Argonaute CLIP defines a deregulated miR-122 bound transcriptome that correlates with patient survival in human liver cancer (Mouse RNA-Seq). Mus musculus

MicroRNA-122, an abundant and conserved liver-specific miRNA, regulates hepatic metabolism and functions as a tumor suppressor, yet systematic and direct biochemical elucidation of the miR-122 target network remains incomplete. To this end, we performed Argonaute crosslinking immunoprecipitation (Ago-CLIP) sequencing in miR-122 knockout and control mouse livers, as well as in matched human hepatocellular carcinoma (HCC) and benign liver tissue to identify miRNA target sites transcriptome-wide in two species. We observed a majority of miR-122 binding on 3’-UTRs and coding exons followed by extensive binding to other genic and non-genic sites. Motif analysis of miR-122 dependent binding revealed a novel G-bulged motif in addition to canonical motifs. A large number of miR-122 targets were found to be species-specific. Upregulation of several common mouse and human targets, most notably BCL9, predicted survival in HCC patients. These results broadly define the molecular consequences of miR-122 downregulation in hepatocellular carcinoma. Overall design: AGO CLIP-seq libraries were generated from four miR-122 KO mice and five floxed littermate controls, all at six weeks of age.

微小RNA-122（MicroRNA-122）是一种丰度较高且保守的肝特异性微小RNA，可调控肝脏代谢并发挥肿瘤抑制因子功能，但目前针对miR-122靶标网络的系统性直接生化阐释仍不完善。为此，我们分别在miR-122基因敲除（miR-122 KO）小鼠及同窝对照小鼠的肝脏中，以及配对的人类肝细胞癌（HCC）与良性肝组织中开展了Argonaute交联免疫沉淀（Ago-CLIP）测序，以在两个物种中实现全转录组水平的miRNA靶位点鉴定。我们发现，miR-122的结合位点多数分布于3'非翻译区（3'-UTRs）与编码外显子，此外还存在大量结合于其他基因位点及非基因位点的情况。对miR-122依赖性结合位点的基序分析显示，除经典基序外，还存在一种新型G凸起基序。大量miR-122靶标被证实具有物种特异性。多个小鼠与人类共有的靶标上调后，其中尤以BCL9最为显著，可用于预测HCC患者的生存情况。上述结果全面阐明了miR-122表达下调在肝细胞癌中的分子效应。整体实验设计：我们从4只6周龄的miR-122 KO小鼠及5只同窝floxed对照小鼠中构建了AGO CLIP-seq文库。