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Zinc finger protein X-linked (ZFX) is a zinc finger protein of Zfy family, which is highly conserved in vertebrates. This transcriptional regulator is not only highly expressed in embryonic stem cells (ESC) and hematopoietic stem cells, but is also upregulated in a number of human cancers where it is functional related to cell proliferation and survival. Hepatocellular carcinoma (HCC) is highly aggressive cancer that commonly resistant to most chemotherapies and displays stemness characteristics. In this study, we examined the expression of ZFX in HCC and its possible functional implications in liver tumorigenesis. Quantitative RT-PCR analysis showed common overexpressions of ZFX in 51.8% HCC tumors when compared with their adjacent nonmalignant liver (n = 43/83; p = 0.004). Inline with the pluripotency role of ZFX, we found silencing of ZFX readily inhibited self-renewal capability (p = 0.0022), colony formation ability (p < 0.0001) and cell proliferation (p < 0.0001) through G0/G1 cell cycle arrest of HCC cells (p = 0.0038). In addition, suppression of ZFX sensitized HCC cells to chemotherapeutic agent cisplatin (p < 0.0001). Further investigations suggested that ZFX bind on the promoter of two important mediators, namely Nanog and SOX-2, activating their expressions in HCC (p < 0.0001). Moreover, in vivo xenograft study demonstrated that overexpression of ZFX would promote the tumor growth (p = 0.031). Taken together, our results show, for the first time, commonly overexpressions of ZFX in HCC, where it likely contributes to the stemness and pluripotent behavior of this highly malignant cancer.

Background & Aims Small non-coding RNAs (ncRNA) are increasingly recognized to play important roles in tumorigenesis. With the advent of deep sequencing, efforts have been put forth to profile the miRNome in a number of human malignancies. However, information on ncRNA in hepatocellular carcinoma (HCC), especially the non-microRNA transcripts, is still lacking. Methods Small RNA transcriptomes of two HCC cell lines (HKCI-4 and HKCI-8) and an immortalized hepatocyte line (MIHA) were examined using Illumina massively parallel sequencing. Dysregulated ncRNAs were verified in paired HCC tumors and non-tumoral livers (n=73) by quantitative reverse transcription-polymerase chain reaction. Clinicopathologic correlations and in vitro functional investigations were further carried out. Results The combined bioinformatic and biological analyses showed the presence of ncRNAs and the involvement of a new PIWI-interacting RNA (piRNA), piR-Hep1, in liver tumorigenesis. piR-Hep1 was found to be upregulated in 46.6% of HCC tumors compared to the corresponding adjacent non-tumoral liver. Silencing of piR-Hep1 inhibited cell viability, motility, and invasiveness, with a concomitant reduction in the level of active AKT phosphorylation. In the analysis of miRNA, we showed for the first time, the abundant expression of miR-1323 in HCC and its distinct association in tumors arising from a cirrhotic background. Furthermore, miR-1323 overexpression in cirrhotic HCC correlated with poorer disease-free and overall survivals of patients ( p Conclusions Our study demonstrated the value of next-generation sequencing in dissecting the ncRNome in cancer. The comprehensive definition of transcriptome unveils virtually all types of ncRNAs and provides new insight into liver carcinogenetic events.

The interstitial chromosome (chr.) 1q21-q22 region is frequently amplified in human cancers, where it has been reported to carry prognostic significance for patients. We attempted to delineate chr. 1q21-q22 for affected gene(s) in hepatocellular carcinoma (HCC) by array-CGH and detected copy number gains of ρ-guanine nucleotide exchange factor-H1 (GEF-H1) as most significant event. Gene expression evaluation in the HCC cohort indicated common up-regulations of GEF-H1 in 64% tumours compared to adjacent non-tumoural liver (64/100; paired t-test p < 0.0001). Moreover, GEF-H1 over-expressions correlated with microvascular invasion and advanced-stage tumours (p < 0.05). High GEF-H1 levels also predict shorter disease-free and overall survival of HCC patients (p < 0.03). Functional knock-down of GEF-H1 by RNAi indicated marked reduction in cell invasion through matrigel and an inhibition of cell migration (p < 0.035), but an effect on cell viability was not apparent. More interestingly, a mesenchymal-epithelial transition (MET) was readily observed in GEF-H1 knock-down cells, where a concomitant re-expression of epithelial markers (E-cadherin and cytokeratin 18) and cell adhesion proteins (α-catenin and γ-catenin) was found but down-regulation of mesenchymal features (N-cadherin, vimentin and fibronectin). This phenotype was accompanied by reduced filamentous actin polymerizations and diminution of the stress fibre formation. In addition, reduced active form of GTP-RhoA, together with its downstream effectors, including cleaved ROCK1 and phosphorylated MLC2, were also detected in GEF-H1-depleted cells. Taken together, our findings underscore a potent oncogenic role for GEF-H1 in promoting the metastatic potentials of HCC, possibly through activation of RhoA signalling and the EMT phenomenon.

Profiling of microRNA expression in human cancers has highlighted downregulation of miR-145 as a common event in epithelial malignancies. Here, we describe recurrent underexpression of miR-145 in hepatocellular carcinoma (HCC) and the identification of a biological pathway by which miR-145 exerts its functional effects in liver tumorigenesis. In a cohort of 80 HCC patients, quantitative reverse transcription polymerase chain reaction corroborated reduced miR-145 expression in 50% of tumors, which also correlated with a shorter disease-free survival of patients. One HCC tumor analyzed with low endogenous miR-145 was propagated as cell line. This in vitro model HKCI-C2 maintained low miR-145 level and upon restoration of miR-145 expression, a consistent inhibitory effect on cell viability and proliferation was readily found. Flow cytometric analysis indicated that miR-145 re-expression could induce G(2)-M cell cycle arrest and apoptosis. Multiple in silico algorithms predicted that miR-145 could target a number of genes along the insulin-like growth factor (IGF) signaling, including insulin receptor substrate (IRS1)-1, IRS2 and insulin-like growth factor 1 receptor. We found protein expression of these putative targets was concordantly downregulated in the presence of miR-145. Luciferase reporter assay further verified direct target association of miR-145 to specific sites of the IRS1 and IRS2 3'-untranslated regions. Subsequent analysis also affirmed miR-145 modulation on the IGF signaling cascade by reducing its downstream mediator, namely the active β-catenin level. Taken together, our study shows for the first time the pleiotropic effect of miR-145 in targeting multiple components of the oncogenic IGF signaling pathway in HCC.

Genomic amplification of regional chromosome 8q24 is a common event in human cancers. In hepatocellular carcinoma (HCC), a highly aggressive malignancy that is rapidly fatal, recurrent 8q24 gains can be detected in >50% of cases. In this study, attempts to resolve the 8q24 region by way of array comparative genomic hybridization for affected genes in HCC revealed distinctive gains of block of proliferation 1 (BOP1). Gene expression evaluation in an independent cohort of primary HCC (n = 65) revealed frequent BOP1 up-regulation in tumors compared with adjacent nontumoral liver (84.6%; P < 0.0001). Significant associations could also be drawn between increased expressions of BOP1 and advance HCC staging (P = 0.004), microvascular invasion (P = 0.006), and shorter disease-free survival of patients (P = 0.02). Examination of expression of C-MYC, a well-known oncogene located in proximity to BOP1, in the same series of primary HCC cases did not suggest strong clinicopathologic associations. Functional investigations by small interfering RNA–mediated suppression of BOP1 in HCC cell lines indicated significant inhibition on cell invasion (P < 0.005) and migration (P < 0.05). Overexpression of BOP1 in the immortalized hepatocyte cell line L02 showed increase cellular invasiveness and cell migratory rate (P < 0.0001). In both gene knockdown and ectopic expression assays, BOP1 did not exert an effect on cell viability and proliferation. Evident regression of the epithelial-mesenchymal transition (EMT) phenotype was readily identified in BOP1 knockdown cells, whereas up-regulation of epithelial markers (E-cadherin, cytokeratin 18, and γ-catenin) and down-regulation of mesenchymal markers (fibronectin and vimentin) were seen. A corresponding augmentation of EMT was indicated from the ectopic expression of BOP1 in L02. In addition, BOP1 could stimulate actin stress fiber assembly and RhoA activation. Conclusion: Our findings underline an important role for BOP1 in HCC invasiveness and metastasis potentials through inducing EMT and promoting actin cytoskeleton remodeling. (HEPATOLOGY 2011;)

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the Polycomb-repressive complex 2 (PRC2) that represses gene transcription through histone H3 lysine 27 trimethylation (H3K27me3). Although EZH2 is abundantly present in various cancers, the molecular consequences leading to oncogenesis remain unclear. Here, we show that EZH2 concordantly silences the Wnt pathway antagonists operating at several subcellular compartments, which in turn activate Wnt/β-catenin signaling in hepatocellular carcinomas (HCC). Chromatin immunoprecipitation promoter array and gene expression analyses in HCCs revealed EZH2 occupancy and reduced expression of Wnt antagonists, including the growth-suppressive AXIN2, NKD1, PPP2R2B, PRICKLE1, and SFRP5. Knockdown of EZH2 reduced the promoter occupancy of PRC2, histone deacetylase 1 (HDAC1), and H3K27me3, whereas the activating histone marks were increased, leading to the transcriptional upregulation of the Wnt antagonists. Combinatorial EZH2 and HDAC inhibition dramatically reduced the levels of nuclear β-catenin, T-cell factor–dependent transcriptional activity, and downstream pro-proliferative targets CCND1 and EGFR. Functional analysis revealed that downregulation of EZH2 reduced HCC cell growth, partially through the inhibition of β-catenin signaling. Conversely, ectopic overexpression of EZH2 in immortalized hepatocytes activated Wnt/β-catenin signaling to promote cellular proliferation. In human HCCs, concomitant overexpression of EZH2 and β-catenin was observed in one-third (61/179) of cases and significantly correlated with tumor progression. Our data indicate that EZH2-mediated epigenetic silencing contributes to constitutive activation of Wnt/β-catenin signaling and consequential proliferation of HCC cells, thus representing a novel therapeutic target for this highly malignant tumor. Cancer Res; 71(11); 4028–39. ©2011 AACR.

Caldesmon (CaD) is an actin-binding protein that is capable of stabilizing actin filaments. Phosphorylation of CaD is widely accepted in the actin cytoskeletal modeling and promotion of cell migration. In this study, we show that CaD is a downstream phosphorylation substrate of PFTK1, a novel Cdc-2-related ser/thr protein kinase. Our study stemmed from an earlier investigation where we demonstrated that PFTK1 kinase conferred cell migratory advantages in human hepatocellular carcinoma (HCC) cells. Here, we showed that PFTK1-knockdown cells exhibited much reduced CaD phosphorylation and consequently caused dissociation of CaD from the F-actin fibers. The cellular localization of CaD was also altered in the absence of PFTK1. Immunofluorescence analysis revealed that PFTK1-abrogated cells exhibited a diffused and blurred appearance of CaD localization, whereas intact co-localization with F-actins was apparent in PFTK1-expressing cells. Without the binding of CaD to actin, disappearance of actin stress fibers was also evident in PFTK1-abrogated cells. In addition, we found that CaD is also commonly up-regulated in HCC tumors when compared to adjacent non-malignant liver (P = 0.022). Taken together, our results highlight a novel biological cascade that involved the phosphorylation activation of CaD by PFTK1 kinase in promoting formation of actin stress fibers.

Homozygous deletion screening has been widely utilized to define tumour suppressor genes (TSGs) in cancers. Although these biallelic deletions are infrequent, their identification has facilitated the discovery of many important TSGs. We have systematically examined the genome of hepatocellular carcinoma (HCC), a highly malignant tumour that is rapidly fatal, for the presence of homozygous deletions. Array-CGH analysis on early passage of HCC cultures and cell lines led us to identify six homozygous deleted (HD) regions. A high concordance between array-CGH and expression of HD genes was demonstrated, where crystallin Lambda1 (CRYL1; located on chromosome 13q12.11) displayed the most frequent down-regulation. We found that reduced mRNA expression of CRYL1 was common in HCC tumours when compared with their adjacent non-tumoural liver (p = 0.0097). Significant associations could also be drawn between repressed CRYL1 and advanced tumour staging, increased tumour size, and shorter disease-free survival of patients (p < 0.037). Moreover, homozygous deletions on CRYL1 could be detected in 36% of HCC cases, where recurrent HDs were identified on exons 1, 5, and 8. Examination of other causal events suggested histone deacetylation and promoter hypermethylation to be likely inactivating mechanisms as well. Re-expression of CRYL1 in the SK-Hep1 cell line, where biallelic loss of CRYL1 was found, induced profound inhibition of cellular proliferation and cell growth (p < 0.0015). By Annexin V staining, CRYL1 restoration readily increased pro-apoptotic cells with an induction of PARP cleavage. Flow cytometry further revealed that CRYL1 could prolong the G(2)-M phase, possibly through interruption of the Cdc2/cyclin B pathway. Given that regional chromosome 13q12-q14 loss is a causal genomic event in HCC tumourigenesis, our finding may have implications for identifying a novel TSG CRYL1 within this important locus.

BRE binds to the cytoplasmic domains of tumor necrosis factor receptor-1 and Fas, and in cell lines can attenuate death receptor-initiated apoptosis by inhibiting t-BID-induced activation of the mitochondrial apoptotic pathway. Overexpression of BRE by transfection can also attenuate intrinsic apoptosis and promote growth of the transfected Lewis lung carcinoma line in mice. There is, however, a complete lack of in vivo data about the protein. Here, we report that by using our BRE-specific monoclonal antibody on the immunohistochemistry of 123 specimens of human hepatocellular carcinoma (HCC), significant differences in BRE expression levels between the paired tumoral and non-tumoral regions (P

Background: It has been proved that cyclo-oxygenase-2 (COX-2) is rapidly induced by inflammatory mediators. However, it is not known whether overexpression of COX-2 in the liver is sufficient to promote activation or secretion of inflammatory factors leading to hepatitis. Aim: To investigate the role forced expression of COX-2 in liver by using inducible COX-2 transgenic (TG) mice. Methods: TG mice that overexpress the human COX-2 gene in the liver using the liver-specific transthyretin promoter and non-TG littermates were derived and fed the normal diet for up to 12 months. Hepatic prostaglandin E 2 (PGE 2 ) content was determined using enzyme immunoassay, nuclear factor kappaB (NF-κB) activation by electrophoretic mobility shift assays, apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labelling and proliferation by Ki-67 immunohistochemistry. Results: COX-2 TG mice exhibited strongly increased COX-2 and PGE 2 , elevated serum alanine aminotransferase level and histological hepatitis. Hepatic COX-2 expression in the TG mice resulted in activation of NF-κB and inflammatory cytokine cascade, with a marked expression of the proinflammatory cytokines tumour necrosis factor (TNF)-α (9.4-fold), interleukin (IL)-6 (4.4-fold), IL-1β (3.6-fold), and of the anti-inflammatory cytokine IL-10 (4.4-fold) and chemokine macrophage inflammatory protein-2 (3.2-fold). The inflammatory response of the COX-2 TG mice was associated with infiltration macrophages and lymphocytes, increased cell proliferation and high rates of cell apoptosis. Administration of the COX-2 inhibitor celecoxib in TG mice restored liver histology to normal. Conclusion: Enhanced COX-2 expression in hepatocytes is sufficient to induce hepatitis by activating NF-κB, stimulating the secretion of proinflammatory cytokines, recruiting macrophage and altering cell kinetics. Inhibition of COX-2 represents a mechanism-based chemopreventive approach to hepatitis.

BRE, brain and reproductive organ-expressed protein, was found previously to bind the intracellular juxtamembrane domain of a ubiquitous death receptor, tumor necrosis factor receptor 1 (TNF-R1), and to down-regulate TNF-alpha-induced activation of NF-kappaB. Here we show that BRE also binds to another death receptor, Fas, and upon overexpression conferred resistance to apoptosis induced by TNF-alpha, anti-Fas agonist antibody, cycloheximide, and a variety of stress-related stimuli. However, down-regulation of the endogenous BRE by small interfering RNA increased apoptosis to TNF-alpha, but nottoetoposide, indicating that the physiological antiapoptotic role of this protein is specific to death receptor-mediated apoptosis. We further demonstrate that BRE mediates antiapoptosis by inhibiting the mitochondrial apoptotic machinery but without translocation to the mitochondria or nucleus or down-regulation of the cellular level of truncated Bid. Dissociation of BRE rapidly from TNF-R1, but not from Fas, upon receptor ligation suggests that this protein interacts with the death inducing signaling complex during apoptotic induction. Increased association of BREwith phosphorylated, sumoylated, and ubiquitinated proteins after death receptor stimulation was also detected. We conclude that in contrast to the truncated Bid that integrates mitochondrial apoptosis to death receptor-triggered apoptotic cascade, BRE inhibits the integration. We propose that BRE inhibits, by ubiquitination-like activity, components in or proximal to the death-inducing signaling complexes that are necessary for activation of the mitochondria.

Mouse Bre, an evolutionarily conserved stress-modulating gene, like its human counterpart, is expressed in multiple alternative transcripts. The main transcript, which is ubiquitously expressed, encodes a protein that binds tumor necrosis factor receptor 1 (TNF-R1) and downregulates TNF-induced activation of NF-kappaB. Alternative splicing of mouse Bre occurs only at the 5' region of the gene, generating either nonfunctional transcripts or transcripts that can encode putative protein isoforms differ at the N-terminal sequence. In contrast, alternative splicing of human BRE occurs at either or both ends of the gene; only the 3' alternative splicing can generate functional transcripts that encode putative protein isoforms differ at the C-terminus, occurrence of the 5' alternative splicing only results in forming nonfunctional transcripts. Unlike the human BRE alternative transcripts which are coexpressed at considerable levels with the main transcript, the mouse counterparts are expressed in a restricted pattern and generally in low abundance except in the heart. Both species, however, share a type of Bre alternative transcripts generated by cryptic splicing at a nonstandard, noncanonical acceptor site. Thus, a highly conserved gene in two species can generate alternative transcripts different in both of the sequence structure and expression pattern, as well as a similar class of transcripts resulting from unconventional transcript processing.

SummaryThe mutagenic effect of hepatitis B (HBV) integration in predisposing risk to hepatocellular carcinoma (HCC) remains elusive. In this study, we performed transcriptome sequencing of HBV-positive HCC cell lines and showed transcription of viral-human gene fusions from the site of genome integrations. We discovered tumor-promoting properties of a chimeric HBx-LINE1 that, intriguingly, functions as a hybrid RNA. HBx-LINE1 can be detected in 23.3% of HBV-associated HCC tumors and correlates with poorer patient survival. HBx-LINE1 transgenic mice showed heightened susceptibility to diethylnitrosamine-induced tumor formation. We further show that HBx-LINE1 expression affects β-catenin transactivity, which underlines a role in activating Wnt signaling. Thus, this study identifies a viral-human chimeric fusion transcript that functions like a long noncoding RNA to promote HCC.

The PFTK1 gene encodes a cdc2-related serine/threonine protein kinase that has been shown to confer cell migratory properties in hepatocellular carcinoma (HCC). However, the prognostic value and biological mechanism by which PFTK1 promotes HCC motility remain largely unknown. Here, we showed from tissue microarray that common upregulations of PFTK1 in primary HCC tumors (n=133/180) correlated significantly with early age onset (⩽40 years), advance tumor grading and presence of microvascular invasion (P⩽0.05). To understand downstream phosphorylated substrate(s) of PFTK1, phospho-proteins in PFTK1 expressing and knockdown Hep3B cells were profiled by two-dimensional-polyacrylamide gel electrophoresis mass spectrometric analysis. Protein identification of differential spots revealed β-actin (ACTB) and transgelin2 (TAGLN2) as the two most profound phosphorylated changes affected by PFTK1. We verified the presence of TAGLN2 serine phosphorylation and ACTB tyrosine phosphorylation. Moreover, reduced TAGLN2 and ACTB phosphorylations in PFTK1-suppressed Hep3B corresponded to distinct actin depolymerizations and marked inhibition on cell invasion and motility. Given that TAGLN2 is a tumor suppressor whose function has been ascribed in cancer metastasis, we examined if TAGLN2 is an intermediate substrate in the biological path of PFTK1. We showed in PFTK1-suppressed cells that knockdown of TAGLN2 over-rode the inhibitory effect on cell invasion and motility, and a recovery on actin polymerization was evident. Interestingly, we also found that unphosphorylated TAGLN2 in PFTK1-suppressed cells elicited strong actin-binding ability, a mechanism that possibly halts the actin cytoskeleton dynamics. Site-directed mutagenesis of TAGLN2 suggested that PFTK1 regulates the actin-binding affinity of TAGLN2 through the S83 and S163 residues, which if mutated can significantly affect HCC cell motility. Taken together, our data propose a novel, oncogene-tumor suppressor interplay, where oncogenic PFTK1 confers HCC cell motility through inactivating the actin-binding motile suppressing function of TAGLN2 via phosphorylation.

Hepatocellular carcinoma (HCC) shows low response to most conventional chemotherapies. To facilitate target identification for novel therapeutic development, we deployed gene expression profiling on 43 paired HCC tumors and adjacent non-tumoral liver, which is also considered as the pre-malignant liver lesion. In conjunction with ontology analysis, a major functional process found to play a role in the malignant transformation of HCC was microtubule-related cellular assembly. We further examined the potential use of microtubule targeting taxane drugs, including paclitaxel and docetaxel, and compared with findings to results from doxorubicin, a common chemotherapeutic agent used in HCC. Recent studies showed that drug delivery by nanoparticles have enhanced efficacy with reduced side effects. In this regard, the nanoparticle albumin-bound (nab)-paclitaxel was also examined. In a panel of HCC cell lines studied, a high sensitivity towards taxane drugs was generally found, although the effect from nab-paclitaxel was most profound. The nab-paclitaxel showed an effective IC50 dose at 15-fold lower than paclitaxel alone or the derivative analogue docetaxel, and ~450-fold less compared to doxorubicin. Flow cytometric analysis confirmed a cell cycle blockade at the G2/M phase and increased apoptosis following nab-paclitaxel treatment. In vivo animal studies also showed that nab-paclitaxel readily inhibited xenograft growth with less toxicity to host cells compared to other anti-microtubule drugs and doxorubicin. Gene silencing of the microtubule regulatory gene STMN1 by RNAi suggested a distinct synergistic effect in the combined treatment with nab-paclitaxel. Our findings in this study highly suggest that the microtubule assembly represents a promising therapeutic target development in HCC.

Cyclooxoygenase (COX)-2 overexpression is involved in gastric carcinogenesis. While high-salt intake is a known risk factor for gastric cancer development, we determined the effects of high salt on gastric chemical carcinogenesis in COX-2 transgenic (TG) mice. COX-2 TG mice were developed in C57/BL6 strain using the full-length human cox-2 complementary DNA construct. Six-week-old COX-2 TG and wild-type (WT) littermates were randomly allocated to receive alternate week of N-methyl-N-nitrosourea (MNU, 240 p.p.m.) in drinking water or control for 10 weeks. Two groups of mice were further treated with 10% NaCl during the initial 10 weeks. All mice were killed at the end of week 50. Both forced COX-2 overexpression and high-salt intake significantly increased the frequency of gastric cancer development in mice as compared with WT littermates treated with MNU alone. However, no additive effect was observed on the combination of high salt and COX-2 expression. We further showed that MNU and high-salt treatment increased chronic inflammatory infiltrates and induced prostaglandin E(2) (PGE(2)) production in the non-cancerous stomach. Whereas high-salt treatment markedly increased the expression of inflammatory cytokines (tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-1 beta and IL-6) in the gastric mucosa, COX-2 overexpression significantly altered the cell kinetics in the MNU-induced gastric cancer model. In conclusion, both high salt and COX-2 overexpression promote chemical-induced gastric carcinogenesis, possibly related to chronic inflammation, induction of PGE(2), disruption of cell kinetics and induction of inflammatory cytokines.

Hepatic COX-2 overexpression is sufficient to induce hepatitis, but its role on liver fibrosis remains unknown. We aim to elucidate possible biological effects of COX-2 in liver fibrosis using both gain-of-function and loss-of-function mouse models. COX-2 transgenic (TG) mice that specifically overexpress the human COX-2 cDNA in the liver, knockout (KO), and wild type (WT) mice were studied in two different murine fibrosis models induced by carbon tetrachloride (CCl(4)) injection or methionine and choline-deficient (MCD) diet. Liver injury was assessed by serum ALT and bilirubin levels and histological examination. Hepatic collagen content was determined by picrosirius red stain morphometry assay and quantitation of hydroxyproline. Hepatic stellate cell (HSC) activation was determined by immunohistochemical analysis of alpha-smooth muscle actin (alpha-SMA). mRNA expression of fibrogenic genes was assayed by real-time quantitative PCR. COX-2 protein was overexpressed in the liver of TG mice compared with WT littermates. CCl(4) or MCD-induced liver fibrotic injury was equally severe in TG and WT mice, as demonstrated by similar elevated levels of hepatic collagen contents. Enhanced COX-2 expression in TG liver did not affect HSC activation and fibrogenic gene expression upon CCl(4) or MCD treatment. Importantly, CCl(4)-treated KO mice did not show significant difference in liver fibrotic damage and fibrogenic gene expression compared with the WT counterparts. This is the first report on the effect of COX-2 in liver fibrosis based on genetic mouse models. The results suggest that COX-2 does not appear to mediate the development of liver fibrosis.

The brain and reproductive organ expressed (BRE) gene encodes a highly conserved stress-modulating protein. To gain further insight into the function of this gene, we used comparative proteomics to investigate the protein profiles of C2C12 and D122 cells resulting from small interfering RNA (siRNA)-mediated silencing as well as overexpression of BRE. Silencing of BRE in C2C12 cells, using siRNA, resulted in up-regulated Akt-3 and carbonic anhydrase III expression, while the 26S proteasome regulatory subunit S14 and prohibitin were down-regulated. Prohibitin is a potential tumour suppressor gene, which can directly interact with p53. We found that cell proliferation was significantly increased after knockdown of BRE, concomitant with reduced p53 and prohibitin expression. In contrast, we observed decreased proliferation and up-regulation of p53 and prohibitin when BRE was overexpressed in the D122 cell line. In total, five proteins were found to be up-regulated after BRE over-expression. The majority of these proteins can target or crosstalk with NF-kappaB, which plays a central role in regulating cell proliferation, differentiation and survival. Our results establish a crucial role for BRE in the regulation of key proteins of the cellular stress-response machinery and provide an explanation for the multifunctional nature of BRE.

Human BRE, a death receptor-associating intracellular protein, attenuates apoptotic response of human and mouse tumor cell lines to death receptor stimuli in vitro. In this report, we addressed whether the in vitro antiapoptotic effect of BRE could impact on tumor growth in vivo. We have shown that the mouse Lewis lung carcinoma D122 stable transfectants of human BRE expression vector developed into local tumor significantly faster than the stable transfectants of empty vector and parental D122, in both the syngeneic C57BL/6 host and nude mice. In vitro growth of the BRE stable transfectants was, however, not accelerated. No significant difference in metastasis between the transfectants and the parental D122 was detected. Thus, overexpression of BRE promotes local tumor growth but not metastasis. We conclude that the enhanced tumor growth is more likely due to the antiapoptotic activity of BRE than any direct effect of the protein on cell proliferation.

BRE, a putative stress-modulating gene, found able to down-regulate TNF-alpha-induced NF-kappaB activation upon overexpression, is now shown in human cells expressed as multiple mRNA isoforms. A total of six isoforms are produced by alternative splicing predominantly at either end of the gene. Predicted from the cDNA sequences of these isoforms, three of them (alpha(a), alpha(b), and alpha(c)) code for BRE of different C-terminus, and the other three (beta(a), beta(b), and beta(c)) may possibly be the nonfunctional counterparts. All human cells examined coexpress all the predominant splice variants, albeit at different ratios. Comparing with normal cells, immortalized human cell lines uniformly express higher levels of BRE. Interestingly, peripheral blood monocytes responded to LPS by down-regulating the expression of all the BRE isoforms, which was however less obvious in the cell line counterpart, THP-1. Isoform alpha(a), which codes for the canonical BRE with a C-terminal peroxisomal targeting sequence, is the most abundant transcript. We propose that the function of BRE and its isoforms is to regulate peroxisomal activities.

Ig genes undergo hypermutation with a nucleotide preference of A over T for mutation on the coding strand. As only with concomitant strand bias can such nucleotide bias be observed, Ig gene hypermutation is generally accepted as a strand-specific process, for which the mechanistic basis remains unknown. It has previously been shown that different non-Ig sequences replacing the LVJ region of an Ig transgene to various extents are targeted for hypermutation with similar mutation frequencies. However, the nucleotide bias characteristic of Ig hypermutation was not found in two of the three such sequences studied. To test whether it is the DNA sequences of the non-Ig substrates that determine the pattern of nucleotide bias in hypermutation or whether the LVJ sequence may contain element(s) that confer strand bias, we have added back all the replaced LVJ sequences to one of the transgenes, L(kappa)-Vgpt*, that expresses no strand bias in hypermutation and studied the outcome. The results show that the gpt sequence in the presence of the complete LVJ sequence hypermutates differently from the same sequence in L(kappa)-Vgpt* where 84% of the LVJ was replaced. The main difference is the resumption of strand bias characteristic of Ig hypermutation. Thus, whether or not a substrate sequence manifests strand bias in hypermutation is not inherently determined by the substrate DNA sequence. This indicates the presence of special element(s) within the LVJ that confer strand bias.

Profilings of microRNA expressions in hepatocellular carcinoma (HCC) have highlighted common upregulations of the miR-183/96/182 cluster. We showed in this study concordant over-expression of miR-183, miR-96 and miR-182 can be frequently detected in primary HCC tumors (59%; n=47/80). In addition, miR-183/96/182 up-regulation correlated with metastatic features including presence of microvascular invasion (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4127. doi:1538-7445.AM2012-4127

Aberrant activation of the canonical Wnt pathway due to accumulation of β-catenin occurs in approximately 70% of hepatocellular carcinomas (HCCs) and contributes to their initiation, development and progression. Genetic mutations in the components of this pathway account for only a subset of HCCs with β-catenin accumulation, suggesting that an alternative mechanism for Wnt/β-catenin activation is prevailing. Enhancer of zeste homolog 2 (EZH2) is known to promote tumorigenesis by down-regulating tumor-suppressor genes; however, whether EZH2 regulates oncogenic pathways is unclear. Using chromatin immunoprecipitation microarray, we uncovered 12 Wnt/β-catenin signal antagonists whose promoters were concordantly occupied by EZH2 and repressive histone modifications in hepatocellular HCC cells. EZH2 over-expressed in 42% (75/179) of human HCCs and significantly associated with β-catenin accumulation. Concomitant inhibition of EZH2 and histone deacetylase transcriptionally activated Wnt/β-catenin signal antagonists, suppressed T-cell factor-dependent transcriptional activity and down-regulated β-catenin transcriptional targets in HCC cells. Conversely, ectopic EZH2 over-expression in nontumorigenic hepatocytes increased cellular proliferation in a β-catenin-dependent manner and promoted tumorigenicity. This study uncovers an EZH2-mediated epigenetic mechanism that leads to Wnt/β-catenin signaling dyregulation in human HCC and suggests that therapeutic interventions targeting EZH2 may confer a clinical benefit in β-catenin-driven malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4881.

Gene expression variations (GEV) among different ethnic groups have been a subject matter for extensive study. Relatively less known is the extent of alternative splicing variations (ASV) in the context of ethnicity. We conducted a transcriptome sequencing study of 20 lymphoblastoid cell lines obtained from Caucasian and Han Chinese, and identified known genes that exhibit differential isoform abundance between the two ethnic groups. Among them hnRNPK, a co-factor of p53 (TP53), could be further replicated in a 39-sample cohort with TaqMan assay. Although within-population novel splice variants are common, inter-population novel splice variants are rare. We further analyzed 5.63billion sequencing reads retrieved from the NCBI Sequence Read Archive and identified potential ethnic-specific transcribed regions.