Effect of small interfering RNA on matrix metalloproteinase-9 expression in vascular endothelial cells stimulated by serum from children with Kawasaki disease
Chen, Z.-H.; Wan, G.-P.; Gu, X.-Q.
Zhonghua Xin Xue Guan Bing Za Zhi 37(9): 837-840
2009
ISSN/ISBN: 0253-3758 PMID: 20128385 Document Number: 634523
To investigate the matrix metalloproteinase-9 (MMP-9) expression in vascular endothelial cells stimulated by the serum obtained from children with Kawasaki disease (KD) during the acute phase in the absence and presence of MMP-9 small interfering RNA (siRNA). MMP-9 siRNA plasmids were constructed and transduced into vascular endothelial cells (ECV-304) by liposomal transfection. ECV-304 were cultured in 6 different conditional media: KD serum + siRNA negative control, normal serum, KD serum + MMP-9 siRNA1 (pSilencer3.1-MMP1), KD serum + MMP-9 siRNA2 (pSilencer3.1-MMP2), KD serum + gamma-globulin, and KD serum. RT-PCR and Western blot were used to detect MMP-9 expression at mRNA and protein levels in ECV-304. The mRNA and protein expression of MMP-9 in ECV-304 cultured with 10% serum from KD patients (2.49 +/- 0.03, 1.20 +/- 0.04) and KD serum + siRNA negative control plasmid (2.45 +/- 0.03, 1.15 +/- 0.03) were significantly higher than those cultured with 10% serum from normal control children (1.21 +/- 0.03, 0.52 +/- 0.03, respectively; all P < 0.01) and the increased MMP-9 expression could be significantly inhibited by MMP-9 siRNA1, MMP-9 siRNA2 and gamma-globulin (100 mg/ml, all P < 0.01). The increase of MMP-9 expression in vascular endothelial cells induced by the serum from KD patients might take part in the formation of coronary artery lesions. Two customized MMP-9 siRNA plasmids (pSilencer3.1-MMP1 and pSilencer3.1-MMP2) can significantly inhibit both MMP-9 mRNA and protein expression.