Assessing the ceRNA hypothesis with quantitative measurements of miRNA and target abundance

Recent studies have reported that competitive endogenous RNAs (ceRNAs) can act as sponges for a miRNA through their binding sites and that changes in ceRNA abundances from individual genes can modulate the miRNA's activity. Consideration of this hypothesis would benefit from knowing the quantitative relationship between a miRNA and its endogenous target sites. Here, we altered intracellular target-site abundance through expression of a miR-122 target in hepatocytes and livers, and analyzed the effects on miR-122 target genes. Target repression was released in a threshold-like manner at high target-site abundance (>1.5x10^5 added target sites per cell), and this threshold was insensitive to the effective levels of the miRNA. Furthermore, in response to extreme metabolic liver disease models, global target-site abundance of hepatocytes did not change sufficiently to affect miRNA-mediated repression. Thus, modulation of miRNA target abundance is unlikely to cause significant effects on gene expression and metabolism through a ceRNA effect. Seventeen mRNA profiles were generated of 1) primary hepatocytes of mice expressing variable levels of a recombinant Adenovirus expressing the transcript of AldolaseA (Ad-AldoA), containing either 1 or 3 sites matching miR-122 or a mutated miR-122 site (no site), 2) primary hepatocytes derived from mice treated with Antagomir-122 (treatment group) or Antagomir-122mm (control group), 3) livers originating of a genetic model (Ldlr deficient mice) causing severe pathological changes in cholesterol metabolism, 4) livers of mice perfused with Insulin or PBS, and 5) livers of mice fed a high-fat or chow diet; most samples were sequenced in duplicate or triplicate by an Illumina HiSeq 2000. One small RNA profile was also generated from livers of mice fed a chow diet by Solexa sequencing.近期已有研究报道，竞争性内源RNA（competitive endogenous RNA, ceRNA）可通过自身结合位点作为微小RNA（microRNA, miRNA）的海绵分子，且单个基因编码的ceRNA丰度变化可调控对应miRNA的活性。要验证这一假说，需明确miRNA与其内源靶位点之间的定量关联。本研究通过在肝细胞及肝脏中表达miR-122靶序列，改变细胞内靶位点丰度，并分析其对miR-122靶基因的影响。当靶位点丰度较高（每细胞添加靶位点超过1.5×10^5个）时，靶基因的抑制效应以阈值依赖性方式解除，且该阈值不受miRNA有效水平的影响。此外，在极端代谢性肝病模型中，肝细胞的整体靶位点丰度未发生足以影响miRNA介导基因抑制的变化。因此，通过调控miRNA靶位点丰度，不太可能通过ceRNA机制对基因表达及代谢产生显著影响。本研究共生成17组mRNA表达谱，涵盖以下5类样本：1) 表达不同滴度重组腺病毒Ad-AldoA的小鼠原代肝细胞，该腺病毒携带醛缩酶A（AldolaseA）转录本，其序列包含1个、3个匹配miR-122的结合位点，或1个突变的miR-122结合位点（无结合位点）；2) 经Antagomir-122（处理组）或Antagomir-122mm（对照组）处理的小鼠原代肝细胞；3) 低密度脂蛋白受体（low-density lipoprotein receptor, Ldlr）缺陷小鼠的肝脏，该遗传模型可导致胆固醇代谢出现严重病理变化；4) 经胰岛素（Insulin）或磷酸盐缓冲液（PBS）灌流的小鼠肝脏；5) 饲喂高脂饲料或普通饲料的小鼠肝脏；绝大多数样本通过Illumina HiSeq 2000平台进行了2~3次重复测序。此外，本研究还通过Solexa测序技术，为饲喂普通饲料的小鼠肝脏生成了1组小RNA表达谱。