A comprehensive analysis of the interactome of miR-21, an established oncomir, by Argonaute-CLIP analysis identifies novel conserved and species-specific targets of miR-21 in human liver and hepatocellular carcinoma

MicroRNAs are ~22 nucleotide RNAs that regulate gene expression at the post-transcriptional level by binding messenger RNA transcripts. MicroRNA (miR)-21 participates in carcinogenesis in hepatocellular carcinoma (HCC) and other malignancies. Here we analyze the miR-21 interactome in HCC by high-throughput sequencing of RNA isolated by Argonaute cross-linking immunoprecipitation (Argonaute-CLIP) in nine HCC cases with matched livers. Argonaute-CLIP identified miR-21 bound to 580 target sites on coding transcripts, of which 332 were located in the coding sequences, 214 in the 39-untranslated region, and 34 in the 59-untranslated region, introns or downstream sequences. Overall, 403 target sites (69.5%) enriched for miR-21 in HCCs relative to liver. We compared expression of miR-21 targets in 377 patients with liver cancer registered in The Cancer Genome Atlas. Ago-CLIP miR-21 enrichment score in HCC distinguished expression of miR-21 targets by their correlation with miR-21 expression. Comparison of the miR-21 interactome in mouse and human livers identified common and species-specific targets. Among targets of miR-21, 27.6% were downregulated in HCC. The miR-21 interactome was predicted to regulate tumor metabolism by inhibiting PPAR alpha/RXR alpha and LXR/RXR signaling. To identify targets of miR-21, we matched Ago-CLIP targets to genes previously reported as aberrantly expressed after anti-miR-21 overexpression in HCC. RMND5A, encoding an E3 ubiquitin ligase, was identified as a potential target of miR-21 regulation and was variably expressed in HCC. Three predicted miR-21 targets, CAMSAP1, DDX1 and MARCKSL1, correlated with HCC patient survival. Collectively, our analysis biochemically identified novel targets of miR-21 likely to play a causal role in hepatocarcinogenesis. Citation Format: Aaron B. Koenig, Juan M. Barajas, Kalpana Ghoshal. A comprehensive analysis of the interactome of miR-21, an established oncomir, by Argonaute-CLIP analysis identifies novel conserved and species-specific targets of miR-21 in human liver and hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5400.