Clinical Biochemistry, Dept. of Clinical Physiopathology, University of Florence, Italy.
Methods. 2013 Jan;59(1):138-46. doi: 10.1016/j.ymeth.2012.09.002. Epub 2012 Sep 16.
Studies on miRNA profiling revealed that a large number of them are significantly deregulated in human cancers. The molecular mechanisms of this deregulation are not totally clarified, even if genetics and epigenetics are frequently involved. Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation in the human genome. A SNP into miRNA gene might affect the transcription of primary miRNA, its processing and miRNA-mRNA interaction. We investigated the distribution of sequence variants of miR-146a, miR-196a2, miR-499 and miR-149 in colorectal cancer (CRC) and their effect on miRNA expression. Each variant was identified with HRM. For miR-499 we demonstrated a significant reduction of its expression in CRC connected to a specific genotype. To evaluate the epigenetic effects on miRNA genes in CRC, we investigated the influence of DNA methylation on miR-34b, miR-34c and miR-9-1 expression. We aimed to verify the relationship between the methylation status of these miRNA genes and their relative expression in tumor samples. For the quantification of DNA methylation we adopted a method based on Differential High Resolution Melting (D-HRM).
miRNA 谱研究表明，大量 miRNA 在人类癌症中存在显著失调。尽管遗传学和表观遗传学经常参与其中，但这种失调的分子机制尚未完全阐明。单核苷酸多态性 (SNP) 是人类基因组中最常见的遗传变异类型。miRNA 基因中的 SNP 可能会影响初级 miRNA 的转录、加工以及 miRNA-mRNA 相互作用。我们研究了 miR-146a、miR-196a2、miR-499 和 miR-149 在结直肠癌 (CRC) 中的序列变异分布及其对 miRNA 表达的影响。每种变体均采用 HRM 进行鉴定。对于 miR-499，我们证明了其在 CRC 中的表达显著降低，与特定基因型有关。为了评估 CRC 中 miRNA 基因的表观遗传效应，我们研究了 DNA 甲基化对 miR-34b、miR-34c 和 miR-9-1 表达的影响。我们旨在验证这些 miRNA 基因的甲基化状态与其在肿瘤样本中的相对表达之间的关系。为了定量 DNA 甲基化，我们采用了基于差分高分辨率熔解 (D-HRM) 的方法。
