Effects Of Long Noncoding RNA Uc.25+ On Apoptosis And Inflammatory Injury Of Human Umbilical Vein Endothelial Cells In High Glucose

Background and Objective:Vascular endothelial cell dysfunction is the major performance ofdiabetic vascular complications,which damage many organs.Studies have shown that high glucose increased reactive oxygen species(ROS)in endothelial cells,decreased the nitric oxide(NO)bioavailability,induced oxidative stress,caused cell dysfunction and apoptosis.P2X7 receptor is an important upstream regulatory site in the process of inflammation and pathological damage.The activation of P2X7receptorhas a vital effect on the release of interleukin 1β(IL-1β)and interleukin 6(IL-6).The increase of intracellular Ca2+and the activation of caspase-9 and caspase-3,promoted the expression of adhesion molecules and chemotaxis molecules,which caused the adhesion and chemotaxis of monocytes and macrophages to human umbilical vein endothelial cells(HUVECs),induced the increase of apoptosis,but also accompanied with the activation of nuclear transcription factor κB(NF-κB).Many studies have shown that expression changes of lncRNAs are closely related to diabetic vascular disease.Our lncRNA and mRNA microarray results showed that lncRNA uc.25+expression was significantly increased in diabetic rats compared with control rats.The aim of this study was to investigate the effect of lncRNA uc.25+on apoptosis and inflammatory injury induced by chronic high glucose in HUVECs.Methods:The HUVECs were randomly divided into control group(Cells were cultured in 5.5mmol/Lglucose for 5 days),high glucose group(Cells were culturedin 33.3 mmol/Lglucose for 5 days),high glucose plus uc.25+short hairpin RNA(shRNA)plasmid group(Cells were transfected with 1μg/mluc.25+shRNA,then cultured with 33.3 mmol/L glucose for 5 days)and high glucose plus shRNA vector group(Cells were transfected with 1μg/mlshRNA vector,then cultured with 33.3 mmol/L glucose for 5 days).The determinations of ROS,Ca2+,adhesion and chemotaxis were detected by green fluorescence or excited by green fluorescence,in order to avoid the impact of green fluorescence on the results,siRNA(50 nmol/L)with the same target sequence of shRNA took the place of shRNA,scramble siRNA(50 nmol/L)was used in place of shRNA vetor plasmid.Fluorescence method was used to detect the intercellular concentration of ROS and Ca2+.Nitric acid reductase assay was used to detect the changes of NO in the culture medium.Adhesion and chemotaxis of THP-1 and HUVEC were detected by Transwell chamber co-culture.DAPI staining was used to detect cell apoptosis.The mRNA expression levels of uc.25+,P2X7receptor,IL-1β,IL-6,caspase-9,caspase-3 and NF-κB were detected by real time PCR.The protein expression levels of P2X7 receptor,IL-1β,caspase-9,caspase-3 and NF-κB were detected by western blotting.The expression of uc.25+in HUVEC was localized by in situ hybridization.RNA-protein immunoprecipitation(RIP)techniques were used to detect uc.25+interacted with proteins.Results:The expression levels of uc.25+in DM patients’ blood and in HUVECs cultured with high glucose were significantly increased(P<0.01).In situ hybridization results showed that uc.25+located in the nucleus and cytoplasm.The ROS content in vascular endothelial cells was significantly increased(P<0.01),Ca2+content significantly increased(P<0.05),the production of NO in the supernatant was decreased(P<0.01).These results indicated that high glucose induced oxidative stress response,and caused apoptosis.After low expression of uc.25+,ROS and Ca2+content decreased,NO production increased,suggesting that uc.25+may be involved in vascular endothelial injury oxidative stress process.Compared with the normal group,the adhesion cell counts of THP-1 to HUVEC were significantly increased(P<0.001),the chemotaxis cell counts were enhanced(P<0.01),suggesting that high glucose enhanced cell adhesion and chemotaxis.The mRNA expression levels of IL-1β,IL-6,caspase-9 and caspase-3 in the high glucose group were increased,and the protein expression levels of IL-1β,caspase-9 and caspase-3 increased significantly,apoptosis in the high glucose group was significantly increased,these results of inflammatory and apoptosis-related factors showed that high glucose induced vascular endothelial cell inflammation and apoptosis.After low expression of uc.25+,the expressions of above related factors were decreased and the nuclear apoptosis was alleviated,suggesting that uc.25+may be involved in the process of inflammation and apoptosis of vascular endothelial injury.The expressions of P2X7 receptor and NF-κB were significantly increased in HUVECs cultured with high glucose(P<0.01).The expressions of P2X7 and NF-κB were significantly inhibited by low expression of uc.25+.RIP results showed that P2X7 and NF-κB were interacted with uc.25+,suggesting that uc.25+maybe combine with P2X7 and NF-κB,involved in vascular endothelial cell apoptosis and inflammatory injury induced by chronic high glucose.Conclusion:LncRNA uc.25+was involved in chronic high glucose induced oxidative stress,inflammatory response,apoptosis of vascular endothelial cells,its mechanism may be related to P2X7/NF-κB signaling pathway.