LINC01116 Is Induced In Hypoxic Vascular Endothelial Cells And Regulates Inflammatory Reaction

ObjectiveOnly 2 percent of human genome RNA transcription encode proteins.The vast majority does not,named the non-coding RNA.It is cataloged by the length,and includes small non-coding RNA?<200nt?which contains miRNA and tRNA;bigger non-coding RNA?>200nt?which contains3ribosome RNA and long non-coding RNA.Nearly 80 percent of non-coding RNA belongs to long non-coding RNA.It does not directly involved in the gene encoding and protein synthesis,but regulates the gene post-transcription,RNA splicing and modification.It has been demonstrasted as an important mediator in occurrence,development,diagnosis,treatment and prognosis of disease.In recent years,it has made a lot of achievements on non-coding RNA,but most of the research was focused on small RNA,long noncoding RNA remained explored.Hypoxia is a common pathophysiologic process in variety of diseases.The research about hypoxic expression of LncRNA was rare.Moreover,most research focus on the role of lncRNA in tumor tissues or cancer cells,the rare is in vascular endothelial cells under hypoxia.Based on our previous analysis on the transcriptomic changes of human umbilical vein endothelial cell?HUVEC?exposed to 1%O₂ for 8h,we found some of differential expressed lncRNA including LINC01116.The aim of the study is to explore the expression of LINC01116 and its function in hypoxic HUVEC.MethodsHUVECs were used in this study and divided into normoxia groups?21%O₂?,hypoxia groups?1%O₂?,normoxia+medicine treatment groups and hypoxia+medicine treatment groups,hypoxia+control siRNA transfection groups and hypoxia+LINC01116 siRNA transfection groups.Hypoxic cells were incubated in a culture chamber with 1%O₂,5%CO₂ and94%N₂.The cells cultured in the chamber with air and 5%CO₂ were served as normoxic controls.1.Real-time quantitative polymerase chain reaction?qRT-PCR?was used to detect mRNA expression of LINC01116.2.Protein level of HIF-1?was enhanced through the treatment of DMOG or DFX,while protein level of HIF-1?was reduced by the treatment of YC-1 or siRNA transfection.3.Western blotting was used to detect protein level of HIF-1?,while mRNA level of GLUT-1 was analyzed to assess the transcriptional activity of HIF-1?pathway.4.Real-time quantitative polymerase chain reaction?qRT-PCR?was used to detect mRNA expression of inflammatory molecules including TNF-??IL-1??ICAM?E-SELECTIN.5.Immunochemistry analysis were used to detect the intracellular distribution of p65,and the percentage of cells with nuclear p65 was calculated.6.Western blotting was used to detect the phosphorylation of p65 in whole cell lysates and protein level of p65 in nuclear extracts.Results1.The effect of hypoxia on LINC01116 expression in HUVECs:the qRT-PCR results showed LINC01116 was significantly upregulated in HUVECs with hypoxic treatment for 4h?6h?8h?12h and 24h?P<0.01?compared with normoxic control.2.The effect of HIF-1 activity on the regulation of LINC01116expression:HIF-1?protein and GLUT-1 mRNA were increased in normoxic cells with dimethyloxalylglycine?DMOG?or desferrioxamine?DFX?treatment,while LINC01116 was upregulated.When HIF-1?protein and GLUT-1 mRNA were decreased in hypoxic cells with YC-1 treatment or with the transfection of siRNA target to HIF-1?,hypoxia induced LINC01116 expression was remarkably attenuated?P<0.01?.3.The effect of LINC01116 on the expression of hypoxia related inflammatory factors:After exposure of hypoxia for 24h,inflammatory factors?TNF-??IL-1??ICAM?E-SELECTIN?were remarkedly increased in HUVEC?P<0.05?.In LINC01116-deficient hypoxic HUVECs,hypoxia induced upregulation of inflammatory factors?TNF-??IL-1??ICAM?E-SELECTIN?were remarkably decreased?P<0.01?.4.The effect of LINC01116 on p65 expression and distribution in hypoxic HUVECs:immunofluorescent stainings analysis revealed that nuclear translocation of p65 was increased after exposure of hypoxia for24h in HUVECs,but it was attenuated in hypoxic HUVECs with siRNA transfection target to LINC01116?P<0.05?.Immunoblotting results showed that the phosphorylation of p65 was markly elevated in HUVECs after exposure of hypoxia for 24h,while it was reduced when LINC01116expression was inhibited by siRNA transfection?P<0.05?.Furthermore,protein expression of p65 was strongly induced in nuclear extracts from hypoxic HUVECs,whereas the induction was attenuated by siRNA transfection trarget to LINC01116?P<0.05?.Conclusion1.Hypoxia upregulates LINC01116 expressionin in HIF-1-dependent way.2.LINC01116 takes part in hypoxia induced inflammatory reaction through regulating inflammatory factors expression and the activity of NF-?B pathway.