Teng Ming-Sheng, Hsu Lung-An, Juan Shu-Hui, Lin Wen-Chi, Lee Ming-Cheng, Su Cheng-Wen, Wu Semon, Ko Yu-Lin
Department of Research, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, New Taipei City, Taiwan.
The First Cardiovascular Division, Department of Internal Medicine, Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Taoyuan, Taiwan.
PLoS One. 2017 Aug 14;12(8):e0183187. doi: 10.1371/journal.pone.0183187. eCollection 2017.
Growth differentiation factor 15 (GDF15) is a strong predictor of cardiovascular events and mortality in individuals with or without cardiovascular diseases. Single nucleotide polymorphisms (SNPs) in microRNA (miRNA) target sites, also known as miRSNPs, are known to enhance or weaken miRNA-mRNA interactions and have been linked to diseases such as cardiovascular disease and cancer. In this study, we aimed to elucidate the functional significance of the miRSNP rs1054564 in regulating GDF15 levels. Two rs1054564-containing binding sites for hsa-miR-873-5p and hsa-miR-1233-3p were identified in the 3' untranslated region (UTR) of the GDF15 transcript using bioinformatics tools. Their activities were further characterized by in vitro reporter assays. Bioinformatics prediction suggested that miRNA binding sites harboring the rs1054564-G allele had lower free energies than those with the C allele and therefore were better targets with higher affinities for both hsa-miR-873-5p and hsa-miR-1233-3p. Reporter assays showed that luciferase activity was significantly decreased by rs1054564-G-containing 3' UTRs for both miRNAs (P < 0.05) and was restored by miRNA inhibitors. Comparing the fold suppression of the two miRNAs, only that of hsa-miR-1233-3p showed significant changes between the rs1054564-G- and C-containing 3' UTRs (P = 0.034). In addition, western blots showed that transfection of both miRNA mimics significantly decreased endogenous GDF15 expression in a melanoma cell line (P < 0.05). Taken together, our findings demonstrate that GDF15 is a target of hsa-miR-873-5p and hsa-miR-1233-3p and that the rs1054564-C allele partially abolishes hsa-miR-1233-3p-mediated translational suppression of GDF15. These results suggest that rs1054564 confers allele-specific translational repression of GDF15 via hsa-miR-1233-3p. Our work thus provides biological insight into the previously reported clinical association between rs1054564 and plasma GDF15 levels.
生长分化因子15（GDF15）是有无心血管疾病个体心血管事件和死亡率的强预测指标。微小RNA（miRNA）靶位点的单核苷酸多态性（SNP），也称为miRSNP，已知可增强或减弱miRNA与mRNA的相互作用，并与心血管疾病和癌症等疾病相关。在本研究中，我们旨在阐明miRSNP rs1054564在调节GDF15水平中的功能意义。使用生物信息学工具在GDF15转录本的3'非翻译区（UTR）中鉴定出两个含有rs1054564的hsa-miR-873-5p和hsa-miR-1233-3p结合位点。通过体外报告基因检测进一步表征了它们的活性。生物信息学预测表明，携带rs1054564-G等位基因的miRNA结合位点比携带C等位基因的结合位点具有更低的自由能，因此对hsa-miR-873-5p和hsa-miR-1233-3p都是具有更高亲和力的更好靶标。报告基因检测显示，对于这两种miRNA，含rs1054564-G的3'UTR均显著降低了荧光素酶活性（P < 0.05），并且通过miRNA抑制剂得以恢复。比较两种miRNA的抑制倍数，只有hsa-miR-1233-3p在含rs1054564-G和C的3'UTR之间显示出显著变化（P = 0.034）。此外，蛋白质免疫印迹显示，转染两种miRNA模拟物均显著降低了黑色素瘤细胞系中内源性GDF15的表达（P < 0.05）。综上所述，我们的研究结果表明GDF15是hsa-miR-873-5p和hsa-miR-1233-3p的靶标，并且rs1054564-C等位基因部分消除了hsa-miR-1233-3p介导的GDF15翻译抑制。这些结果表明，rs1054564通过hsa-miR-1233-3p赋予GDF15等位基因特异性的翻译抑制作用。因此，我们的工作为先前报道的rs1054564与血浆GDF15水平之间的临床关联提供了生物学见解。
