Open Access Methodology article

Downregulation of protease activated receptor expression and cytokine production in P815 cells by RNA interference

Liya Qiao1†, Huiyun Zhang1†, Shandong Wu2† and Shaoheng He1,3*

Author Affiliations

1 Allergy and Inflammation Research Institute, Shantou University Medical College, 22 Xin-ling Road, Shantou, Guangdong 515041, PR China

2 Zhejiang University Medical College, Science and Research Building, Block C, Hangzhou 310013, PR China

3 Clinical Research Center, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, PR China

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BMC Cell Biology 2009, 10:62 doi:10.1186/1471-2121-10-62

Published: 7 September 2009

Abstract

Background

Protease-activated receptors (PAR) are seven transmembrane G-coupled receptors comprising four genes (PAR-1 ~ PAR-4). Mast cell has been identified to be able to express PARs and release an array of cytokines upon activation. Recently, it was reported that interleukin (IL)-12 could regulate the expression of PARs in mast cells, and tryptase could induce IL-4 and IL-6 release from mast cells. In order to further investigate the issues, RNA interference (RNAi) technique was employed and small interfering RNAs (siRNA) of PARs were transfected in P815 cells.

Results

The results showed that siRNAs for PAR-1, PAR-2 and PAR-4 significantly downregulated expression of PAR-1, PAR-2 and PAR-4 mRNAs and proteins in P815 cells at 24, 48 and 72 h following transfection. siRNA PAR-1.2 and siRNA PAR-4.2 significantly reduced IL-12 induced upregulation of PAR-1 and PAR-4 expression, respectively when P815 cells were transfected with them for 48 h. siRNA PAR-2.3 blocked IL-12 induced downregulation of PAR-2 expression on both mRNA and protein levels. It was also observed that siRNA PAR-2.3 and siRNA PAR-1.2 reduced trypsin induced IL-4 release by approximately 92.6% and 65.3%, and SLIGKV-NH2 induced IL-4 release by 82.1% and 60.1%, respectively. Similarly, siRNA PAR-2.3 eliminated tryptase-induced IL-4 release by 75.3%, and siRNA PAR-1.2 diminished SFLLR-NH2 induced IL-4 release by 79.3%. However, siRNA PAR-1.2, siRNA PAR-2.3 and siRNA PAR-4.3 at 10 nM did not show any effect on tryptase-induced IL-6 release from P815 cells.

Conclusion

In conclusion, siRNAs of PARs can modulate PAR expression and PAR related cytokine production in mast cells, confirming that PARs are likely to play a role in allergic reactions.