The Regulatory Mechanisms Of MiR-30 In TGF-β Induced Podocyte Injury And The Function Of MiR-30 In Podocyte Macrophage-like Polarization

MicroRNAs(miRNAs)are a class of small endogenous non-coding RNA that widely exist in eukaryotes.Generally,miRNAs mediate messenger RNA degradation or translational repression by targeting the 3’-untranslated region(3’UTR)of target mRNA through pairing interaction.Recent studies have shown that miRNAs are widely involved in regulating cellular processes,including cell proliferation and apoptosis,adipogenic differentiation,epithelial-to-mesenchymal transition.miRNAs play critical roles in biological growth,development and disease pathology.It also plays indispensable roles in maintaining podocyte homeostasis.Our laboratory have found that miR-30s are abundant in podocyte and are reduced in glomeruli in focal segmental glomerular sclerosis(FSGS).MiR-30s protect podocyte from apoptosis and maintain the function of cytoskeleton both in vivo and in vitro.It’s generally accepted that TGFβ is a key regulator ofkidney pathogenesis and induces miR-30 family downregulation in podocyte.MiRNA mature after Drosha and Dicer shear.The transcriptio n start site is not clear,so its transcriptional regulation is largely unknown Therefore,in part one we investigated the molecular mechanism of TGF-β regulating miR-30d in podocyte injury.Recent researches have revealed that podocytes have features of immune cells,and display expression of surface markers of antigen presenting cells.Podocytes express various cytokines under the stimulis of inflammation.Podocytes can remove antigens in blood by surface receptors,and are considered similar to macrophage as professional antigen presenting cells which indicating that podocytes can directly involved in the immune response during development of glomerular disease.However,whether miR30s are involved in immune response is unknown Macrophage can be roughly divided into proinflammatory Ml and anti-inflammatory M2 penotypes and play diferent roles in inflammatory diseases.Through the analysis ofmiR-30 target genes,we found that the 3’UTR of SOCS3 was a candidate which was an important inhibitor of inflammatory sigialing and promoted macrophages M1 polarization.Therefore,in part two we intend to explore whether miR-30 regulateds podocytes M1 polarization through SOCS3 and the molecular mechanisms.Part One:TGF-β induces miR-30d down-regulation and podocyte injury through Smad2/3 and HDAC3 associated transcriptional repressionObjective:Previous research has indicated teat the microRNA-30 family plays important roles in maintaining kidney homeostasis.Patients with focal segmental glomerulosclerosis(FSGS)have reduced miR-30 levels in glomerulus.TGF-βrepresses miR-30s in podocytes,which leads to podocyte cytoskeleton damage and apoptosis.In this study,we aim to investigate the mechanism by which TGF-β represses miR-30d in vitro and explore potential target for the treatment ofpodocyte injury.Methods:Cultured human immortalized podocytes and HEK293 cells were used.Cells were treated with TGF-β in concentration gradient and time gradient and then qRT-PCR was used to detect miR-30s expression.We cloned miR-30d promoter region.and constructed a luciferase report plasmid.Then we constructed 5’ and 3’-truncted reporter plasmids based on the putative SBE.Through the luciferase report system,we detected miR-30d promoter activity.qRT-PCR and luciferase report system was used to detect the reverse function of TSA on miR-30d downregulation induced by TGF-β.For specific Histones deacetylase function,we used western blot to teste the protein levels of part of the HD AC family and found that HD AC3 was specifically upregula ted by TGF-β.Then we constructed the HDAC3 interference plasmid and observed miR30d expression and the activity of miR-30d promoter under the action of HDAC3 selective inhi-bitors or HD AC 3 interference.To verify the function of HDAC3,ChIP was performed to test the combination ofHDAC3 and NCoR with miR-30d promoter region.and immune co-precipitation(co-IP)was used to detect protein interactions between Smad2/3 and HDAC3,NCoR and HDAC3.Afterwards ChIP assay was used to observe the the effect of TSA and HD AC 3 selective inhibitor on inhibiting the combination between corepressor complex and miR-30d promoter.Finally,western blot was performed to detect the protein level of miR-30 target gene p53 and Notchl with TSA or RGFP966 pretreatment.Annexin V flow cytometry was used to detect podocyte apoptosis with RGFP966 pretreatment.Immunofluorescence was used to test the protective effect of TSA or RGFP966 on podocyte cytoskeleton.Results:We first verified that TGF-β downregulated miR-30 family expression which could be reversed by TSA.Bioinformatics analysis showed that several Smad binding elements were included in miR-30d promoter closed to the transcription start site.We cloned miR-30d promoter(-1799/+229)and luciferase report system displayed that TGF-β inhibited miR-30d promoter activity.Truncted reporter plasmids showed that repression of miR-30d by TGF-β was based on fragment closed to transcription start site(-383/+229)which was blocked by histone-deacetylase inhibition.TGF-βspecifically enhanced HDAC3 levels.Knockdown of HDAC3 by shRNA or a selective inhibitor RGFP966 significantly relieved the repression ofmiR-30d expression and the promoter transcription which indicated that HD AC 3 was involved in miR-30d regulation by TGF-β.Further study showed that TGF-β promoted HD AC 3 association with Smad2/3 and NCoR and caused their accumulation at the putative Smad binding site on the miR-30d promoter,which was prohibited by TSA or RGFP966.Above results indicated that TGF-β could induce Smad2/3-HDAC3-NCoR complex to downregulate miR-30d.Finally,we found that TSA or RGFP966 treatment reversed TGF-β-induced up-regulation of miR-30d targets Notchl and p53 and alleviated the podocyte cytoskeleton damage and apoptosis.Conclusion:This study uncovered the new mechanism of TGF-β induced podocyte injury through miRNA.Namely,TGF-β could induce Smad2/3-HDAC3NCoR transcriptioinal repression complex to accumulate to the promoter of miR-30d thus downregulated miR-30d downregulation and induced podocyte injury.The results highly reminds that inhibition of HDAC3 activity great therapeutic potential in TGF-β/miR-30 mediated podocyte injury.Part Two:The function of miR-30 in podocyte macrophage-like polarization by downregulating SOCS3Objective:Under inflammmatory conditions,podocytes display enhanced expression surface markers of antigen presenting cells.Inflammatory stimulis LPS and TGF-β downregulate miR-30 in podocytes.We suspected that podocytes could act as macrophages which have alternative phenotypes.We also find that there is miR-30 target sepuence in 3’UTR of SOCS3 mRNA.So part two we aim to explore whether podocytes have the potency of macrophage-like polarization and the roles of miR-30s in podocytes macrophage-like Ml phenotypic polarization.Methods:To verify whether podocytes behaved like marcrophages under M1/M2 stimulati,RT-PCR was used to detect Ml and M2 markers.qRT-PCR was performed to detect miR-30 expression.Immunohistochemistry was performed to show SOCS3 expression in podocyte injury-associated glomerular.Through TargetScan and miRbase,we found that SOCS3 was a perfect target of miR-30s.To prove the prediction,we overexpressed and sponged miR-30 to detect SOCS3 levels and constructed SOCS3 3’UTR luciferase report plasmid.Western blot and 3×SIE luciferase report assay was used to detect the association between miR-30 and SOCS3JAK/STAT pathway.Finally,wound bealing assay was used to reflect podocyte migration.Results:We first detect Ml markers in cultured podocytes with Ml stimuli LPS/TNFα,and found that IL-6 and NOS2 was upregulated in a dose and time dependent manner.Similarly,Arginase 1 and IL-10 was upregulated with the treatment of M2 stimuli Dexamethasone.LPS downregulated miR-30s,and dexamethasone could reverse it and inhibited Ml miomarker.Overexpression of SOCS3 inhibited M1 biomarker ans podocytes migration induced by LPS.Overexpression of miR-30 could inhibit production of Ml markers and podocytes migration and phagocytosis induced by LPS.Moreover,miR-30 could inhibit NF-kB signaling induced by LPS.SOCS3was upregulated in glomerulis of FSGS patients andmiR-30 sponge mice with spontaneous proteinuria.Western blot and luciferase report assay confirmed that SOCS3 was a target of miR-30.We further found that miR-30 could stimulate STAT3 activity by targeting SOCS3.Finally,we found that miR-30 inhibited podocytes inflammation,migration and phagocytosis induced by LPS through targeting SOCS3.Conclusion:This study revealed the new insight that podocytes could polarize to M1 and M2 phenotype like macrophages.miR-30 could mitigate podocyte M1 polarization induced by LPS through directly targeting SOCS3.Targeting miR-30 or SOCS3 may be an effective regulator to interfere podocyte inflammation.