Study On The Mechanism Of MiR-101a-3p In Regulating Emt In The Renal Fibrosis After Acute Kidney Injury By Targeting COL10A1 And TGF?R1

Objective:Acute kidney injury?AKI?is a group of clinical syndromes,in which ischemia-reperfusion injury,hypoxia,nephrotoxic drugs,sepsis,trauma and imbalance of humoral distribution can all cause damage of kidney,eventually lead to renal function loss,which is characterized by oliguria or anuria.Even with short-term recovery of renal function,kidneys that have experienced AKI have a greater risk of long-term Chronic kidney disease?CKD?than those who have never experienced AKI.CKD is a chronic disease,in which a variety of cytokines and signal path are involved,and the pathogenesis is complicated.renal fibrosis plays a necessary role in the development of CKD.How to prevent renal fibrosis after AKI has attracted more and more attention,which is of great significance to solve the socio-economic problems of CKD.The level of miR-101-3p?miR-101?has been reported to increase significantly in the serum of patients with AKI,which has a protective effect in AKI.the expression and role of miR-101 in the renal fibrosis after AKI remain unclear.Epithelial-mesenchymal transition?EMT?involves changes in cell morphology,loss of polarity and enhancement of motility.EMT plays an important role in the process of renal fibrosis.Inhibition of EMT can effectively alleviate renal fibrosis and inhibit the progression of CKD.The bioinformatics predicts that COL10A1 and TGF?R1bind to miR-101-3p respectively,and these two gene promote EMT and participate in the process of fibrosis.we speculated that miR-101 may inhibit EMT by targeting COL10A1 and TGF?R1 and play a protecting role in the renal fibrosis after AKI.The purpose of this study was to clarify the role of miR-101 in the renal fibrosis after AKI,to explore the mechanism which miR-101 acts on renal EMT,and elucidate its specific molecular mechanism.This study will reveal the biological mechanism in the renal fibrosis after AKI from a new perspective of miR-101,and provide more theoretical basis for the clinical treatment in the renal fibrosis after AKI.Methods:the first part,animal experiment,building animal experimental model,testing the expression of miR-101 in the renal fibrosis after AKI,predicting and verifying the changes of target genes:8 to 12 weeks C57BL/6 male mice applying ischemia-reperfusion?I/R?establish animal model,the C57BL/6 mice were average divided into two groups:control group?Sham group?and model group?I/R group?.The mice were anesthetized by intraperitoneal injection of pentobarbital sodium.The ischemia was performed for 35 min on a 38?heating pad by clamping of left renal hilus,and then the left kidney was re-perfused by releasing the clamp.Mice were killed at 42 days after I/R injury and left kidneys were collected.The experiment of Sham-operated control mice was performed without clamping of left renal hilus.Experiment grouping:Sham group,I/R group.1.HE staining was used to detect the morphological changes of renal tissues;2.Collagen formation in renal tissue was detected by Masson staining;3.Serum creatinine?Scr?was detected by enzymatic method,and serum urea?BUN?level was detected by urease colorimetry.4 real-time PCR was used to detect the expression of miR-101a-3p in renal tissues.5.Targetscan was used to predict the target genes that could bind to miR-101.6.dual luciferase reporter genes verified the targeted binding relationship between miR-101 and COL10A1,TGF?R1.The second part,animal experiments,detecting the effect and mechanism of miR-101 in the renal fibrosis after AKI:The mmu-miR-101,and its negative control?LV-NC?were obtained from the company.For overexpression,the miR-101 was amplified and subcloned into lentivirus vector.An empty lentivirus vector was used as a negative control.Lentivirus was then introduced into mice by intravenous injection?1×109 TU/ml,150?l per mouse?at 24 h after I/R injury.Mice were killed at 42 days after I/R injury and renal tissues were collected.Experimental grouping:I/R group,I/R+NC group,I/R+mmu-miR-101 group.1 the expression levels of miR-101 in three groups were detected by real-time PCR;2 HE staining was used to detect the morphological changes of renal tissues;3 Collagen formation in renal tissue was detected by Masson staining;4 The expression of?-SMA and Collagen 1 in renal tissue was detected by immunofluorescence method,and the positive expression area was quantitatively analyzed;5 real-time PCR was used to detect the expression level of?-SMA in renal tissue;6 The level of?-SMA protein in renal tissue was detected by Western blot;7 Effects of miR-101 on EMT:1)protein levels of E-cadherin and Vimentin in renal tissues were detected by Western blot;2)mRNA levels of E-cadheri and Vimentin in renal tissues were detected by real-time PCR;8 effects of miR-101on COL10A1 and TGF?R1 expression in renal tissues:1)mRNA levels of COL10A1 and TGF?R1 in renal tissues were detected by real-time PCR.2)protein levels of COL10A1 and TGF?R1 in renal tissues were detected by Western blot.The third part,cell experiments,?I?The effect of miR-101 on hypoxia-induced HK-2 cells:HK-2 cells were cultured in Dulbecco's modified Eagle's medium,containing 10%fetal bovine serum.For hypoxic culture,cells were placed in a hypoxic incubator at 37?with 1%O₂ and 5%CO₂ for 48 h.Control cells?normoxic cells?were incubated for 48 h under normoxic conditions?21%O₂,5%CO₂,37??.Experimental grouping:Normoxia,Hypoxia,Hypoxia+NC,Hypoxia+hsa-miR-101mimics.1.Effect of miR-101 on hypoxia-induced EMT in HK-2 cells:1)real-time PCR was used to detect the mRNA level of hsa-mi R-101 in the cells;2)E-cadherin,Vimentin and?-SMA levels were detected by Western blot;3)the expression of E-cadherin and Vimentin was detected by immunofluorescence assay.2.Effects of miR-101 on COL10A1 and TGF?R1 in HK-2 cells:1)mRNA levels of COL10A1and TGF?R1 were detected by real-time PCR.2)protein levels of COL10A1 and TGF?R1 were detected by Western blot.???The effect of COL10A1 on EMT in hypoxia-induced HK-2 cell:human COL10A1 siRNA and control NC siRNA were designed and chemically synthesized.HK-2 cells were transfected with si-COL10A1or si-NC,After transfection,hypoxia cells were cultured in an incubator containing 1%O₂ and normoxic cells were cultured in an incubator containing 21%O₂.Experimentalgrouping:Normoxia,Hypoxia,Hypoxia+si-NC,Hypoxia+si-COL10A1.1.Protein levels of COL10A,E-cadherin,Vimentin and?-SMA were detected by Western blot;2.mRNA levels of COL10A1,E-cadherin,Vimentin,and?-SMA were detected by real-time PCR.Results:the first part,42 days after I/R injury,mice renal tubular expansion,more inflammatory cells infiltration,part of epithelial cells atrophy and collapse,mesenchymal cells and extracellular matrix components increase,collagen in glomeruli and renal tubular interstitial increase with the performance of more focal fibrosis.The process of renal fibrosis is accompanied by loss of renal function.miR-101 was down-regulated in the renal fibrosis after AKI.The TargetScan predicted the binding sites of COL10A1 and TGF?R1 on mi R-101,COL10A1 and TGF?R1 were up-regulated in the I/R group,contrary to the trend of miR-101.Finally,the targeting relationship between COL10A1,TGF?R1 and miR-101 was verified by using double luciferase reporter gene experiment.The results showed that miR-101significantly down-regulated the luciferase activity of COL10A1 and TGF?R1-3'UTR in the wild group compared with the NC group,with statistically significant difference,while the luciferase activity of TGF?R1 and COL10A1-3'UTR mutant group showed no difference.The second part,1.Overexpression of miR-101 reduces renal fibrosis after AKI.miR-101 expression was up-regulated in the kidney tissues of the I/R+miR-101 group.HE staining and masson staining of renal tissue sections showed that after over expressing mi R-101,renal tubular and glomerular structure are more clear,basement membrane are integrity,inflammatory cells and Collagen deposition are reduced.after overexpression miR-101 Collagen-1-positive area and?-SMA-positive area is reducing,shows overexpression of miR-101 effectively attenuated kidney fibrogenesis after AKI.2.Overexpression of miR-101 alleviates renal EMT.E-cadherin and vimentin were used to quantitatively assess the EMT.miR-101overexpression lead to a significant increase in the mRNA and protein expression levels of the epithelial marker E-cadherin,while the expression levels of the mesenchymal marker vimentin were reduced.3.miR-101 inhibited the expression of COL10A1 and TGF?R.The overexpression of miR-101 down-regulated the mRNA and protein expression levels of COL10A1 and TGF?R1 in renal tissues.suggesting that miR-101 contributes to regulating COL10A1 and TGF?R1 expressions in the renal fibrosis after AKI.The third part:1.miR-101 alleviates EMT in HK-2 cells.Under hypoxia conditions?1%O₂ and 5%CO₂?,HK-2 cells were arranged in disorder,the intercellular space became wider,with the appearance of spindle long muscle fibroblasts,while the control cells showed ovoid epithelioid cell morphology.miR-101 expression was up-regulated under hypoxic conditions,which was significantly higher in hypoxia+hsa-miR-101 mimics than that in the hypoxia+NC group.HK-2 cells incubated under hypoxic conditions down-regulated expression of E-cadherin and up-regulated expression of vimentin and?-SMA,compared with those in normoxic HK-2 cells,These results indicated that the epithelial cells had more fibroblast-like cells during EMT,and hypoxia might promote EMT.Besides,miR-101 expression led to an increase of the E-cadherin expression and a reduction of vimentin and?-SMA expression in HK-2 cells under hypoxic conditions.These results suggested that miR-101 suppressed EMT process in hypoxia-induced HK-2 cells.2.The effects of miR-101 overexpression on COL10A1 and TGF?R1 were investigated:The overexpression of miR-101 down-regulated the mRNA and protein expression levels of COL10A1 and TGF?R1 in renal tissues as indicated by RT-PCR and western blotting,suggesting that miR-101 contributes to regulating COL10A1 and TGF?R1expressions in the renal fibrosis after AKI.3.After knockdown of COL10A1,E-cadherin increased,while Vimentin and?-SMA decreased,indicating that reducing the expression of COL10A1 could reduce the EMT level of hypoxia-induced HK-2cells.Conclusion:1.Renal interstitial fibrosis after AKI is an important pathological manifestation of chronic outcome of kidney maladaptive repair.2.Up-regulation of miR-101 can reduce renal fibrosis after AKI and inhibit the expression of COL10A1 and TGF TGF?R1.3.miR-101 inhibited COL10A1 and TGF?R1and alleviated EMT in HK-2 cells.Moreover,down-regulation of COL10A1 could reverse EMT of HK-2 cells.