Endogeneous mRNA level fluctuations in various brain tumor cells

Background: microRNAs (miRNAs) are approximately 21 nucleotide non-coding transcripts capable of regulating gene expression. The most widely studied mechanism of regulation involves binding of the miRNA to a target mRNA, usually in its 3’ untranslated region (UTR). As a result, translation of the target mRNA is inhibited and sometimes the mRNA itself can be de-stabilized. The inhibitory effects of miRNAs have been linked to many diverse cellular processes including malignant proliferation and apoptosis, development and differentiation, metabolic processes and neural plasticity. We asked whether endogenous fluctuations in a set of mRNA and miRNA profiles contain correlated changes that are statistically distinguishable from the many other fluctuations in the data set. Methodology/Principal Findings: Biopsies from 12 human primary brain tumors were used to extract RNA. These samples were used to determine genome-wide mRNA expression levels by microarray analysis and a miRNA profile by real-time reverse transcription PCR (RT-PCR). Correlation coefficients were determined for all possible mRNA-miRNA pairs and the distribution of these correlations compared to the random distribution. An excess of high positive and negative correlation pairs were observed at the tails of these distributions. Most of these highest correlation pairs do not contain sufficiently complementary sequences to predict a target relationship; nor do they lie in physical proximity to each other. However, by examining pairs in which the significance of the correlation coefficients is modestly relaxed, negative correlations do tend to predict targets and positive correlations do tend to predict physically proximate pairs. A subset of high correlation pairs were experimentally validated by over-expressing or suppressing a miRNA and measuring the correlated mRNAs. Conclusions/Significance: Sufficient information exists within a set of tumor samples to detect endogenous correlations between miRNA and mRNA levels. Based on the validations of the correlations the causal arrow for these correlations is likely to be directed from the miRNAs to the mRNAs. From these data sets, we inferred and validated a tumor suppression pathway linked to miR-181c. Keywords: cell type comparison Microarray data were obtained from the 12 tumors using Affymetrics HG-U133A array platforms. Tissue from glioma resections were obtained and collected at UCLA under approval from the Institutional Review Board. Categorization into high or low grade was based on the formal pathology report, with WHO grades III and IV being termed "high grade" and grades I and II termed "low grade". Histological diagnoses fell into the following categories: two low grade anaplastic mixed gliomas, one low grade oligodendroglioma, six high grade glioblastomas, two high grade gliosarcomas, and one high grade glioblastoma/gliosarcoma.

背景：微小RNA（microRNAs, miRNAs）是一类长度约21个核苷酸的非编码转录本，具备调控基因表达的能力。目前研究最为广泛的调控机制为：miRNA与靶mRNA结合，结合位点通常位于靶mRNA的3'非翻译区（3' untranslated region, UTR）。结合后会抑制靶mRNA的翻译过程，部分情况下还会使mRNA本身的稳定性下降。miRNA的这种抑制效应与诸多细胞过程密切相关，包括恶性增殖、细胞凋亡、发育与分化、代谢过程以及神经可塑性。本研究旨在探究：一组mRNA与miRNA表达谱中的内源性波动，是否存在统计学上可与数据集内其他诸多波动相区分的相关性变化。

方法与主要结果：选取12例人脑原发性脑瘤活检组织提取RNA，通过微阵列分析检测全基因组mRNA表达水平，通过实时逆转录聚合酶链反应（real-time reverse transcription PCR, RT-PCR）构建miRNA表达谱。计算所有可能的mRNA-miRNA配对的相关系数，并将该相关系数的分布与随机分布进行对比。在这些分布的尾部区域，观察到显著过多的高正相关与高负相关配对。其中绝大多数相关性最强的配对，其序列间互补性不足以预测靶标关系，且二者在物理位置上也不相邻。不过，当适当放宽相关系数的显著性阈值时，负相关配对能够较好地预测靶标关系，而正相关配对则更倾向于对应物理位置相邻的基因对。我们通过过表达或抑制某一miRNA，并检测其对应的关联mRNA表达变化，对部分高相关性配对进行了实验验证。

结论与意义：肿瘤样本数据集内包含足够信息，可用于检测miRNA与mRNA水平之间的内源性相关性。通过对相关性的验证可知，这些相关性的因果方向大概率为从miRNA指向mRNA。基于本数据集，我们推断并验证了一条与miR-181c相关的肿瘤抑制通路。

关键词：细胞类型比较 本研究的微阵列数据来自12例肿瘤样本，采用Affymetrics HG-U133A阵列平台。胶质瘤切除组织由加州大学洛杉矶分校（UCLA）在机构审查委员会批准下获取并收集。肿瘤分级（高分级或低分级）依据正式病理报告确定：世界卫生组织（World Health Organization, WHO）Ⅲ级和Ⅳ级归为“高分级”，Ⅰ级和Ⅱ级归为“低分级”。组织病理学诊断分类如下：2例低级别间变性混合性胶质瘤，1例低级别少突胶质细胞瘤，6例高级别胶质母细胞瘤，2例高级别胶质肉瘤，以及1例高级别胶质母细胞瘤/胶质肉瘤。