| BACKGROUND AND OBJECTIVEAutophagy is a self-digestive process in the cell that participates in a variety of life activities,including nutritional hunger,the defense of intracellular pathogens,removing the damaged organelles,biological macromolecules degradation,and so on.Apoptosis has been called type I programmed cell death.It plays an important role in the different developmental processes and contributes to maintaining the balance between cells and tissues.Autophagy is closely related to apoptosis and both of them play important roles in regulating the fate of cells.Oxidative stress damage induced by high concentrations of glucose in the blood leads to apoptosis and senescence of vascular endothelial cells,and also affects autophagy and inflammation of endothelial cells.Studies have found that endothelial cell damage destroys endothelial integrity and barrier function,which is one of the important factors in the initiation of vascular diseases.Inhibition of high glucose-induced autophagy and apoptosis of vascular endothelial cells is important for maintaining the normal functions of the endothelium.The transcript of more than 200 bases from a non-protein-encoding gene,which regulates cell proliferation,differentiation,autophagy,apoptosis,inflammation,etc.called long noncoding RNA(1ncRNA).At present,only a few IncRNAs have been shown to be involved in high glucose-induced autophagy and apoptosis of vascular endothelial cells,and a large number of IncRNAs involved in this process urgently need to be identified and studied.Moreover,discovering novel IncRNAs which are capable of regulating high glucose-induced endothelial damage has important significance and application value for the diagnosis and treatment of vascular complications caused by hyperglycemia.In the previous study,we have found that a small chemical molecule,3-benzyl-5-([2-nitrophenoxy]methyl)-dihydrofuran-2(3H)-one(3BDO),can inhibit serum and FGF2-starvation-induced vascular endothelial cell apoptosis;3BDO can activate MTOR and inhibit autophagy in vascular endothelial cells;use of 3BDO,we have identified that TGFB2-OT1(TGFB2 overlapping transcript 1)promotes vascular endothelial cell autophagy and inflammation induced by lipopolysaccharide(LPS)and oxidized low density lipoprotein(oxLDL).Here,we intend to discover new IncRNAs that regulating high glucose-induced autophagy and apoptosis in vascular endothelial cells by using chemical small molecule 3BDO and lncRNA microarray analysis.RESULTSIn this study,we utilized human umbilical vein endothelial cells(HUVEC)as a cell model.We intended to explore new IncRNAs that regulate high glucose-induced vascular endothelial cell autophagy and apoptosis by utilizing a small chemical molecule,3-benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3h)-one(3BDO),and illuminate the mechanism of newly identified long non-coding RNAs in the regulation of vascular endothelial cell autophagy and apoptosis.Firstly,after treatment with high glucose(30 mM)or high glucose + 3BDO,the effect of 3BDO on high glucose-induced vascular endothelial cell autophagy and apoptosis were analyzed by immunofluorescence,acridine orange staining and western blot.Results showed that high glucose increased the number of LC3B puncta in vascular endothelial cells,promoted the formation of acidic autophagic vacuoles,up-regulated the protein level of LC3B-II,did not affect the protein level of autophagy substrate SQSTM1,and promoted the cleavage of PARP1 protein.3BDO treatment could reduce the number of high glucose-induced LC3B puncta and acidic autophagic vacuoles in cells,and down-regulate the protein levels of LC3B-? and cleaved-PARP1.Therefore,3BDO inhibited vascular endothelial cell autophagy and apoptosis caused by high glucose.Next,we used lncRNA microarray analysis,real-time PCR and bioinformatics analysis to find new lncRNA which might be regulated by high glucose and 3BDO.LncRNA CA7-4 was most significantly downregulated after treatment of vascular endothelial cells with 3BDO in all detected lncRNAs with elevated expression levels caused by high concentrations of glucose;real-time PCR analysis confirmed that high glucose upregulated and 3BDO downregulated the level of lncRNA CA 7-4.Before exploring the function of lncRNA CA7-4,we detected the expression level and subcellular localization of lncRNA CA 7-4 in cells.The data revealed that lncRNA CA 7-4 highly expressed in HUVEC,A549 and Hela cells;bioinformatics prediction,RNA-fluorescence in situ hybridization and nuclear/cytoplasmic RNA separation analysis showed that lncRNA CA7-4 located in cytoplasm and nucleus;nuclear/cytoplasmic RNA separation analysis showed that high glucose increased,3BDO reduced the level of lncRNA CA 7-4 in the cytoplasm without affecting the level of lncRNA CA 7-4 in the nucleus.To explore the role of lncRNA CA 7-4 in regulating autophagy and apoptosis of vascular endothelial cells,we constructed lncRNA CA7-4 full-length sequence overexpression plasmid(pcDNA3.1-CA47-4)and lncRNA CA 7-4 specific interfering RNA,and also detected the transfection efficiency of pcDNA3.1-CA7-4 plasmid and lncRNA CA 7-4 interfering RNA by using real-time PCR(qPCR).After overexpression and knockdown of lncRNA CA7-4,we detected the autophagy and apoptosis of high glucose-induced and normal cultured vascular endothelial cells by cellular immunofluorescence assay,hoechst 33258 staining,western blot and other experimental methods;after overexpression of lncRNA CA 7-4,vascular endothelial cells were treated with small chemical molecule 3BDO to detect the regulation of lncRNA CA 7-4 function by 3BDO.Results showed that the number of LC3B puncta and the level of LC3B-? protein in the cells increased after overexpression of lncRNA C.A 7-4;the level of LC3B-II protein decreased after knockdown of IncRNA CA 7-4,suggesting that IncRNA CA7-4 promoted vascular endothelial cell autophagy.In addition,we obtained the consistent results in hyperglycemic treatment and normal cultured vascular endothelial cells;after overexpression of IncRNA CA7-4,the protein levels of BAX,cleaved-PARP1 and cleaved-CASP3 increased.Meanwhile,the hoechst 33258 staining result also proved that overexpression of CA 7-4 promoted vascular endothelial cell apoptosis,whereas CA 7-4 knockdown reversed the results.We obtained the consistent results in hyperglycemic treatment and normal cultured vascular endothelial cells.Therefore,IncRNA CA7-4 promoted autophagy and apoptosis of vascular endothelial cells.After clarifying the regulation of vascular endothelial cell autophagy and apoptosis by IncRNA CA 7-4,we next investigated the mechanism of IncRNA CA 7-4 regulation in this process.Results of bioinformatics prediction,real-time PCR,dual luciferase reporter gene assay and RNA-binding protein imunoprecipitation(RIP)experiments showed that IncRNA CA7-4 could regulate vascular endothelial cell autophagy and apoptosis by decoying MIR877-3P and MIR5680 as a competitive endogenous RNA.LncRNA CA 7-4 could directly bind to M1R877-3P and MIR5680,and negatively regulate the levels of them,while 3BDO up-regulated the levels of MIR877-3P and MIR5680.Therefore,IncRNA CA 7-4 could function as endogenous competitive RNA.In order to further explore whether IncRNA CA 7-4 could regulate autophagy and apoptosis of vascular endothelial cells through MIR877-3P and MIR5680,we overexpressed and knocked down MIR877-3P and MIR5680,respectively,to study their roles in vascular endothelial cell autophagy and apoptosis.In vascular endothelial cells,mimics and inhibitors of MIR877-3P and MIR5680 were transfected respectively,then analyzing the effect of MIR877-3P and M1R5680 on cell autophagy and apoptosis by morphological observation,western blot,TUNEL assay,hoechst 33258 staining and cellular immunofluorescence assay.Results proved that MIR877-3P and MIR5680 weakened autophagy and apoptosis in high glucose treated and normal cultured vascular endothelial cells.Studies have shown that microRNA regulates the expression of target genes by binding to the 3'UTR of target gene mRNA.We predicted the possible target genes for MIR877-3P and MIR5680 by using the bioinformatics websites,and then detected the binding of MIR877-3P and MIR5680 to their target genes,as well as the regulatory effects of MIR877-3P and MIR5680 on the target genes by using the dual luciferase reporter gene assay,qPCR analysis and western blot.Results demonstrated that MIR877-3P directly targeted CTNNBIP1 3'UTR and inhibited the expression of CTNNBIP1 at the mRNA and protein le-vels;MIR5680 directly bonded with DPP43'UTR,and decreased DPP4 at the mRNA and protein levels.Next,CTNNBIP1 and DPP4 were knocked down respectively to detect their effects on autophagy and apoptosis of vascular endothelial cells induced by high glucose.Western blot results showed that knockdown of CTNNNBIP1 and DPP4 could weaken the autophagy and apoptosis of vascular endothelial cells induced by high glucose.Moreover,we found the possible downstream target proteins of CTNNBIP and DPP4 through literature search:CTNNB1 and AMPK.Then the levels of CTNNB1 and p-AMPK were detected after MIR877-3P,MIR5680 and IncRNA CA 7-4 overexpression and knockdown,or after 3BDO treatment.As the results showed that MIR877-3P and 3BDO increased the CTNNB1 protein level downstream of CTNNBIP1,and IncRNA CA7-4 negatively regulated the CTNNB1 level;MIR5680 and 3BDO inhibited the phosphorylation of AMPK in the downstream of DPP4,while lncRNA CA7-4 increased the phosphorylation level of AMPK.In conclusion,high glucose upregulated the level of IncRNA CA 7-4 in vascular endothelial cells,and 3BDO inhibited the effect of high glucose;IncRNA CA 7-4 promoted vascular endothelial cell autophagy and apoptosis;as a competitive endogenous RNA.IncRNA CA7-4 negatively regulated levels of MIR877-3P and MIR5680;MIR877-3P and MIR5680 targeted the 3'UTRs of CTNNBIP1 and DPP4.respectively,downregulated the expression of CTNNBIP1 and DPP4,increased the level of CTNNB1 and decreased the phosphorylation of AMPK,thereby inhibited autophagy and apoptosis of vascular endothelial cells. |
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