The Gene Expression Characteristics Of MiR-199b-5p Overexpression In Renal Podocyte

BackgroundPodocyte injury is a common pathophysiological change in variety of renal diseases.It is widely found in renal diseases such as minimal change nephropathy,focal segmental glomerulosclerosis and IgA nephropathy.Many factors,such as heredity,mechanics,stress and toxin can cause podocyte injury.Sustainable damage in podocytes can cause glomerular reduction,eventually leading to renal function decreased.MicroRNA(miRNA)are a class of noncoding RNA that regulate target gene expression at the posttranscriptional level.By inducing post-transcriptional silencing of target genes,miRNA regulate many biological processes.miRNA play an important role in podocytes and are necessary for the maintenance of podocyte homeostasis.They mainly participate in the regulation of pathological and physiological processes in kidney.This study intends to investigate the function of miR-199b-5p in podocyte apoptosis and the gene expression profiles in podocyte overexpressing miR-199b-5p for the potential molecular mechanism of podocyte apoptosis,and provide theoretical basis for the diagnosis and treatment of renal diseases caused by podocyte injury.Methods1.Mouse podocytes were cultured in vitro and miR-199b-5p mimics and miRNA-199b-5p overexpressed negative control(Negative Control,NC)were transiently transfected into mouse podocytes by lipofectamine 2000.2.Quantitative real-time PCR(qRT-PCR)was used to measure the expression of miR-199b-5p in podocytes.3.Flow cytometry was used to detect the effect of miR-199b-5p on podocyte apoptosis.4.The differential gene expression in podocytes transfected with miR-199b-5p mimics were analyzed by high-throughput sequencing.The function of the target genes was predicted via Gene Ontology(GO)analysis and pathway analysis.Results1.qRT-PCR results showed that miR-199b-5p expression in podocytes of miR-199b-5p mimics group was higher than that in control group(P<0.05).2.Flow cytometry indicated that the rate of cellular apoptosis in control group was(32.97 ± 1.45)%.The apoptosis rate in transfected mimics group(25.51 ± 0.94)%was lower compared with control group(P<0.05).3.High-throughput sequencing revealed that 106 genes were apparently down-regulated after miR-199b-5p overexpressing in podocytes.4.Gene enrichment analysis showed that the target genes of miR-199b-5p could inhibit podocyte apoptosis through NF-kB and TNF signaling pathways,and regulate the stability of actin cytoskeleton and other biological processes.ConclusionsOverexpression of miR-199b-5p could inhibit podocyte apoptosis,suggesting that this gene might play a protective role in the process of podocyte injury by regulating the target gene expression.