Arabkari Vahid, Clancy Eoin, Dwyer Róisín M, Kerin Michael J, Kalinina Olga, Holian Emma, Newell John, Smith Terry J
Molecular Diagnostics Research Group, School of Natural Sciences and National Centre for Biomedical Engineering Science (NCBES), NUI Galway, Galway, Ireland.
Discipline of Pathology, School of Medicine, Lambe Institute for Translational Research, NUI Galway, Galway, Ireland.
Pathol Oncol Res. 2020 Apr;26(2):833-844. doi: 10.1007/s12253-019-00627-y. Epub 2019 Mar 6.
MicroRNAs, as small non-coding regulatory RNAs, play crucial roles in various aspects of breast cancer biology. They have prognostic and diagnostic value, which makes them very interesting molecules to investigate. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is the gold standard method to analyse miRNA expression in breast cancer patients. This study investigated two RT-qPCR methods (absolute and relative) to determine the expression of ten miRNAs in whole blood samples obtained from luminal A breast cancer patients compared to healthy controls. Whole blood samples were collected from 38 luminal A breast cancer patients and 20 healthy controls in Paxgene blood RNA tubes. Total RNA was extracted and analysed by relative and absolute RT-qPCR. For relative RT-qPCR, miR-16 was used as an endogenous control. For absolute RT-qPCR, standard curves were generated using synthetic miRNA oligonucleotides to determine the absolute copy number of each miRNA. Of the ten miRNAs that were analysed, the absolute RT-qPCR method identified six miRNAs (miR-16, miR-145, miR-155, miR-451a, miR-21 and miR-486) that were upregulated and one miRNA (miR-195) that was downregulated. ROC curve and AUC analysis of the data found that the combination of three miRNAs (miR-145, miR-195 and miR-486) had the best diagnostic value for luminal A breast cancer with an AUC of 0.875, with 76% sensitivity and 81% specificity. On the other hand, the relative RT-qPCR method identified two miRNAs (miR-155 and miR-486) that were upregulated and miR-195, which was downregulated. Using this approach, the combination of three miRNAs (miR-155, miR-195 and miR-486) was showed to have an AUC of 0.657 with 65% sensitivity and 69% specificity. We conclude that miR-16 is not a suitable normalizer for the relative expression profiling of miRNAs in luminal A breast cancer patients. Compared to relative quantification, absolute quantification assay is a better method to determine the expression level of circulating miRNAs in Luminal A breast cancer.
微小RNA作为小型非编码调节RNA，在乳腺癌生物学的各个方面发挥着关键作用。它们具有预后和诊断价值，这使得它们成为非常值得研究的分子。逆转录定量聚合酶链反应（RT-qPCR）是分析乳腺癌患者中miRNA表达的金标准方法。本研究调查了两种RT-qPCR方法（绝对定量和相对定量），以确定从腔面A型乳腺癌患者获得的全血样本中十种miRNA的表达，并与健康对照进行比较。在帕克斯基因血液RNA管中从38例腔面A型乳腺癌患者和20例健康对照中采集全血样本。提取总RNA并通过相对定量和绝对定量RT-qPCR进行分析。对于相对定量RT-qPCR，使用miR-16作为内参。对于绝对定量RT-qPCR，使用合成的miRNA寡核苷酸生成标准曲线，以确定每种miRNA的绝对拷贝数。在分析的十种miRNA中，绝对定量RT-qPCR方法鉴定出六种上调的miRNA（miR-16、miR-145、miR-155、miR-451a、miR-21和miR-486）和一种下调的miRNA（miR-195）。对数据进行ROC曲线和AUC分析发现，三种miRNA（miR-145、miR-195和miR-486）的组合对腔面A型乳腺癌具有最佳诊断价值，AUC为0.875，敏感性为76%，特异性为81%。另一方面，相对定量RT-qPCR方法鉴定出两种上调的miRNA（miR-155和miR-486）和下调的miR-195。使用这种方法，三种miRNA（miR-155、miR-195和miR-486）的组合显示AUC为0.657，敏感性为65%，特异性为69%。我们得出结论，miR-16不是腔面A型乳腺癌患者中miRNA相对表达谱分析的合适标准化物。与相对定量相比，绝对定量测定是确定腔面A型乳腺癌中循环miRNA表达水平的更好方法。
