ADAR1-dependent miR-3144-3p editing simultaneously induces MSI2 expression and suppresses SLC38A4 expression in liver cancer.

Aberrant adenosine-to-inosine (A-to-I) RNA editing, catalyzed by adenosine deaminase acting on double-stranded RNA (ADAR), has been implicated in various cancers, but the mechanisms by which microRNA (miRNA) editing contributes to cancer development are largely unknown. Our multistage hepatocellular carcinogenesis transcriptome data analyses, together with publicly available data, indicated that ADAR1 was the most profoundly dysregulated gene among RNA-editing enzyme family members in liver cancer. Targeted inactivation of ADAR1 inhibited the in vitro tumorigenesis of liver cancer cells. An integrative computational analyses of RNA-edited hotspots and the known editing frequency of miRNAs suggested that the miRNA miR-3144-3p was edited by ADAR1 during liver cancer progression. Specifically, ADAR1 promoted A-to-I editing of canonical miR-3144-3p to replace the adenosine at Position 3 in the seed region with a guanine (ED_miR-3144-3p(3_A < G)) in liver cancer cells. We then demonstrated that Musashi RNA-binding protein 2 (MSI2) was a specific target of miR-3144-3p and that MSI2 overexpression was due to excessive ADAR1-dependent over-editing of canonical miR-3144-3p in liver cancer. In addition, target prediction analyses and validation experiments identified solute carrier family 38 member 4 (SLC38A4) as a specific gene target of ED_miR-3144-3p(3_A < G). The ectopic expression of both ADAR1 and the ED_miR-3144-3p(3_A < G) mimic enhanced mitotic activities, and ADAR1 suppressed SLC38A4 expression in liver cancer cells. Treatments with mouse-specific ADAR1-, MSI2-siRNA-, or SLC38A4-expressing plasmids suppressed tumorigenesis and tumor growth in a mouse model of spontaneous liver cancer. Our findings suggest that the aberrant regulation of ADAR1 augments oncogenic MSI2 effects by excessively editing canonical miR-3144-3p and that the resultant ED_miR-3144-3p(3_A < G) simultaneously suppresses tumor suppressor SLC38A4 expression, contributing to hepatocellular carcinogenesis.

Serum Proteins, HMMR, NXPH4, PITX1 and THBS4; A Panel of Biomarkers for Early Diagnosis of Hepatocellular Carcinoma.

The high morbidity rate of hepatocellular carcinoma (HCC) is mainly linked to late diagnosis. Early diagnosis of this leading cause of mortality is therefore extremely important. We designed a gene selection strategy to identify potential secretory proteins by predicting signal peptide cleavage sites in amino acid sequences derived from transcriptome data of human multistage HCC comprising chronic hepatitis, liver cirrhosis and early and overt HCCs. The gene selection process was validated by the detection of molecules in the serum of HCC patients. From the computational approaches, 10 gene elements were suggested as potent candidate secretory markers for detecting HCC patients. ELISA testing of serum showed that hyaluronan mediated motility receptor (HMMR), neurexophilin 4 (NXPH4), paired like homeodomain 1 (PITX1) and thrombospondin 4 (THBS4) are early-stage HCC diagnostic markers with superior predictive capability in a large cohort of HCC patients. In the assessment of differential diagnostic accuracy, receiver operating characteristic curve analyses showed that HMMR and THBS4 were superior to α-fetoprotein (AFP) in diagnosing HCC, as evidenced by the high area under the curve, sensitivity, specificity, accuracy and other values. In addition, comparative analysis of all four markers and AFP combinations demonstrated that HMMR-PITX1-AFP and HMMR-NXPH4-PITX1 trios were the optimal combinations for reaching 100% accuracy in HCC diagnosis. Serum proteins HMMR, NXPH4, PITX1 and THBS4 can complement measurement of AFP in diagnosing HCC and improve identification of patients with AFP-negative HCC as well as discriminate HCC from non-malignant chronic liver disease.