Preliminary Study On The Expression And Function Of Runx1 In Vascular Endothelial Cells Under Hypoxia

ObjectiveVascular endothelium,located in the inner layer of vascular wall,is a barrier between circulating macromolecules and tissues.It is involved in regulating a variety of life activities,including inflammation,coagulation,hematopoiesis,endocrine regulation and humoral transport,by sensing and responding to changes in the internal environment.Endothelial cell response induced by hypoxia plays an important role in vascular dysfunction caused by inflammatory diseases,tumors and various factors.Therefore,it is important to understand the hypoxic response mechanism of endothelial cells for the prevention and treatment of hypoxia-related diseases.Based on the previous analysis on the transcriptomic changes of human umbilical vein endothelial cell（HUVEC）exposed to 1%O₂ for 8h,we found that the expression of Runx1（Runt-related transcription factor 1）increased significantly,but its role in the hypoxic response of vascular endothelial cells remains unclear.The aim of this study is to reveal the expression of Runx1 in HUVEC under hypoxia and to explore the role of Runx1 in the regulation of endothelial cell injury and inflammatory molecules secretion.MethodsAccording to the experimental design,HUVECs were used in this sthdy,the normal oxygen conditions were set at 37℃、21%O₂、5%CO₂、74%N₂,and the anoxic conditions were 37℃、1%O₂、5%CO₂、74%N₂.The cells were divided into 21%O₂ HUVEC group and 1%O2 HUVEC group,21%O₂ HUVEC+solvent control group and 21%O₂ HUVEC+medicine treatment group,1%O₂ HUVEC+interference sequence control group（Negative control,si-NC）group and 1%O₂ HUVEC+small interfering RNA（siRNA）group.Normoxic HUVEC was cultured in normal incubator（37℃、21%O₂、5%CO₂、74%N₂）as control group,and hypoxic HUVEC was cultured in hypoxic chamber（37℃、1%O₂、5%CO₂、74%N₂）.1.Quantitative Real-time polymerase chain reaction（qRT-PCR）and Western blot（WB）were used to detect the expression level of target genes and proteins.2.The expression of hypoxia-inducible factor-1 alpha（HIF-1α）in normoxic cells was increased by Proline hydroxylase inhibitor dimethyloxalyl glycine（DMOG）and desferrioxamine（DFX）,and inhibited by small interfering RNA.3.Western blot was used to detect the level of HIF-1α,and the transcriptional activity of HIF-1 was reflected by the mRNA level of glucose transporter-1（Glut1）.4.The level of reactive oxygen species（ROS）reflects the oxidative stress of cells,the activity of lactate dehydrogenase（LDH）in culture supernatant reflects the damage of cell membrane,and the relative expression level of Bcl-2/Bax,a protein related to apoptosis,reflects the activation of apoptotic pathway by Western blot.5.The expression and secretion of hypoxia-related inflammatory molecules were detected by qRT-PCR and enzyme-linked immunosorbent assay（ELISA）.Results1.The effect of hypoxia on Runx1 expression in HUVECs:The results of qRT-PCR showed that Runx1 was continuously over-expressed in HUVEC at 2h,4h,6h,8h,12h and 24h after hypoxia treatment,and the difference was significant compared with normoxia control group（P<0.05）.2.The effect of up-regulation or down-regulation of HIF-1a expression on Runx1 expression in normoxic HUVECs:After treatment with DMOG and DFX,the expression of HIF-1αprotein and Glut1 mRNA in normoxic HUVEC were increased,and the expression of Runx1 was remarkably increased（P<0.05）.After treatment with small interfering RNA（siHIF-1α）,the expression of HIF-1αprotein and Glut1 mRNA in hypoxic HUVEC cells decreased,while the expression of Runx1 decreased significantly（P<0.05）.3.The effect of Runx1 on hypoxic HUVECs injury:LDH activity test showed that LDH activity in cell culture supernatant increased significantly at 6,12 and 24 hours of hypoxia（p<0.05）.After interfering with the expression of Runx1（siRunx1）,LDH activity in HUVEC decreased significantly at 6h,12h and 24h of hypoxia（P<0.05）.The results of ROS detection showed that ROS in cell culture supernatant increased significantly at 24 hours of hypoxia,and the difference was statistically significant compared with the normoxia groups（P<0.05）.After interfering with the expression of Runx1（siRunx1）,ROS in HUVEC decreased significantly at 24 hours of hypoxia（P<0.05）.Western blot analysis of total protein showed that after 24 hours of hypoxia,the expression of Bcl-2protein decreased,while the expression of Bax protein increased significantly.The results showed that the ratio of Bcl-2/Bax protein decreased significantly after hypoxia（p<0.05）.After siRunx1 interference treatment,the ratio of Bcl-2/Bax protein in hypoxic HUVECs increased significantly（p<0.05）,compared with the interference sequence control group（NC）.4.The effect of Runx1 on the expression of hypoxia-related inflammatory molecules in hypoxic HUVECs:At 24 hours of hypoxia,the levels of TNF-α,IL-1βand ICAM in HUVECs were significantly increased（P<0.05）.After interfering with the expression of Runx1,the levels of the above-mentioned inflammatory molecules decreased significantly（P<0.05）.Conclusion1.Hypoxia induces the expression of Runx1 in vascular endothelial cells in HIF-1-dependent manner.2.Runx1 participates in hypoxic vascular endothelial cell injury,possibly through mediating oxidative stress and cell apoptosis.3.Runx1 is involved in the expression and secretion of inflammatory molecules in vascular endothelial cells induced by hypoxia and mediated the hypoxic inflammatory response in vascular endothelial cells...