Expression Levels Of HBx Research Articles

HBX protein promotes LASP-1 expression through activation of c-Jun in human hepatoma cells.

LIM and SH3 domain protein 1 (LASP-1) is known to participate in the progression of hepatocellular carcinoma (HCC). We previously showed that ectopic expression of hepatitis B virus (HBV) X protein (HBX) enhanced the expression of LASP-1, which promoted proliferation and migration of HCC cells. Here, we further demonstrated the molecular mechanism underlying upregulation of LASP-1, mediated by HBX, in HBV-infected HCC cells. Through a luciferase activity assay, we discovered that the LASP-1 promoter region regulated by HBX contained an AP-1 binding element in human hepatoma cells. Interestingly, c-Jun, one subunit of AP-1, was mainly responsible for activation, mediated by HBX, of the LASP-1 promoter. Furthermore, HBX was shown not only to interact with phosphorylated c-Jun in HCC cells but also to activate c-Jun by increasing the activation of PI3-K/JNK signaling. Chromatin immunoprecipitation (ChIP) assay demonstrated that HBX was capable of binding to the LASP-1 promoter with c-Jun. Further, the expression levels of HBX were shown to be significantly positively correlated with that of LASP-1 and phosphorylatedc-Jun in HBV-related HCC tissues by immunohistochemistry analysis. In addition, the N-terminus of HBX was found to be responsible for the activation of c-Jun, as well as the expression of LASP-1. Taken together, these results suggest that HBX contributes to LASP-1 expression via the activation of c-Jun to increase the promoter activity of LASP-1 in HBV-related HCC cells.

HBx protein-mediated ATOH1 downregulation suppresses ARID2 expression and promotes hepatocellular carcinoma.

Hepatitis B virus X protein plays a crucial role in the pathogenesis of hepatocellular carcinoma. We previously showed that the tumor suppressor ARID2 inhibits hepatoma cell cycle progression and tumor growth. Here, we evaluated whether hepatitis B virus X protein was involved in the modulation of ARID2 expression and hepatocarcinogenesis associated with hepatitis B virus infection. ARID2 expression was downregulated in HBV‐replicative hepatoma cells, HBV transgenic mice, and HBV‐related clinical HCC tissues. The expression levels of HBx were negatively associated with those of ARID2 in hepatocellular carcinoma tissues. Furthermore, HBx suppressed ARID2 at transcriptional level. Mechanistically, the promoter region of ARID2 gene inhibited by HBx was located at nt‐1040/nt‐601 and contained potential ATOH1 binding elements. In addition, ectopic expression of ATOH1 or mutation of ATOH1 binding sites within ARID2 promoter partially abolished HBx‐triggered ARID2 transcriptional repression. Functionally, ARID2 abrogated HBx‐enhanced migration and proliferation of hepatoma cells, whereas depletion of ATOH1 enhanced tumorigenecity of HCC cells. Therefore, our findings suggested that deregulation of ARID2 by HBx through ATOH1 may be involved in HBV‐related hepatocellular carcinoma development.

Hepatitis B virus X protein promotes interleukin-7 receptor expression via NF-κB and Notch1 pathway to facilitate proliferation and migration of hepatitis B virus-related hepatoma cells.

BackgroundInterleukin-7 receptor (IL-7R) is involved in the abnormal function of solid tumors, but the role and regulatory mechanisms of IL-7R in HBV-related hepatocellular carcinoma (HCC) are still unclear.MethodsGene and protein expression levels of IL-7R were examined in hepatoma cells transfected with hepatitis B virus (HBV) plasmids and in hepatoma cells transfected with the multifunctional nonstructural protein X (HBX). The expression of HBX and IL-7R was measured by immunohistochemical analysis in HBV-related HCC tissues. The role of NF-κB and Notch1 pathways in HBX-mediated expression of IL-7R in hepatoma cells was examined. Activation of IL-7R downstream of intracellular signaling proteins AKT, JNK, STAT5, and the associated molecules CyclinD1 and matrix metalloproteinase-9 (MMP)-9, was assessed in HBX-positive cells with or without treatment with IL-7R short hairpin RNA (shRNA). Additionally, the role of IL-7R in HBX-mediated proliferation and migration of hepatoma cells was investigated.ResultsThe expression of IL-7R was increased in hepatoma cells transfected with HBV plasmids; HBX was responsible for the HBV-mediated upregulation of IL-7R. Compared to adjacent tissues, the expression of HBX and IL-7R was increased in HBV-related HCC tissues. Additionally, the relative expression levels of HBX were associated with IL-7R in HBV-related HCC tissues. The activation of NF-κB pathways and expression of Notch1 were increased in hepatoma cells transfected with HBX, and inhibition of NF-κB and Notch1 pathways significantly decreased HBX-mediated expression of IL-7R. The activation of AKT and JNK and the expression of CyclinD1 and MMP-9 were increased in HBX-positive cells. When cells were treated with IL-7R shRNA, the activation of AKT and JNK, as well as the expression of CyclinD1 and MMP-9, were significantly inhibited. Additionally, IL-7R was responsible for HBX-induced proliferation and migration ability of hepatoma cells.ConclusionsOur data demonstrate that HBX can upregulate IL-7R via NF-κB and Notch1 pathways to facilitate the activation of intracellular pathways and expression of associated molecules, and contribute to proliferation and migration of hepatoma cells.

E3 Ubiquitin Ligase Siah-1 is Down-regulated and Fails to Target Natural HBx Truncates for Degradation in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a common worldwide malignancy with high morbidity and mortality. Hepatitis B viral (HBV)-encoded X protein (HBx) and natural HBx variants play important roles in HBV-associated HCC development. HBx is an unstable protein that can be degraded in vivo. Our previous study found that the E3 ubiquitin ligase Siah-1 could target HBx for poly-ubiquitylation and proteasomal degradation and attenuate the transcriptional activity of HBx. However, in HCC patients, high expression levels of HBx and HBx variants are frequently observed and are associated with HCC progression. The mechanism underlying their up-regulation is largely unknown. In this study, we screened for Siah-1 mutations in 270 HCC samples and 9 HCC cell lines, and examined Siah-1 mRNA and protein expression in a subset of paired HCC specimens. Our results demonstrate that Siah-1 is highly conserved, as no somatic mutation was identified, with the exception of one synonymous transition from G to A at codon 67. Both the mRNA and protein levels of Siah-1 were significantly down-regulated in HCC tissues compared with their adjacent normal counterparts. We constructed three natural HBx truncates that were identified in our HCC cases. We found that Siah-1 failed to decrease the stability of these HBx variants and was unable to inhibit the transcriptional activity of these HBx truncates at heat shock elements (HSEs). Moreover, Siah-1 had weaker association with three HBx mutants than full length HBx. Therefore, our findings suggest that down-regulation of Siah-1, but not its mutations, and natural HBx variants resistant to Siah-1-induced degradation may be a novel mechanism for HCC development.

WISP-1: a novel mediator of hepatitis B virus-related hepatocellular carcinoma

To explore the effect of hepatitis B virus (HBV) X protein (HBX) on expression of the host gene Wnt induced secreted protein-1 (WISP-1) that is related to the pathogenic process of hepatocellular carcinoma (HCC). Tumor and paratumor tissues were collected from HCC patients, and normal liver tissues were collected from healthy controls. Immunohistochemistry was used to evaluate the in vivo presence and expression levels of HBX and WISP-1 in the three tissue types. HepG2 cells stably transfected with pc-DNA3.1(+)-HBX or with pc-DNA3.1(+) only (G0, control) were generated and used to examine in vitro the HBX-induced changes in WISP-1 expression at the mRNA and protein levels by reverse transcription polymerase chain reaction and western blotting, respectively. The HCC tissues showed significantly higher rates of positivity for WISP-1 expression than the non-tumor controls (76.6% vs. paratumor: 23.4% or normal tissues: 0%, x2= 35.967, P less than 0.01). HBX increased WISP-1 expression in HepG2 cells at both the mRNA (1170.33 +/- 41.26 vs. G0: 265.34 +/- 27.47, t = 31.63, P less than 0.01) and protein (240.33 +/- 11.37 vs. G0: 40.33 +/- 7.09, F = 600.57, P less than 0.01) levels. HBV may up-regulate expression of the host gene WISP-1 through its X protein and thus promote the development of HCC.

Hepatitis B virus X protein upregulates Lin28A/Lin28B through Sp-1/c-Myc to enhance the proliferation of hepatoma cells

Hepatitis B virus X protein (HBx) plays critical roles in the pathogenesis of hepatocellular carcinoma (HCC). Here, we were interested in knowing whether the oncogene Lin28A and its homolog Lin28B are involved in the hepatocarcinogenesis mediated by HBx. We showed that the expression levels of Lin28A and Lin28B were increased in clinical HCC tissues, HepG2.2.15 cell line and liver tissues of p21-HBx transgenic mice. Interestingly, the expression levels of HBx were positively associated with those of Lin28A/Lin28B in clinical HCC tissues. Moreover, the overexpression of HBx resulted in the upregulation of Lin28A/Lin28B in hepatoma HepG2/H7402 cell lines by transient transfection, suggesting that HBx was able to upregulate Lin28A and Lin28B. Then, we examined the mechanism by which HBx upregulated Lin28A and Lin28B. We identified that the promoter region of Lin28A regulated by HBx was located at nt -235/-66 that contained Sp-1 binding element. Co-immunoprecipitation showed that HBx was able to interact with Sp-1 in HepG2-X cells. Moreover, chromatin immunoprecipitation (ChIP) demonstrated that HBx could bind to the promoter of Lin28A, which failed to work when Sp-1 was silenced. Electrophoretic mobility shift assay (EMSA) further identified that HBx was able to interact with Sp-1 element in Lin28A promoter via transcription factor Sp-1. In addition, we found that c-Myc was involved in the activation of Lin28B mediated by HBx. In function, Lin28A/Lin28B played important roles in HBx-enhanced proliferation of hepatoma cells in vitro and in vivo. In conclusion, HBx activates Lin28A/Lin28B through Sp-1/c-Myc in hepatoma cells. Lin28A/Lin28B serves as key driver genes in HBx-induced hepatocarcinogenesis.

Hepatitis B virus X protein promotes hepatoma cell proliferation via upregulation of MEKK2

To investigate the mechanism underlying the increase of hepatoma cell proliferation by hepatitis B virus X protein (HBx). HepG2, H7402 and HepG2.2.15 cells, which constitutively replicated hepatitis B virus were used. The effects of HBx on hepatoma cell proliferation were examined using 5-ethynyl-2-deoxyuridine (EdU) incorporation assay and MTT assay. The expression level of MEKK2 was measured using RT-PCR, Western blot and luciferase reporter gene assay. The activity of activator protein 1 (AP-1) was detected using luciferase reporter gene assay. The phosphorylation levels of JNK and c-Jun were measured using Western blot. The expression levels of HBx and MEKK2 in 11 clinical hepatocellular carcinoma (HCC) tissues were measured using real time PCR and Western blot. In addition, the expression of MEKK2 in 95 clinical HCC tissues was examined using immunohistochemistry. HBx significantly enhanced HepG2-X cell proliferation. In HepG2-X, H7402-X and HepG2.2.15 cells, the expression level of MEKK2 was remarkably increased. In HepG2.2.15 cells, HBx was found to activate JNK and AP-1, which were the downstream effectors of MEKK2 in HepG2-X and HepG2.2.15 cells. In 11 clinical HCC tissues, both HBx and MEKK2 expression levels were remarkably increased, as compared to those in the corresponding peritumor tissues. In 95 clinical HCC tissues, the rate of detection of MEKK2 was 85.3%. HBx promotes hepatoma cell proliferation via upregulating MEKK2, which may be involved in hepatocarcinogenesis.

Antisense regulation of expression and transactivation functions of the tumorigenic HBx and c-myc genes

Earlier we have shown that the X- myc transgenic mice develop hepatocellular carcinoma (HCC) due to co-expression of c-Myc and HBx protein of hepatitis B virus [R. Lakhtakia, V. Kumar, H. Reddi, M. Mathur, S. Dattagupta, S.K. Panda, Hepatocellular carcinoma in a hepatitis B ‘x’ transgenic mouse model: a sequential pathological evaluation. J. Gastroenterol. Hepatol. 18 (2003) 80–91]. With the aim to develop therapeutic strategies for HCC, we constructed several mono- and bicistronic antisense recombinants against HBx and c-myc genes to regulate their expression as well as transactivation function in a human hepatoma cell line. A dose-dependent inhibition in the expression levels of HBx and c-Myc was observed with monocistronic constructs. Likewise, the bicistronic recombinants also blocked the expression as well as transactivation functions of cognate genes with equal efficacy. Further, expression of the constituent genes from the X- myc transgene could also be inhibited by these antisense constructs in cell culture. Thus, our study points towards clinical implications of antisense regulation of tumor-promoting genes in the management of HCC.

Specific inhibition of gene expression and transactivation functions of hepatitis B virus X protein and c- myc by small interfering RNAs

With a view to developing therapeutic strategies against hepatocellular carcinoma (HCC), we have recently shown that co-expression of c- myc and the X protein of hepatitis B virus (HBx) resulted in the development of HCC in the X- myc transgenic mice [Lakhtakia et al., J. Gastroenterol. Hepatol. 18 (2003) 80–91]. We now show in cell culture-based studies that small interfering RNA (siRNA) corresponding to HBx and c- myc can regulate expression and transactivation of the target genes. Expression vectors for small hairpin RNAs (shRNAs) against two different regions each of the HBx and c- myc open reading frames were constructed and their regulatory effects were investigated in COS-1 cells. A dose-dependent specific inhibition in the expression levels of HBx and c- myc was observed with individual shRNAs. Further, the recombinantly expressed shRNAs also blocked the transactivation functions of their cognate genes. Though each shRNA worked at a different efficiency, the inhibitory effects with two different shRNAs were cumulative. These results appear promising for developing a siRNA-based therapy for HCC.

Activating mechanism of transcriptor NF-kappaB regulated by hepatitis B virus X protein in hepatocellular carcinoma.

To investigate the mechanism and significance of NF-kappaB activation regulated by hepatitis B virus X protein (HBx) in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC). The expression levels of HBx, p65, IkappaB-alpha and ubiquitin were detected by immunohistochemistry in HCC tissue microarrays (TMA) respectively, and IkappaB-alpha was detected by Western blot in HCC and corresponding liver tissues. The percentage of informative TMA samples was 98.8% in 186 cases with a total of 367 samples. Compared with corresponding liver tissues (60.0%), the HBx expression was obviously decreased in HBV-associated HCC (47.9%, u=2.24, P<0.05). On the contrary, the expressions of p65 (20.6% vs 45.3%, u=4.85, P<0.01) and ubiquitin (8.9% vs 59.0%, u=9.68, P<0.01) were notably elevated in HCC. In addition, IkappaB-alpha had a tendency to go up. Importantly, positive relativity was observed between HBx and p65 (chi2=10.26, P<0.01), p65 and IkappaB-alpha (chi (2)=16.86, P<0.01), IkappaB-alpha and ubiquitin (chi2=8.90, P<0.01) in HCC, respectively. Both active and non-active forms of NF-kappaB are increased in HBV-associated HCC. Variant HBx is the major cause of the enhancement of NF-kappaB activity. The activation always proceeds in nucleus and the proteasome complexes play an important role in the activation.