The Effect Of SIRT6 On The Proliferation And Migration Of Human Umbilical Vein Endothelial Cells Under Hypoxia

Background: Ischemic heart disease(IHD)refers to heart disease caused by coronary artery lesions resulting in myocardial ischemia and hypoxia.IHD is a common cardiovascular disease that occurs mostly in adults over 40 years old.In recent years,the onset of the disease has become younger and has become one of the major diseases that threaten human health.IHD treatment principles include improving coronary blood supply and reducing myocardial oxygen consumption to improve patient symptoms and quality of life,preventing myocardial infarction and death to prolong survival.At present,therapeutic angiogenesis has become a new approach to IHD treatment.Therapeutic angiogenesis can prevent ventricular remodeling and protect heart function by promoting angiogenesis in the ischemic area,increasing blood flow,improving microcirculation,and reducing infarct size.Angiogenesis is regulated by many factors,so finding targets to promote angiogenesis will help to further clarify its molecular mechanism and open up new ideas for the treatment of IHD.As an anti-aging factor,silent information regulator 6(SIRT6)plays an important role in protecting the stability of the genome,anti-inflammatory and oxidative stress.SIRT6 is bidirectional for tumor angiogenesis and is related to tumor type.SIRT6 is involved in regulating cardiovascular diseases.But whether SIRT6 can participate in the regulation of IHD angiogenesis has not been reported.Objective: 1.The effect of hypoxia on the expression of SIRT6 in human umbilical vein endothelial cells(HUVECs);2.To investigate the effect of SIRT6 on the proliferation and migration of HUVECs under hypoxia conditions by interfering with the expression level of SIRT6 in HUVECs.Methods: 1.HUVECs were cultured and identified in vitro;2.Ad-SIRT6,Ad-GFP,sh-SIRT6,and sh-GFP infected HUVECs with different concentrations.Immunofluorescence was used to determine the optimal multiplicity of infection(MOI)value for infection,and the levels of SIRT6 were detected with western blot;3.HUVECs were cultured under hypoxic conditions(95% N2 and 5% CO2)for24 hours.Subsequently,real time-PCR was used to detect SIRT6 m RNA expression of HUVECs cultured under normoxia and hypoxia for 24 hours;4.After adenovirus was successfully transfected,the experiment was divided into two groups:Ad-SIRT6(SIRT6 overexpression group),Ad-GFP(overexpression control group).The effect of SIRT6 on the proliferation of HUVECs under hypoxic conditions was examined by CCK8 assay;5.The cells were divided into two groups as above,and the effect of SIRT6 on the migration of HUVECs under hypoxic conditions was evaluated by transwell assay.Results: 1.HUVECs grew on a single layer and adhered to the wall,showing a spindle shape.Immunofluorescence results showed that cell surface markers VWF and CD31 were positively expressed,confirming that the cultured cells were HUVECs;2.The best adenovirus infection efficiency was SIRT6 overexpression adenovirus 5(MOI value),overexpression control adenovirus5(MOI value),SIRT6 sh RNA adenovirus 10(MOI value),and sh RNA control adenovirus 5(MOI value).Western blot was used to further detect up-regulation and down-regulation of SIRT6 protein expression,the differences were statistically significant(P <0.05);3.Real time-PCR results showed that compared with the normoxic group,SIRT6 m RNA expression level in the hypoxic group was significantly reduced(P <0.05);4.CCK8 assay results showed that in hypoxic state,the proliferation level of HUVECs in SIRT6 overexpression adenovirus group was significantly increased compared with the overexpression control adenovirus group(P <0.05);5.Transwell assay results showed that under hypoxic conditions,the number of cells passing through the chamber in SIRT6 overexpression adenovirus group increased significantly compared with the overexpression control adenovirus group(192.3 ± 6.8 vs 68 ± 8.9;P<0.05).Conclusions: 1.The expression of SIRT6 in HUVECs was inhibited under the the condition of hypoxia;2.SIRT6 can promote the proliferation and migration of HUVECs under hypoxic conditions,suggesting that SIRT6 may become the target of angiogenesis in IHD.