A functional polymorphism at miR-491-5p binding site in the 3'-UTR of MMP-9 gene confers increased risk for atherosclerotic cerebral infarction in a Chinese population.

Background: and purpose: Matrix metalloproteinases 9 (MMP-9) has been reported to play a critical role in the pathophysiology of atherosclerotic cerebral infarction (ACI). Here we assessed association of MMP-9 polymorphisms with ACI susceptibility and the function of SNPs through microRNA mediated regulation.

Methods: Genotyping was performed using MALDI-TOF mass spectrometry. Reporter gene plasmids with the MMP-9 3'UTR carrying either the mutant or the wild-type MMP-9 allele were constructed. Also, we constructed pcDNA-3.1-miR-491-5p recombinant plasmid, which transiently co-transfected human umbilical vein endothelial cells (HUVEC) with the reporter plasmids. Reporter plasmids, miR-491-5p mimics and inhibitor were transfected into HUVE cells line by lipofectamine. MMP-9 mRNA expression in HUVEC was detected by RT-PCR and protein level by ELISA.

Results: The rs1802908 and rs2664517 polymorphisms were not observed in all subjects from Hunan Han Chinese. No significant difference in genotype distribution of rs20544 and rs9509 between cases and controls were observed (p＞0.05). The rs1056628CC genotype had a significantly increased risk for ACI as compared with carries of the rs1056628 A allele (total χ(2) = 12.041, P = 0.002). Reporter gene assay revealed that the rs1056628 A allele showed lower reporter activity than the rs1056628C allele. Hsa-miR-491-5p had effect on modulation of MMP-9 gene in vitro. The rs1056628 A→C variant in the 3'-UTR of the MMP-9 increased MMP-9 protein expression in cultured HUVECs.

Conclusions: Our data suggested that the rs1056629A→C variation contributes to an increased risk of ACI by increasing MMP-9 expression through affecting binding of miR-491 to the polymorphic site in the 3'-UTR of MMP-9.