The Relationship Between MicroRNA Targeting FosB And IgA Nephropathy And Its Relationship With Damp-heat Constitution

ObjectiveTo study the expression of Finkel-Biskis-Jinkins murine osteosarcoma viral oncogene homolog B(FosB)in the serum of patients with IgA nephropathy(IgAN)and in human podocytes and renal tubular epithelial(HK-2)cells;to study the effect of FosB on the proliferation and apoptosis of human podocytes and HK-2 cells,and to explore the effect of FosB on the inflammatory response of human podocytes and HK-2 cells.Look for the miRNA targeting FosB,study the role of the miRNA in the progression of IgAN,and explore the relationship between the miRNA and the damp-heat constitution.MethodsWe used the serum of 20 IgAN patients and 20 healthy controls recruited in the previous research to extract and purify pIgAl from them to make pIgA1-HMC conditioned medium,human podocytes and human HK-2 cells were cultured in pIgAl-HMC conditioned medium to form IgAN cell models and control cell models.The expression level of FosB in the serum of IgAN patients and IgAN cell models detected by qRT-PCR and Western blott.Prepare FosB recombinant lentiviral plasmid(pFosB),transfect human podocytes and human HK-2 cells with pFosB and control plasmid(pLVX)respectively,and detect the changes of FosB expression in each group by qRT-PCR and Western blot.MTT method was used to detect the proliferation of each group of cells.FITC/PI double staining method and flow cytometry analysis technology were used to detect the apoptosis and cycle of human podocytes and human HK-2 cells.Western blot was used to detect the expression of anti-apoptotic proteins in each group of cells;and the release and expression of inflammatory factors in each group of cells were detected by ELISA and qRT-PCR.The bioinformatics software predicts that miR-27a-3p and FosB 3’UTR have complementary binding sites,and the luciferase reporter gene assay verifies the targeted regulatory relationship between miR-27a-3p and FosB.The expression level of miR-27a-3p in the serum of IgAN patients and IgAN cell models detected by qRT-PCR.Different doses of miR-27a-3p inhibitor were transfected into HEK 293T cells,and the mRNA and protein expression of FosB were detected by qRT-PCR and Western blotting experiments,which proved that miR-27a-3p has a regulatory effect on FOSB.Subsequently,the synthetic miR-27a-3p inhibitor was transfected into human podocytes and HK2 cells,and the proliferation,apoptosis,and expression of anti-apoptotic proteins and inflammatory factors in each group of cells were evaluated.Using the results of high-throughput sequencing data of the serum of the pre-study Normal and Damp-heat testers,miR-27a-3p was used as the main research object,and the data was statistically analyzed.ResultsCompared with the control group,the expression of FosB was significantly lower in IgAN clinical serum samples and IgAN cell models(P<0.001).The expression of FosB in human podocytes and HK2 cells transfected with pFosB was significantly increased(P<0.001).The proliferation activity of human podocytes and HK2 cells in the pFosB transfection group was significantly increased(P<0.001),the expression of anti-apoptotic proteins Bcl-2,Mcl-1 and Bcl-xl were up-regulated,and the apoptotic rate was significantly decreased(P<0.001),the expression of inflammatory cytokines bFGF,IL-6,MCP-1,RANTES and TGF-β decreased.The luciferase reporter gene assay showed that compared with the miR-NC group,the luciferase activity of the miR-27a-3p mimic transfected group was significantly reduced(P<0.001),while the miR-27a-3p mutant transfected The luciferase activity of the stained group is not affected.The expression of miR-27a-3p in IgAN clinical serum samples and IgAN podocytes and HK2 cell models was significantly increased(P<0.001).With the increase of the dose of miR-27a-3p inhibitor,the expression levels of FosB protein and mRNA increased significantly,and the expression of miR-27a-3p and FosB in HEK 293T cells was negatively correlated.Compared with the control group,the proliferation activity of human podocytes and HK2 cells transfected with miR-27a-3p inhibitor was significantly increased(P<0.001),and the expression of anti-apoptotic proteins Bcl-2,Mcl-1 and Bcl-xl Up-regulation,the apoptosis rate decreased significantly(P<0.001),and the expression of inflammatory cytokines bFGF,IL-6,MCP-1,RANTES and TGF-β decreased.The statistical analysis of the high-throughput sequencing data obtained in the previous study found that the average expression level of miR-27a-3p in the damp-heat group was higher than that in the normal group,but it was not statistically significant(P>0.05).It may be due to the sample size being too small,or the physical identification is affected by subjective factors and causing errors.ConclusionThe expression of FosB in the serum of IgAN patients,IgAN podocytes and HK2 cell models is reduced.FosB can regulate the proliferation,apoptosis and inflammation of human podocytes and HK2 cells.The expression of miR-27a-3p is increased in the serum,IgAN podocytes and HK2 cell models of patients with IgA nephropathy.MiR-27a-3p targets and regulates FosB,which can inhibit FosB protein and mRNA expression.By regulating the expression of FosB,miR-27a-3p can inhibit the proliferation of human podocytes and HK2 cells,and promote cell apoptosis and the release of cellular inflammatory factors.The relationship between miR-27a-3p and damp-heat constitution needs further study.