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CRISPR-Cas9 mediated gene knockout in human coronary artery endothelial cells reveals a pro-inflammatory role of TLR2.
Cell Biology International 2018 Februrary
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.
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