Deep sequencing and proteomics analysis reveal globally dysregulated expression of microRNA and their target genes in rat kidneys treated by carcinogenic dose of Aristolochic Acid

MicroRNAs (miRNAs) play an important role in carcinogenesis by regulating the protein translation from their targeted messenger RNAs (mRNA). Aristolochic Acid (AA) is a potent carcinogen that can induce kidney tumors in both human and experimental animals. Although the expression and roles of miRNAs have been extensively studied in cancer tissues or tissues treated by carcinogens, it is still unclear how the three levels of gene expression, including miRNA, mRNA and protein, are regulated and coordinated during the early stage of carcinogenesis. Here, we treated rats with 10 mg/kg AA or vehicle control for 12 weeks and the kidney tissues were quickly isolated and frozen immediately after the treatment. Eight kidney samples (4 for the treatment and 4 for the control) were used for analyzing miRNA and mRNA expressions with deep sequencing, and protein expressions with proteomics. MiRNA expressions were significantly changed by AA treatment. The treated samples were well separated from the control samples in both principal component analysis (PCA) and hierarchical clustering analysis (HCA). Quantitative measurements of six miRNAs using TaqMan real-time PCR were consistent with the deep sequencing results in terms of both direction and magnitude of gene expression change. Sixty-three miRNAs (adjusted p value < 0.05 and fold change > 1.5), 6,794 mRNAs (adjusted p value < 0.05 and fold change > 2.0), and 800 proteins (fold change > 2.0) were significantly altered by AA treatment. By combining the results of a computational target predicting algorithm, and the mRNA and protein expression data, 143 genes were found by the differentially expressed miRNAs (DEMs). The DEM-targeted genes are mainly related to cancer, cell growth, which reflects the carcinogenic processes in the AA-treated rat kidneys.微小核糖核酸（MicroRNAs, miRNAs）可通过靶向调控信使核糖核酸（messenger RNAs, mRNA）的蛋白质翻译过程，在肿瘤发生过程中发挥重要作用。马兜铃酸（Aristolochic Acid, AA）是一种强效致癌物，可在人类及实验动物体内诱发肾脏肿瘤。尽管目前针对癌组织或致癌物处理组织中的miRNAs表达与功能已有大量研究，但在肿瘤发生早期阶段，miRNA、mRNA与蛋白质这三个层面的基因表达如何被调控并协同发挥作用，目前仍不明确。 本研究以10 mg/kg剂量的AA或溶剂对照处理大鼠12周，处理结束后迅速分离肾脏组织并即刻冷冻保存。我们选取8份肾脏样本（处理组与对照组各4份），通过深度测序分析miRNA与mRNA的表达水平，并借助蛋白质组学技术检测蛋白质表达情况。 研究结果显示，AA处理可显著改变miRNA的表达谱。在主成分分析（Principal Component Analysis, PCA）与层次聚类分析（Hierarchical Clustering Analysis, HCA）中，处理组样本均与对照组样本实现良好分离。通过TaqMan实时荧光定量PCR对6种miRNA进行定量检测，其表达变化的方向与幅度均与深度测序结果一致。 经AA处理后，共有63种miRNA（校正后P值<0.05且倍数变化>1.5）、6794种mRNA（校正后P值<0.05且倍数变化>2.0）以及800种蛋白质（倍数变化>2.0）的表达水平发生显著改变。结合计算靶标预测算法的结果以及mRNA与蛋白质表达数据，我们通过差异表达miRNAs（Differentially Expressed miRNAs, DEMs）筛选得到143个靶标基因。这些DEMs的靶基因主要与癌症、细胞生长等过程相关，反映了AA处理大鼠肾脏中的致癌进程。