Exploration Of Target Genes Related To Coronary Atherosclerotic Plaque Formation And Research On Its Function And Mechanism

Background and objection:Coronary heart disease(CHD) is a common disease caused by a variety of reasons, the incidence of CHD is rising. The formation of atherosclerotic plaques in the coronary artery and its rupture and thrombosis, is the main cause of acute cardiovascular events. Now in addition to research on obesity, smoking, high blood pressure, diabetes, hyperlipidemia and other traditional risk factors for the formation of coronary atherosclerosis plaque, research on target genes related to the formation of atherosclerotic plaque is becoming more and more significant. Myocyte enhancer factor 2A(MEF2A) gene is the first gene found to be the cause of coronary heart disease (CHD) and Myocardial infarction (MI), then more and more research about CHD and MI caused by MEF2A gene mutations were carried out. We investigated a group of families with a phenomenon of familial coronary heart disease morbidity. We found all of the sick family members have a deletion of 6 bases "CAGCCG" on MEF2A gene exon 11. MEF2A gene function abnormalities led to the occurrence of CHD and MI.Inflammation play a key role in the formation and development of atherosclerotic plaque, it can induce macrophages secrete a variety of inflammatory cytokines, such as TNF-a, IL-6 and so on. Macrophages in atherosclerotic plaque play an important role in regulating inflammation. Classically activated macrophages (M1) promote the development of inflammation while alternatively activated macrophages (M2) has a strong anti-inflammatory properties. Regulating the functions of macrophages can restrain the formation and progression of atherosclerotic plaque, arginase-1 (Arg-1) can improve the activity of smooth muscle cell proliferation, inhibit inflammation and highly expressed in M2 macrophages, and have important functions in regulation of macrophage differentiation and function. Arg-1 can inhibit macrophage inflammatory reaction, reduce tumor necrosis factor-a (TNF-α) generation and restrain monocyte chemotactic migration, also can decrease inflammatory response in atherosclerotic plaque and improve stability of plaque. Our previous researches have fully confirmed the role of Arg-1 in coronary atherosclerotic plaque. Through the exploration of upstream regulation gene of Arg-1, we can find new genes associated with atherosclerosis. Through exploration of new genes and studying its functions, we can find new intervention targets to atherosclerosis, and it is of great significance for the treatment of diseases.Based on the above research, we put forward the following hypothesis:(1)By screening transcription factors binding with activity area of Arg-1 promoter, to find new target gene similar to MEF2A that related to the formation of coronary atherosclerosis plaque.(2)To explore the function and mechanism of new gene related to atherosclerosis plaque inflammation reaction.This study was based on the research of dysfunction of MEF2A gene in familial coronary atherosclerotic heart disease, and the study of Arg-1 before. Aiming to discover new target genes related to regulation of plaque inflammation, and to study its function and mechanism, in order to find new preventive and therapeutic targets related to coronary atherosclerosis plaque inflammation.Methods:1. Cell culture:Lipopolysaccharide (LPS) acts as a proinflammatory inducer, interferon (IFN)+lipopolysaccharide (LPS) and interleukin-4 (IL-4) acts as macrophage polarization type inducer.2. PGL3 vector construction:We generated Arg-1 promoter-pGL3 constructs from-560 bp,-510 bp,-460 bp,-410 bp,-360 bp,-310 bp,-260 bp with serial deletions of the Arg-1 promoter.3. Cell transfection:Human aortic smooth muscle cells(HASMCs) were transfected with Argl-Pgl3 plasmid, Argl-siRNA, respectively. Raw 264.7 cells were transfected with ILF3-lentivirus, and then the cells were used to the next step of experiment.4. Luciferase activity detection:After preparating of cell lysis solution, we detected the high activity fragment of luciferase according to the luciferase kit manual processing and testing promptly with a photometer.5. Purification and analysis of binding proteins on Arg-1-460bp to-410bp promoter region:Proteins combined on the promoter region of Arg-1-460 bp to-460 bp were purified with DNA binding protein purification kits,, then samples were sent to company to analyze protein after trypsin digestion and desalting treatment, analyzing the active binding proteins.6. Chromatin immunoprecipitation, (ChIP):We verified whether ILF-3 directly bound with the activity fragment of the Arg-1 promoter according to the kit manual operation.7. Real-time fluorescence quantitative RT-PCR detection:ILF-3, Arg-1 and inflammatory factors mRNA expression were detected by RT-PCR after total RNA were extracted.8. Western blot:β-actin, GAPDH, ILF-3, Arg-1, IL-10, TNF-a, IL-6, P38/phospho-P38, JNK/phospho-JNK, ERK/phospho-ERK, nuclear factor κb (NF-κB) P65 protein expression were detected by Western blot after total protein were extracted.9. Immunohistochemistry analysis:Human coronary atherosclerotic plaque paraffin sections were disposed by Immunohistochemistry analysis, and then the distribution of ILF-3 in plaque was observed.10. Immunofluorescence analysis:We observed the distribution of ILF-3 in Raw 264.7 cells and the location of ILF-3 and Arg-1 in coronary artery and coronary atherosclerotic plaques by useing immunofluorescence staining and confocal laser technology.Results:1. ILF-3 directly binds to Arg-1 promoter-460 bp to-410 bp area and adjusts the transcription process of Arg-1. We analyzed proteins binding on promoter by mass spectrometer. The mass spectrometer analysis showed ILF-3 binds to Arg-1 promoter, and ChIP analysis verified this phenomenon again. The mRNA and protein expression of Arg-1 levels were reduced 70% after ILF-3 suppression by useing ILF-3-siRNA (p<0.01).2. Time and dose dependent regulation of ILF-3 and inflammatory factors induced by LPS in Raw 264.7 cells. The protein expression of ILF-3 was increased at first but then decreased in a dose-dependent manner,1000 ng/ml showed a downtrend response. LPS also had a time-dependent regulation to ILF-3, Arg-1 and IL-10 expression. Expression of ILF-3 began to elevate at 2h and peaked at 4h, then declined till 72h, the expression of Arg-1 increased at 2h, peaked at 12h and IL-10 peaked at 4h, then Arg-1 and IL-10 decreased.3. The expression level of ILF-3 in macrophages and atherosclerotic plaque.In Raw 264.7 cells, ILF-3 were mainly distributed in the nucleus, especially in the nucleolus. Western blot and laser confocal microscopy results showed that ILF-3 expressed at a low level in M1 macrophages and a high level in M2 macrophages.In atherosclerosis plaque, ILF-3 were found highly-expressed in plaque and mainly in macrophages. Immunofluorescence double staining of ILF-3 and Arg-1 showed ILF-3 and Arg-1 were co-localized in human coronary artery atherosclerotic plaque, and both were highly expressed in macrophages.4. ILF-3 overexpression inhibited LPS-induced pro-inflammatory factors and up-regulates anti-inflammatory factors in macrophages.LPS induction increased the lever of TNF-a, IL-6 and IL-1β. TNF-a, IL-6 and IL-1β were evidently reduced, while IL-10 and Arg-1 were increased compared with the LPS group after ILF-3 overexpression.5. Overexpression of ILF-3 inhibited the activity of mitogen-activated protein kinase (MAPK) and NF-κB signal pathways.LPS could increase the protein expression of p-P38, p-JNK, p-ERK, and P65 compared with static cells. ILF-3 overexpression significantly attenuated expression of LPS-induced P65 and phosphorylation of P38 and JNK, but did not reduce the expression of p-ERK. The results suggested that ILF-3 was involved in the regulation of MAPK and NF-κB signaling pathways induced LPS.Conclusion and significance:1. ILF-3 acting as transcription factor of Arg-1 can regulate the transcription of Arg-1, maybe a new target gene for coronary atherosclerosis plaque inflammation.2. ILF-3, highly expressed in M2 macrophages, can enhance function of M2 macrophages.3. ILF-3 inhibits the secretion of macrophage inflammatory factors induced by LPS and promotes the secretion of anti-inflammatory factors.4. ILF-3 inflammation suppression effects are mediated by suppression of p-P38 /MAPK, p-JNK/MAPK and NF-κb signal pathway.5. We confirm that the ILF-3 is the transcription factor of Arg-1, and systemmically elaborate the function, mechanism of ILF-3 in inflammation, and the distribution of ILF-3 in coronary atherosclerotic plaque and macrophages. ILF-3 is probably to be a new preventive and therapeutic target relevant to coronary atherosclerotic plaque inflammation, and is of great significant for the study of coronary heart disease.