The Effect Of SOCS1 Silencing By RNA Interference On Apoptosis In Human Umbilical Vein Endothelial Cells

OBJECTIVETo determine whether SOCS1 is expressed in human umbilical vein endothelial cell; to determine whether the expression of SOCS1 in the endothelial cell could be inhibited effectively by RNA interference ; to study the effect of SOCS1 silencing on endothelial cell apoptosis induced by anoxia/ reoxygenation.METHODS1. After Human Umbilical Vein Endothelial Cell(HUVEC) were cultured , SOCS1 expression was determined by RT-PCR.2. Four different pairs of siRNA(siRNA-1,siRNA-2,siRNA-3,siRNA-4)were designed and synthesized to silence SOCS1.3. The negative control siRNAs labelled with fluorescence(5'FAM) were divided into four groups according to different concentrations(25nmol/L,50 nmol/L,75 nmol/L,100 nmol/L) and transfected into cells with liposome.Then fluorescent microscope was employed to determine the concentration at which siRNAs were transfected most effectively.4. HUVEC were divided into eight groups: four groups transfected by the different siRNAs already designed, GAPDH positive control group (GAPDH-siRNA-PC), negative control group (siRNA-NC), mock control group(Mock) and blank control group(Blank).In these groups, these siRNAs at the optimal concentration were transfected. After 48 hours, RNAs were extracted.5. The selected siRNA as well as the negative control siRNA at the optimal concentration was transfected into HUVEC. After 24 hours, these cells were divided into four groups:â‘ . negative control siRNA;â‘¡. negative control siRNA +anoxia(20hours)/ reoxygenation(4hours);â‘¢. SOCS1 siRNA;â‘£.SOCS1 siRNA+ anoxia(20hours)/ reoxygenation(4hours).Then, Caspase-3,Bax protein expression in endothelial cells were detected by Western Blot. RESULTS1. There was high SOCS1 expression in the cultured HUVEC that was not interfered by any factor.2. It was the 50nmol/L that the siRNAs were transfected most effectively.3. The expressions of SOCS1 in the siRNA-1,siRNA-2,siRNA-3 and siRNA-4 group decreased compared to siRNA-NC,Mock and Blank group (P < 0.05) and were significantly down-regulated compared to GAPDH-siRNA-PC(P<0.01). In addition, siRNA-3 had the optimal silencing efficiency in four different pairs of siRNAs (P<0.05). The expressions of SOCS1 in siRNA-NC,Mock and Blank group decreased compared to GAPDH-siRNA-PC (P < 0.05).There was no statistical significance among siRNA-NC,Mock and Blank group(P>0.05).4. After RNA interference and anoxia/ reoxygenation, Caspase-3 and Bax protein levels in SOCS1 siRNA+anoxia/ reoxygenation group increased compared to other three groups ( P<0.05 ) . Caspase-3 and Bax protein levels in siRNA-NC+anoxia/reoxygenation group increased compared to siRNA-NC group(P<0.05). Caspase-3 protein level in siRNA-NC+anoxia/reoxygenation group increased compared to SOCS1 siRNA group(P<0.05). There was no statistical significance of Caspase-3 protein expression between siRNA-NC and SOCS1 siRNA group(P>0.05). Bax protein level in SOCS1 siRNA group was higher than that in siRNA-NC group(P<0.05).CONCLUSIONS1. For the first time,we find that there is high SOCS1 expression in the cultured HUVEC that is not interfered by any factor.2. The expression of SOCS1 in the endothelial cell could be inhibited effectively by RNA interference.3. SOCS1 silencing can exacerbate endothelial cell apoptosis induced by anoxia/ reoxygenation..