| Objective: To investigate the expression of miRNA-155 in foam cell, plaque tissue and serum of patients with atherosclerosis and give futher study on the molecular mechanism of mi R-155 involved in the process of atherosclerosis.Methods: 1. Constitutive expression of seven miRNAs were detected by quantitative real-time polymerase chain reaction(q RT-PCR) in foam cell to screen the most significantlyelevated in ox LDL-treated THP-1 cells;2.To confirm that the expression of mi R-155 was increased inpatients with AS, we measured the level of mi R-155 in the plasma of patients with AS and in the normal controlgroup; the relative expression level ofmi R-155 in 17 pairs of atherosclerotic lesions and normal veins from the same patients were also detected by q RT-PCR; 3. To confirm that if the relative expression trend of mi R-155 was changed with the the lipid deposition amount or time, THP-1 was first stimulated with PMA to differentiate into macrophages and then the macrophages were treated with ox-LDL for different concerntration of different time; 4.Foam cells were transfected with mi R-155 mimic or inhibitor for for 0, 6, or 12 hours, the secretion of TNF-α was detected by ELISA and Oil Red O staining was performed to determine the effect of mi R-155 on foam cell formation;5. Several computational analyses available in online miRNA target databases, including Mir DB, Targetscan, and Pic Tar to identify potentialdirect mRNA targets of mi R-155, luciferase reporter assay was performed to identify the potential relationship between mi R-155 and CARHSP1; 6.To verify that CARHSP1 is required for TNF-α mRNA stabilization in foam cells, we transfected CARHSP1 overexpression plasmid or CARHSP1 si RNA into THP-1 cells, and the cells were then treated with ox LDL to form foam cells.the level of TNF-α protein production was measured by ELISA and Oil Red O staining was performed to determine the effect of CARHSP1 on foam cell formation. 7. After transfection the mi R-155 mimic or inhibitor, western blot was used to detect the protein level of CARHSP1 and downstream inflammation factor. 8. Using the TRANSFAC database tool to find the binding sites of NF-κB p65 which were located in the promoter of mi R-155, the Ch IP assay and luciferase reporter assay were performed to detect which putative binding site within the mi R-155 promoter binds to NF-κB p65.Results:1. Among these miRNAs, mi R-155 was the most significantly elevated in ox LDL-treated THP-1 cells;2. the mi R-155 level was indeed upregulated in patients with AS, the mi R-155 level was significantly increased in the atherosclerotic lesions compared with the normal veins, the expression of mi R-155 is increased by ox LDL stimulation in a dose- and time-dependent manner;3. The relative expression of mi R-155 was upregulated with the increase of lipids amount and the extension of treat time; 4.mi R-155 attenuates lipid uptake and suppresses the inflammatory response by repressing the expression of TNF-α in foam cell formation; 5.several computational analyses available in online miRNA target databases, including Mir DB, Targetscan, and Pic Tar predicted that CARHSP1 was one of the target gene of mi R-155, transfection with the mi R-155 mimic and a CARHSP1 3’-UTR reporter vector resulted in a significant reduction of luciferase activity;6.CARHSP1 knockdown down-regulates TNF-α protein production and reduces the level of TNF-α mRNA, CARHSP1 overexpression increases the level of TNF-α mRNA and up-regulates TNF-α protein production, these results suggest that CARHSP1 is required for TNF-α mRNA stabilization; 7. CARHSP1 is a functional target of mi R-155, which directly targets its 3’UTR, and that CARHSP1 serves as a mediator of mi R-155 by regulating the production of TNF-α and thereby affecting foam cell formation; 8.NF-κB, which is activated by TNF-α in THP-1 cells, is a transcriptional enhancer of mi R-155.Conclusion: Our findings shed light on the mechanisms that increased mi R-155 levels relieve chronic inflammation and atherosclerosis-associated foam cell formation by signaling through the mi R-155–CARHSP1–TNF-α pathway. |
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