The Effect Of LncRNA-ARAP1-AS2/ARAP1 On High Glucose Induced Cytoskeleton Rearrangement And EMT In HK-2 Cells

Objective: Diabetic nephropathy(DN)is one of the serious complications of diabetes,affecting nearly 40% patients with type 1 or type 2 diabetes.However,the underlying molecular mechanism leading to DN has not yet been fully elucidated.Long non-coding RNA(lncRNA)is an important component of epigenetic regulation and plays an important role in the pathogenesis of DN.Antisense lncRNA can participate in the development of disease by regulating the sense transcript.ARAP1(ArfGAP with RhoGAP domain,ankyrin repeat and PH domain 1)genes are located near T2 D risk alleles.Studies have shown that changes in protein levels are associated with cytoskeleton reorganization and Golgi apparatus remodeling and the endocytic trafficking of membrane receptors.In this study,human renal tubular epithelial cells(HK-2)were cultured in high glucose,observeing the expression changes of lncRNA-ARAP1-AS2(lncRNA-ARAP1 antisense RNA 2)and ARAP1.The ARAP1-AS2 overexpression plasmid was used to measure whether ARAP1-AS2 was involved in the damage of HK-2 cells in high glucose by regulating ARAP1.In addition,knockdown of ARAP1 expression to explore the mechanism of ARAP1 on HK-2 cell injury in high glucose,so that to discover the new molecular mechanism of DN,and provide a new strategy for the clinical treatment of DN.Methods: 1.HK-2 cells were cultured in vitro and divided into normal glucose group(NG)and high glucose group(HG).After 48 hours,using RT-PCR to detect lncRNA-ARAP1-AS2,ARAP1.The expression of ARAP1 was detected by Western blot and immunofluorescence.2.Divding the cells into 4 groups: negative control with normal glucose group(NC-NG),negative control with high glucose group(NC-HG),ARAP1 knockdown with normal glucose group(ARAP1-shRNA-NG),ARAP1 knockdown with high glucose group(ARAP1-shRNA-HG),active Cdc42 level was detected by Cdc42-GTP Pulldown assay,cytoskeleton was observed by phalloidin fluorescent staining,cell migration was observed by Transwell and Wound Healing test,and detecting the EMT and fibrosis index by Western blot.so as to explicit the role of ARAP1 knockdown in high glucose-induced HK-2 cell injury.3.Divding the cells into 4 groups: negative control with normal glucose group(NC-NG),negative control with high glucose group(NC-HG),ARAP1-AS2 overexpressed with normal glucose group(ARAP1-AS2-up-NG),ARAP1-AS2 overexpression with high glucose group(ARAP1-AS2-up-HG),RT-PCR and Western blot to detect the effect of ARAP-AS2 overexpression on ARAP1 and HK-2 cell-epithelial-mesenchymal transition(EMT).Results: 1.Culturing HK-2 cells for 48 hours,RT-PCR,Western blot and immunofluorescence showed that ARAP1-AS2 and ARAP1 expression were up-regulated in high glucose(P<0.05).2.RT-PCR and Western blot showed that pLKO.1-shRNA3 had a more significant knockdown effect on ARAP1(P<0.05).3.Cdc42-GTP Pulldown assay showed that in HK-2 cells,the level of active Cdc42 increased under high glucose state,and knockdown of ARAP1 decreased the level of Cdc42-GTP(P<0.05).4.Phalloidin fluorescent staining showed that high glucose caused the cytoskeleton reorganization and the formation of pseudopodia of HK-2 cells.Decreasing ARAP1 expression inhibited cytoskeletal rearrangement in high glucose.5.Transwell and Wound Healing test showed that culturing in high glucose for 48 hours,HK-2 cells migration increased.Knocking down ARAP1 can reduce the migration of cells in high glucose state,while knocking down ARAP1 has no significant effect on the cells migration in normal glucose.6.Western blot showed that α-SMA,FN,Collagen IV increased expression,E-cadherin expression decreased in the group of high glucose.Knockdown of ARAP1 expression in high glucose,the expression of α-SMA,FN and Collagen IV was decreased,and E-cadherin was increased(P<0.05).7.Overexpression of ARAP1-AS2 mimicked the damage of HK-2 cells in high glucose state,the expression of ARAP1 and α-SMA increased,and the expression of E-cadherin decreased(P<0.05).Conclusions: 1.ARAP1-AS2 and ARAP1 expression were increased in HK-2 cells under high glucose condition.2.In HK-2 cells,the level of Cdc42-GTP increased,cytoskeleton reorganization,and cell migration increased under high glucose condition.Knockdown of ARAP1 expression can reduce the level of Cdc42-GTP,cytoskeleton rearrangement,pseudopod formation,thereby reducing cell migration,EMT and fibrosis.Inhibition of ARAP1 expression may be a new target for the treatment of renal tubular injury in diabetes.3.ARAP1-AS2 may participate in the EMT process of HK-2 cells by positively regulating the expression of ARAP1..