Long Non-Coding RNA MALAT1 Mediated High Glucose-induced HK-2 Cell EMT And Injury

ObjectivesWe cultured HK-2 cells(human proximal tubular epithelial cells)in vitro and investigated mRNA level of MALAT1 in HK-2 cells treated with high glucose.Furthermore,we examined whether the up-regulated MALAT1 induced by high glucose mediated EMT and injury in HK-2 cells.Lastly,we attempted to study whether Wnt/?-catenin was involed in these processes.Methods1.Detection the expression of MALAT1 in HK-2 cells under high glucose condition:HK-2 cells were cultured with normal glucose,mannitol and high glucose for 12 h,24 h,36 h,and 48 h.RT-qPCR was used to detect the mRNA level of MALAT1.2.MALAT1 mediated EMT and injury in high glucose-treated HK-2 cells:We transfected MALAT1 siRNA and negative control siRNA into HK-2 cells.Western blot was used to detect the protein level of E-Cadherin and a-SMA,RT-qPCR was used to exam the mRNA level of E-Cadherin,?-SMA,KIM-1,and NGAL and the protein levels of KIM-1 and NGAL were tested by ELISA.3.The mechanism under up-regulated-MALAT1-mediated EMT and injury in HK-2 cells:After we transfecte MALATl siRNA into HK-2 cells,we detected the protein levels of?-catenin?c-Myc?cyclin D1 and the location of ?-catenin in HK-2 cells using immunofluorescence.The EMT,injury,and Wnt/?-catenin related markers were detected when the cells were treated with LiCl or DKK-1.Last,we added LiCl or DKK-1 into transfected-cell and exmined the expression of EMT markers.Results1.MALAT1 was up-regulated in high glucose-treated HK-2 cells.Treatment with HG significantly increased the mRNA level of MALAT1 in a time-dependent manner and its mRNA level peaked at 36 h.The expression of MALAT1 was not changed in mannitol-treated cells.2.Up-regulated MALAT1 induced EMT and injury in HK-2 cells.High glucose induced decreased E-Cadherin and increased ?-SMA,KIM-1,and NGAL both at protein and mRNA levels.After the cells were transfected with MALAT1 siRNA,the changes described before could be partly reversed.3.Up-regulated MALAT1 activated Wnt/?-catenin pathway in high glucose-treated HK-2 cells.Western blot showed that high glucose and LiC1 could up regulated the protein ofCyclin D1,c-Myc,? ?-catenin,whereas DKK-1 could reverse these effect of high glucose.After we transfected MALAT1 siRNA into HK-2 cells,the up-regulation of Cyclin D1,c-Myc,? ?-catenin induced by high glucose could be partly inhibited,whereas negative control siRNA showed no difference compared with high glucose group.4.Up-regulated MALAT1 mediated EMT in HK-2 cells through Wnt/?-catenin pathway.Western blot results demonstrated that LiCl significantly induced decreased E-Cadherin and increased a-SMA even though MALAT1 was knockdown in HK-2 cells,and DKK-1 could further alleviate HG-induced EMT on the base of knocking down MALAT15.Up-regulated MALAT1 mediated-injury in HK-2 cells was not associated with Wnt/p-catenin pathway.Our data showed that LiCl exposure depressed KIM-1 and NGAL to an even lower level than that of the normal glucose and mannitol groups.Meanwhile,DKK-1 treatment could not decrease the expression of KIM-1 and NGAL under high glucose condition,which indicated that activation of the Wnt/?-catenin pathway might play a protective role in injury of HK-2.ConclusionOur results indicated that up-regulated MALAT1 mediated EMT and injury in high glucose-treated HK-2 cells,in which MALAT1 might induce EMT through activation of Wnt/?-catenin pathway.However,activation of the Wnt/?-catenin pathway might play a protective role in injury of HK-2.Our results provide a framework for developing novel nephroprotective therapies by targeting MALAT1.