Effect Of MicroRNA-155 On TRIF Signaling Pathway In Atherosclerosis

Objective:Clarify whether miR-155 can midiate TRIF signaling pathway, regulate the balance of pro-inflammatory and anti-inflammatory response, and provide new ideas and methods for the prevention and treatment of atherosclerosis.Methods:1. OxLDL (20μg/ml) stimulated RAW263.7 cells for Oh,6h,12h,24h. We collected the cell culture for real-time PCR and western blot. Expression in miR-155, SOCS1, TRIF mRNA was detected by RT-PCR under different induction time. Western blot detecte different intervention time effects on the protein concentration of TRIF and SOCS1.2. SOCS1 adenovirus infected RAW264.7 cells for 48 hours, RT-PCR was used to analyze the concentration of miR-155, SOCS1, TRIF mRNA after transfection. Using Western blot analysis the protein concentration of TRIF and SOCS1 after transfection.3. High-fat feeding on ApoE-/-mice to establish a mouse model of atherosclerosis. Intravenously injecte with saline formulated antagomir control and antagomir miR-155. Divided into C57BL/6 mice in ordinary feeding group, ApoE-/-mice in high-fat feding group, ApoE-/-mice in high-fat feding with antagomir NC group, ApoE-/-mice in high-fat feding with antagomir miR-155 group. Collect thoracoabdominal aortic tissue samples of mice.4. Paraffin embedded the thoracic-abdominal aorta, use HE staining to test the shape of aortic plaques, Masson staining to check collagen deposition within plaques.5. Using Western blot analysis the protein concentration of TRIF and SOCS1.6. Statistical analysis:the experimental data are presented as mean μg standard deviation (x μg S), with P<0.05 as significant difference. SPSS 17.0 software is used for the statistical analysis.Results:1. In oxLDL on RAW264.7 cells, RT-PCR results showed that, the expression of miR-155 and TRIF with the intervention time increased gradually increased (P< 0.05), and SOCS1 expression is decreased gradually (P< 0.05), the difference has statistical significance; at the same time, Western blot results showed that, TRIF protein concentration with the intervention time increased increased (P< 0.05), and SOCS1 protein concentration is with the intervention time increased decreased (P< 0.05).2. After SOCS1 adenovirus transfection, RT-PCR results show that SOCS1 expression level was significantly higher (P< 0.05), suggesting that transfection was successful, the expression of trif decreased significantly (P< 0.05), miR-155 expression changes is not obvious (P> 0.05); shown in the Western blot results and transfected SOCS1 expression level was significantly higher (P< 0.05), whereas trif expression was significantly reduced (P< 0.05), the difference is significant.3. High fat diet in mice, HE staining showed that the atherosclerosis model was established successfully.4. After the intervention of antagomir miR-155, Masson staining showed that the number of collagen fibers in interference group than the control group increased, suggesting that antagomir miR-155 plaque stability, the intervention successed. Western blot results showed that the SOCS1 expression in the intervention group was significantly higher than the control group (P< 0.05), while the expression of TRIF in the intervention group were significantly lower than the control group (P< 0.05), the difference was statistically significant.Conclusion Objective:1. MiR-155 and TRIF are involved in macrophage inflammatory stimulation by oxLDL, and miR-155 can regulate TRIF by inhibite SOCS1 in inflammation.2. After SOCS1 adenovirus transfection, miR-155 did not change significantly, while TRIF significantly reduced, further confirmed that SOCS1 can negative feedback regulation of TRIF.3. High fat diet ApoE-/- mice to establish a mouse model of atherosclerosis, the expression of TRIF was significantly increased, while the expression of SOCS1 was significantly decreased, suggesting that TRIF is involved in atherosclerosis.4. By antagomir miR-155 successfully established miR-155 expression inhibition of atherosclerotic mouse model; miR-155 inhibition and its molecular targets of SOCS1 expression level was significantly higher, further regulation of TRIF and TRIF appear expression significantly decreased, indicated that miR-155 promotes inflammation by inhibiting the target molecules of SOCS1, and regulate the trif mediated signal transduction pathway again.