The Association Of MMP-9Gene3'UTR Polymorphism And Atherosclerotic Cerebral Infarction And Polymorphism Regulation Mechanism Of MiRNA-491-mediated

Background and aims:Stroke is one of the leading causes of death in the world. Most stroke patients are classified as having ischemic stroke (IS). The causes of IS are very diverse, such as:hypertension, diabetes, hyperlipidemia, vasculitis, antiphospholipid antibodies (aPL), the angiotensin-1converting enzyme (ACE), and atherosclerosis, whereas atherosclerosis resulting in cerebral or carotid arterial stenosis/occlusion plays the most important role in the occurrence of IS. Matrix metalloproteinase9(MMP-9) plays a pivotal role in early atherosclerosis, vascular remodeling, and development of arterial plaque rupture. The potentially functional MMP-9gene polymorphism may contribute to the susceptibility of Atherosclerotic cerebral infarction (ACI), We aimed to investigate the association between the interaction of3'UTR single-nucleotide polymorphisms (rs20544,rs1056628,rs1802908, rs2664517and rs9509) of the MMP-9gene and ACI in a Han population of Hunan of China.Materials and methods:A case-control study composed of340proven patients with ACI and309sex-matched and ethnically matched control participants were enrolled in the study. We recruited all subjects from March2008to March2011. Matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was used for detection of genotype and allele frequencies. Then We construct-ed reporter plasmids with the mutant and wild-type MMP-9(CDS and full lenghth of3'UTR) linked to Luciferase(PGL3-MMP-9-wil and PGL3-MMP-9mut2182A/C). Besides, we also we constructed pcDNA-3.1/miR-491recombinant plasmid, which transiently co-transfected human umbilical vein endothelial cells(HUVEC) with PGL3-MMP-9-wil and mut respectively. Using a dual-luciferase reporter system to experim-ental validation whether or not miR-491interacts with the3'UTR of MMP-9wil and mut mRNA. Furthermore, We synthesized the mir-491-5p mimics and inhibitors, which transiently co-transfected HUVEC with PGL3-MMP-9-wil and mut recombinant plasmids respectively. The expression of MMP-9was detected by reverse transcriptase PCR (RT-PCR) and enzyme linked immunosorbent assay (ELISA).Results:1) All SNPs were in Hardy-Weinberg equilibrium. We found the MMP-9SNPs rs20544,rs1056628and rs9509, but not found SNPs rs1802908and rs2664517in the control and patient population.2) The AA, AC and CC genotype frequencies of MMP-9SNP rs1056628were0.594/0.332/0.074and0.709/0.262/0.029in the patient and control population respectively; A/C allele frequencies were0.760/0.240and0.840/0.160respectively; C allele and CC genotype frequency of ACI were significantly higher than those in the control group (P<0.01). 3) The CC,CT and TT genotype frequencies of MMP-9SNP rs20544were0.471/0.405/0.124and0.489/0.382/0.129in the patient and control population respectively; C/T allele frequencies were0.674/0.326and0.680/0.320respectively; There were no significant difference between the two group (P>0.05).4) The TT, TC and CC genotype frequencies of MMP-9SNPrs9509were0.491/0.418/0.091and0.466/0.447/0.087in the patient and control population respectively; T/C allele frequencies were0.700/0.300and0.689/0.311respectively; There were no significant difference between the two group (P>0.05).5) Successfully transfected with the recombinant plasmid. MiR-491led to a significant reduction of relative luciferase activity in MMP-9wil group compared to MMP-9mut group (P<0.05), these findings conform the predicted binding of miR-491to the3'UTR of MMP-9and demonstr-ate a reduction expression of MMP-9due to MMP-9SNP rs1056628changed.6) In the wild-type and mutant group, MiR-491does not significantly downregulate MMP-9mRNA level. However, compared with wild-type group, MiR-491significantly upregulates MMP-9protein level in MMP-9mutant group (P<0.01)Conclusions:1) The SNPs of rs1802908and rs2664517may not exist among Han Chinese in Hunan. 2) It is probably that the Polymorphism of MMP-9rs20544C→T and rs9509T→C are independent on the occurrence of ACI.3) The MMP-9SNPs of rs1056629A->C may be the risk factor of ACI among Han Chinese in Hunan.4) The mechanism that underlie the association between MMP-9SNP rs1056628with ACI is miR-491significantly upregulates MMP-9protein level compared with the group that without SNP rs1056628changed.