The Mechanisms Of LncRNA NORAD Regulating Functions Of Endothelial Cells Through MiR590-3p Under Hypoxia

Objective:As continuously improved life quality,ischemic heart diseases have been one of leading causes of death worldwide.Recently,the researches have found that myocardial ischemia could induce angiogenesis which improves the blood supply in the ischemic area.Effective treatment of reperfusion can reduce myocardial necrosis in the early stage to maintain normal heart function,due to increased blood supply.It is also important to prevent heart failure caused by late left ventricular remodelling.Although the treating methods have developed rapidly for decades,the therapeutic effects of ischemic heart diseases are still unsatisfactory,leading to more explorations of new treatment strategy.Among them,proangiogenic therapy has gained more attention because its roles in promoting effects of myocardial reperfusion treatment and increasing the function of ischemic myocardium.However,the mechanism of angiogenesis is still not completely clear.According to the existing results,it is a complex process involving multiple factors and stages.Apart from hypoxia-inducible factors,growth factors,stem cells and progenitor cells,non-coding RNAs have also been proved to take part in the process of angiogenesis.In human genome,there are?98%of genes transcribed into non-coding RNAs.In recent years,the function and effects of long non-coding RNAs?lncRNAs?have attracted more attention.It was found that lncRNAs play a regulatory role in various biological processes,including gene transcription,histone modification and DNA methylation.Besides,lncRNAs are closely related to the development of cardiovascular system,the maintenance of its physiological function and also the alteration of its pathological conditions.For example,the changes of lncRNA expression levels can be seen in the pathological conditions like myocardium infarction,heart failure,and hypertrophic cardiomyopathy.RNAs interact with each other,forming a complex network to regulate gene expressions at the post-transcriptional level.Supported by a series of experimental evidence,a competing endogenous RNA?ceRNA?hypothesis was proposed in 2011,which mainly indicated that various RNA transcripts shared the same miRNA response elements?MREs?,thereby competitively binding to corresponding miRNAs and forming ceRNAs.In a recent study,a highly conserved lncRNA during evolution has been discovered that is named NORAD?noncoding RNA activated by DNA damage?because it is induced by DNA damage.NORAD is abundant in the cytoplasm and is well-expressed in many tissues and cell lines.Inactivation of NORAD can lead to cellular mitotic defects,tetraploid cell increases and alteration of chromosome amount,even in diploid cells,suggesting an unstable phenotype of chromosome.These results inform that lncRNA NORAD may affect cell mitosis,cell proliferation and other related function.Thus far,the functional understanding of lncRNA NORAD is still limited.Therefore,this present work aimed to study the effects of NORAD on the function of endothelial cells under hypoxia and its possible mechanism.We also identified some key factors involving this process and provided novel targets and strategy for treatments of ischemic heart diseases.Methods:The primary human umbilical vein endothelial cells?HUVECs?were purchased from Shanghai Zhongqiaoxinzhou Biotech Co.,Ltd.After sub-culturing,cells between the third and tenth generation were selected to do further experiments.In hypoxia experiments,the effects of hypoxic environment on HUVEC cell viability,migration,tube formation and expression of some indicators were explored.Normoxic and hypoxic culturing environments were set up using a tri-gas incubator,in which normoxia condition was 5%CO₂,21%O₂ at 37°C and hypoxia condition was 1%O₂,5%CO₂,94%N₂at 37°C.Twenty-four hours after culturing under hypoxia or normoxia,cell viability of HUVECs was detected by CCK-8 assay.Transwell assay was used to check cell migration,Matrigel tube-formation assay was performed to check tube formation ability of HUVECs.Real-time PCR was adopted to measure the mRNA expression levels of lncRNA NORAD,miR-590-3p and HIF-1?.The protein levels of HIF-1?was evaluated by Western Blot.In transfection experiments,all cells were cultured for 24 h under hypoxia after transfection.To detect the regulation of NORAD on endothelial cell function under hypoxic condition,HUVECs were transfected with shNORAD,and co-transfected with shNORAD+NORAD_1-2000-OE.Afterward,real-time PCR was performed to detect the expression levels of NORAD and miR590-3p and their reversed alterations.Besides,corresponding restored changes of cell viability,migration and tube formation ability were also checked.In order to verify that NORAD regulated HUVEC functions through regulating miR590-3p,endothelial cells were transfected with miR590-3p mimics and co-transfected with shNORAD+miR590-3p inhibitor,followed by same detection as above.Dual luciferase reporter assay was used to confirm the direct bind effects of NORAD and miR590-3p.To study the effects of NORAD/miR590-3p axis on their downstream targets,HUVECs were transfected with shNORAD and co-transfected with shNORAD+miR590-3p,real-time PCR and ELISA assay were used to check the expression levels and alterations of VEGFA,FGF1 and FGF2.Dual luciferase reporter assay was also used to verify the binding effects of miR590-3p on VEGFA,FGF1 and FGF2.Results:In hypoxia experiments,after culturing for 24 h under hypoxia and normoxia condition,CCK-8 results showed that HUVEC cell viability was increased under hypoxia.Besides,cell migrated capacity and tube forming ability were also enhanced under hypoxic condition from the results of transwell and tube formation assays,respectively.Moreover,real-time PCR and Western Blot data showed that under hypoxia,the expression levels of NORAD and HIF-1?were up-regulated while the miR590-3p were down-regulated.In transfection experiments,we predicted that miR590-3p may be a binding target of NORAD through bioinformatics methods.HUVECs co-transfected with shNORAD+NORAD_1-2000-OE showed that up-regulated miR590-3p by NORAD knockdown only was decreased.Co-transfected cells also reversed the inhibitory effects of cell viability,cell migration and tube formation ability caused by shNORAD transfection.These results indicated that lncRNA NORAD could regulate partial functions of HUVECs under hypoxic condition.Meanwhile,cells transfected with mi R590-3p mimics showed similar effects with NORAD knockdown.Co-transfection of shNORAD+miR590-3p inhibitor could reverse the increased mi R590-3p expression and relieve the inhibitory effects of cell viability,migration and tube formation ability.Dual luciferase assay showed that cells co-transfected with miR590-3p mimics and wildtype NORAD had lower luciferase activity,but mutant NORAD had no obvious effect on it.These results informed that NORAD regulated HUVEC cell functions through mi R590-3p under hypoxia.Furthermore,VEGFA,FGF1 and FGF2 were also predicted as potential targets of miR590-3p through bioinformatics,which were also verified by dual luciferase reporter assay.Besides,the expression levels of VEGFA,FGF1 and FGF2 were decreased in NORAD knockdown HUVECs,which can be reversed by further co-transfection of shNORAD+miR590-3p inhibitor.Thus,these figures suggested that NORAD regulated VEGFA,FGF1 and FGF2 expression through miR590-3p under hypoxic condition.Conclusion:Under the hypoxic condition,NORAD was up-regulated,leading to increased HUVEC cell viability,migration and tube formation ability.Moreover,NORAD negatively regulated miR590-3p expression to further inhibit the expression of VEGFA,FGF1 and FGF2,thereby affecting HUVEC functions.Therefore,the present work indicates that lncRNA NORAD-mi R590-3p-VEGFA/FGF1/FGF2 axis plays a significant role in the process of angiogenesis under hypoxia.