The Inhibition Of Human Microvascular Endothelial Cells(HMEC-1) By Simultaneous Interdiction Of SP1 And HIF1 Under Hypoxia Condition

Background:Retinal neovascular disease is one of the significant diseases of blindness and is harm to eyesight.The main reason for neovascularization is known that local hypoxia state of retina to induce angiogenesis and its mechanism is becoming hot topic.Studies showed that hypoxia stimulates the activation of many transcription factors and vascular endothelial cells,which leads to angiogenesis.Besides to the vascular endothelial growth factor,special protein 1(SP1)and hypoxic inducible factor 1-α(HIF1-α)also can lead to angiogenesis.According to recent researches,SP1 and HIF1-α bind the promoter region of VEGF,which can regulate and control the expression of VEGF;We also find that adenosine levels associated with the activation of Human microvascular endothelial cells,because SP1 and HIF1-α can regulate the key enzyme of adenosine: adenosine triphosphatase(CD39)and 5’-nucleotidase(CD73).Our study research that double genes knockdown of SP1 and HIF1-α can restrain the proliferation,migration and invasion ability of human microvascular endothelial cell(HMEC-1)more than single gene knockdown of SP1 or HIF1-α and traditional anti-VEGF drugs(Ranibizumab Injections).This study will provide a new treatment for retinal neovascular diseases.Object:1.Under hypoxia model,the expression of SP1 and HIF1-α increase in HMEC-1,and decrease by SP1 or/and HIF1-α knockdown.2.Double genes knockdown of SP1 and HIF1-α can restrain the proliferation,migration and invasion ability of HMEC-1 more than single gene knockdown of SP1 or HIF1-α.3.Double genes knockdown of SP1 and HIF1-α decrease the activity of HMEC-1 through inhibiting the methods of both VEGF-pathway and CD39-CD73-adenosine-pathway.4.Double genes knockdown of SP1 and HIF1-α restrain the proliferation,migration and invasion ability of HMEC-1 more than traditional anti-VEGF clinical drugs(Ranibizumab Injections).Method:1.Hypoxia group(hypoxia incubator: 37 ℃,volume fraction of 1% O2,94% N2 and 5% CO2)and normoxia group(normoxia incubator: 37 ℃ and 5% volume fraction of CO2),incubate for 12 hours and 24 hours,respectively.We tested and compared the expression of SP1 and HIF1-α in HMEC-1 by Western blot in hypoxia group,normoxia group,gene knockdown group.2.Experiment are divided into hypoxia group,normoxia group,si RNA-Control group(Control-si),si RNA-SP1 group(SP1-si),si RNA-HIF1-α group(HIF1-α-si),si RNA-double knockdown group(SP1-si + HIF1-α-si),incubate for 24 hours.We tested and compared the restrained effect of HMEC-1 in every group by CCK8,EDU,Transwell-migration,Transwell-invasion.3.Experiment are divided into siRNA-Control group(Control-si),si RNA-SP1 group(SP1-si),si RNA-HIF1-α group(HIF1-α-si),si RNA-double knockdown group(SP1-si + HIF1-α-si),incubate for 24 hours.We tested and compared the expression of VEGF,CD39,CD73 in HMEC-1 by Western blot and immunofluorescence.And we tested the concentration of adenosine in HMEC-1 by High performance liquid meter.4.Experiment are divided into si RNA-double knockdown group(SP1-si + HIF1-α-si),anti-VEGF group(0.5mg/ml Ranibizumab Injections),incubate for 24 hours.We tested and compared the restrained effect of HMEC-1 in every group by CCK8,EDU,Transwell-migration,Transwell-invasion.Result:1.Under hypoxia model,the expression levels of SP1 and HIF1-α are higher in hypoxia group than normoxia group,and depend on time.siRNA SP1 or/and HIF1-α can effectively inhibit its expression in cells under hypoxia condition.2.Under hypoxia model,the cells activation effects are higher in hypoxia group than normoxia group.And the inhibition effects of activation of si RNA-double knockdown group are higher than the siRNA-SP1 or siRNA-HIF1-α group.3.Under hypoxia model,SP1 and/or HIF1-α si RNA,can effectively restrain expression of VEGF,CD39,CD73 and adenosine,and the inhibition effect s of siRNA-double knockdown group are higher than the siRNA-SP1 or si RNA-HIF1-α group.4.Under hypoxia model,SP1 and HIF1-α si RNA group have more obviously restrained effects on HMEC-1 than Anti-VEGF group.Conclusion:Above results show that hypoxia situation can increase the expression levels of SP1 and HIF1-α in HMEC-1.SP1 or/and HIF1-α gene knockdown can decrease the cells activation effects(proliferation,migration and invasion ability of HMEC-1)through both VEGF-pathway and CD39-CD73-adenosine-pathway and double knockdown SP1 and HIF1-α are more effective than single knockdown SP1 or HIF1-α and traditional anti-VEGF clinical drugs(o.5mg/ml Ranibizumab Injections).So our study proves that both SP1 and HIF1-α genes play important roles in hypoxia condition induced activation effects of HMEC-1,and the further study about double targets knockdown effects can be researched to make it become the new treatment of retinal neovascular diseases.