MiR-21 Attenuates Contrast-induced Renal Cell Apoptosis By Targeting PDCD4

BackgroundWith the rapid development of coronary angiography or percutaneous coronary interventions and the increase use of contrast-enhanced Computed Tomography,contrast-induced acute kidney injury(CI-AKI)has become a third most common cause of hospital-acquired renal failure,which significantly associated with increased renal and cardiovascular morbidity,mortality and societal healthcare burdens.Currently in clinical,stains,intravenous saline and limited the dose of contrast medium were used to reduce the renal injury.However,the incidence of CI-AKI was 1%-7%in common patients and would up to 50%in patients with heart failure,chronic kidney disease or diabetes mellitus.Therefore,it is important to analyze the physiopathologic mechanism of CI-AKI.Recently studies indicated that contrast medium(CM)-induced renal epithelial cell apoptosis is a major underlying cause of renal failure and associated with renal function.Therefore,it is important to analyze the molecular mechanism of apoptosis,which might benefit for preventing the CI-AKI in the future.MicroRNAs(miRNAs)are a class of small,non-coding RNA molecules with a length of 18-25 nucleotides,which suppress the gene expression by inhibition of translation or facilitation of mRNA by binding to the 3'-untranslated region(3'-UTR)with completely or not fully combine.MiRNAs plays an important role in normal biological functions such as cell growth,proliferation,differentiation,and apoptosis.With the feature of tissue-specific and stable expression in body fluids,it has become a new biomarker to identify the disease state,such as renal carcinoma,ischemia reperfusion injury and nephrotoxic drugs induced acute renal injury.Specifically,the gene sequencing of miR-21 was same in human and mouse.In addition,miR-21 has been find to regulate renal cell apoptosis and fibrosis,which were in involved in the development of CI-AKI.However,different disease model may contribute to various pathogenesis and the expression of miR-21.Until known,the change of miR-21 and whether it involved in the apoptosis process in renal tissue after CM exposure remain unknown.Programmed cell death 4(PDCD4)expressed in proliferating cells where it is known to suppress tumorigenesis and induce apoptosis.In addition,low expression level of PDCD4 can promote the proliferation,invasion and metastasis of tumor cells,which were involved in the development of CI-AKI.Target gene prediction using Targetscan and microRNA.org databases indicated that PDCD4 is a potential target of miR-21,since the PDCD4 3' UTR harboured the miR-21.Based on these characteristics,we speculated that miR-21 may regulated PDCD4 involved in renal tubular epithelial cells apoptosis induced by CM exposure.ObjectiveEstablished the model of low-osmolar induced renal cell injury in vitro and evaluated the concentration of miR-21,PDCD4 and apoptosis-related protein.Through the gain-and loss-of function experiments to analyze the effect of miR-21 in human renal proximal tubular epithelial(HK-2)cells under CM treatment and to investigate the association between miR-21 and PDCD4 expression.Methods1.HK-2 cells were treated with various concentrations of Ultravist for 2 h,2.5 h and 3 h.State of the cells were observed during the cell culture.Cytotoxicity and cell apoptosis were measured.2.HK-2 ells were transfected with miR-21-mimics,miR-21-inhibitor and scrambled control,respectively.After 48 h,cells were treated with Ultravist.The efficiency of mimic or inhibitor was confirmed by qRT-PCR.3.HK-2 cells were transfected with siRNA against PDCD4 or negative control siRNA using Lipofectamine 2000 according to the manufacturer's instructions.Expression of gene were detected by qRT-PCR.4.Terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)assay and flow cytometry(FCM)were used to detect and calculate the cell apoptosis.5.Proteins,such as Bcl-2,Bax and PDCD4,were detected by western blotting.Results1.CM decreased the activity of HK-2 cells and increased the floating cell with the increase of concentration and contact time.2.CM induces apoptosis and inhibits miR-21 expression in HK-2 cells.Apoptosis rate was increased following CM treatment,as determined by the TUNEL assay(P<0.05).Consistent with this observation,the expression of the pro-apoptotic factor Bax was increased whereas that of the anti-apoptotic factor Bcl-2 was decreased under these conditions(P<0.01).Furthermore,compared to untreated cells,miR-21 level was down-regulated by CM treatment as determined by qRT-PCR(P<0.05).3.MiR-21 overexpression inhibits CM-induced apoptosis in HK-2 cells.MiR-21 level was increased in cells transfected with mimic and reduced in inhibitor-treated cells,demonstrating successful transfection.A western blot analysis revealed that Bax expression was down-regulated whereas that of Bcl-2 was up-regulated upon transfection miR-21 mimic(P<0.05);the converse was observed in cells transfected with miR-21 inhibitor(P<0.05).Furthermore,overexpression of miR-21 mimic decreased CM-induced apoptosis whereas miR-21 inhibitor had opposite effect,as determined with TUNEL assay.4.MiR-21 inhibits HK-2 cell apoptosis by binding to the PDCD4 3' UTR.Target gene prediction using Targetscan and microRNA.org databases indicated that PDCD4 is a potential target of miR-21,since the PDCD4 3' UTR harboured the miR-21.PDCD4 expression was up-regulated in cells in the presence of CM(P<0.05).However,this effect was abrogated by overexpression of miR-21 mimic as compared to cells transfected with negative control miR-21 mimic or those that were untransfected.Moreover,PDCD4 expression was increase in cells transfected with miR-21 inhibitor as compared to the CM-only group,whereas the level was reduced upon transfection of miR-21 mimic,suggesting that miR-21 attenuates apoptosis by inhibiting PDCD4 expression.Furthermore,luciferase activity in cells transfected with pGL3-PDCD4-3' UTR-WT was reduced by about 50%relative to that in cells transfected with the MUT construct(P<0.01),suggesting that miR-21 directly binds the PDCD4 transcript and thereby regulates its expression.5.PDCD4 play an important role in the CM-induced cell injury.PDCD4 mRNA and protein levels were decreased upon transfection of siRNA-PDCD4(P<0.05).This was accompanied by down-regulation of Bax and up-regulation of Bcl-2 relative to cells treated with CM only(P<0.05).ConclusionsThe analysis indicated that CM induced the renal cell apoptosis,decreased the level of miR-21 and increased the concentration of PDCD4.Moreover,miR-21 protected renal cells against CM-induced apoptosis by directly regulating PDCD4.These results provide a basis for the development of therapeutic strategies to treat CI-AKI.Whether the miR-21/PDCD4 pathway has this function in vivo will be investigated in future studies.