Research On The Function And Mechanism Of Trim72 In Rats With Chronic Heart Failure

Myocardial fibrosis including atrial fibrillation, ventricular dilatation, myocardial ischemia, cardiac shock, can lead to such as heart disease, even cause serious damage to the health of mankind. Renin angiogenesis aldosterone system(RAAS) and transforming growth factor beta is the main pathogenesis of myocardial fibrosis, especially TGF-beta 1 is known and the most closely relationship between tissue fibrosis of growth factors, and regulate cell growth and differentiation, and promote cell proliferation, inhibit inflammation and other biological function.. Trim72 a total of 7 exons(also known as MG53, Gene ID: 365377), encoding 477 amino acids, protein molecular weight for 53 KD. The role of Trim72 in chronic heart failure and molecular mechanism is still not clear, therefore, based on SD rats model, the main research Trim72 expressed in heart failure in the organization, the cardiac fibroblasts proliferation and migration ability of molecular mechanisms.Part 1 Based on the bioinformatics analysis of differentiallyexpressed genes in heart failureObjective:Using GEO public database to express the spectral data, combined with bioinformatics methods to analyze genetic variations in chronic heart failure, involved in biological processes and signaling pathways, finding and heart failure is closely related to the gene.Methods: In this part of the study, GEO heart failure expression profile data for the analysis of public data materials, differentially expressed genes in heart failure was analyses by EC and TAC software, further combined with bioinformatics methods to analyze differences in the genes involved in biological processes and signaling pathways, mRNAmRNA express network using Cytoscape for network building. Using the ClueGO plug-ins do KEGG signaling pathways of gene sets, GeneOntology, Reactom pathway enrichment analysis, etc.Results: After the for expression of matrix, using R Limma in package analysis of differentially expressed genes between the two, a total of 950 different genes, 430 genes, raised 520 genes.GO enrichment analysis 950 differentially expressed genes in the biological process, and found that they significantly enriched in cell proliferation, cell effort), reshaping extracellular matrix(ECM), cell skeleton development, stimulate response(response to stree),signal transduction,regulation ofsignaling), metabolic regulation, regulation of metabolic process) and biological processes.Combining the KEGG database, the Pathway signal Pathway enrichment analysis of differentially expressed genes, the Pathway of bioinformatics analysis results show that the genetic difference between enrichment in the TGF signaling Pathway, the Hippo signaling Pathway, PI3 k AKT signaling Pathway and Wnt signaling Pathway and Cytokine receptor interation signaling Pathway and so on.Conclusions: Trim72 may play an important role in the occurrence of heart failure.Part 2: The expression of Trim72 in SD rat with heart failureObjective:SD rats with chronic heart failure model was constructed, and the research on expression of Trim72 in SD rats with chronic heart failure model of the heart tissue, preliminarily Trim72 myocardial fibrosis in chronic heart failure and processes the role, for Trim72 in provides the theory basis for the diagnosis and treatment of chronic heart failure.Methods: Based on the results of the bioinformatics analysis, we predict the Trim72 play an important role in the heart development, this part USES the abdominal aortic stenosis induced SD rats with chronic heart failure, the ultrasonic echocardiography, HE and Masson staining method identification of heart failure model, the use of real time PCR, western blot and immunohistochemistry detection technology, such as heart failure specimens and the specimens of normal tissues Trim72 mRNA and protein expression level of the situation.Results: By abdominal aortic stenosis method successfully copied in SD rats with chronic heart failure, echocardiography and Masson staining, according to the results of model building a successful SD rats with chronic heart failure.Immunohistochemical results show that the heart of the middle and lower Trim72 in normal SD rats express or express, in SD rats with chronic heart failure heart high expression.Conclusions: Trim72 highly expressed in SD rats with chronic heart failure heart high, has the potential to be an auxiliary diagnostic markers of chronic heart failure.Part 3: The effect of silencing Trim72 expression on biologicalbehavior in myocardial fibroblastsObjective:Study on silencing Trim72 expression ability of cardiac fibroblasts proliferation and migration, the influence of clear Trim72 role in cardiac fibroblasts.Methods: The trypsin and collagenase digestion heart tissue of SD rats, differential centrifugation separation stick wall myocardial fibroblasts, morphological observation and immunofluorescence method to identify myocardial fibroblasts. In the original generation of CFs cell cultivation for the model, with a small interference RNA technology Trim72 silencing CFs cells expression, the experiment is divided into three groups: blank group, negative control group, siRNA experimental group. After siRNA transfection 48 and 72 h, Real Time PCR and Western blot detection Trim72 respectively in the interference effect of mRNA and protein level. CCK-8 experimental detection silencing Trim72 CFs cell proliferation after change, Transwell detect silence after Trim72 CFs cell migration.Results: By trypsin and collagenase digestion heart tissue of SD rats, differential centrifugation separation stick wall myocardial fibroblasts, in containing 10% fetal bovine serum DMEM diet culture medium culture 24 h under the condition of normal cells, that can be observed under inverted microscope to observe the varied forms of cell cell body is bigger, flat, form more for spindle, no spontaneous pulsation. Cytoplasmic transparent, light color, seem to be very thin, larger nucleus shows ovoid, middle and cells in good condition, rapid growth.Visible under the fluorescent microscope cells are polygonal spindle. The rats myocardial fibroblasts positive for SMA antigen expression, cell purity is more than 97%.This can be identified as myocardial fibroblasts.SiRNA 48 and 72 h after transfection, Real Time PCR and Western blot results show that compared with the blank group and the negative control group, experimental group siRNA silence Trim72 effectively in the expression of mRNA and protein level. CCK-8 and Transwell results show that compared with the blank group and the negative control group, experimental group CFs cell proliferation and migration ability significantly decreased.Conclusions: Silencing of Trim72 expression can restrain the CFs cell proliferation and migration ability.Part 4: The molecular mechanism of cardiac fibroblasts cellproliferation and migration induced by Trim72Objective:Preliminary research the molecular mechanism of cardiac fibroblasts cell proliferation and migration induced by Trim72, targeting for Trim72 regulation of signaling pathways and downstream target genes.Methods: In this part, using gene expression profile chip finding Trim72 involved in signaling pathways and downstream target genes, first of all, we use the early screening Trim72-siRNA transfection CFs cells, silent Trim72 expression of Trizol extract transfection after 48 h of total RNA, using gene expression profile of rats after Trim72 expression gene chip analysis expression of the change., then apply EC and TAC combined with bioinformatics software, of differentially expressed genes in Gene Ontology and Pathway analysis of signal channel, looking for Trim72 involved in biological processes.Results: The results of gene expression profile shows that, after Trim72-siRNA inhibiting Trim72 expression in myocardial fibroblasts, significant changes in a total of 265 genes, of which 123 gene expression, gene expression by GO bioinformatics analysis results show that the differentially expressed genes significantly enriched in Cell adhesion, Skeletal system development, Heart development and Extracellular matrix organization and other biological processes, Pathway of bioinformatics analysis results show that the main enrichment of differentially expressed genes in the TGF-beta signaling Pathway, Cell adhesion molecules(CAMs), the Hippo signaling Pathway and Calcium signaling Pathway and other signaling pathways.Conclusions: Integration of differentially expressed and GO, the result of the Pathway as a result, we speculated that Trim72 may through the TGF- beta signaling Pathway pathways play a role, qRT PCR and Western blot results also got further validation.These results suggest that Trim72 may through the pathway and/or gene expression changes and myocardial fibroblasts proliferation and migration ability.