Role of miR-148a in Hepatitis B Associated Hepatocellular Carcinoma

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 b-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 cotransfected with reporter gene mutants in the 39UTR of PTEN mRNA. Introduction of anti-miR-148a also resulted in depressed Akt signaling by HBx and URG11, resulting in decreased expression of b-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 associate

Hepatitis Bx Antigen Stimulates Expression of a Novel Cellular Gene, URG4, that Promotes Hepatocellular Growth and Survival

Hepatitis B virus encoded X antigen (HBxAg) may contribute to the development of hepatocellular carcinoma (HCC) by up-or downregulating the expression of cellular genes that promote cell growth and survival. To test this hypothesis, HBxAg-positive and-negative HepG2 cells were constructed, and the patterns of cellular gene expression compared by polymerase chain reaction select cDNA subtraction. The full-length clone of one of these upregulated genes (URG), URG4, encoded a protein of about 104 kDa. URG4 was strongly expressed in hepatitis 13-infected liver and in HCC cells, where it costained with HBxAg, and was weakly expressed in uninfected liver, suggesting URG4 was an effector of HBxAg in vivo. Overexpression of URG4 in HepG2 cells promoted hepatocellular growth and survival in tissue culture and in soft agar, and accelerated tumor development in nude mice. Hence, URG4 may be a natural effector of HBxAg that contributes importantly to multistep hepatocarcinogenesis

Enhanced Cell Survival of Gastric Cancer Cells By a Novel Gene URG4

Upregulated gene 4 (URG4), a novel gene located on 7 chromosome (7p13), was found to contribute to hepatocarcinogenesis. However, the role of URG4 in the gastric carcinogenesis still remains unclear. In the present study, URG4 was found by immunohistochemistry to be upregulated in human gastric cancer tissues compared with matched adjacent nonneoplastic tissues. The proliferating cell nuclear antigen index is higher in gastric cancer tissues with high URG4 expression than in those with low URG4 expression. The growth of GES-1 cells, which are immortalized human gastric epithelial mucosa cells with baseline URG4 expression, was accelerated by URG4 induction. Downregulation of URG4 through URG4 small interfering RNA (siRNA) in SGC7901 and MKN28 cells, which had high endogenous URG4 expression, suppressed cell proliferation in both of these cells. URG4-siRNA also inhibited the proliferation of SGC7901 and MKN28 cells in soft agar and tumor formation in nude mice. Overexpression of URG4 in GES cells upregulated cyclin D1, whereas repression of URG4 in SGC7901 and MKN28 cells downregulated cyclin D1. The data suggested that URG4 played an important role in the development of human gastric cancer by regulating the expression of cyclin D1 and might be used as a potential therapeutic target for gastric cancer

Differentially expressed miRNAs in HepG2X and HepG2URG11 compared to HepG2CAT cells by miRNA array.

a<p>fold-changes for each miRNA in HepG2X and HepG2URG11 cells were calculated relative to the corresponding miRNA levels in HepG2CAT cells.</p

Characteristics of HBxAg and miR-148a in tumor and nontumor compartments from HBV-infected HCC patients.

<p>a. Sex 1  =  male; 2  =  female.</p><p>b. Hepatitis b surface antigen (HBsAg) presence in serum: 0  =  no, 1  =  yes.</p><p>c. NT  =  non-tumor; T  =  tumor.</p><p>d. Intensity of HBxAg staining was evaluated as 0 (no signal) through 3 (intense signal).</p><p>e. Tissue distribution of HBxAg: S  =  scattered, L  =  lobular, D  =  diffuse.</p><p>f. Negative ΔΔCt value  =  elevated expression of miR-148a in tumor; positive ΔΔCt value  =  elevated expression of miR-148a in non-tumor liver.</p><p>g. Fold change is calculated as 2^ΔΔCt. The average fold change for elevated miR-148a was calculated by adding up the fold-change from each patient with a positive ΔΔCt value divided by the number of patients with a positive ΔΔCt value. The positive ΔΔCt values listed in this table are plotted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035331#pone-0035331-g001" target="_blank">Figure 1</a> and represent elevated miR-148a levels in liver compared to tumor. Parallel calculations were performed to determine the average fold change among patients with negative ΔΔCt values, which are plotted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035331#pone-0035331-g001" target="_blank">Figure 1</a> as elevated miR-148a levels in tumor compared to liver.</p

Role of miR-148a in tumorigenesis.

<p>Role of miR-148a in tumorigenesis.</p

The relationship between URG11 and PTEN.

<p>(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.</p

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

<p>(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. *  =  <i>P</i> < 0.05 and **  =  <i>P</i> < 0.01 in the Student’s t test.</p