Expression of <i>Notch1</i>, <i>Jagged1</i> and <i>Dll4</i> mRNA in biopsy from HBV-infected HCC patients.

<p>Total RNA isolated from dissected biopsy (n = 8) from tumor or non-tumor lesions were analysed for mRNA expression of <i>Notch1</i> (A), <i>Jagged1</i> (B) and <i>Dll4</i> (C) by qPCR.</p

Hepatitis B Virus HBx Activates Notch Signaling via Delta-Like 4/Notch1 in Hepatocellular Carcinoma

<div><p>Hepatitis virus B (HBV) infection is one of the major causes of hepatocellular carcinomas (HCC). HBx protein encoded in HBV genome is one of the key viral factors leading to malignant transformation of infected cells. HBx functions by interfering with cellular functions, causing aberration in cellular behaviour and transformation. Notch signalling is a well-conserved pathway involved in cellular differentiation, cell survival and cell death operating in various types of cells. Aberration in the Notch signalling pathways is linked to various tumors, including HCC. The role of HBx on the Notch signalling in HCC, however, is still controversial. In this study, we reported that HBV genome-containing HCC cell line HepG2 (HepG2.2.15) expressed higher Notch1 and Delta-like 4 (Dll4), compared to the control HepG2 without HBV genome. This upregulation coincided with increased appearance of the cleavage of Notch1, indicating constitutively activated Notch signalling. Silencing of HBx specifically reduced the level of Dll4 and cleaved Notch1. The increase in Dll4 level was confirmed in clinical specimens of HCC lesion, in comparison with non-tumor lesions. Using specific signalling pathway inhibitors, we found that MEK1/2, PI3K/AKT and NF-κB pathways are critical for HBx-mediated Dll4 upregulation. Silencing of HBx clearly decreased the level of phosphorylation of Akt and Erk1/2. Upon silencing of <i>Dll4</i> in HepG2.2.15, decreased cleaved Notch1, increased apoptosis and cell cycle arrest were observed, suggesting a critical role of HBx-Dll4-Notch1 axis in regulating cell survival in HCC. Furthermore, clonogenic assay confirmed the important role of Dll4 in regulating cell survival of HBV-genome containing HCC cell line. Taken together, we reported a link between HBx and the Notch signalling in HCC that affects cell survival of HCC, which can be a potential target for therapy.</p></div

Effects of specific inhibitors on expression of Notch1 and Dll4 and effects of HBx silencing on activation of MAPK, NF-κB and PI3K/Akt pathways in HepG2.2.15.

<p>(A) HepG2.2.15 were treated with indicated doses of inhibitors for 24 hr. Cell lysates from were analysed by Western blot. β-actin was used as loading control. BAY11-7082, LY294002, SB203580 and U0126 are pathway specific inhibitors of NF-κB, PI3K/Akt, MAPK/p38 and MAPK/ERK, respectively. (B-C) HepG2.2.15 was transiently transfected with siHBx or control scramble siRNA for 48 hr. Cell lysates from were analysed by Western blot. β-actin was used as loading control.</p

Protein profiles of Notch receptors, Notch ligands and the expression of Notch target gene <i>Hes1</i> in HepG2 and HepG2.2.15.

<p>(A) Cell lysates from HepG2 or HepG2.2.15 were analysed by Western blot. β-actin was used as loading control. (B) Total RNA from three cell lines were analyzed for expression profiles of <i>Hes1</i> by qPCR. The results are representative of at least two independent experiments where *** indicates statistical significance at the <i>p</i> <0.001 level.</p

Effects of silencing <i>Dll4</i> on the level of cleaved Notch1 in HepG2.2.15.

<p>HepG2.2.15 was transiently transfected with siRNA specific for <i>Dll4</i> or control scramble siRNA as described in materials and methods. Total RNA was analysed for mRNA expression of <i>Notch1</i>, <i>Dll4</i> and <i>Hes1</i> (A) by qPCR. The results are representative of at least two independent experiments where ** indicates statistical significance at the <i>p</i><0.01 level. Cell lysates were analysed for cleaved Notch1, Notch1 and Dll4 by Western blot. β-actin was used as loading control (B).</p

Expression profiles of mRNA of Notch ligands in THLE-2, HepG2 and HepG2.2.15.

<p>Total RNA from three cell lines were analyzed for expression profiles of <i>Jagged 1</i> and <i>Jagged2</i> (A, B) and <i>Dll1</i>, <i>Dll3</i>, <i>Dll4</i> (C-E) by qPCR. The results are representative of at least two independent experiments where *** indicates statistical significance at the <i>p</i> <0.001 level.</p

Effect of silencing <i>Dll4</i> on cell cycle progression and apoptosis of HepG2.2.15.

<p>HepG2.2.15 was transiently transfected with siRNA specific for <i>Dll4</i> or control scramble siRNA as described in materials and methods. (A) Cell viability was measured by MTS assay. The results are representative of at least two independent experiments where * and *** indicates statistical significance at the <i>p</i> <0.05 and <i>p</i><0.001 level, respectively. (B) Cell cycle analysis was carried out and the percentages of cells in each phase was calculated. The results are representative of at least two independent experiments where *** indicates statistical significance at the <i>p</i> <0.001 level. (C) Apoptosis was detected by Annexin V binding assay. The results are representative of at least two independent experiments where * indicates statistical significance at the <i>p</i> <0.05 level. (D) HBV genome per cell was measured in control or <i>Dll4</i> silencing HepG2.2.15 as described in materials and methods.</p

Effects of <i>HBx</i> silencing on mRNA expression of Notch ligands in HepG2.2.15.

<p>HepG2.2.15 was transiently transfected with siRNA specific for <i>HBx</i> or control scramble siRNA as described in materials and methods. Total RNA from three cell lines were analyzed for mRNA expression of <i>Jagged1</i> (A), <i>Jagged 2</i> (B) <i>Dll1</i> (C) <i>Dll3</i> (D), <i>Dll4</i> (E) by qPCR. The results are representative of at least two independent experiments where ** and *** indicates statistical significance at the <i>p</i> <0.01 and <i>p</i><0.001 level, respectively.</p

Effects of <i>HBx</i> silencing on mRNA and protein expression of HBV viral genes and Notch receptors in HepG2.2.15.

<p>HepG2.2.15 was transiently transfected with siRNA specific for <i>HBx</i> or control scramble siRNA as described in materials and methods. Total RNA or proteins from HepG2.2.15 transfected with scramble or <i>HBx</i> siRNA were analyzed for viral RNA expression or Western blot (A). The relative level of HBsAg (B) and HBeAg (C) was investigated by chemiluminescent microparticle immunoassay (CMIA). S/CO is sample RLU/Cutoff RLU. The level of <i>Notch 1–4</i> mRNA was analyzed by qPCR (D-G). The results are representative of at least two independent experiments where *, ** and *** indicates statistical significance at the <i>p</i><0.05, <i>p</i> <0.01 and <i>p</i><0.001 level, respectively.</p

Effects of <i>HBx</i> silencing in HepG2.2.15 and HBx overexpression in HepG2 on expression of Notch receptors and ligands.

<p>(A) HepG2.2.15 was transiently transfected with siRNA specific for <i>HBx</i> or control scramble siRNA as described in materials and methods. Cell lysates from were analysed by Western blot. β-actin was used as loading control. (B-C) HepG2 was transiently transfected with the control pEGFP vector or pEGFP-HBx for 24 hr. Cells were sorted for GFP⁺ cells. Total RNA from three cell lines were analyzed for mRNA expression of Notch receptors, Notch ligands and <i>Hes1</i> by qPCR. The results are representative of at least two independent experiments where *, ** and *** indicates statistical significance at the <i>p</i> <0.05, <i>p</i><0.01 and <i>p</i><0.001 level, respectively.</p