Role of miR-148a in hepatitis B associated hepatocellular carcinoma

Abstract

Hepatitis B virus encoded X antigen (HBx) is a trans-regulatory protein that alters the activity of selected transcription factors and cytoplasmic signal transduction pathways. HBx transcriptionally up-regulates the expression of a unique gene, URG11, which in turn transcriptionally up-regulates β-catenin, thereby contributing importantly to hepatocarcinogenesis. HBx and URG11 also alter the expression of multiple microRNAs, and by miRNA array analysis, both were shown to promote the expression of miR-148a. Elevated miR-148a was also seen in HBx positive liver samples from infected patients. To study the function of miR-148a, anti-148a was introduced into HepG2 and Hep3B cells stably expressing HBx or stably over-expressing URG11. Anti-miR-148a suppressed cell proliferation, cell cycle progression, cell migration, anchorage independent growth in soft agar and subcutaneous tumor formation in SCID mice. Introduction of anti-miR-148a increased PTEN protein and mRNA expression, suggesting that PTEN was targeted by miR-148a. Anti-miR-148a failed to suppress PTEN expression when co-transfected with reporter gene mutants in the 3'UTR of PTEN mRNA. Introduction of anti-miR-148a also resulted in depressed Akt signaling by HBx and URG11, resulting in decreased expression of β-catenin. Thus, miR-148a may play a central role in HBx/URG11 mediated HCC, and may be an early diagnostic marker and/or therapeutic target associated with this tumor type.

Figure 1. Relationship between HBx, URG11 and miR-148a expression levels.

(A) Endogenous levels of miR-148a were determined in the indicated cell lines, and their levels normalized to miR-148a in CAT expressing cells. (B) Cells were transiently transfected with URG11 siRNA (white bars), anti-miR-148a (light gray bars, positive control) or control miRNA (dark gray bars), and the levels of endogenous miR-148a determined. (C) Parallel experiments as in panel A except that HepG2RG11 and Hep3BURG11 cells were used. (D) Relative levels of URG11 protein in cells transiently transfected with URG11 specific siRNA or control siRNA. The results show one of three experiments.

Figure 2. Expression of miR-148a in tumor and non-tumor liver tissues.

Each bar represents data collected from one patient. The difference in miRNA expression between tumor (T) and non-tumor (NT) was determined by qRT-PCR and determination of ΔΔCT, where ΔΔCt  =  ΔCt of miR-148a in tumor – ΔCt of miR-148a in non-tumor. U6 was used for normalization. Negative values indicate that miR-148a levels were higher in tumor compared to adjacent non-tumor. Positive values indicate that miR-148a levels were higher in NT (liver) compared to T. ^aHBx NT, HBx T: HBx staining in tumor (T) and nontumor (NT) is scored as follows: 0  =  negative, 1  =  up to 25% of cells stained positive, 2  =  25–50% of cells stained positive, 3  =  > 50% cells stained positive. ^bDx NT refers to diagnosis of lesions in nontumor liver. These are as follows: 0  =  no significant lesions, 1  =  chronic hepatitis, 2  =  cirrhosis. ^cedmon T refers to the Edmondson classification of cellular differentiation within tumor nodules. They are as follows: 1  =  Edmondson I-II, 2  =  Edmondson II, 3  =  Edmondson II-III, 4  =  Edmondson III. dVein inv T refers to venous invasion of the tumor nodule, where 0  =  no evidence for invasion, and 1  =  presence of venous invasion. eEncap T refers to tumor encapsulation, where 0  =  none and 1  =  encapsulation.

Figure 3. Effect of anti-miR-148a on cell phenotype.

HepG2CAT, HepG2X and HepG2URG11 cells were (A) transiently transfected with anti-miR-148a or (B) stably transduced with recombinant lentivirus encoding anti-miR-148a. In both cases, cell growth was evaluated by MTT assay on days 1, 2 and 3 of culture. All values were normalized to cells treated only with transfection reagent and are presented as a ratio of test:control O.D. values. (C) Serum starved cells were analyzed by flow cytometry on day 3 after the addition of serum. The percentage of cells in G1,S,G2/M phases are indicated in each panel.

Figure 4. Effect of anti-miR148a on cell migration and anchorage independent growth.

(A) Migration of HepG2CAT, HepG2X, and HepG2URG11 cells stably expressing anti-miR-148a or control anti-miR. The top portion of this panel shows the results of a single experiment, while the graphical representation below is the average from 3 independent experiments. The black bar in the photomicrographs is 50um. (B) Summary of soft agar assays for HepG2CAT, HepG2X and HepG2URG11 cells stably expressing anti-miR-148a or control anti-miR. The average number of colonies reported here was from three independent experiments performed in triplicate. *  =  P < 0.05; **  =  P < 0.01 in the Student’s t test.

Figure 5. Effect of anti-miR-148a on PTEN expression.

(A) Homology between the PTEN 3′UTR and anti-miR-148a. (B) PTEN protein levels in cells stably expressing anti-miR-148a or anti-miR control were determined by western blotting. (C) HepG2CAT, HepG2X, HepG2URG11 cells were transiently co-transfected with anti-miR-148a or anti-miR-Ctrl and a PTEN 3′UTR expression plasmid. Firefly (test) and Renilla (control) luciferase activities were measured after 48hr. The ratios were calculated from the Firefly luciferase: Renilla luciferase readings. *  =  P < 0.05 and **  =  P < 0.01 in the Student’s t test.

Figure 6. Effect of anti-miR-148a upon the expression levels of Akt, GSK3β, and β-catenin.

The indicated cell lines were stably transfected with anti-miR-148a (or control miRNA) and then analyzed for total and active β-catenin, p-GSK3β, as well as total and p-Akt by western blotting. The relative amounts of each, normalized to β-actin, are presented below each blot.

Figure 7. The relationship between URG11 and PTEN.

(A) URG11, PTEN, p-PTEN and PI3K protein expression in the indicated cultures transiently transfected with siURG11 or control siRNA. The numbers listed under each blot represent the band intensities after normalization to β-actin. (B) PTEN promoter activity (indicated by firefly luciferase activity) and phRL_null (the renilla luciferase control) were transiently co-transfected into the indicated cell lines. Luciferase activities were measured 48 hrs later. (C) PTEN promoter activity was assayed in cells 48 hrs after transient transfection with siURG11 or control siRNA. Each of the experiments in this figure was repeated at least three times.

Figure 8. Model of miR148a associated signaling pathway.

miR148a increases β-catenin expression by inhibiting PTEN. See the text for additional details.