Effects Of SRC-1 Knock-Down On The Functions Of Vascular Endothelial Cells In Vitro

Objective Steroid receptor coactivator-1(SRC-1)has been approved to be widely expressed in vivo and possess comprehensive functions.SRC-1 can promote the progression of various cancers,influence the reproductive system,maintain the balance of energy metabolism,and play a special role in the cardiovascular system.SRC-1 can inhibit the neointima formation so as to protect the blood vessels.Besides,loss of SRC-1 will induce the synthesis disorder of ATP which results in the decrease of myocardial contractility and function and even heart failure.What's more,SRC-1 gene polymorphisms may be the potential reason for the coronary artery aneurysms formation in the patients with Kawasaki disease.As a kind of cell that owns active metabolic function,the vascular endothelial cells(VECs)have diverse functions,such as acting as a barrier,secreting vasoconstriction and vasodilation factors,balancing vascular homeostasis and participating in inflammation.Endothelial cell dysfunction(ECD)has a close relationship with the development of various cardiovascular diseases such as hypertension and atherosclerosis.Our research aimed to investigate the effects of SRC-1 knock-down on the functions of vascular endothelial cells in vitro.Methods Equivalent amounts of human umbilical vein endothelial cells(HUVECs)were seeded in six-well plates and cultivated overnight.HUVECs were transfected with siRNAs(siCtrl,siSRC-1-1 and siSRC-1-2)by Lipofectamine 2000 for 8 hours,then cultivated in complete culture solution(including recombinant human basic fibrobrast growth factor,bFGF,10ng/ml)for 62 hours.The concentrations of endothelin-1(ET-1)and nitric oxide(NO)in the supernatant were determined by enzyme-linked immuno sorbent assay(ELISA)and detection kit respectively.The proteins levels such as SRC-1,von Willebrand factors(v WF),endothelial nitric oxide synthase(eNOS),p65,p-p65,p38 and p-p38 were measured by Western blot.The mRNAs levels such as SRC-1 and endothelin-1(ET-1)were detected by qRT-PCR.Results Western blot showed that siRNAs significantly knocked down the protein levels of SRC-1 both in SRC-1-1 and SRC-1-2 [siCtrl(1.03)vs siSRC-1-1(0.30),P<0.001;siCtrl(1.03)vs siSRC-1-2(0.29),P<0.001].qRT-PCR indicated that the mRNA levels of SRC-1 in siSRC-1-1 and siSRC-1-2 were significantly reduced [siCtrl(1.00)vs siSRC-1-1(0.67),P<0.001;siCtrl(1.00)vs siSRC-1-2(0.73),P<0.01].Both of the results of Western blot and qRT-PCR confirmed that we had successfully knocked down the expression of SRC-1 in HUVECs.Western blot showed that vWF levels in siSRC-1-1 and siSRC-1-2 were higher than that in siCtrl [siCtrl(1.05)vs siSRC-1-1(1.76),P<0.01;siCtrl(1.05)vs siSRC-1-2(2.11),P<0.001].ELISA revealed that knock-down of SRC-1 augmented ET-1 [siCtrl(3.13)vs siSRC-1-1(19.52),P<0.001;si Ctrl(3.13)vs siSRC-1-2(17.95),P<0.01].qRT-PCR also showed that knock-down of SRC-1 increased ET-1 mRNA [siCtrl(1.00)vs siSRC-1-1(3.35),P<0.01;siCtrl(1.00)vs siSRC-1-2(3.44),P<0.01].There were no changes of NO after knocking down SRC-1 [siCtrl(31.83)vs siSRC-1-1(30.62),P>0.05;siCtrl(31.83)vs siSRC-1-2(30.25),P>0.05].No differences of eNOS,p65,p-p65,p38 and p-p38 were seen in siCtrl,siSRC-1-1 or siSRC-1-2 after knocking down SRC-1 [eNOS: siCtrl(1.01)vs siSRC-1-1(0.97),P>0.05;siCtrl(1.01)vs siSRC-1-2(1.06),P>0.05;p-p65/p65: siCtrl(0.97)vs siSRC-1-1(0.96),P>0.05;siCtrl(0.97)vs siSRC-1-2(0.93),P>0.05;p-p38/p38: siCtrl(0.98)vs siSRC-1-1(0.94),P>0.05;siCtrl(0.98)vs siSRC-1-2(1.05),P>0.05].Conclusion SRC-1 has a protective effect on the VECs and may adjust vasodilatation.In short,SRC-1 can regulate the VECs' functions,and loss of SRC-1 will lead to vascular endothelial cell dysfunction.