Effect Of MicroRNA-15a On Epithelial-to-mesenchymal Transition Induced By High Glucose In Peritoneal Mesothelial Cells

Background Ultrafiltration failure(UFF)caused by peritoneal fibrosis(PF)is currently a challenge for peritoneal dialysis(PD),which is the leading cause of PD patients dropping out.Previous studies have shown that peritoneal mesothelial cells transdifferentiation,inflammatory cells infiltration,neoangiogenesis and extracellular matrix deposition caused by long-term PD are important pathophysiological basis of peritoneal fibrosis,of which peritoneal mesothelialcells transdifferentiation and neoangiogenesis play the most important role.Vascular endothelial growth factor is a class of protein that selectively promotes the formation of vascular endothelial cells.Previous studies have confirmed that VEGF is closely related to the development of peritoneal mesothelial cells transdifferentiation and neoangiogenesis.The HPMCs that experienced EMT produce more VEGF,the peritoneal mesothelial cells of PD patients who have ultrafiltration failure produce more VEGF,induced VEGF expression in peritoneal mesothelial cells promoting peritoneal neovascularization,resulting in ultrafiltration failure.Therefore,the up-regulation of VEGF expression is a key factor in the development of peritoneal fibrosis and peritoneal ultrafiltration failure,but its molecular mechanism is not clear.Micro RNAs(mi RNAs,mi Rs)is a class of endogenous non-coding small RNAs in eukaryotes that is about 20-22 nucleotides in length and regulate the expression of the target gene at post-transcriptional levels.Micro RNAs regulate the expression of the target protein by completely or incompletely paired with the base of the 3 'UTR of the target mRNA,resulting in degradation of the target mRNA,or inhibition of protein translation of the target mRNA.Micro RNAs play an important role in cell growth,differentiation,apoptosis and tumorigenesis.Our previous study found that mi R-15 a was down-regulated in high glucose-induced peritoneal mesothelial cell transdifferentiation and negatively correlated with VEGF expression.We found that VEGF may be the target gene of mi R-15 a by bioinformatics analysis.Therefore,we propose that mi R-15 a is involved in the development of high glucose-induced peritoneal fibrosis via targeting VEGF.Objective This study is designed to detect the expression of mi R-15 a,VEGF and fibrosis factors such as E-cadherin,MMP9,?-SMA,Col-IV,FN in human peritoneal mesothelial cells stimulated with high glucose,to explore whether mi R-15 a involves in the epithelial-to-mesenchymal transition induced by high glucose in HPMCs,and to verify whether mi R-15 a modulates high glucose induced epithelial-to-mesenchymal transition in HPMCs by targeting VEGF.Methods HPMCs were cultured in vitro.1.To observe the effect of high glucose on the expression of mi R-15 a,VEGF,E-cadherin,MMP9,?-SMA,Col-IV,FN in HPMCs.In vitro cultured HPMCs were divided into the following four groups :(1)Normal control group(glucose concentration is 17.5mmol/L);(2)Isotonic control group(17.5mmol/L glucose+ 107.5mmol/L mannitol);(3)75mmol/L glucose group;(4)125mmol/L glucose group.HPMCs were cultured for 48 hours,morphological changes of cells were observed by light microscope.The expression of mi R-15 a and VEGF mRNA were detected by quantitive real-time PCR,the expression of VEGF,E-cadherin,?-SMA,MMP9,Col-IV,FN were detected by Western Blot.2.Luciferase reporter assay is carried out to verify VEGF is the target of mi R-15 a.Constructed wild-type and mutant VEGF 3'UTR-pmir GLO plasmid,then co-transfected the plasmid with mi R-15 a mimics into HPMCs.The luciferase activity were examined according to the manufacturer's instructions.3.To detect the effect of down-regulation of mi R-15 a or up-regulation of micro RNA 15 a on the expression of mi R-15 a,VEGF,E-cadherin,MMP9,?-SMA,Col-IV,FN in HPMCs.HPMCs were transfected with mi R-15 a mimics or inhibitor,transfection control group and normal control group were set up.HPMCs were cultured for 48 hours,The expression of mi R-15 a and VEGF mRNA were detected by quantitive real-time PCR,the expression of VEGF,E-cadherin,MMP9,?-SMA,Col-IV,FN were detected by Western Blot.4.To detect the effect of overexpression of mi R-15 a on the expression of VEGF,E-cadherin,?-SMA,MMP9,Col-IV,FN under high glucose conditions.The HPMCs were divided into the following four groups:(1)normal control group;(2)high glucose group(glucose concentration is 125mmol/L);(3)transfected with mi R-15 a mimics under high glucose condition(125mmol/L glucose+mi R-15 a mimics);(4)transfected with mi R-control under high glucose condition(mi R-control +125mmol/L glucose).The expression of mi R-15 a and VEGF mRNA were detected by quantitive real-time PCR,the expression of VEGF,E-cadherin,MMP9,?-SMA,Col-IV,FN were detected by Western Blot.Results 1.The effect of high glucose on the expression of mi R-15 a,VEGF,E-cadherin,MMP9,?-SMA,Col-IV,FN.Normal HPMCs showed a characteristic cobblestone-like appearence,then converted into a fibroblast-like morphology after continuous stimulation with high glucose for 48 hours.High glucose condition significantly decreased the expression of mi R-15 a in dose-dependent manner compared with normal control group,the expression of VEGF mRNA and protein were significantly increased in high glucose condition;the expression of E-cadherin were signifcantly decreased,the expression of MMP9,?-SMA,Col-IV,FN were significantly increased in high glucose group(P<0.05),all of them changed in dose-dependent manner.2.VEGF is the target of mi R-15 a The results of dual luciferase reporter assay showed that when HPMCs co-transfected with wild-type VEGF3'UTR and mi R-15 a mimics,the luciferase activity decreased significantly(P<0.05).3.The effect of overexpression or inhibition of mi R-15 a on the expression of VEGF,E-cadherin,MMP9,?-SMA,Col-IV,FN.The expression of mi R-15 a were increased in mi R-15 a mimics transfection group(P<0.05),whereas did not change in mi R-15 a inhibitor transfection group compared with normal control group.The protein and mRNA expression of VEGF were decreased in mi R-15 a mimics transfection group,whereas increased in mi R-15 a inhibitor transfection group(P<0.05).The expression of E-cadherin were increased in mi R-15 a mimics transfection group,whereas decreased in mi R-15 a inhibitor transfection group(P<0.05).The expression of ?-SMA,MMP9,Col-IV,FN were decreased in mi R-15 a mimics transfection group,whereas increased in mi R-15 a inhibitor transfection group(P<0.05).The above changes were not observed in transfection control group.4.The effect of overexpression of mi R-15 a on the expression of VEGF,E-cadherin,MMP9,?-SMA,Col-IV,FN under high glucose condition.The high glucose condition significantly decreased the expression of mi R-15 a and E-cadherin,increased the expression of VEGF,MMP9,?-SMA,Col-IV,FN(P<0.05)compared with normal control group.Compared with high glucose group,the expression of mi R-15 a were increased in high glucose mi R-15 a mimics transfection group,the mRNA and protein expression of VEGF were decreased,whereas the expression of E-cadherin were increased,the expression of MMP9,?-SMA,Col-IV,FN were decreased in high glucose mi R-15 a mimics transfection group(P<0.05).Conclusion 1.High glucose induced endothelial-to-mesenchymal transition in HPMCs,high glucose decreased the expression of mi R-15 a.High glucose increased the expression of VEGF,which was negatively correlated with mi R-15 a.2.VEGF is a target of mi R-15 a.3.Overexpression of mi R-15 a inhibits the expression of VEGF in HPMCs,which suggests that mi R-15 a influence the endothelial-to-mesenchymal transition via VEGF in HPMCs.4.Overexpression of mi R-15 a could reverse the endothelial-to-mesenchymal transition induced by high glucose in HPMCs.