Study On The Role Of The MiR-146a On LPS-induced CXCL16 Expression

Objective: Endothelial inflammation characterizes the early stages of atherosclerosis.CXCL16 is a protein that functions as both a chemokine and adhesion molecule,playing a crucial role in the pathogenesis of atherosclerosis.(1)To observe the effects of LPS,a major inducer of inflammation,on CXCL16 expression in endothelial cells.(2)To investigate whether miR-146 a,a negative regulator of atherosclerosis,participates in this process.Methods: HUVECs were cultured in vitro,and incubated in culture dishes for treatment with LPS in subsequent experiments.Bioinformatic analysis was used to predict the potential target genes of miR-146 a,including the web tools Targetscan.For the overexpression or inhibition of miR-146 a activity,HUVECs were transfected with miR-146 a mimics or inhibitors,respectively.The expression of miR-146 a was quantified using qRT-PCR,the effects of transfection were evaluated by FAM fluorescence and real-time PCR.For the examination of CXCL16,TLR4 and NF-κB production,ELISA and Western blotting were used,and cell viability was detected using the cell counting kit assay.Results:(1)HUVECs were treated with serial dilutions of LPS(0-10 μg/mL)for 24 hours or with constant doses of LPS(1 μg/mL)for 0-48 h.Cell viability was significantly changed with treatment of 1,3 and 10 μg/mL of LPS,and the percentage of viable cells decreased at 6,12,24,and 48 h.Treatment with 1 μg/mL of LPS resulted in a significant induction of CXCL16 expression in HUVECs and the supernatant.(2)HUVECs were treated with LPS as described in Fig 1,TLR4 and phospho-NF-κB levels were significantly increased by the 1 μg/mL LPS treatment,and LPS induced significant increases in TLR4 and phospho-NF-κB expression in the 24-h treatment condition.Next,we blocked TLR4 using a 10 μM concentration of the TLR4 inhibitor TAK-242 for 12 h.TAK-242 inhibited LPS-induced CXCL16 expression in HUVECs and supernatant,implicating the involvement of the TLR4/NF-κB pathway.(3)We measured miR-146 a levels after exposing HUVECs to 1 μg/mL LPS for 24 h.LPS treatment significantly induced in the expression of miR-146 a to levels approximately threefold higher than baseline.HUVECs were later transfected with miR-146 a mimics and inhibitors to either increase or decrease miR-146 a expression,respectively.Transfection of miR-146 a mimics dramatically reduced the LPS-induced increase in CXCL16 expression,whereas the miR-146 a inhibitor further increased its expression level.(4)We transfected miR-146 a mimics or inhibitors into HUVECs to investigate their effects on TLR4 protein expression.miR-146 a overexpression inhibited LPS-induced TLR4 and NF-κB protein expression,whereas the inhibition of miR-146 a increased their expression.This finding occurred in a similar manner to the negative regulation of CXCL16 by miR-146 a.HUVECs were transfected with miR-146 a inhibitors for 24 h followed by the addition of the TLR4 inhibitor TAK-242(10uM)for 12 h.The results showed that TAK-242 blocked the ability of miR-146 a inhibitors to promote CXCL16 expression in LPS treated cells.Similarly,miR-146 a increased TLR4 and NF-κB expression,but the effect was blocked by TLR4 inhibition.Conclusion:(1)LPS treatment induced CXCL16 expression in HUVECs and the secretion of a soluble form of CXCL16 in the supernatant.(2)LPS induced CXCL16 expression through the TLR4/NF-κB signaling pathway.(3)LPS triggers significant upregulation of miR-146 a,and miR-146 a inhibits LPS-induced CXCL16 expression in a TLR4-dependent manner.