Up-regulation Of The Transcriptional Activity Of Human ENOS Promoter By SiRNA Targeting To P38α

AIM:To investigate regulatory effects of p38a signaling pathway on the transcription activity of a human endothelial nitric oxide synthase (eNOS) promoter through p38a-targeting siRNAto silence p38a gene in a human vascμlar endothelial cell line (HUVEC-12). METHODS:To establish a dual-luciferase assay model, the cells were cotransfected with a pGL2-eNOS plus siRNA-p38a, as well as a pRL-TK vector as an internal control. The transcription activity of human eNOS promoter was determined through a dual-luciferase reporter gene system. Western blot was used to analyze the expression of P-p38a protein from the HUVEC-12 cells treated by p38α-targeting siRNA. Total RNA was extracted by QIAGEN RNeasy Mini Kit. Reverse transcription polymerase chain reaction (RT-PCR) was carried out to analyze the mRNA expression level of eNOS. Western blot was used to determine the expression of eNOS and p-eNOS protein from the cells treated by p38a-targeting siRNA. The expression and distribution change of eNOS protein were detected by immunofluorescence after p38a genes were silenced by p38a-targeting siRNA. At last, the nitric oxide level from the supernatant of the cells treated by p38α-targeting siRNA was determined by the Griess Reaction. RESULTS:The activity of human eNOS promoter was upregulated by p38a-targeting siRNA, but downregulated by LPS stimulation. Compared with the control, the expression of P-p38a was obviously weakened by interference of p38a-targeting siRNA, while the corresponding activity of eNOS promoter was upregulated. The mRNA expression level of eNOS from the cells treated by p38a-targeting siRNA was increased. The expression of P-eNOS protein was increased by interference of p38a-targeting siRNA, but the expression of unphosphorylated eNOS was not siganificantly altered. It seemed that relative fluorescence intensity of eNOS protein from the cells treated by p38a-targeting siRNA was higher than the control. Compared with the control, the concentration of NO in the supernatant from HUVEC-12 cells was enhanced by interference of p38a-targeting siRNA.CONCLUSION:The siRNA interfering with p38a signal can up-regulate the transcriptional activity of the human eNOS promoter, resulting in the increase of NO production.