Eriksen Anne Haahr Mellergaard, Andersen Rikke Fredslund, Pallisgaard Niels, Sørensen Flemming Brandt, Jakobsen Anders, Hansen Torben Frøstrup
Danish Colorectal Cancer Center South, Center of Clinical Excellence, Vejle Hospital, Vejle, Denmark.
Institute of Regional Health Research, University of Southern Denmark, Odense, Denmark.
PLoS One. 2016 Mar 3;11(3):e0150593. doi: 10.1371/journal.pone.0150593. eCollection 2016.
MicroRNAs (miRNAs) play important roles in regulating biological processes at the post-transcriptional level. Deregulation of miRNAs has been observed in cancer, and miRNAs are being investigated as potential biomarkers regarding diagnosis, prognosis and prediction in cancer management. Real-time quantitative polymerase chain reaction (RT-qPCR) is commonly used, when measuring miRNA expression. Appropriate normalisation of RT-qPCR data is important to ensure reliable results. The aim of the present study was to identify stably expressed miRNAs applicable as normaliser candidates in future studies of miRNA expression in rectal cancer.
We performed high-throughput miRNA profiling (OpenArray®) on ten pairs of laser micro-dissected rectal cancer tissue and adjacent stroma. A global mean expression normalisation strategy was applied to identify the most stably expressed miRNAs for subsequent validation. In the first validation experiment, a panel of miRNAs were analysed on 25 pairs of micro dissected rectal cancer tissue and adjacent stroma. Subsequently, the same miRNAs were analysed in 28 pairs of rectal cancer tissue and normal rectal mucosa.
From the miRNA profiling experiment, miR-645, miR-193a-5p, miR-27a and let-7g were identified as stably expressed, both in malignant and stromal tissue. In addition, NormFinder confirmed high expression stability for the four miRNAs. In the RT-qPCR based validation experiments, no significant difference between tumour and stroma/normal rectal mucosa was detected for the mean of the normaliser candidates miR-27a, miR-193a-5p and let-7g (first validation P = 0.801, second validation P = 0.321). MiR-645 was excluded from the data analysis, because it was undetected in 35 of 50 samples (first validation) and in 24 of 56 samples (second validation), respectively. Significant difference in expression level of RNU6B was observed between tumour and adjacent stromal (first validation), and between tumour and normal rectal mucosa (second validation).
We recommend the mean expression of miR-27a, miR-193a-5p and let-7g as normalisation factor, when performing miRNA expression analyses by RT-qPCR on rectal cancer tissue.
微小RNA（miRNA）在转录后水平调控生物学过程中发挥着重要作用。在癌症中已观察到miRNA失调，并且miRNA正作为癌症管理中诊断、预后和预测的潜在生物标志物进行研究。在测量miRNA表达时，常用实时定量聚合酶链反应（RT-qPCR）。对RT-qPCR数据进行适当的标准化对于确保可靠结果很重要。本研究的目的是确定在未来直肠癌miRNA表达研究中可作为标准化候选物的稳定表达的miRNA。
我们对十对经激光显微切割的直肠癌组织和相邻基质进行了高通量miRNA谱分析（OpenArray®）。应用全局平均表达标准化策略来鉴定最稳定表达的miRNA，以便后续验证。在首次验证实验中，对25对显微切割的直肠癌组织和相邻基质分析了一组miRNA。随后，在28对直肠癌组织和正常直肠黏膜中分析了相同的miRNA。
从miRNA谱分析实验中，miR-645、miR-193a-5p、miR-27a和let-7g在恶性组织和基质组织中均被鉴定为稳定表达。此外，NormFinder证实这四种miRNA具有高表达稳定性。在基于RT-qPCR的验证实验中，标准化候选物miR-27a、miR-193a-5p和let-7g的平均值在肿瘤与基质/正常直肠黏膜之间未检测到显著差异（首次验证P = 0.801，第二次验证P = 0.321）。miR-645被排除在数据分析之外，因为它分别在50个样本中的35个（首次验证）和56个样本中的24个（第二次验证）中未被检测到。在肿瘤与相邻基质之间（首次验证）以及肿瘤与正常直肠黏膜之间（第二次验证）观察到RNU6B表达水平的显著差异。
当通过RT-qPCR对直肠癌组织进行miRNA表达分析时，我们推荐将miR-27a、miR-193a-5p和let-7g的平均表达作为标准化因子。
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