Effects Of MicroRNA-let-7a On Epithelial-mesenchymal Transition In High Glucose-incubated Renal Tubular Epithelial Cells

Background:Diabetic kidney disease（DKD）is one of the major microvascular complications of diabetes.With its high prevalence and complex pathogenesis,DKD is the main cause of end-stage renal disease（ESRD）.The current treatment of DKD includes blood pressure control with angiotensin converting enzyme inhibitors（ACEI）or angiotensin receptor blockers（ARB）and strict blood glucose control,which has been successful in delaying the progression of renal damage.Despite treatment,there is substantial residual risk of disease progression with existing therapies.Therefore,there is an urgent need to better understand the molecular mechanisms driving diabetic kidney disease to help identify new therapies that slow progression and reduce associated risks.The histopathological changes in DKD include glomerular hypertrophy,mesangial cell expansion,glomerulosclerosis,tubulointerstitial fibrosis（TIF）,etc.The renal tubular epithelial-mesenchymal transition（EMT）is an important factor for TIF.Therefore,seeking methods to block or reverse EMT is important for the treatment of DKD.MicroRNAs（mi RNAs）is a class of single-stranded,non-coding RNA molecules of about 20-25 nucleotides in length.mi RNAs is a recognized epigenetic regulator and gene expression regulator involved in the occurrence and progression of many diseases.Studies have shown that mi RNAs is a key factor in regulating DKD,and it may be involved in podocyte apoptosis,extracellular matrix（ECM）accumulation,glomerular hypertrophy,and fibrosis.Studies have shown that the expression of microRNA-let-7a decreases in kidney tissues of the diabetic kidney disease rat,as well as in rat mesangial cells.MicroRNA-let-7a may participate in the progression of DKD.However,no scholars have investigated the relationship between microRNA-let-7a and EMT in renal tubular epithelial cells.After high glucose continuously stimulated NRK-52E cells for 48 hours,we found significant changes in cell morphology,decreased expression of E-cadherin protein,and increased expression ofα-SMA protein,which proved that high glucose promoted renal tubular epithelial-mesenchymal transition.The expression of TGF-β1 protein and p-p38MAPK protein was increased,indicating that TGF-β1 protein and p-p38MAPK protein may participate in EMT,and p38MAPK may be located downstream of TGF-β1.In conclusion,exploring the effect of microRNA-let-7a and TGF-β1/p38MAPK pathway on EMT is expected to discover new diagnostic and therapeutic targets for DKD.Objective:1.To observe changes of microRNA-let-7a expression in NRK-52E cells cultured with high glucose,and investigate the relationship between EMT and microRNA-let-7a expression.2.Hypoxpression or overexpression of microRNA-let-7a,investigating whether microRNA-let-7a regulates EMT via TGF-β1/p38MAPK pathway.Methods:Object of study:NRK-52E cells.1.Cells are randomly divided into NC,HG,NC+ML,HG+ML,HG+IL,HG+ML HG+MNC,HG+INC and NC+MNC.The expression of microRNA-let-7a was gauged by RT-q PCR.U6 was selected for reference genes,and the relative expression abundance was represented by 2^-△△CTvalue.2.The expression of E-cadherin protein andα-SMA protein was determined by Western blot.β-Tubulin as reference protein.3.Detecting the the expression of TGF-β1 protein、p38MAPK protein and p-p38MAPK protein by Western blot.Results:1.RT-q PCR results showed that the microRNA-let-7a was significantly decreased in HG.2.Western blot results:compared with the NC group,the expression of E-cadherin protein was decreased in HG;the expression ofα-SMA protein,TGF-β1 protein and p-p38MAPK protein were increased in HG.Compared with HG,the expression of E-cadherin protein was increased in HG+ML;the expression ofα-SMA protein,TGF-β1protein and p-p38MAPK protein were decreased in HG+ML.Conclusions:1.After Incubating NRK-52E cells with 50 mmol/L high glucose medium for 48h,microRNA-let-7a expression levels were downregulated and EMT occurred in renal tubules.2.NRK-52E cells were cultured with 50 mmol/L high glucose medium for 48h,overexpression or hypoxpression of microRNA-let-7a was able to affect the expression of E-cadherin protein,α-SMA protein.This result suggests that microRNA-let-7a may regulate the EMT.Overexpression of microRNA-let-7a maybe can reverse the EMT.3.Overexpression or hypoxpression of microRNA-let-7a was able to affect the expression ofα-SMA protein,TGF-β1 protein.This result suggests that microRNA-let-7a maybe can regulates EMT via the TGF-β1/p38MAPK pathway.