RNA Interference Decreases PAR-2 Expression And Function In P815 Cells

In rencent years, protease-activated receptors (PARs) have been indentified as receptors for serine proteinases. PARs are a subfamily of seven transmembrane G-protein-coupled receptors. Among them, PAR-1, PAR-3 and PAR-4 is a receptor of thrombin[2,3,4]; PAR-1, PAR-2 and PAR-4 is a receptor of trypsin; PAR-2 is receptors of tryptase.RNA interference (RNAi) is a process in which double-stranded RNA (dsRNA) induces the postranscriptional degradation of homologous transcripts. RNAi can be initiated by exposing cells to dsRNA either via transfection or endogenous expression. With unprecedented speed, RNA interference (RNAi) has advanced from its basic iscovery in lower organisms to becoming a powerful genetic tool.RNA interference (RNAi) is an alternative strategy to examine the function of a receptor that has not been used, so far, in P815. In our study, we synthesis 3 small Interference RNA (siRNA)for PAR-1, PAR-2 and PAR-4 to analyze the effect of PARs gene silencing on P815. After being interferected, P815 cells were treated and analyzed by real time RT-PCR to detect expression of PRAs at mRNA level, flowcytometry and confocal microscopy to detect expression of PRAs at protein level. After the PARs interfected 40 hours by siRNA, P815 cells were exposed to PAR-AP, trypsin, tryptase and thrombin, culture supernatants were collected at 16 hours.Concentrations of IL-4, IL-6 and IL-13 in culture supernatants were quantified using commercial specific ELISA kits.The results showed that: PAR-1 coulde not be inhibited by siRNA PAR1-1 ,3nm siRNA PAR1-2 had the max inhibition effect for PAR-1 at 48 hours. siRNA PAR2-3 had the max inhibition effect for PAR-2. All three siRNA of PAR-4 could decresaes PAR-4 expression in mRNA and protein lever and keeped on 72 houres, siRNA PAR4-3 had the max inhibition effect for PAR-4 at 48 houres.After siRNA inhibits PAR-1,PAR-2 and PAR-4 experssion in P815, trypsin and tryptase could not promoted the IL-4 release. Trypsin and tryptase maybe effect P815 by PARs and thrombin by PAR-1 and PAR-4. The inhibit of PAR-1 by siRNA in P815 cells inhienced IL-6 release, trypsin, tryptase and thrombin induced IL-6 were most likely through activation of PAR-1 and PAR-2. The inhibit of PAR-4 by siRNA in P815 cells downregulatedIL-6 release. When PAR-1, PAR-2 and PAR-4 were inhibited by siRNA, PAR-AP, trypsin, tryptase and thrombin coulde not promoted the IL-4 release, these serine protease maybe effected IL-13 in P815 cell by PAR-1,PAR-2 and PAR-4.