(ICAM-1)、白介素6(IL-6)和白介素8(IL-8)的表达水平。免疫印迹法检测髓系分化主要反应基因88(MyD88)、磷酸化IRAK-1(pIRAK-1)和磷酸化核因子-κB(pNF-κB)的表达情况。我们的结果表明，经TLR2激动剂处理后，TLR2-KO细胞中ICAM-1、IL-6和IL-8的表达水平低于野生型细胞。此外，MyD88、pIRAK-1和pNF-κB的Western blotting显示，这些促炎分子在TLR2KO细胞中的表达水平比野生型细胞在TLR2激动剂刺激下的表达水平低得多。我们认为TLR2可能通过引入促炎症分子MyD88、pIRAK-1和pNF-κB来影响HCAECs的炎症反应。
Endothelial inflammatory responses promote the development and progression of atherosclerosis. It was reported that Toll‐like receptors 2 (TLR2) is associated with endothelial inflammation. However, the effect of TLR2 on inflammatory responses in human coronary artery endothelial cells (HCAECs) remains largely unknown. Here, we tested the hypothesis that TLR2 can enhance inflammatory reactions in HCAECs after stimulated by TLR2 agonist. First, we used CRISPR‐Cas9 technology to knockout TLR2 gene in HCAECs. Then, TLR2‐KO and wild type HCAECs were treated with TLR2 agonist peptidoglycan (PGN). The expression levels of intercellular cell adhesion molecule‐1 (ICAM‐1), interleukin‐6 (IL‐6), and interleukin‐8 (IL‐8) were analyzed by real‐time PCR, Western blot, and ELISA. The expression status of myeloid differentiation primary response gene 88 (MyD88), phosphorylated IRAK‐1 (pIRAK‐1) and phosphorylated NF‐κB (pNF‐κB) were detected by Western blot. Our results show that after treated with TLR2 agonist, the expression levels of ICAM‐1, IL‐6, and IL‐8 were downregulated in TLR2‐KO cells compared to those of wild type cells. Further, Western blots of MyD88, pIRAK‐1, and pNF‐κB show that the expression levels of these pro‐inflammatory molecules were much lower in TLR2‐KO cells compared to that of wild type cells by stimulating with TLR2 agonist. We suggest that TLR2 may affect inflammatory reaction in HCAECs by introducing pro‐inflammatory molecules like MyD88, pIRAK‐1, and pNF‐κB.