Study On The Regulation Effect And Mechanism Of MicroRNA-155 On Atherosclerosis Inflammation

Study Background and PurposeAcute coronary syndrome(ACS) is a clinical syndrome with pathological base of complete or incomplete thrombotic occlusion caused by coronary atherosclerotic plaque rupture or erosion. ASC is a serious type of coronary heart disease with high mortality. The risk mainly comes from unstable plaque, pathologically called vulnerable plaque, which is characterized by a thin fibrous cap and a large lipid core with shoulder patches abundant in inflammatory cytokines pruducted by foam macrophage. Clinical and pathological studies have shown that continuous stimulation of chronic inflammation in the blood vessels plays a key role in atherosclerosis. Monocytes differentiate to macrophages after they enter into vascular endothelium, then they transform into foam cells after phagocytosis of lipids, releasing a great deal of inflammatory cytokines and accumulating in plaques. It is an important factor in promoting the development of plaque. The current idea is that less monocyte recruitment and alleviated inflammation is a key link in retard progression of atherosclerosis and stable atherosclerotic vulnerable plaque. Therefore, immune-modulatory molecules of inflammation play a critical role in atherosclerosis regulation.Micro RNAs(mi RNAs) are known to be involved in the regulation of inflammation and immune responses. mi RNAs, non-coding RNA molecules with 18-22 nucleotides in length, exert post-transcriptional horizontal regulation in gene expression by inhibiting protein translation of its target molecule or causing m RNA degradation. Studies have shown that mi R-155 has significantly-increased level during atherosclerosis, and play a regulatory role for atherosclerosis through different target molecules. Meanwhile, micro RNA-155(mi R-155) is known to be an inflammatory regulator by inhibiting the expression of its target molecules. However, the effect and mechanism of mi R-155 on atherosclerotic inflammation and stability of plaque remain unclear and further studies are required.To this end, this paper plans to first screen the mi R-155 target molecules that is related to atherosclerosis inflammation and experimentally verifie them. Then observe the effect of mi R-155 and its target molecules on ox LDL-stimulated macrophage inflammatory. Finally, interfere the mi R-155 expression in mice with atherosclerosis through antagomir, and observe whether the inhibition of mi R-155 expression is beneficial to alleviate the atherosclerotic inflammation and improve plaque stability, so as to provide the foundation for the research of atherosclerosis treatment.Methods1. Bioinformatics analysis and identification of mi R-155 target molecules in THP-1 cells(1) Use the target analysis software Target Scan and Miranda to make predictions, combined with literature review, and screen the mi R-155 target genes that are related to atherosclerotic inflammation;(2) Construct targets dual luciferase reporter vectors, which are co-transfected to PMA-induced THP-1 cells with mi R-155 mimic(overexpression of mi R-155) or inhibitor(inhibiting the expression of mi R-155), to verify whether mi R-155 is capable of binding with the 3’-UTR of the target gene;(3) Make real-time andquantitative PCR and Western blot tests to detect the influences of mi R-155 on m RNA and protein expression of target molecules.2. mi R-155 and si RNA interfere the effect of target molecules on ox LDL-stimulated THP-1 macrophage inflammatory:(1) Samples are received after 24 h of PMA-abduction, 24 h stimulation of ox LDL(50μg / ml) to THP-1 macrophages, 24 h of transfection of mi R-155 mimic/inhibitor, and target molecules of mi R-155;(2) Check expression changes of inflammatory cytokines TNF-α, IL-1β, chemokines CCL2, CCL4 CCL7 with quantitative PCR and ELISA methods;(3) Use Western blot to detect the phosphorylation levels of IκBα and STAT3, and ELISA to detect the level of NF-κB activation.3. The effect of mi R-155 inhibition on atherosclerotic inflammation and plaque progression:(1) Establish a mouse model of atherosclerosis with inhibition of mi R-155 expression by using antagomir-155: Apo E-/- mice(6 weeks old) with high-fat diet for 12 weeks, group the mice into two of mi R-155 control group and mi R-155 inhibition group, 16 mice one group, and intravenously injecte with saline formulated antagomir control and antagomir-155 respectively for three days. Three weeks later put them to death, and then collect peripheral blood, bone marrow mononuclear cells(BMMCs) and thoracoabdominal aortic tissue to detect the indexes.(2) Separate and collecte the thoracic-abdominal aorta(spanning from the aortic arch to the iliac artery branch), paraffin embedded, use HE staining to test the shape of aortic plaques, MASSON staining to check collagen deposition within plaques, immunohistochemical method to detect the expression of plaque macrophages(CD68), smooth muscle(SMA) and predicted mi R-155 target molecules.(3) mi R-155 levels detect by quantitative PCR.(4) Real-time and quantitative PCR and ELISA to test the expression changes of chemokine TNF-α, IL-1β, CCL2, CCL4 and CCL7.(5) Western blot to detect phosphorylation levels of STAT3 and IκBα, and ELISA to detect the level of NF-κB activation.4. Statistical analysis: the experimental data are presented as mean ± standard deviation(mean ± SD). Statistics comparison use One-way ANOVA, with P <0.05 as significant difference. SPSS 15.0 software is used for the statistical analysis.Results1. cytokine signal transduction inhibitor-1(suppressor of cytokine signaling-1, SOCS1) and sirtuin1(SIRT1) are predicted to be mi R-155 target genes by bioinformatics software;2. By constructing dual luciferase reporter vector containing the mi R-155 targets and GFP reporter vector, mi R-155 is confirmed to integrate with the predicted binding site on SOCS1 and SIRT1 m RNA; mi R-155 can significantly inhibite the m RNA and protein levels of SOCS1, SIRT1(P <0.05).3. The effects mi R-155 exerts on ox LDL-stimulated THP-1 macrophage inflammation:(1) RT-PCR results show that mi R-155 expression in ox LDL-stimulated THP-1 macrophages was significantly up-regulated(P <0.01).(2) RT-PCR and ELISA results show that m RNA and protein levels of TNF-α, IL-1β, CCL2, CCL4 and CCL7 are significantly reduced with mi R-155 inhibition(P<0.01), NF-κB transcriptional activity is also significantly decreased(P<0.05); when mi R-155 is overexpressed and after si RNA interferes SOCS1 and SIRT1, m RNA of TNF-α, IL-1β, CCL2, CCL4 and CCL7 and protein levels are significantly increased(P<0.01), and NF-κB transcriptional activity is also significantly higher(P <0.05).(3) Western Blot results show that when mi R-155 inhibited, IκBα and STAT3 phosphorylation levels are significantly decreased; when mi R-155 is overexpressed and si RNA interferes SOCS1, IκBα and STAT3 phosphorylation levels increase significantly; in addition, when mi R-155 is in inhibition, p65 acetylation is at a lower level, but after mi R-155 overexpression and si RNA interference to SIRT1, p65 acetylation levels increase.(4) Transwell experiments results show that inhibition of mi R-155 expression can reduce migration of THP-1 macrophages, whereas mi R-155 overexpression and si RNA’s interference to SOCS1 and SIRT1, the migration of THP-1 macrophages increased(P<0.05).3. The effects of mi R-155 inhibition on inflammation and plaque progression in atherosclerosis mouse model:(1) Tail vein injection of antagomir-155, real-time and quantitative PCR shows that mi R-155 is significantly reduced in the mice serum, vascular tissue and BMMCs(P<0.01), successfully establishing the mi R-155-inhibited atherosclerosis mouse model.(2)After the suppression of mi R-155 by antagomir-155, the secretion of TNF-α, IL-1β, CCL2, CCL4 and CCL7 expression decreased significantly in serum(P<0.01), and significantly reducing plaque macrophages and increasing collagen and smooth muscle cells(P<0.05) at the same time.(3) After antagomir-155 takes effect, SOCS1 and SIRT1 increase in atherosclerotic plaques and vascular tissue, with reduced STAT3 and IκBα phosphorylation levels and lower NF-κB transcriptional activity(P<0.05).Conclusions1. Predict and verify that hsa-SOCS1 and hsa-SIRT1 are target molecules of hsa-mi R-155; in addition, mi R-155 may cause SOCS1 and SIRT1 inhibition by mediating degradation of their m RNA.2. mi R-155 promotes ox LDL-stimulated THP-1 macrophage inflammation, whose mechanism may include: mi R-155 promotes STAT3 and NF-κB signaling pathways by inhibiting its target molecules SOCS1 and SIRT1, so as to increase inflammation cytokines and chemokines with increased migration of monocyte-macrophage cells.3. With antagomir-155 intervention to mi R-155 expression, the Apo E-/- mouse model of atherosclerosis that inhibits mi R-155 expression is successfully established; mi R-155 inhibition helps to alleviate the atherosclerosis inflammation and improve atherosclerotic plaque stability of the Apo E-/- mouse model, of which the mechanism may include inhibiting the activation of STAT3 and NF-κB pathway by increasing its target molecule SOCS1, SIRT1 expression.