ADAR1 promotes miR-3144-3p editing to simultaneously induce oncogenic MSI2 and suppress tumor suppressor SLC38A4 in liver cancer

Background: Aberrant adenosine-to-inosine (A-to-I) RNA editing, catalyzed by adenosine deaminase acting on double-stranded RNA (ADAR), is implicated in various cancers, but the mechanisms by which microRNA (miRNA) editing contributes to cancer development are currently largely unknown. Method: Our multi-step hepatocellular carcinogenesis transcriptome data analyses, together with publicly available data, indicated that ADAR1 is the most dysregulated gene among the RNA editing enzyme families in hepatocellular carcinoma (HCC). Targeted inactivation of ADAR1 inhibits in vitro tumorigenesis of HCC cells. Integrative computational analyses of RNA editing hotspots and the editing frequency of miRNAs suggested miR-3144-3p a potential mRNA edited by ADAR1 in hepatocellular carcinogenesis. Result: Through direct sequencing of ADAR1-knocked-down or -overexpressing cells, we demonstrated that ADAR1 promoted A-to-I editing of the canonical miR-3144-3p to change position 3 adenosine in the seed region to guanine (ED_miR-3144-3p(3_A<G)) in HCC cells. We then demonstrated that Musashi RNA-binding protein 2 ( MSI2 ) is a specific target of miR-3144-3p, and that MSI2 overexpression is due to ADAR1-dependent over-editing of the canonical miR-3144-3p in HCC cells. Notably miR-3144-3p treatments showed similar to in vitro anti-tumor activities of ADAR1-specifc siRNA in HCC. In addition, target prediction analyses and validation experiments identified solute carrier family 38 member 4 ( SLC38A4 ) as specific target gene for ED_miR-3144-3p(3_A<G). We also demonstrated that ectopic expressions of both ADAR1 and ED_miR-3144-3p(3_A<G) mimics enhanced mitotic activities and that ADAR1 suppressed SLC38A4 in HCC cells. Treatments with mouse-specific ADAR1-, MSI2-siRNA or SLC38A4-expressing plasmids suppressed tumor incidence and growth in a spontaneous mouse HCC model. Conclusions: : Our findings suggest that aberrant regulation of ADAR1 augments oncogenic MSI2 via overediting the canonical miR-3144-3p, and the resultant ED_miR-3144-3p(3_A<G) simultaneously suppresses tumor suppressor SLC38A4, thereby contributing to hepatocellular carcinogenesis.