The Role And Mechanism Of Sublytic C5b-9-stimulated Up-regulated MiR-3546 In Regulating GMCs Apoptosis In Thy-1 Nephritis Rats

Part Ⅰ MicroRNA expression profile in the renal tissue of Thy-1N rats and in the GMCs stimulated with sublytic C5b-9 as well as the correlation between miR-3546 expression and sublytic C5-9-induced GMCs apoptosisObjective:To examine the change of microRNAs(miRNAs)expression profile both in the renal tissues of rats with Thy-1 nephritis(in vivo)and in the rat glomerular mesangial cells(GMCs)incubated by sublytic C5b-9(in vitro).Meanwhile,to identify the miRNAs which are co-upregulated both in vivo and in vitro and related to GMCs apoptosis exposed to sublytic C5b-9,and to further analyze the correlation between miR-3546 and GMCs apoptosis.Methods:Rat Thy-1 nephritis(Thy-1N)model was established firstly,and the rat GMCs was cultured and stimulated with sublytic C5b-9.The expression profile of miRNAs both in vivo and in vitro at 0h,1h,2h and 3h was detected and analyzed by miRNA array.For co-upregulated miRNAs botn in vivo and in vitro,the qPCR experiment was used to further verify these miRNAs expression phase at 0h,0.5h,1h,2h,3h and 6h.Moreover,for the really co-upregulated miRNA both in miRNA array and in qPCR detection,the mimics of these miRNAs were synthesized and transfected into GMCs for 24h and then transfection efficiency and caspase 3 expression level was determined using fluorescence microscope,flow cytometry and western blot(WB).Furthermore,the dose-dependent caspase 3 activation in the GMCs transfected with with 7 miRNAs mimics of three different concentrations(40nM、80 nM and 160 nM)was detected using WB.Finally,the percentages of apoptotic GMCs after sublytic C5b-9 stimulation for 3h or miR-3546 mimic transfection for 24h or miR-3546 inhibitor transfection for 24h following by sublytic C5b-9 treatment for 3h were measured by flow cytometry.Results:Rat Thy-1N model was reproduced and rat GMCs was cultured.MiRNA array analysis both in vivo and in vitro showed that,there were 43 upregulated miRNAs and 21 downregulated miRNAs in vivo;and there were 62 upregulated miRNAs and 31 downregulated miRNAs in vitro.Based on the overlap of upregulated miRNAs both in vivo and in vitro,we found that 17 miRNAs were co-upregulated.Thereafter,among these 17 miRNAs,14 co-upregulated miRNAs in vivo and 11 co-upregulated miRNAs in vitro were further confirmed by qPCR.Next,to identify the GMCs apoptosis-related co-upregulated miRNAs,we synthesized 11 miRNAs mimics and then transfected GMCs with these mimics respectively,followed with caspase 3 detection using WB.The results displayed that the mimics of miR-150-3p,miR-328a-5p,miR-409-3p,miR-3546 and miR-3584-5p could obviously increase the levels of cleaved caspase 3 expression in the GMCs.In the following dose-dependent experiments,the result showed that miR-3546 mimic could markedly elevate cleaved caspase 3 expression in the best dose-dependent manner.Thus,we transfected GMCs with miR-3546 mimic or inhibitor,following with sublytic C5b-9 treatment for 3h and examined the percentage of GMCs apoptosis by flow cytometry,and the data revealed that the numbers of GMCs apoptosis were remarkably increased by sublytic C5b-9 stimulation or miR-3546 mimic transfection,but were significantly decreased after miR-3546 inhibitor transfection following with sublytic C5b-9 incubation.Conclusion:In the renal tissues of Thy-1N rats(in vivo)and in the cultured GMCs treated with sublytic C5b-9(in vitro),the expression changes of many miRNAs were exhibited.Among co-upregulated miRNAs both in vivo and in vitro,miR-3546 can not only markedly increase the expression level of cleaved caspase 3,but also obviously enhance GMCs apoptosis,indicating that miR-3546 upregulation plays a promoting role in sublytic C5b-9-induced GMCs apoptosis.Part Ⅱ Prediction and verification for target gene SOX4 of upregulated miR-3546 in the GMCs induced by sublytic C5b-9 stimulationObjective:To predict and verify the target gene SOX4 of miR-3546,and to detect the expression phase of SOX4 in the renal tissue of Thy-1N rats(in vivo)and in the GMCs stimulated by sublytic C5b-9(in vitro).Meanwhile,to determine the role of miR-3546 in regulating SOX4 gene expression.Methods:Bioinformatics tools(TargetScan,miRDB and PANTHER)were used to predict and analyze target genes of miR-3546 following with RT-PCR detection.Among predicted target genes,transcription factor SOX4,selected as a putative target gene of miR-3546,its expression level in the renal tissue of Thy-1N rats(in vivo)and in the GMCs exposed to sublytic C5b-9(in vitro)was examined using qPCR and WB at fixed time and in different treatment groups.Next,miR-3546 mimic or inhibitor was synthesized and transfected into GMCs for 24h(treated or untreated with sublytic C5b-9 for 3h),and then the level of SOX4 mRNA and protein was detected by qPCR and WB.Moreover,the luciferase reporter plasmids of SOX4 wild type(WT)and seed region mutation SOX4 3’ untranslated region(3’UTR)werel constructed and transfected into GMCs or HEK-293T cells for 48h followed with sublytic C5b-9 stimulation for 3h or co-transfected with miR-3546 mimic,then the SOX4 3’ UTR activity was measured by luciferase reporter assay.Results:Based on the predicting,analyzing and RT-PCR detection,transcription factor SOX4 was selected as a putative target gene of miR-3546.Thereafter,qPCR and WB experiments were performed and the results showed that SOX4 expression level was significantly down-regulated both in vivo and in vitro,minimum at 2h(mRNA)and 3h(protein)in vivo,and minimum at 3h(mRNA and protein)in vitro.Compared with the miR-3546 expression phase,SOX4 expression phase was opposite to miR-3546.Additionally,the GMCs transfected with miR-3546 mimic could not only significantly increase miR-3546 level,but also markedly reduce SOX4 mRNA and protein level.On the contrary,the GMCs transfected with miR-3546 inhibitor following by sublytic C5b-9 treatment for 3h,not only miR-3546 expression was greatly suppressed,but also SOX4 expression was obviously increased.Furthermore,the GMCs treated with sublytic C5b-9 could remarkably reduce WT SOX4 3’UTR luciferase activity,while there was no influence on the SOX4 3’UTR mutation.Meantime,the HEK-293T cells with miR-3546 mimic transfection also markedly lessened luciferase activity upon co-transfection with WT SOX4 3’UTR,but not for the SOX4 3’UTR mutation.Conclusion:SOX4 acted as a putative target gene of miR-3546,its expression was notably decreased both in vivo and in vitro,and the SOX4 expression phase was opposite to miR-3546.The GMCs transfected with miR-3546 mimic or miR-3546 inhibitor could really reduce or elevate the SOX4 expression level respectively.Because the SOX4 3’UTR activity could be markedly suppressed by sublytic C5b-9 stimulation and miR-3546 mimic,hence,our findings indicate that SOX4 indeed is a target gene of miR-3546 regulation.Part Ⅲ Survivin expression both in vivo and in vitro,survivin gene transcription triggered by SOX4 as well as the regulatory effect of miR-3546/SOX4/survivin on sublytic C5-9-induced GMCs apoptosisObjective:To detect survivin expression level in the renal tissues of Thy-1N rats(in vivo)and in the GMCs incubated by sublytic C5b-9(in vitro).Meantime,to explore the role and mechanism of survivin gene transcription triggered by SOX4 and sublytic C5b-9-mediated GMCs apoptosis regulated by miR-3546/SOX4/survivin axis.Methods:The mRNA and protein level of survivin in the renal tissue of Thy-1N rats(in vivo)and in the GMCs stimulated by sublytic C5b-9(in vitro)at 0h,0.5h,1h,2h,3h and 6h was first detected using qPCR and WB.Next,the plasmids of SOX4 overexpression(pIRES2/SOX4)and SOX4 short hairpin RNA(shSOX4)were constructed,and then transfected into GMCs respectively for 48h followed with(or without)sublytic C5b-9 stimulation for 3h.Thereafter,the expression levels of SOX4 and survivin were measured using qPCR and WB.Meanwhile,the expression of survivin was also examined in the GMCs after miR-3546 mimic and miR-3546 inhibitor transfection respectively,or miR-3546 mimic and pIRES2/SOX4 co-transfection.Subsequently,the full-length(FL)and 4 truncated promoter plasmids of survivin were constructed,and GMCs was transfected with the above-mentioned promoter plasmids for 48h followed with sublytic C5b-9 incubation for 3h or accompanied with pIRES2/SOX4 transfection,and then the luciferase activities of survivin promoter in the GMCs were detected.Besides,ChIP-PCR and ChIP-qPCR assays were performed to determine the binding site of SOX4 to survivin promoter and the effect of sublytic C5b-9 stimulation and SOX4 overexpression on SOX4 binding to the site of survivin promoter.Finally,WB and flow cytometry were used to detect the expression of cleaved caspase 3 and the percentage of apoptotic GMCs after SOX4 and survivin gene overexpression or knockdown followed with or without sublytic C5b-9 stimulation for 3h,or miR-3546 overexpression accompanied with SOX4 and survivin overexpression.Results:The gene expression of survivin was significantly decreased and bottomed at 3h both in vivo and in vitro.The expression phase of survivin was very similar to that of SOX4.Meanwhile,SOX4 overexpression in GMCs could obviously increase survivin expression,but knockdown of SOX4 gene markedly suppressed survivin synthesis.In addition,miR-3546 overexpression also greatly reduced survivin expression,but miR-3546 overexpression accompanied with pIRES2/SOX4 in GMCs could significantly elevate survivin production.Contrarily,the GMCs transfected with miR-3546 inhibitor could markedly inhibit survivin decrease upon sublytic C5b-9 stimulation.Afterwards,the luciferase assay verified that the promoter activity of survivin could be remarkably downregulated in response to sublytic C5b-9 treatment,but greatly enhanced by SOX4 overexpression.Furthermore,ChIP-PCR experiment manifested that the survivin promoter region(-1278～-853nt)contained SOX4 binding site,and ChIP-qPCR further exhibited that the GMCs exposed to sublytic C5b-9 could result in the decrease of SOX4 binding to the region(-1278～-853nt)of survivin promoter,and SOX4 overexpression could increase the level of SOX4 binding to the survivin promoter.Meantime,WB and flow cytometry showed that the expression of cleaved caspase 3 and the number of GMCs apoptosis were decreased or increased in the GMCs with pIRES2/SOX4 and pIRES2/survivin transfection or shSOX4 and shsurvivin transfection following with sublytic C5b-9 stimulation.Moreover,miR-3546 overexpression in GMCs accompanied with pIRES2/SOX4 or pIRES2/survivin co-transfection could markedly rescue the GMCs apoptosis mediated by miR-3546 mimic.Conclusion:The expression level of survivin was significantly decreased in the renal tissues of Thy-1N rats and in the sublytic C5b-9-treated GMCs,and the mechanism of survivin downregulation was related to the suppression of SOX4 expression by upregulated miR-3546.Because SOX4 could bind to survivin promoter,which activated survivin gene transcription,thus SOX4 down-regulation led to the decrease of survivin expression,and on the contrary,survivin downregulation promoted GMCs apoptosis triggered by sublytic C5b-9.Together,these results suggest that during the process of GMCs apoptosis in the early stage of Thy-1N,the change of miR-3546/SOX4/survivin axis really affects the GMCs apoptosis triggered by sublytic C5b-9 complex.