| Objective Myocardial fibrosis is an important pathological change of ventricular remodeling in patients with hypertension,and it is an important pathophysiological basis in patients with cardiovascular disease.However,the specific molecular mechanism of ventricular remodeling remains unclear.To investigate the differences of Osteopontin(OPN)expression,the activation,proliferation and apoptosis of fibroblast,and Type I collagen(COLL-1)and alpha smooth muscle actin(α-SMA)expression in fibroblasts stimulated by different concentrations of uric acid.Hypertension-induced ventricular remodeling is an important pathological change in myocardial disease.Dr.Hu’s previous study found that HLA-B may be the hub gene of hypertension combined with ventricular remodeling.In this study,we intend to use advanced simulation algorithms and clinical samples of patients with hypertension combined with ventricular remodeling to verify the core role of HLA-B in this disease.Materials and Methods Cultured primary human cardiac fibroblasts(HCFs)were divided into four groups according to different concentrations of uric acid(UA): UA 0 mg/dl group,UA 5mg/dl group,UA 10 mg/dl group and UA 15mg/dl group.After 48 hours stimulation,the following experiments were carried out: the m RNA expression of OPN,α-smooth muscle actin(α-SMA)and Coll-1 was detected by quantitative polymerase chain reaction(qPCR);the protein expression of OPN,α-SMA and Coll-1was detected by Western blotting(WB);the viability of fibroblasts was detected by luminescence cell growth activity test kit.The cell proliferation toxicity of cells was detected by Cell Counting Kit-8(CCK-8).The ability of cells migration was detected by Trans-well test.After 48 hours stimulation,the apoptosis of cells was detected by Propidium Iodide staining kit.Firstly,the expression of HLA-B in patients with hypertension combined with ventricular remodeling was verified by Perl and LIMMA based on Gene Expression Omnibus(GEO)database,and then the expression of HLA-B in the disease was verified by RT-qPCR by collecting clinical samples of patients with hypertension combined with ventricular remodeling.Modular verification and disease correlation analysis of the core role of HLA-B in hypertension combined with ventricular remodeling were carried out by Weighted Gene Co-expression Network Analysis(WGCNA).The functional enrichment of Differentially expressed genes(DEGs)related to hypertension combined with ventricular remodeling with HLA-B as the hub gene was verified by Metascape and GSEA,and HLA-B as an important hub gene related to ventricular remodeling was analyzed by CTD database.Further predict the competing endogenous RNA(ce-RNA)network of HLA-B as the hub gene of hypertension combined with ventricular remodeling,and identify the candidate small molecule drugs related to HLA-B and hypertension combined with ventricular remodeling.Results After 48 hours of treatment with different concentrations of uric acid,the expressions of OPN,α-SMA and Coll-1 in cardiac fibroblasts in UA 5mg/dl group,UA 10 mg/dl group and UA 15mg/dl group were higher than those in UA 0 mg/dl group in qPCR and WB,and the highest significance was found in UA 10 mg/dl group(P<0.001).The results of cell growth activity test showed that the growth and proliferation activity of cardiac fibroblasts in UA 5mg/dl group,UA 10 mg/dl group and UA 15mg/dl group was significantly higher than that in UA 0 mg/dl group(P<0.001),and the trend of cell proliferation activity also showed an inverted U curve.The results of CCK-8 showed that the proliferative toxicity of UA 5 mg/dl group and UA 10 mg/dl group was significantly higher than that of UA 0 mg/dl group(P=0.004).The results of Trans-well chamber migration assay showed that the cell migration ability of UA 5mg/dl group,UA 10 mg/dl group and UA 15mg/dl group was enhanced,and that of UA 10 mg/dl group was the most significant.The results of Propidium Iodide staining detection after 72 hours of stimulation with different concentrations of uric acid showed that apoptosis was reduced in UA 5mg/dl group,UA 10 mg/dl group and UA 15mg/dl group,especially in UA 10 mg/dl group.Based on Dr.Hu’s previous study,significant hub genes were found in peripheral blood samples of hypertensive patients with ventricular remodeling: HLA-B,CTSL,RHOG,FLNA,MYD88,CAPNS1 and TIMP1.Among them,the change of HLA-B is the most significant.In this study,through HLA-B differential expression analysis,it was found that the expression of HLA-B in hypertension with ventricular remodeling group was significantly higher than that in non-left ventricular remodeling group(log FC=-0.596,p = 0.037).Furthermore,the same expression trend was obtained by PCR analysis: the relative expression level of HLA-B in ventricular remodeling group was significantly higher than that in control group(P <0.05).By WGCNA analysis,the brown module contains HLA-B gene,and the correlation coefficient between brown module and ventricular remodeling is 0.67.In the HLA-B high expression group,the enrichment analysis of gene ontology(GO)showed that it was mainly manifested in cardiac phospholipid metabolism,cell migration and cardiac development.The results of enrichment analysis by Kyoto Encyclopedia of Gene and Genome(KEGG)showed that it was mainly manifested in Mitogen Activated Protein Kinases(MAPK)signal pathway and cell autophagy.ROC analysis showed that HLA-B could be used as a predictive target for ventricular remodeling with high specificity and sensitivity(area under the curve = 0.735,P =0.014).Conclusions(1)Uric acid can up-regulate the expression of OPN in cardiac fibroblasts,promote the proliferation and activation of fibroblasts,and increase the secretion of collagen.(2)Uric acid can up-regulate the expression of OPN in cardiac fibroblasts,delay the apoptosis of fibroblasts,and prolong the survival time of fibroblasts.(3)The effect of uric acid on fibroblasts promoting fibrosis was not always dose dependent,but showed an inverted U-shaped curve.(4)There is a correlation between HLA-B and hypertension combined with ventricular remodeling,which may be used as a new gene marker for clinical diagnosis of this disease. |
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