Laboratory of Cancer Genetics, School of Medical Sciences, University of Campinas, Rua Vital Brasil, 50, Distrito de Barão Geraldo, Campinas, São Paulo, CEP: 13083-888, Brazil.
Department of Internal Medicine, School of Medical Sciences, University of Campinas, Campinas, São Paulo, Brazil.
Sci Rep. 2020 Oct 12;10(1):17039. doi: 10.1038/s41598-020-73675-z.
We conducted a two-stage association study on patients with oropharynx (OP) squamous cell carcinoma (SCC) and healthy controls to identify single nucleotide variants (SNVs) located at the microRNA (miR)-binding sites of carcinogenesis genes associated with risk and prognosis of the disease. In stage 1, 49 patients and 49 controls were analyzed using Genome-Wide Human SNV Arrays to identify variants in the 3'-untranslated region (3'-UTR) of carcinogenesis-related genes, and one SNV was selected for data validation in stage 2 by TaqMan assays in 250 OPSCC patients and 250 controls. The ERP29 c.*293A > G (rs7114) SNV located at miR-4421 binding site was selected for data validation among 46 SNVs. The ERp29 and miR-4421 levels were evaluated by quantitative-PCR and Western blotting. Interaction between miR-4421 with 3'-UTR of ERP29 was evaluated by luciferase reporter assay. Event-free survival (EFS) was calculated by Kaplan-Meier and Cox methods. ERP29 GG variant genotype was more common in OPSCC patients than in controls (6.4% vs 3.6%, p = 0.02; odds ratio: 5.67; 95% confidence interval (CI) 1.27-25.26). Shorter EFS were seen in the base of tongue (BT) SCC patients with GG genotype (0.0% vs 36.2%, p = 0.01; hazard ratio: 2.31; 95% CI: 1.03-5.15). Individuals with ERP29 AG or GG genotypes featured lower levels of ERP29 mRNA (p = 0.005), ERp29 protein (p < 0.001) and higher levels of miR-4421 (p = 0.02). The miR-4421 showed more efficient binding with 3'-UTR of the variant G allele when compared with wild-type allele A (p = 0.001). Our data suggest that ERP29 rs7114 SNV may alter the risk and prognosis of OPSCC due to variation in the ERp29 production possibly modulated by miR-4421.
我们进行了一项两阶段的关联研究，纳入了口咽（OP）鳞状细胞癌（SCC）患者和健康对照者，以鉴定位于致癌基因 miRNA（miR）结合位点的单核苷酸变异（SNV），这些 SNV 与疾病的风险和预后相关。在第一阶段，我们使用全基因组人类 SNV 阵列分析了 49 例患者和 49 例对照者，以鉴定致癌基因 3'-非翻译区（3'-UTR）中的变异，然后通过 TaqMan 分析在第二阶段选择一个 SNV 在 250 例 OPSCC 患者和 250 例对照者中进行数据验证。选择位于 miR-4421 结合位点的 ERp29 c.*293A>G(rs7114)SNV 进行 46 个 SNV 的数据验证。通过定量 PCR 和 Western 印迹评估 ERp29 和 miR-4421 的水平。通过荧光素酶报告基因测定评估 miR-4421 与 ERp29 3'-UTR 的相互作用。通过 Kaplan-Meier 和 Cox 方法计算无事件生存（EFS）。OPSCC 患者中 ERp29 GG 变异基因型比对照组更常见（6.4% vs 3.6%，p=0.02；优势比：5.67；95%置信区间（CI）1.27-25.26）。舌底（BT）SCC 患者 GG 基因型的 EFS 更短（0.0% vs 36.2%，p=0.01；风险比：2.31；95%CI：1.03-5.15）。具有 ERp29 AG 或 GG 基因型的个体 ERp29 mRNA 水平较低（p=0.005），ERp29 蛋白水平较低（p<0.001），miR-4421 水平较高（p=0.02）。与野生型 A 等位基因相比，miR-4421 与变异 G 等位基因的 3'-UTR 结合更有效（p=0.001）。我们的数据表明，由于 ERp29 产生的变化，可能受 miR-4421 调节，ERP29 rs7114 SNV 可能改变 OPSCC 的风险和预后。
