Impact Of MicroRNA-346on Glucose-induced Epithelial-mesenchymal Transition In Mice Podocyte

BackgroundDiabetic nephropathy (DN) is one of the most common microvascularcomplications of diabetes. The morbidity of DN is increasing which leads to thegrowing incidence of end-stage renal disease (ESRD).The pathogenesis of DN iscomplex which involving multiple signaling pathways and a variety of proteins.Itspathogenesis is still not clear now. In clinical, the manifestations of DN is proteinuriawhich leads to glomerulosclerosis and renal fibrosis. Podocyte, a highly differentiatedcell, has physiological significance in maintaining the integrity of filtration membrane.Numerous studies showed that the central link of glomerular injury was the impair ofpodocytes, various physicochemical, biological factors could affect the function ofpodocytes, resulted in the development of proteinuria.MicroRNAs (miRNAs) are recently discovered endogenous non-coding smallRNAs which modulate gene expression through binding to the3’-untranslated region(3’-UTR) of target miRNA that inducing mRNA suppression or degradation at thepost-transcriptional level. miRNAs play an important role in diverse physiologicaland pathological processes.Recent studies suggested that the abnormal expression ofmiRNAs were involved in the occurrence and developmented of DN, studies showedthat the miR-346regulated the expression of GSK-3β and activated Wnt/beta -Catenin pathway which could regulate human bone marrow mesenchymal stem celltransdifferentiatio. However, the expression and function of miR-346in podocytescultured by high glucose remain unclear. Glycogen synthase kinase3β (GSK-3β) playan important role in transdifferentiation. Our previous studies had shown that the highglucose could induce epithelial-mesenchymal transition of mice podocytes. GSK-3βplayed an important role in this process.Further study should be done to understandthe relation between miR-346and GSK-3β.ObjectiveThe study explores whether miR-346is involved in the epithelial-mesenchymaltransition of mice podocytes induced by high glucose,making clear that the relationbetween miR-346and GSK-3β,at the same time the role of miR-346in thedevelopment and progression of DN.Methods1．The expression of miR-346in podocytes is under different concentration ofglucose. Podocytes were cultured36hours with normal glucose (5.6mmol/L) andnormal glucose+44.4mmol/L mannitol （achieveing isoosmolality）,differentconcentration of high glucose （12.5mmol/L,25mmol/L）.The expression of miR-346was detected by RT-PCR.2．Testing whether miR-346affects the nephrin and α-SMA of podocytes bytransfecting podocytes which cultured in high glucose（25mmol/L）with an miR-346mimic and an miR-346inhibitor.3． Testing whether miR-346directly regulate the GSK-3β expression bytargeting the3’-UTR of GSK-3β mRNA,luciferase assays were performed.4. Testing whether miR-346affects the GSK-3βof podocytes by transfectingpodocytes which cultured in high glucose （5.6mmol/L）with an miR-346mimic andan miR-346inhibitor. Results1．miR-346in podocytes changes under different glucose.Compared with the normal glucose (5.6mmol/L) cultured podocytes, miR-346was highly expressed in podocytes in glucose of12.5mmol/L.However,Theexpression of miR-346in glucose of25mmol/L is lower expressed in podocytes thanglucose of12.5mmol/L. These data showed that miR-346is highly upregulated incertain amount of glucose concentration range, However, the expression of miR-346is downregulated when the glucose exceeds the range.2．The change of nephrin protein and α-SMA protein in podocytes transfected withmiR-346mimic/inhibitor.Compared with the high glucose (25mmol/L) control group, nephrin proteinproduction was significantly elevated and α-SMA protein was markedlydownregulated in podocytes transfected with the miR-346mimic.In contrast,themiR-346inhibitor significantly downregulated the level of nephrin protein andupregulated the level of α-SMA protein.3．Luciferase report testifys whether the direct target gene of miR-346is GSK-3βTo testify wherther GSK-3β is a direct target gene of miR-346,ligated into themultiple cloning site of the plasemia to yield GSK-3β3’-UTR Luciferase activitywhich was measured using the Dual-Luciferase Reporter Assay System andnormalized to the expression of the control Renilla contrast.there is no difference inthe luciferase activity between the experimental group and control group.Theseresults demonstrated that the GSK-3β3’-UTR sequence is not recognized bymiR-346and that GSK-3β is not a direct target of miR-346.4. The change of GSK-3β protein in podocytes transfected with miR-346mimic/inhibitor.Compared with the (5.6mmol/L) control group, GSK-3β protein was markedlydownregulated in podocytes transfected with the miR-346mimic. the level ofGSK-3β protein was not changed in podocytes transfected with the miR-346inhibitor. Conclusion1．miR-346may be involved in the occurrence and development in the earlyprocess of DN.2． Upregulating expression of miR-346attenuates high glucose-inducedepithelial-mesenchymal transition of podocytes.3．The protective effect of miR-346is mediated by GSK-3β in podocytes.