Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. Thus, a better understanding of molecular aberrations involved in HCC pathogenesis is necessary for developing effective therapy. It is well established that cancer cells metabolize energy sources differently to rapidly generate biomass. Glucose-6-phosphate-dehydrogenase (G6PD), the rate-limiting enzyme of the Pentose Phosphate Pathway (PPP), is often activated in human malignancies to generate precursors for nucleotide and lipid synthesis. Here, we determined the clinical significance of G6PD in primary human HCC by analyzing RNA-seq and clinical data in The Cancer Genome Atlas. We found that the upregulation of G6PD correlates with higher tumor grade, increased tumor recurrence, and poor patient survival. Notably, liver-specific miR-122, which is essential for metabolic homeostasis, suppresses G6PD expression by directly interacting with its 3′UTR. Luciferase reporter assay confirmed two conserved functional miR-122 binding sites located in the 3′-UTR of G6PD. Furthermore, we show that ectopic expression of miR-122 and miR-1, a known regulator of G6PD expression coordinately repress G6PD expression in HCC cells. These miRNAs also reduced G6PD activity in HepG2 cells that express relatively high activity of this enzyme. Collectively, this study provides evidence that anti-HCC efficacy of miR122 and miR-1 could be mediated, at least in part, through inhibition of PPP by suppressing the expression of G6PD.

MicroRNAs are ~22 nucleotide RNAs that regulate gene expression at the post-transcriptional level by binding messenger RNA transcripts. miR-21 is described as an oncomiR whose steady-state levels are commonly increased in many malignancies, including hepatocellular carcinoma (HCC). Methods known as cross-linking and immunoprecipitation of RNA followed by sequencing (CLIP-seq) have enabled transcriptome-wide identification of miRNA interactomes. In our study, we use a publicly available Argonaute-CLIP dataset (GSE97061), which contains nine HCC cases with matched benign livers, to characterize the miR-21 interactome in HCC. Argonaute-CLIP identified 580 miR-21 bound target sites on coding transcripts, of which 332 were located in the coding sequences, 214 in the 3′-untranslated region, and 34 in the 5′-untranslated region, introns, or downstream sequences. We compared the expression of miR-21 targets in 377 patients with liver cancer from the data generated by The Cancer Genome Atlas (TCGA) and found that mRNA levels of 402 miR-21 targets are altered in HCC. Expression of three novel predicted miR-21 targets (CAMSAP1, DDX1 and MARCKSL1) correlated with HCC patient survival. Analysis of RNA-seq data from SK-Hep1 cells treated with a miR-21 antisense oligonucleotide (GSE65892) identified RMND5A, an E3 ubiquitin ligase, as a strong miR-21 candidate target. Collectively, our analysis identified novel miR-21 targets that are likely to play a causal role in hepatocarcinogenesis.