RESUMO
MicroRNA-29b has been reported to epigenetically regulate proatherogenic genes in response to oxLDL. Since transcription factors and epigenetic regulations are important mechanisms to regulate gene expression, we investigated whether these mechanisms are involved in oxLDL-induced microRNA-29b upregulation. First, we confirmed that microRNA-29b expression was increased in the aorta of mice fed with a high-fat diet, which was consistent with our previous in vitro findings. Next, we found that oxLDL only activated the microRNA-29b-1/microRNA-29a cluster gene on chromosome 7 but not the other distinct microRNA-29b gene located on chromosome 1. Using the promoter reporter assay and chromatin immunoprecipitation, activator protein-1 (AP-1) was shown to bind to the microRNA-29b-1 promoter. We further identified the signaling pathway of LOX-1/Ca(2+)/ROS/ERK/c-Fos was involved in oxLDL-mediated microRNA-29b overexpression after treating with the MAPTAM (Ca(2+) chelator), NAC (ROS scavenger), U0126 (ERK inhibitor) and c-Fos (one of the AP-1 proteins) shRNA, respectively. To investigate epigenetic regulations, we found that microRNA-29b promoter contained no CpG islands for DNA methylation. Therefore we investigated whether histone modifications influence microRNA-29b promoter activity. We showed that down-regulation of HDAC1 and the modifications on histone 3 lysine 4 (H3K4) and H3K9 significantly affected microRNA-29b expression. Furthermore, knockdown of c-Fos expression attenuated the effect of oxLDL-induced histone modifications on the microRNA-29b gene expression. Taken together, our data suggest that both transcription factor activation and histone modifications are important regulatory mechanisms of oxLDL-induced atherogenic process. This article is part of a Special Issue entitled OxLDL causes both epigenetic modification and signaling regulation on the microRNA-29b gene: Novel mechanisms for cardiovascular diseases.
Assuntos
Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Epigênese Genética , Lipoproteínas LDL/metabolismo , MicroRNAs/genética , Transdução de Sinais , Acetilação , Animais , Células Cultivadas , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histonas/metabolismo , Humanos , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Depuradores Classe E/metabolismoRESUMO
MicroRNAs (miRNAs), small noncoding RNAs, can control gene expression by binding to their target genes for degradation and/or translational repression. Epigenetic mechanisms are defined as heritable changes in gene expression that do not involve coding sequence modifications. Both mechanisms play an important role in maintaining physiological functions and are also related to disease development. However, few studies report that miRNA-mediated epigenetic regulations are involved in atherosclerosis. In the present study, oxidized low-density lipoprotein (oxLDL) significantly increased primary human aortic smooth muscle cell (HASMC) migration through MMP-2/MMP-9 up-regulation associated with decreased DNA methylation levels. Either mRNA or protein level of DNA methyltransferase 3b (DNMT3b) showed a dose-dependent down-regulation in oxLDL-mediated HASMCs. Knockdown DNMT3b expression enhanced oxLDL-induced DNA demethylation levels of MMP-2/MMP-9. The expression of miRNA-29b (miR-29b), directly targeting DNMT3b, was up-regulated by oxLDL treatment in a dose-dependent manner. OxLDL-mediated MMP-2/MMP-9 up-regulation, DNMT3b down-regulation, and DNA demethylation were all attenuated after knockdown miR-29b expression by antagomiR-29b. We find that oxLDL can up-regulate miR-29b expression, resulting in DNMT3b down-regulation in HASMCs and epigenetically regulated MMP-2/MMP-9 genes involved in cell migration. These results show that miRNA-mediated epigenetic regulation may be a novel mechanism in atherosclerosis.
Assuntos
Doenças Cardiovasculares/metabolismo , Epigênese Genética/genética , Lipoproteínas LDL/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , MicroRNAs/genética , Aorta/citologia , Western Blotting , Doenças Cardiovasculares/genética , Movimento Celular/efeitos dos fármacos , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Espectrometria de Massas , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , DNA Metiltransferase 3BRESUMO
PURPOSE: We showed previously that single nucleotide polymorphism (SNP) rs662702 in PAX6 may be located in a microRNA-328 binding site that causes susceptibility to high myopia. Our study was done to elucidate the role of PAX6 and its relationship with microRNA-328 in myopia. METHODS: A luciferase assay was used to confirm microRNA-328 binding to the PAX6 locus. Clones containing each allele of rs662702 were created and tested for their binding affinity to microRNA-328. Because a low level of PAX6 is a risk factor for myopia, we tested whether knockdown of PAX6 affects retinal pigment epithelial (RPE) cells and scleral cells, as well as expression of myopia-related genes. We also tested for the effect of retinoic acid (RA) on microRNA-328 expression, since RA-responsive elements are predicted to lie in the microRNA-328 promoter. RESULTS: MicroRNA-328 was shown to bind to the wild-type, but not mutant 3' untranslated region (UTR) of PAX6. The risk C allele of rs644242 had strong response to microRNA-328 but the protective T allele did not respond to microRNA-328. Down-regulation of PAX6 in RPE increased RPE proliferation, but reduced scleral cell proliferation. In addition, transforming growth factor (TGF)-ß3 in the RPE and matrix malleoproteinase-2 (MMP2) in the sclera were increased, while collagen I and integrin ß1 in the sclera were decreased. RA dose-dependently increased microRNA-328 expression and, in turn, suppressed PAX6 expression. CONCLUSIONS: We elaborated the relationship among myopia development, SNP rs662702, microRNA-328 and RA. The data imply that reduction of miR-328 and/or RA can be potential strategies for myopia prevention or treatment.