Regulatory Function of MicroRNA-149 on Thromboxane A2 Receptor Gene Expression in Asthmatics

Abstract

Background and objectives: The regulation of inflammatory responses in human diseases is mediated by the coordinated control of inflammatory gene expression at the transcriptional level as well as at the post-transcriptional level. One mechanism of post-transcriptional regulation is through control by microRNAs, which bind to the 3′ untranslated region (3′ UTR) of target genes and regulate mRNA stability or translation. The aim of this genetic association study was to identify miR-149 polymorphisms and evaluate their relationship with asthma with a view to further understanding the pathogenesis of asthma.

Subjects and methods: One hundred and eighty-four patients with asthma and 168 healthy normal controls (NC) were enrolled in this study. Single nucleotide polymorphisms (SNPs) in miR-149 (rs2292832) were genotyped using a TaqMan® allelic discrimination assay. Plasma miR-149 expression was evaluated using quantitative RT-PCR with a TaqMan® MicroRNA Reverse Transcription Kit and TaqMan® MicroRNA Assays. The expression of the thromboxane A2 receptor (TBXA2R) gene in peripheral blood mononuclear cells (PBMCs) was measured by quantitative RT-PCR. Human Embryonic Kidney 293T (HEK293T) cells were transfected with an EGFP-tagged TBXA2R 3′UTR plasmid construct and a miR-149 mimic or a miR-149 inhibitor using lipofectamine. EGFP expression was evaluated by measuring fluorescence intensity.

Results: Asthma patients had a significantly lower frequency of the miR-149 C allele compared to that of the NC group (P = 0.014). The expression levels of plasma miR-149 in asthma patients were significantly lower than that of the NC group (P = 0.002). Plasma miR-149 expression levels showed a negative correlation with both total IgE (r = -0.320; P = 0.027) and peripheral eosinophil count (r = -0.311, P = 0.030). The expression of the TBXA2R gene in PBMCs was significantly higher in asthma patients compared to that in the NC group (P = 0.024). Transfection with the miR-149 mimic significantly suppressed TBXA2R expression (P = 0.04), whereas transfection with the miR-149 inhibitor significantly increased TBXA2R expression (P = 0.02).

Conclusion: These findings suggest that the miR-149 C-allele (rs2292832) is associated with the phenotype of asthma and that miR-149 negatively regulates TBXA2R expression. Therefore, miR-149 may contribute to the pathogenesis of asthma.