Abstract 1928: Comprehensive identification of miR-122 targetome in the liver and HCC by HITS-CLIP analysis reveals conserved regulation of AMPK signaling in hepatocarcinogenesis
Authors: Barajas Juan 
Published: July 2016

Abstract
miR-122, a conserved liver-specific miRNA, is critical for maintaining liver homeostasis. Loss of miR-122 is associated with loss of hepatic phenotype, metastasis and poor prognosis. Recent in vivo work has established miR-122 as a bona fide tumor suppressor, with poorly differentiated, Afp-positive, high grade, spontaneous HCC observed in liver-specific (LKO) and germ-line (KO) miR-122 knockout mice. We hypothesized that the pathophysiology exhibited by KO mice would be mediated through de-repressed targets in KO livers and therefore, dysfunctional regulation of specific pathways involved in liver homeostasis and carcinogenesis. Although the role of miR-122 in the maintenance of liver homeostasis is well established, systematic and direct biochemical elucidation of the miR-122 target network remains incomplete. To this end, we performed Ago-dHITS-CLIP analysis in livers of 6 week-old wild type (WT) and KO mice to identify miRNA targetome in vivo, which revealed ∼1800 miR-122 binding sites, with over 30 fold enrichment for the miR-122 seed sequence sites in WT compared to KO livers. Surprisingly, a majority of miR-122 binding sites were observed on coding exons (40%) followed by 3’-UTRs, introns and 5’-UTRs. Motif analysis of miR-122 dependent binding sites lacking the seed sequence revealed a novel G-bulged motif in addition to the canonical miR-122 seed sequences. Moreover, the hepatic gene expression profile of these mice measured by RNA-seq showed up regulation of ∼2500 genes and down regulation of ∼1200 genes in KO livers compared to WT livers. A comparison of HITS-CLIP and RNA-seq data revealed that majority of miR-122 targets harboring canonical binding sites at their 3’UTRs as well as some coding regions, are significantly upregulated in miR-122 KO livers without notable alteration in the expression of or G-bulged targets at the RNA level. HITS-CLIP analysis of several human HCC and matching benign liver tissues also identified both canonical and G-bulged targets; where the majority of both are located in the coding regions followed by 3’UTRs. Association of miR-122 targets with Argonaute was precipitously reduced in HCCs expressing lower miR-122 levels than respective benign livers. Notably, though miR-122 is completely conserved and highly expressed in human and mouse livers, a large number of identified targets seem to be species-specific. Ingenuity Pathway Analysis of enriched targets revealed conserved pathway specific regulation in mouse and human livers. Pathways commonly associated with critical roles in liver carcinogenesis and liver metabolism, such as AMPK signaling and PI3/AKT pathways, were the major targets of miR-122-mediated regulation. This study suggests that miR-122 functionality is conserved across species.
Citation Format: Juan Barajas. Comprehensive identification of miR-122 targetome in the liver and HCC by HITS-CLIP analysis reveals conserved regulation of AMPK signaling in hepatocarcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1928.