Effect And Mechanism Of MiR-30a/Notch1 Axis On Podocyte Injury In Lupus Nephritis

Background:Systemic Lupus Erythematosus(SLE),which occurs in women of childbearing age,is an autoimmune disease characterized by the production of multiple autoantibodies and involves multiple organs throughout the body.Among them,lupus nephritis(LN)formed by renal involvement is the most common and has serious consequences.However,traditional therapeutic drugs such as glucocorticoids and immosuppressants have limited clinical efficacy and many side effects,so the treatment of lupus nephritis urgently needs to seek new targets.Podocytes are intrinsic cells of the kidneys that attach to the lateral side of the glomerular basement membrane and,together with vascular endothelial cells and glomerular basal membranes,form the glomerular blood filtration barrier.LN disease often involves renal podocytes and affects the glomerular filtration barrier,and such patients present with large amounts of proteinuria.Studies have shown that the Notch1 signaling pathway expresses silencing in mature podocytes,when the podocytes are damaged,the Notch1 signaling pathway is abnormally activated,and is involved in promoting the pathological process of podocyte damage,inhibition of the Notch1 signaling pathway in podocytes can improve the pathological features of podocyte damage,including lupus nephritis.MicroRNAs(miRNAs)are a class of small,highly conserved non-coding RNAs with a length of 18-25 bp,and miRNAs inhibition the translation of mRNAs by binding to the 3’ UTR of downstream target messenger RNAs(mRNAs)is one of the important ways in which they regulate gene expression at the post-transcriptional level.MiRNAs are involved in the occurrence and development of lupus nephritis,and the use of miRNAs as a biological indicator to diagnose lupus nephritis and as a new target for the treatment of lupus nephritis is one of the current research hotspots.The expression level of serum miRNAs in SLE patients screened for SLE in the previous study of the research group found that compared with the systemic lupus erythematosus without lupus nephritis(N-LN),the serum miRNA-30a expression in the LN group decreased significantly,and the biogenesis analysis suggested that Notch1 was a potential target of miR-30a.Therefore we suspect that miR-30a may be involved in podocyte damage in lupus nephritis by modulating the Notch 1 pathway.Objective:This study investigates the mechanism of miR-30a and Notch1 on podocyte damage when faced with Immunoglobulin G derived from sera of patients with lupus nephritis(IgG-LN).Methods:30 patients with primary SLE(including 15 cases of LN and 15 cases of N-LN)were selected as the disease group,28 cases of healthy control(HC)were used as the control group,and the expression level of serum miR-30a in all subjects was detected by RT-qPCR.Renal podocytes were incubated with serum-derived immunoglobulin G and cell viability was detected using CCK8 experiments,and podocyte markers(nephrin,podocin)and Notchl expression levels were detected by WB,RT-qPCR experiments.Predict and detect binding of miR-30a and its downstream Notch1 mRNA using bioinformatics analysis and luciferase reporter gene experiments.Podocytes transfection of the Notchl intracellular domain1 plasmid vector(NICD1 vector)regulated Notchl overexpression,transfection of miR-30a mimics or inhibitors(miR-30a inhibitors)upregulated or downregulated miR-30a expression.Results:1.The expression level of miR-30a in the SLE group was significantly lower than that in the HC group(P<0.05),and the expression level of miR-30a in the LN group was significantly lower than that in the N-LN group(P<0.01).The expression level of serum miR-30a was negatively correlated with anti-dsDNA antibody content in SLE patients(r=-0.429,P<0.05),and with 24h urinary protein quantification in LN patients(r=-0.645,P<0.01).2.Compared with Immunoglobulin G derived from sera of healthy controls(IgG-CON),the cell viability,podocyte markers nephrin and podocin expression,and miR-30a expression were decreased in podocytes cultured in IgG-LN.3.Luciferase reporter gene assay verified the targeted binding of miR-30a and Notch1.In podocytes,the expression of Notch1 was downregulated or upregulated following the expression of miR-30a was upregulated or downregulated.The upregulation of miR-30a mitigated the damage of podocytes incubated with IgG-LN,and the overexpression of Notch1 reversed the protective effect of miR-30a on podocytes.Conclusions1.The decrease in miR-30a expression in the serum of patients with lupus nephritis is related to the quantification of 24h urine protein in patients with LN,the increase in podocyte damage in IgG-LN incubation,and the decrease in miR-30a expression of induced podocytes,suggesting that miR-30a is related to damage to renal podocytes in lupus nephritis.miR-30a strengthens the resistance of podocytes to damage caused by IgG-LN with Notch1.2.miR-30a strengthens podocyte resistance to damage caused by serum lgG in patients with LN by inhibiting Notch1,and the effect of miR-30a on Notch1 may be through the 3’UTR of its mRNA.