The role of miR-122 in the dysregulation of glucose-6-phosphate dehydrogenase (G6PD) expression in hepatocellular cancer

Abstract

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.

Figure 1

G6PD is altered in human HCC. (a) G6PD mRNA levels in tumors from liver cancer patients. Normalized mRNA levels from benign livers (n = 50) and liver tumors (n = 377) quantified using RNA-seq were queried from The Cancer Genome Atlas. Significance was determined using Welch’s T-Test function in R (p-value = 1.082346e-40). (b) Dot-plot of G6PD mRNA levels in different tumor grades (ANOVA, P-value = 6.79e-07). Comparison between means using Tukey Honest Significance Test in R revealed significant difference in G6PD mRNA levels with increasing tumor grade (Supplementary Table 1). (c) Kaplan-Meier curve of G6PD overall survival in liver cancer patients (n = 318, patients with missing data or surviving past 5 years were removed from the analysis). Patients were stratified by high expression (top 50th percentile), and low expression (bottom 50th percentile). (d) Kaplan-Meier curve of tumor recurrence in liver cancer patients with respect to G6PD levels. Analysis was done as described in (c).

Figure 2

G6PD is a novel target of miR-122. (a) Schematic depicting miR-122 sites located on the human G6PD 3′-UTR. (b,c) G6PD normalized expression (log2(normalized count +1)) and RSEM expression (log2) of miR-122 (b) and miR-1 (c) were plotted per tumor sample. miRNA and mRNA expression data in liver cancer patients was queried from The Cancer Genome Atlas using Xena UCSC Browser (xena.ucsc.edu; xenabrowser.net). Regression coefficients and corresponding p-values are shown.

Figure 3

Validation of G6PD as a novel miR-122 target. (a) Luciferase reporter assay of G6PD 3′-UTR (wild type or mutant) driven Renilla luciferase activity normalized to firefly luciferase after transfection H293T cells with scrambled (NC) or miR-122 mimic RNA (miR-122). (b,c) G6PD mRNA levels in HCC cells transfected with in miR-122 mimic or scrambled (NC) RNA (b), and in Huh-7 cells transfected with miR-122 anti-sense oligo (miR-122 KD) or negative control oligo (NC) (c). mRNA levels were measured by RT-qPCR.

Figure 4

miR-1 and miR-122 levels are negatively correlated in human liver cancer. Scatterplot and linear regression of miR-122 and miR-1 levels in liver cancer patients. The LIHC-TCGA data downloaded from UCSC Xena Cancer browser (xenabrowser.net) was plotted and linear regression coefficients were calculated using the lm() function in R. Overall regression coefficient between miR-122 and miR-1 was calculated as −0.212 (p-value = 1.22 × 107). Data was further stratified by G6PD mRNA levels (high = dark blue, low = light blue). Individual coefficients and p-values were calculated for each condition using the lm() function in R and are shown on the plot. Density plots were used to visualize the distribution of miR-1 (top panel) and miR-122 (right panel) levels in the context of G6PD mRNA levels. Kolmogorov–Smirnov test was used to calculate significant differences in the relative distribution of miRNA and G6PD mRNA levels (miR-122 p-value = 1.94 × 10⁻⁸, miR-1 p-value = 0.09263).

Figure 5

miR-1 and miR-122 reppress G6PD expression by direct targeting. (a) G6PD was validated as a miR-1 and miR-122 target using luciferase reporter assay. H293-T cells were transfected with control RNA (NC), miR-122, miR-1, or combination of both, along with psi-CHECK2 vector harboring human G6PD 3′-UTR and Firefly luciferase (an internal control). After 48 hours, luciferase activities were measured in cell extracts per manufacture’s protocol (Promega). (b,c) G6PD immunoblot and RT-qPCR analysis shows depletion of G6PD protein level (b) and mRNA level (c) in HCC cells transfected with miRNA mimics (50 nM) for 48hrs. (* indicates p-value < 0.05, p-value was calculated using Student T-test). Cropped immunoblot images in (b) were obtained from the same gel. Full immunoblot images are found in supplemental material.

Figure 6

G6PD expression and activity are suppressed by miR-122 and miR-1 in HepG2 cells. HepG2 cells transfected with scrambled miRNA (NC), miR-1, miR-122, combo (miR-1 and miR-122), G6PD siRNA (siG6PD) or negative control siRNA (NC) for 48 hours. (a) G6PD activities in these cells were determined by measuring NADPH absorbance at 341 nm and calculating the NADP⁺/NADPH ratios. Asterisks indicates p-value < 0.05, p-value was calculated using two-tailed Student T-test). (b) Immunoblotting of G6PD protein was performed in the lysates. G6PD levels were normalized to that of vinculin. (c,d) Cell viability measured using CellTiter-Glo® Luminescent kit. Cell viability over time was determined as change in luminescence from 0-hour time point. Cropped immunoblot images in (b) were obtained from the same gel. Full immunoblot images are found in supplemental material.