Santos Jéssica Silva Dos, Zunta Gabriella Lucatto, Negrini Amanda Binatto, Ribeiro Marina Silva Guinda, Martinez Carlos Augusto Real, Ribeiro Marcelo Lima, Lourenço Gustavo Jacob, Ortega Manoela Marques
Laboratory of Cell and Molecular Tumor Biology and Bioactive Compounds, São Francisco University (USF), Bragança Paulista, Brazil.
Department of Surgery and Proctology, São Francisco University (USF), Bragança Paulista, Brazil.
Tumour Biol. 2020 May;42(5):1010428320923856. doi: 10.1177/1010428320923856.
The aim of this study was to evaluate the association of single-nucleotide variant n.60G>C (rs2910164) of microRNA (miR)-146a, related to suppressing of BRCA1/2 DNA repair protein, with the risk and survival of colorectal cancer patients, as well as miR-146a and levels and miR binding efficiency. The genotypes were identified in 125 colorectal cancer patients and 276 controls using TaqMan polymerase chain reaction assay. The miR-146a and levels were assessed by quantitative-polymerase chain reaction protocols. Primary precursor of miR-146a containing G (wild-type) and C (variant) allele were cloned into pcDNA.3.3 vector and co-transfected in HT-29 colorectal cancer cell line. Luciferase reporter assay was performed to assess miR-146a binding to 3'-untranslated region in HT-29. The differences between groups were calculated using chi-square or Fisher's exact test, logistic regression, and Mann-Whitney test. The prognostic impact of single-nucleotide variant genotypes on overall survival was evaluated by Kaplan-Meier estimate and Cox regression. The GC or CC genotypes prevalence was similar in patients and controls (50.4% vs 50.7%, = 0.74). However, patients with tumors in advanced stage with miR-146a GG genotype had 2.41 more chance of dying than GC or CC genotypes. In addition, tumor tissues of patients with GG genotype presented higher miR-146a ( = 0.02) and lower ( = 0.01) and ( < 0.0001) levels when compared to those with GC or CC genotypes. In fact, pcDNA.3.3-miR-146a-G presented increased binding capacity to the 3'-untranslated region of ( = 0.001) compared to pcDNA.3.3-miR-146a-C. In addition, the G allele altered the binding affinity between miR-146a and its 3'-untranslated region target ( < 0.001), thus enhancing suppression of expression. Our results suggest that single-nucleotide variant rs2910164 does not influence the colorectal cancer risk in Brazilian patients; however, the GG genotype could act as a factor of worse prognosis in patients with advanced disease due to suppression of modulated by miR-146a.
本研究旨在评估与BRCA1/2 DNA修复蛋白抑制相关的微小RNA（miR）-146a的单核苷酸变体n.60G>C（rs2910164）与结直肠癌患者的风险和生存情况，以及miR-146a水平和miR结合效率之间的关联。使用TaqMan聚合酶链反应分析法在125例结直肠癌患者和276例对照中鉴定基因型。通过定量聚合酶链反应方案评估miR-146a水平。将含有G（野生型）和C（变体）等位基因的miR-146a初级前体克隆到pcDNA.3.3载体中，并在HT-29结直肠癌细胞系中共转染。进行荧光素酶报告基因检测以评估miR-146a与HT-29中3'-非翻译区的结合情况。使用卡方检验或Fisher精确检验、逻辑回归和Mann-Whitney检验计算组间差异。通过Kaplan-Meier估计和Cox回归评估单核苷酸变体基因型对总生存的预后影响。患者和对照中GC或CC基因型的患病率相似（50.4%对50.7%，P = 0.74）。然而，处于晚期且具有miR-146a GG基因型的肿瘤患者死亡几率比GC或CC基因型高2.41倍。此外，与GC或CC基因型患者相比，GG基因型患者的肿瘤组织中miR-146a水平更高（P = 0.02），而[具体基因或蛋白名称]水平更低（P = 0.01）以及[另一具体基因或蛋白名称]水平更低（P < 0.0001）。事实上，与pcDNA.3.3-miR-146a-C相比，pcDNA.3.3-miR-146a-G对[具体基因或蛋白名称] 3'-非翻译区的结合能力增强（P = 0.001）。此外，G等位基因改变了miR-146a与其3'-非翻译区靶标的结合亲和力（P < 0.001），从而增强了对[具体基因或蛋白名称]表达的抑制。我们的结果表明，单核苷酸变体rs2910164不影响巴西患者的结直肠癌风险；然而，由于miR-146a对[具体基因或蛋白名称]的抑制作用，GG基因型可能是晚期疾病患者预后较差的一个因素。
