| BackgroundMorbidity and mortality rate of coronary heart disease have been continous increasing in recent years and become serious threaten to human health. Atherosclerosis is a major risk factor of cardiovascular disease. Previous research have illustrated that microRNA play a key role as gene regulator in formation and development of atherosclerotic plaques. In development of atherosclerotic plaques and cardiovascular disease, microRNA participate in dysfunction of endothelium and macrophage, proliferation and migration of vascular smooth muscle cells and formation of foam cell.Research on expression status of mir-142-5p in atherosclerotic plaques has not been reported. Macrophage was involved in several progresses of innate immunity, including clearing apoptotic cell, avoiding cell apoptosis and tissue restructuring and release of inflammatory factors. Inflammatory play an important role in pathological process of CHD. MicroRNAs have been reported to participate in phylogenesis,differentiation and activiation of inflammatory cells. Previous research of mir-142-5p have focused on tumors, immunity diseases and stem cells. Research of mir-142-5p in CHD have not been reported at present.Research objectives(1) To discuss the changes of mir-142-5p expression level.(2)To investigate expression of mir-142-5p in macrophage further.(3)To explore target gene of mir-142-5p and the affect to apoptosis of macrophage.Methods1.1Construction of animal model8weeks old male ApoE-/-mice was used in present research. The mice was changed to high-fat diet after3days’basal diet for2weeks.Then we performed right common carotid artery tubulization. After high-fat diet for8weeks, the mice were divided to three groups(12mice each groups):control group, stabilized plague group, vulnerable plague group. The vulnerable plague group was given Pisaj syndrome noise interference for4weeks. Then all the mice were anesthesia and executed to obtain blood sample.1.2MicroRNA biochip detectionSend the flash-frozen carotid sample to Kangcheng-bio for a microRNA chip detection to identity the change of of mir-142-5p expression in atherosclerotic plaques.1.3The change of mir-142-5p expression in human macrophage.The expression level of mir-142-5p in ox-LDL stimulated cells was detected with RT-PCR technique.1.4Tracing for target gene of mir-142-5p.TGF-β2was predicted to be target gene of mir-142-5p. Inhibitor of mir-142-5p and mimics were transfected into human macrophage and detect the influence on TGF-β2by Western-bolt and RT-PCR.1.5mir-142-5p influences apoptosis of macrophage.Inhibitor of mir-142-5p mimics and inhibitor of TGF-β2were transfected into human macrophage. Function of human macrophage was observed by Annexin V-PE Apoptosis Detection Kit.1.6Statistics analysisData analysis was conducted with SPSS17.0. All data were presented as mean6standard error of the mean, and statistical comparisons were made with a paired t test and ANOVA tests. Differences were considered significant if P<0.05.Results2.1Expression of mir-142-5in plagueBiochip detection of microRNA results show expression level of mir-142-5p in stabilized plague group is6.8folds higher than blank control; expression level of mir-142-5p is2.7folds higher than vulnerable plagues group.2.2Expression of mir-142-5p in macrophage Human endothelial cells, smooth muscle cells and macrophage was cultured, then stimulated with ox-LDL50ng/ml for24hours before microRNA was extracted. RT-PCR was used to analyze expression of the3cell lines. In endothelial cells and smooth muscle cells, mir-142-5p expression of ox-LDL stimulated group decrease1.5folds compared with non-stimulated group. According to biochip detection result, mir-142-5p expression in macrophage cells is5.4folds higher than blank control.2.3Prediction of mir-142-5p target geneAs processed with database-based target gene prediction software, TGF-β2is the most probable target gene of mir-142-5p. Inhibitor of mir-142-5p and mimics was transfected into human macrophage cells. Protein and RNA were extracted and analyzed by western blot. Both results show TGF-β2is target gene of mir-142-5p.2.4mir-142-5p affects apoptosis of human macrophage cellsApoptosis of the blank control group was not observed. Cell apoptosis of ox-LDL group was increased(Apoptosis cell percentage:6.83±0.17%).Cell apoptosis of mir-142-5p mimics+ox-LDL group(Apoptosis cell percentage:5.27±0.19%);TGF-β2inhibitor mir-142-5p inhibitor+ox-LDL group (Apoptosis cell percentage:1.38±0.27%) and TGF-02inhibitor+ox-LDL group (Apoptosis cell percentage:2.74±0.21%) was also more slight than the other two groups. Cell apoptosis of N.C+ox-LDL group(Apoptosis cell percentage:6.49±0.25%) was also increased. Mir-142-5p restrains apoptosis of marque cells partly through TGF-β2.(P<0.05)Conclusions1. Expression of mir142-5p is at a high level in atherosclerotic plaques.2.mir142-5p is over expressed in human macrophage cells in CHD; Accords with the trend of the testing results of the chip, has the inhibitory effect of ox-LDL induced apoptosis.3. TGF-P2is target gene of mir142-5p and regultates apoptosis of human macrophage cells. |
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