MiR-877-3p Induces Tubular Epithelial Cell Apoptosis Through Targeting Bcl-2 In Diabetic Kidney Disease

BackgroundDiabetic kidney disease(DKD)is one of the most important microvascular complications in diabetic patients and is the main cause of end-stage renal disease(ESRD)in western countries.Previous studies have suggested that diabetic kidney disease is mainly due to glomerular lesions in high glucose conditions that result in the production of proteinuria.Although changes in the glomerulus are important,studies have suggested that the tubules not only negatively respond to the glomerulus,thereby affecting the glomerular lesions,but that because the renal tubules are more susceptible to hemodynamics,lesions are more likely to occur.Therefore,focusing on the effect of renal tubular disease on DKD has a key role in delaying its progression.Progressive forms of kidney disease exhibit common pathological processes such as fibrosis and renal cell apoptosis.The renal proximal tubules,which are susceptible to various metabolic and hemodynamic factors,play a key role in apoptosis.Apoptosis is the process of strict control of multiple genes.These genes are very conserved among species,such as Bcl-2 family,caspase family,and so on.Bcl-2 in the Bcl-2 family is the first recognized anti-apoptosis factor that inhibits Caspase-3 activation and prevents apoptosis.In recent years,microRNA(miRNA)is an endogenous short non-coding RNA with a length of 22 bases,which has attracted wide attention in the development of DKD.Studies have confirmed that miRNA plays a regulatory role in various physiological and pathological processes such as cell proliferation,differentiation,and apoptosis.At present,more than 2,000 miRNAs have been found in the human genome,and studies have shown that miRNAs are associated with diabetic kidney disease,such as miR-30c and miR-26a.At the early stage of the research group suggested that through the nalysis of the differential expression profiles of urine exosomes in patients with type 2 diabetic kidney disease,miR-877-3p in patients with DKD was 4.26 times more than that of type 2 diabetes patients.In recent years,studies have shown that,miR-877-3p can affect the progress of IgA nephropathy,but also can inhibit cell proliferation.However,there is no study about miR-877-3p in the process of DKD.Therefore,this study aims to explore the role of miR-877-3p in renal tubular epithelial cell apoptosis and its mechanism in diabetic kidney disease.Method1.Animal experimentAccording to C57BL/6J mice(n = 12)induced by STZ combined with high fat diet to establish diabetic kidney disease model by research group.Meanwhile,normal mice(n = 12)were used as the control group.Diabetic group and control mice were sacrificed at 6w and 12w.Kidney tissue was frozen at-80 ?.2.Cell cultureThe human renal tubular epithelial cell line(HK-2)was obtained from the cell bank of Chinese Academy of Sciences,Shanghai.HK-2 was cultured in DMEM medium containing 1.0g/L glucose containing 10%FBS and incubated in a humidified atmosphere of 5%C02 at 37?.The medium is changed every 48h.3.Cell transfection(1)miRNA mimic and inhibitor transfectionThe day before transfection,cells were seeded in 12-well plates and the cell density reached 70%-80%upon transfection.50nM miRNA mimic and inhibitor were transfeced into HK-2 cells using Lipofectamine@3000.(2)Plasmid and mimic cotransfectionThe day before transfection,the cells were seeded in a 24-well plate and the cell density reached 70%-80%upon transfection.50nM miRNA mimic and 1?g luciferase reporter plasmid were transfected into HK-2 cells.4.RNA extraction and qRT-PCRTrigol was used to extract total RNA from mouse kidney and HK-2 cells.Using M-MLV reverse transcriptase reagent reversed transcription of RNA.SYBR method was used to detect the gene expression level by real-time fluorescence quantitative PCR.The relative value of each gene was calculated by the 2-??Ctmethod using GAPDH as an internal control.5.Western BlotRIPA lysis buffer extracted HK-2 cells and kidney total protein.BCA method was used for the determination of protein concentration.Equal amount of proteins(20?g)were electrophoresed using 12%SDS-PAGE gels and then transferred to PVDF membrane.The PVDF membrane was blocked and incubated with primary antibodies overnight at 4? shaker.After washing with TBST buffer,add secondary antibody(1:5000)for 2h at room temperature.After being washed again in TBST,the color was developed with enhanced chemiluminescence and the results were scanned,and the target band gray values were analyzed with Image J software.6.Luciferase reporter assayThe 3'UTR region vector of wild-type and mutated-type Bcl-2 were constructed.RLU values were determined on a multifunctional microplate reader using the Promega Dual Luciferase reporter assay kit.7.Statistical AnalysisAll data were analyzed using SPSS 20.0 statistical software.All results are expressed as x±SEM.Two samples were too distributed using independent samples t test,and multiple sets of data were used one-way ANOVA.LSD was used to compare the mean of variance and the variance analysis of Welch method.P values less than 0.05 was considered significant.Result1.The expression of miR-877-3p in renal cortex of DM mice induced by STZ combined with high fat diet Compared with the control group,the expression of miR-877-3p in renal cortex of DM group increased obviously at 6W,and further increased at 12W of DKD.2.The expression of miR-877-3p in high glucose-induced HK-2 cells.The expression level of miR-877-3p in HK-2 cells gradually increased with the stimulation of 30mmol/L high glucose stimulation time,reaching the peak at 48h.This further indicates that miR-877-3p may be involved in the pathogenesis of DKD.3.miR-877-3p regulated high glucose-induced apoptosis through the target gene Bcl-21)Target gene identificationa)Targetscan bioinformatics software was used to showed the binding site of miR-877-3p in the 3'UTR region of Bcl-2 gene.The luciferase reporter assay confirmed that miR-877-3p directly targets the 3UTR of Bcl-2.b)qRT-PCR assays demonstrated that HK-2 cells transfected with miR-877-3p mimic down-regulated Bcl-2 expression,and Bcl-2/Bax decreased severely;that miR-877-3p inhibitor up-regulated Bcl-2 expression,and Bcl-2/Bax increased.2)miR-877-3p regulated HK-2 cell apoptosis.a)qRT-PCR assays demonstrated that inhibitors of miR-877-3p down-regulated Bax in the presence of 30 mmol/L high glucose,but up-regulated Bcl-2.Conclusion1.In early DKD mouse kidney cortex the expression of miR-877-3p increased.2.The effect of high glucose on the expression of miR-877-3p in HK-2cells was time-dependent.MiR-877-3p could promote the apoptosis of renal tubular epithelial cells in the progression of DKD.3.Luciferase reporter gene experiments confirmed that miR-877-3p targets Bcl-2.Overexpression of miR-877-3p down-regulated Bcl-2 expression,up-regulated of Bax.4.miR-877-3p regulates cell apoptosis3 by changing the expression of Bcl-2/Bax.