Using the promoter methylation assay, we have shown that MDGA2 (MAM domain containing glycosylpho... more Using the promoter methylation assay, we have shown that MDGA2 (MAM domain containing glycosylphosphatidylinositol anchor 2) is preferentially methylated in gastric cancer. We analysed its biological effects and prognostic significance in gastric cancer. MDGA2 methylation status was evaluated by combined bisulfite restriction analysis and bisulfite genomic sequencing. The effects of MDGA2 re-expression or knockdown on cell proliferation, apoptosis and the cell cycle were determined. MDGA2 interacting protein was identified by mass spectrometry and MDGA2-related cancer pathways by reporter activity and PCR array analyses. The clinical impact of MDGA2 was assessed in 218 patients with gastric cancer. MDGA2 was commonly silenced in gastric cancer cells (10/11) and primary gastric cancers due to promoter hypermethylation. MDGA2 significantly inhibited cell proliferation by causing G1-S cell cycle arrest and inducing cell apoptosis in vitro, and suppressed xenograft tumour growth in both...
We found that carbonic anhydrase IV (CA4), a member of the carbonic anhydrases, is silenced in co... more We found that carbonic anhydrase IV (CA4), a member of the carbonic anhydrases, is silenced in colorectal cancer (CRC). We analysed its epigenetic inactivation, biological effects and prognostic significance in CRC. The biological functions of CA4 were determined by in vitro and in vivo tumorigenicity assays. The CA4 co-operator was identified by immunoprecipitation and mass spectrometry. CA4 downstream effectors and signalling pathways were elucidated by promoter luciferase assay, electrophoretic mobility shift assay and chromatin immunoprecipitation. The clinical impact of CA4 was assessed in 115 patients with CRC. CA4 was silenced in all nine CRC cell lines and 92.6% of CRC tumours. The promoter hypermethylation contributed to the inactivation of CA4, and it was detected in 75.7% of the patients with CRC. After a median follow-up of 49.3 months, multivariate analysis showed that the patients with CA4 hypermethylation had a recurrence of Stage II/III CRC. The re-expression of CA4 ...
Using whole genome sequencing, we identified gene amplification of solute carrier family 12 membe... more Using whole genome sequencing, we identified gene amplification of solute carrier family 12 member 5 (SLC12A5) located at 20q13.12 in colorectal cancer (CRC). We analysed its amplification, overexpression, biological effects and prognostic significance in CRC. SLC12A5 amplification status was evaluated by fluorescence in situ hybridisation (FISH). The effects of SLC12A5 re-expression or knockdown were determined in proliferation, apoptosis, invasion and metastasis assays. SLC12A5 target genes and related pathways were identified by reporter activity and cDNA microarray analyses. Clinical impact of SLC12A5 overexpression was assessed in 195 patients with CRC. Amplification of SLC12A5 was verified in 78 out of 191 (40.8%) patients with primary CRC by FISH, which was positively correlated with its protein overexpression (p<0.001). Biofunctional investigation of SLC12A5 revealed that SLC12A5 significantly increased cell proliferation, G1-S cell cycle transition, invasion/migration ab...
Dapper homolog (DACT) 2 is one of the Dact gene family members, which are important modulators of... more Dapper homolog (DACT) 2 is one of the Dact gene family members, which are important modulators of Wnt signaling pathway. We aim to clarify its epigenetic inactivation, biological function and clinical implication in colon cancer. DACT2 was silenced in five out of eight colon cancer cell lines, but robustly expressed in normal colon tissues. The loss of DACT2 expression was regulated by promoter hypermethylation. Restoring DACT2 expression in colon cancer cell lines suppressed tumor cell growth by inducing cell apoptosis and inhibiting cell proliferation both in vitro and in vivo. Moreover, DACT2 overexpression effectively reduced lung metastasis of colon cancer cells in nude mice. These effects by DACT2 were attributed to inhibition of Wnt/β-catenin signaling. Reexpression of DACT2 significantly suppressed the transcriptional activity of both wild-type β-catenin and degradation-resistant form mutant β-catenin (S33Y). DACT2 could actively shuttle into and out of nuclei, with its pred...
Peroxisome proliferator-activated receptor alpha (PPARα) ligands have been reported to suppress c... more Peroxisome proliferator-activated receptor alpha (PPARα) ligands have been reported to suppress cancer growth. However, the role of PPARα in hepatocarcinogenesis remains unclear. We investigated the functional significance of PPARα in HCC. PPARα-knockout (PPARα-/-) mice were more susceptible to diethylnitrosamine (DEN)-induced HCC at 6 months compared with wild-type (WT) littermates (80% versus 43%, P < 0.05). In resected HCCs, TUNEL-positive apoptotic cells were significantly less in PPARα-/- mice than in WT mice (P < 0.01), commensurate with a reduction in cleaved caspase-3 and caspase-7 protein expression. Ki-67 staining showed increased cell proliferation in PPARα-/- mice (P…
The mechanisms by which Epstein-Barr virus (EBV) contributes to the development of gastric cancer... more The mechanisms by which Epstein-Barr virus (EBV) contributes to the development of gastric cancer are unclear. We investigated EBV-associated genomic and epigenomic variations in gastric cancer cells and tumors. We performed whole-genome, transcriptome, and epigenome sequence analyses of a gastric adenocarcinoma cell line (AGS cells), before and after EBV infection. We then looked for alterations in gastric tumor samples, with (n = 34) or without (n = 100) EBV infection, collected from patients at the Prince of Wales Hospital, Chinese University of Hong Kong (from 1998 through 2004), or the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China (from 1999 through 2006). Transcriptome analysis showed that infected cells expressed 9 EBV genes previously detected in EBV-associated gastric tumors and 71 EBV genes not previously reported in gastric tumors. Ten viral genes that had not been reported previously in gastric cancer but were expressed most highly in EBV-infected cells also were expressed in primary EBV-positive gastric tumors. Whole-genome sequence analysis identified 45 EBV-associated nonsynonymous mutations. These mutations, in genes such as AKT2, CCNA1, MAP3K4, and TGFBR1, were associated significantly with EBV-positive gastric tumors, compared with EBV-negative tumors. An activating mutation in AKT2 was associated with reduced survival times of patients with EBV-positive gastric cancer (P = .006); this mutation was found to dysregulate mitogen-activated protein kinase signaling. Integrated epigenome and transcriptome analyses identified 216 genes transcriptionally down-regulated by EBV-associated hypermethylation; methylation of ACSS1, FAM3B, IHH, and TRABD increased significantly in EBV-positive tumors. Overexpression of Indian hedgehog (IHH) and TraB domain containing (TRABD) increased proliferation and colony formation of gastric cancer cells, whereas knockdown of these genes reduced these activities. We found 5 signaling pathways (axon guidance, focal adhesion formation, interactions among cytokines and receptors, mitogen-activated protein kinase signaling, and actin cytoskeleton regulation) to be affected commonly by EBV-associated genomic and epigenomic alterations. By using genomic, transcriptome, and epigenomic comparisons of EBV infected vs noninfected gastric cancer cells and tumor samples, we identified alterations in genes, gene expression, and methylation that affect different signaling networks. These might be involved in EBV-associated gastric carcinogenesis.
B cell CLL/lymphoma 6 member B (BCL6B) is a novel tumor suppressor silenced in human cancer. In t... more B cell CLL/lymphoma 6 member B (BCL6B) is a novel tumor suppressor silenced in human cancer. In this study, we investigated the functional role and underlying mechanisms of BCL6B in hepatocellular carcinoma (HCC). BCL6B was expressed in normal HCC tissues, but its expression was suppressed in 6 out of 9 HCC cell lines. Loss of BCL6B expression was associated with promoter hypermethylation. Ectopic expression of BCL6B in HepG2 and Huh7 cell lines inhibited colony formation (P <0.05), cell viability (P <0.01), and tumorigenicity in nude mice (P <0.05). BCL6B expression also induced apoptosis (P <0.05), an effect associated with activation of the caspase cascade and cleavage of PARP. Stable expression of BCL6B in MHCC97L cells suppressed cell migration (P <0.05) and invasion (P <0.05), and significantly reduced the incidence and severity of lung metastasis in an orthotopic HCC mouse model. The anti-metastatic effect of BCL6B was mediated by up-regulation of cell adhes...
expression of cytoskeletal markers of quiescent stellate cells. Gene-expression profiling identif... more expression of cytoskeletal markers of quiescent stellate cells. Gene-expression profiling identified IL-8 signalling and the wnt-β-catenin pathway as the key canonical pathways affected. Organotypic co-culture of PSCs and cancer cells demonstrated an indirect effect of treated PSCs on cancer cell morphology, proliferation (decrease) and apoptosis (increase) and confirmed the engagement of the wnt-β-catenin signalling pathway. There was less βcatenin signalling (TOPFlash reporter assays) in cancer cells co-cultured with treated quiescent PSCs, which was mediated by an increased expression of sFRP4 (secreted frizzledrelated protein 4) resulting in decreased invasion of cancer cells. Additionally, in contrast to vehicle treated orgnaotypic cultures, PSCs exposed to ATRA did not invade into the extracellular matrix gel but formed a "wall" at the cancer-matrix junction, blocking cancer cell invasion. Conclusion: Targeting the desmoplastic stroma with agents such as ATRA interferes with the, for tumour progression important, tumour-stroma cross-talk and offers exciting opportunities for combinatorial therapy for pancreatic cancer.
In flowering plants, tapetum degeneration is proposed to be triggered by a programmed cell death ... more In flowering plants, tapetum degeneration is proposed to be triggered by a programmed cell death (PCD) process during late stages of pollen development; the PCD is thought to provide cellular contents supporting pollen wall formation and to allow the subsequent pollen release. However, the molecular basis regulating tapetum PCD in plants remains poorly understood. We report the isolation and characterization of a rice (Oryza sativa) male sterile mutant tapetum degeneration retardation (tdr), which exhibits degeneration retardation of the tapetum and middle layer as well as collapse of microspores. The TDR gene is preferentially expressed in the tapetum and encodes a putative basic helix-loop-helix protein, which is likely localized to the nucleus. More importantly, two genes, Os CP1 and Os c6, encoding a Cys protease and a protease inhibitor, respectively, were shown to be the likely direct targets of TDR through chromatin immunoprecipitation analyses and the electrophoretic mobility shift assay. These results indicate that TDR is a key component of the molecular network regulating rice tapetum development and degeneration.
Recent 16S ribosomal RNA gene (rRNA) molecular profiling of the stomach mucosa revealed a surpris... more Recent 16S ribosomal RNA gene (rRNA) molecular profiling of the stomach mucosa revealed a surprising complexity of microbiota. Helicobacter pylori infection and non-steroidal anti-inflammatory drug (NSAID) use are two main contributors to gastritis and peptic ulcer. However, little is known about the association between other members of the stomach microbiota and gastric diseases. In this study, cloning and sequencing of the 16S rRNA was used to profile the stomach microbiota from normal and gastritis patients. One hundred and thirty three phylotypes from eight bacterial phyla were identified. The stomach microbiota was found to be closely adhered to the mucosa. Eleven Streptococcus phylotypes were successfully cultivated from the biopsies. One to two genera represented a majority of clones within any of the identified phyla. We further developed two real-time quantitative PCR assays to quantify the relative abundance of the Firmicutes phylum and the Streptococcus genus. Significantly higher abundance of the Firmicutes phylum and the Streptococcus genus within the Firmicutes phylum was observed in patients with antral gastritis, compared with normal controls. This study suggests that the genus taxon level can largely represent much higher taxa such as the phylum. The clinical relevance and the mechanism underlying the altered microbiota composition in gastritis require further functional studies.
The basic/helix-loop-helix (bHLH) transcription factors and their homologs form a large family in... more The basic/helix-loop-helix (bHLH) transcription factors and their homologs form a large family in plant and animal genomes. They are known to play important roles in the specification of tissue types in animals. On the other hand, few plant bHLH proteins have been studied functionally. Recent completion of whole genome sequences of model plants Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) allows genome-wide analysis and comparison of the bHLH family in flowering plants. We have identified 167 bHLH genes in the rice genome, and their phylogenetic analysis indicates that they form wellsupported clades, which are defined as subfamilies. In addition, sequence analysis of potential DNA-binding activity, the sequence motifs outside the bHLH domain, and the conservation of intron/exon structural patterns further support the evolutionary relationships among these proteins. The genome distribution of rice bHLH genes strongly supports the hypothesis that genome-wide and tandem duplication contributed to the expansion of the bHLH gene family, consistent with the birth-and-death theory of gene family evolution. Bioinformatics analysis suggests that rice bHLH proteins can potentially participate in a variety of combinatorial interactions, endowing them with the capacity to regulate a multitude of transcriptional programs. In addition, similar expression patterns suggest functional conservation between some rice bHLH genes and their close Arabidopsis homologs. Figure 3. NJ phylogenetic tree of the OsbHLH members. This tree indicates the predicted DNA-binding activities, the intron distribution pattern, and the conservative sequence out of the bHLH domain. The unrooted tree, constructed using MEGA 3.0, summarizes the evolutionary relationships among the 167 members of the OsbHLH protein family. The proteins are named according to OsbHLH numbers (see Supplemental Fig. 1; ). The colorful dots on the nodes indicate the bootstrap values of the tree, which is built by the maximum parsimony method. The variation rates across the amino acid positions were shown by the length of the branch. The tree shows the 22 phylogenetic subfamilies (A-V) with high predictive value. The bootstrap values lower than 50 are not shown in the phylogenetic tree. The markers in front of the OsbHLH numbers indicate the predicted DNAbinding activity of each protein, i.e. the roundish marker indicates putative G-box binders, the square marker indicates putative non-G-box but E-box binders, the triangle marker indicates putative non-E-box binders (i.e. possible DNA-binding capacity but no predicted recognition of an E box), and the upside-down triangle marker indicates putative non-DNA binders (see for categories). The colors of these markers indicate the numbers and positions of the introns localized in the bHLH domain of each (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 15: 63-78 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403-410 Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389-3402 Atchley WR, Fitch WM (1997) A natural classification of the basic helixloop-helix class of transcription factors. Proc Natl Acad Sci USA 94: 5172-5176 Atchley WR, Terhalle W, Dress A (1999) Positional dependence, cliques, and predictive motifs in the bHLH protein domain. J Mol Evol 48: 501-516
Using microRNA (miRNA) expression array, we identified that miR-7 was deregulated in colorectal c... more Using microRNA (miRNA) expression array, we identified that miR-7 was deregulated in colorectal cancer (CRC). We studied the biological role and molecular target of miR-7 in CRC. miR-7 was downregulated in six out of seven colon cancer cell lines. Ectopic expression of miR-7 suppressed colon cancer cell proliferation (P&lt;0.05), induced apoptosis (P&lt;0.05) and caused cell-cycle arrest in G1 phase (P&lt;0.05). The tumor suppressive function of miR-7 was further confirmed in nude mice (P&lt;0.05). The 3&#39;-untranslated region (3&#39;UTR) of Yin Yang 1 (YY1) mRNA contains an evolutionarily conserved miR-7 binding site using in silico searches, luciferase reporter assay and western blot analysis confirmed that miR-7 directly bound to YY1 3&#39;UTR to negatively regulate the protein expression of YY1 in colon cancer cell lines HCT116 and LOVO. Intriguingly, knock-down of YY1 in three colon cancer cell lines (HCT116, LOVO and DLD1) consistently suppressed cell proliferation (P&lt;0.01) and induced apoptosis (P&lt;0.01), indicating the opposite functions of miR-7 and YY1 in CRC. Consistent with these data, ectopic expression of YY1 promoted cell growth by increasing proliferation (P&lt;0.01) and suppressing apoptosis (P&lt;0.001). The tumorigenic ability of YY1 was further confirmed in vivo in xenograft-nude mouse model (P&lt;0.01). In addition, pathway analyses revealed that the oncogenic effect by YY1 was associated with inhibiting p53 and modulating its downstream effectors p15, caspase cascades and C-Jun, and activating Wnt signaling pathway through activating β-catenin, anti-apoptotic survivin and fibroblast growth factor 4. Furthermore, multivariate analysis revealed that patients with YY1 protein high expression had a significant decrease in overall survival, and Kaplan-Meier survival curves showed that these patients had significantly shorter survival than others (P&lt;0.0001). In conclusion, MiR-7 is a novel miRNA with tumor suppressive function in colon cancer by targeting oncogenic YY1. YY1 promotes colon cancer growth through inhibiting p53 and promoting Wnt signaling pathways and serves as an independent prognostic biomarker for CRC patients.
Using genome-wide promoter methylation analysis, we identified a disintegrin-like and metalloprot... more Using genome-wide promoter methylation analysis, we identified a disintegrin-like and metalloprotease with thrombospondin type 1 motif 9 (ADAMTS9) is methylated in cancer. We aim to clarify its epigenetic inactivation, biological function and clinical implication in gastric cancer. ADAMTS9 was silenced in 6 out of 8 gastric cancer cell lines. The loss of ADAMTS9 expression was regulated by promoter hypermethylation and could be restored by demethylation agent. Ectopic expression of ADAMTS9 in gastric cancer cell lines (AGS, BGC823) inhibited cell growth curve in both the cell lines (Po0.0001), suppressed colony formation (Po0.01) and induced apoptosis (Po0.001 in AGS, Po0.01 in BGC823). Moreover, conditioned culture medium from ADAMTS9-transfected cell lines significantly disrupted the human umbilical vein endothelial cell tube formation capacity on Matrigel (Po0.01 in AGS, Po0.001 in BGC823). The in vivo growth of ADAMTS9 cells in nude mice was also markedly diminished after stable expression of ADAMTS9 (Po0.001). On the other hand, ADAMTS9 knockdown promoted cell proliferation (Po0.001). We further revealed that ADAMTS9 inhibited tumor growth by blocking activation of Akt and its downstream target the mammalian target of rapamycin (mTOR). ADAMTS9 also reduced phosphorylation of mTOR downstream targets p70 ribosomal S6 kinase, eIF4E-binding protein and downregulated hypoxia-inducible factor-1a. Therefore, this is the first demonstration that ADAMTS9 is a critical tumor suppressor of gastric cancer progression at least in part through suppression of oncogenic AKT/mTOR signaling. Moreover, promoter methylation of ADAMTS9 was detected in 29.2% (21/72) of primary gastric tumors. Multivariate analysis showed that patients with ADAMTS9 methylation had a poorer overall survival (relative risk (RR) ¼ 2.788; 95% confidence interval, 1.474-5.274; P ¼ 0.002). Kaplan-Meier survival curves showed that ADAMTS9 methylation was significantly associated with shortened survival in gastric cancer patients (P ¼ 0.001, log-rank test). In conclusion, ADAMTS9 acts as a functional tumor suppressor in gastric cancer through inhibiting oncogenic AKT/mTOR signaling pathway. Methylation of ADAMTS9 is an independent prognostic factor of gastric cancer.
Aberrant expression and altered function of transcription factors (TFs) have vital roles in many ... more Aberrant expression and altered function of transcription factors (TFs) have vital roles in many aspects of tumor development and progression. In this study, we investigated the functional significance of a TF, Yin Yang1 (YY1) in tumorigenesis of endometrioid endometrial carcinoma (EEC). We demonstrated that YY1 is upregulated in EEC cell lines and primary tumors; and its expression is associated with tumor stages. Depletion of YY1 inhibits EEC cell proliferation and migration both in vitro and in vivo, whereas overexpression of YY1 promotes EEC cell growth. These results suggest that YY1 functions as an oncogenic factor in EEC. Transcriptome analysis revealed a significant effect of YY1 on critical aspects of EEC tumorigenesis through inhibition of APC expression. Further mechanistic investigation uncovered a new epigenetic silencing mode of APC by YY1 through recruitment of EZH2 and trimethylation of histone 3 lysine 27 on its promoter region. Moreover, YY1 overexpression was found to be a consequence of miR-193a-5p downregulation through direct miR-193a-5p-YY1 interplay. Our results therefore establish a novel miR-193a-5p-YY1-APC axis, which contributes to EEC development, and may serve as future intervention target.
expression of cytoskeletal markers of quiescent stellate cells. Gene-expression profiling identif... more expression of cytoskeletal markers of quiescent stellate cells. Gene-expression profiling identified IL-8 signalling and the wnt-β-catenin pathway as the key canonical pathways affected. Organotypic co-culture of PSCs and cancer cells demonstrated an indirect effect of treated PSCs on cancer cell morphology, proliferation (decrease) and apoptosis (increase) and confirmed the engagement of the wnt-β-catenin signalling pathway. There was less βcatenin signalling (TOPFlash reporter assays) in cancer cells co-cultured with treated quiescent PSCs, which was mediated by an increased expression of sFRP4 (secreted frizzledrelated protein 4) resulting in decreased invasion of cancer cells. Additionally, in contrast to vehicle treated orgnaotypic cultures, PSCs exposed to ATRA did not invade into the extracellular matrix gel but formed a "wall" at the cancer-matrix junction, blocking cancer cell invasion. Conclusion: Targeting the desmoplastic stroma with agents such as ATRA interferes with the, for tumour progression important, tumour-stroma cross-talk and offers exciting opportunities for combinatorial therapy for pancreatic cancer.
Using genome-wide promoter methylation assay, B cell CLL/lymphoma 6 member B (BCL6B) was found to... more Using genome-wide promoter methylation assay, B cell CLL/lymphoma 6 member B (BCL6B) was found to be preferentially methylated in cancer. A study was undertaken to examine the epigenetic regulation, biological function and clinical significance of BCL6B in gastric cancer (GC). BCL6B promoter methylation was evaluated by combined bisulfite restriction analysis and sequencing. The biological functions of BCL6B were determined by cell viability, colony formation, flow cytometry and in vivo tumorigenicity assays. The molecular targets of BCL6B were identified by cDNA expression array. BCL6B was silenced or downregulated in all nine GC cell lines and readily expressed in normal gastric tissues. Loss of BCL6B expression was regulated by promoter hypermethylation. Re-expression of BCL6B in GC cell lines inhibited colony formation, suppressed cell viability, induced apoptosis and restrained the tumorigenecity in nude mice. These effects were associated with upregulation of the pro-apoptosis genes tumour necrosis factor receptor superfamily member 1A, caspase-8, caspase-9, caspase-3 and caspase-7 and nuclear enzyme poly (ADP-ribose) polymerase, downregulation of the pro-proliferation genes S100 calcium binding protein A4 and vascular endothelial growth factor A, and induction of the tumour suppressor genes ataxia telangiectasia mutated homologue and p53. BCL6B hypermethylation was detected in 49.0% (102/208) and 66.3% (67/101) of two independent cohorts of patients with GC, respectively. BCL6B methylation was an independent factor for the survival of patients with GC (p=0.001 for cohort I, p=0.02 for cohort II). BCL6B plays a pivotal role as a potential tumour suppressor in GC. Detection of methylated BCL6B may serve as an independent biomarker for the prognosis of GC.
pathogenecity of disease-specific genes of H. pylori. Results; 14 TOF spots were significantly el... more pathogenecity of disease-specific genes of H. pylori. Results; 14 TOF spots were significantly elevated in H. pylori from duodenal ulcer compared to strains from gastric ulcer; bII 5012, ATP synthase, EF-Tu, acetyl Co-A transferase, PGK, etc, whereas 26 spots were significantly in strains from gastric ulcer, including trigger factors, Hsp, proline peptidase, pilt like protein, etc. Interestingly, 30 spots were significantly increased in strain from gastric cancer compared to gastric ulcer, including EF.Tu, adenylate cyclase, pyruvate ferrodoxin oxidoreductase, gln A MCP, zinc protease, ASPS etc. Biomarker validations performed through immunoblotting with proteomes from H. pylori and sera from patients include Zinc protease and Hsp. Conclusion: In summary, putative biomarker might come from the authentic proteomes of H. pylori, of which we could identify zinc protease and Hsp could be candidates for potential biomarker to discriminate pathogenecity of H. pylori.
are unknown. METHODS: Using a genetic approach, we have assessed the impact of Tff2 deficiency in... more are unknown. METHODS: Using a genetic approach, we have assessed the impact of Tff2 deficiency in the gp130 Y757F/Y757F mouse model of gastric tumourigenesis. To investigate TFF2 transcriptional silencing in human cancer, gene expression and promoter methylation analysis was conducted upon ninety-one gastric mucosal specimens encompassing different stages of gastric cancer development: H. pylori-positive gastritis, pre-neoplastic adjacent to cancer showing intestinal metaplasia (IM), cancer and normal controls. RESULTS: Antral tumour size in gp130 Y757F/Y757F /Tff2 -/compound mutant mice was increased 54% and 33% at 6 and 12 weeks respectively compared to gp130 Y757F/Y757F single mutant littermates. Increased tumour load was positively correlated with epithelial cell proliferation and negatively correlated with transcription of gastric tumour suppressor genes, Tff1 and Gastrokines (Gkn)1 and Gkn2. In addition to effects on tumourigenesis, the gp130 Y757F/Y757F /Tff2 -/fundus displayed pervasive glandular atrophy, characterised by loss of oxyntic and zymogenic lineages, expansion of the mucous neck cell layer, and increased type-1 T-helper (Th1) cytokine release, recapitulating the pre-neoplastic atrophy induced by H. pylori in humans. In translational studies we present novel data showing that the TFF2 promoter is frequently and progressively methylated during human gastric tumourigenesis. TFF2 methylation is positively correlated with transcriptional silencing, and discriminates normal, H. pyloriinfected/IM and cancer tissue by hierarchical cluster analysis. Genome demethylation restored TFF2 expression in gastric cancer cell lines confirming that TFF2 silencing is methylation dependent. CONCLUSIONS: TFF2 is a novel tumour suppressor gene that regulates gastric differentiation and exerts anti-proliferative effects in gastric tumourigenesis. Promoter methylation is a major TFF2 silencing mechanism that operates during pre-neoplastic progression with potential therapeutic value in early detection and/or prognostic outcome.
Using the promoter methylation assay, we have shown that MDGA2 (MAM domain containing glycosylpho... more Using the promoter methylation assay, we have shown that MDGA2 (MAM domain containing glycosylphosphatidylinositol anchor 2) is preferentially methylated in gastric cancer. We analysed its biological effects and prognostic significance in gastric cancer. MDGA2 methylation status was evaluated by combined bisulfite restriction analysis and bisulfite genomic sequencing. The effects of MDGA2 re-expression or knockdown on cell proliferation, apoptosis and the cell cycle were determined. MDGA2 interacting protein was identified by mass spectrometry and MDGA2-related cancer pathways by reporter activity and PCR array analyses. The clinical impact of MDGA2 was assessed in 218 patients with gastric cancer. MDGA2 was commonly silenced in gastric cancer cells (10/11) and primary gastric cancers due to promoter hypermethylation. MDGA2 significantly inhibited cell proliferation by causing G1-S cell cycle arrest and inducing cell apoptosis in vitro, and suppressed xenograft tumour growth in both...
We found that carbonic anhydrase IV (CA4), a member of the carbonic anhydrases, is silenced in co... more We found that carbonic anhydrase IV (CA4), a member of the carbonic anhydrases, is silenced in colorectal cancer (CRC). We analysed its epigenetic inactivation, biological effects and prognostic significance in CRC. The biological functions of CA4 were determined by in vitro and in vivo tumorigenicity assays. The CA4 co-operator was identified by immunoprecipitation and mass spectrometry. CA4 downstream effectors and signalling pathways were elucidated by promoter luciferase assay, electrophoretic mobility shift assay and chromatin immunoprecipitation. The clinical impact of CA4 was assessed in 115 patients with CRC. CA4 was silenced in all nine CRC cell lines and 92.6% of CRC tumours. The promoter hypermethylation contributed to the inactivation of CA4, and it was detected in 75.7% of the patients with CRC. After a median follow-up of 49.3 months, multivariate analysis showed that the patients with CA4 hypermethylation had a recurrence of Stage II/III CRC. The re-expression of CA4 ...
Using whole genome sequencing, we identified gene amplification of solute carrier family 12 membe... more Using whole genome sequencing, we identified gene amplification of solute carrier family 12 member 5 (SLC12A5) located at 20q13.12 in colorectal cancer (CRC). We analysed its amplification, overexpression, biological effects and prognostic significance in CRC. SLC12A5 amplification status was evaluated by fluorescence in situ hybridisation (FISH). The effects of SLC12A5 re-expression or knockdown were determined in proliferation, apoptosis, invasion and metastasis assays. SLC12A5 target genes and related pathways were identified by reporter activity and cDNA microarray analyses. Clinical impact of SLC12A5 overexpression was assessed in 195 patients with CRC. Amplification of SLC12A5 was verified in 78 out of 191 (40.8%) patients with primary CRC by FISH, which was positively correlated with its protein overexpression (p<0.001). Biofunctional investigation of SLC12A5 revealed that SLC12A5 significantly increased cell proliferation, G1-S cell cycle transition, invasion/migration ab...
Dapper homolog (DACT) 2 is one of the Dact gene family members, which are important modulators of... more Dapper homolog (DACT) 2 is one of the Dact gene family members, which are important modulators of Wnt signaling pathway. We aim to clarify its epigenetic inactivation, biological function and clinical implication in colon cancer. DACT2 was silenced in five out of eight colon cancer cell lines, but robustly expressed in normal colon tissues. The loss of DACT2 expression was regulated by promoter hypermethylation. Restoring DACT2 expression in colon cancer cell lines suppressed tumor cell growth by inducing cell apoptosis and inhibiting cell proliferation both in vitro and in vivo. Moreover, DACT2 overexpression effectively reduced lung metastasis of colon cancer cells in nude mice. These effects by DACT2 were attributed to inhibition of Wnt/β-catenin signaling. Reexpression of DACT2 significantly suppressed the transcriptional activity of both wild-type β-catenin and degradation-resistant form mutant β-catenin (S33Y). DACT2 could actively shuttle into and out of nuclei, with its pred...
Peroxisome proliferator-activated receptor alpha (PPARα) ligands have been reported to suppress c... more Peroxisome proliferator-activated receptor alpha (PPARα) ligands have been reported to suppress cancer growth. However, the role of PPARα in hepatocarcinogenesis remains unclear. We investigated the functional significance of PPARα in HCC. PPARα-knockout (PPARα-/-) mice were more susceptible to diethylnitrosamine (DEN)-induced HCC at 6 months compared with wild-type (WT) littermates (80% versus 43%, P < 0.05). In resected HCCs, TUNEL-positive apoptotic cells were significantly less in PPARα-/- mice than in WT mice (P < 0.01), commensurate with a reduction in cleaved caspase-3 and caspase-7 protein expression. Ki-67 staining showed increased cell proliferation in PPARα-/- mice (P…
The mechanisms by which Epstein-Barr virus (EBV) contributes to the development of gastric cancer... more The mechanisms by which Epstein-Barr virus (EBV) contributes to the development of gastric cancer are unclear. We investigated EBV-associated genomic and epigenomic variations in gastric cancer cells and tumors. We performed whole-genome, transcriptome, and epigenome sequence analyses of a gastric adenocarcinoma cell line (AGS cells), before and after EBV infection. We then looked for alterations in gastric tumor samples, with (n = 34) or without (n = 100) EBV infection, collected from patients at the Prince of Wales Hospital, Chinese University of Hong Kong (from 1998 through 2004), or the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China (from 1999 through 2006). Transcriptome analysis showed that infected cells expressed 9 EBV genes previously detected in EBV-associated gastric tumors and 71 EBV genes not previously reported in gastric tumors. Ten viral genes that had not been reported previously in gastric cancer but were expressed most highly in EBV-infected cells also were expressed in primary EBV-positive gastric tumors. Whole-genome sequence analysis identified 45 EBV-associated nonsynonymous mutations. These mutations, in genes such as AKT2, CCNA1, MAP3K4, and TGFBR1, were associated significantly with EBV-positive gastric tumors, compared with EBV-negative tumors. An activating mutation in AKT2 was associated with reduced survival times of patients with EBV-positive gastric cancer (P = .006); this mutation was found to dysregulate mitogen-activated protein kinase signaling. Integrated epigenome and transcriptome analyses identified 216 genes transcriptionally down-regulated by EBV-associated hypermethylation; methylation of ACSS1, FAM3B, IHH, and TRABD increased significantly in EBV-positive tumors. Overexpression of Indian hedgehog (IHH) and TraB domain containing (TRABD) increased proliferation and colony formation of gastric cancer cells, whereas knockdown of these genes reduced these activities. We found 5 signaling pathways (axon guidance, focal adhesion formation, interactions among cytokines and receptors, mitogen-activated protein kinase signaling, and actin cytoskeleton regulation) to be affected commonly by EBV-associated genomic and epigenomic alterations. By using genomic, transcriptome, and epigenomic comparisons of EBV infected vs noninfected gastric cancer cells and tumor samples, we identified alterations in genes, gene expression, and methylation that affect different signaling networks. These might be involved in EBV-associated gastric carcinogenesis.
B cell CLL/lymphoma 6 member B (BCL6B) is a novel tumor suppressor silenced in human cancer. In t... more B cell CLL/lymphoma 6 member B (BCL6B) is a novel tumor suppressor silenced in human cancer. In this study, we investigated the functional role and underlying mechanisms of BCL6B in hepatocellular carcinoma (HCC). BCL6B was expressed in normal HCC tissues, but its expression was suppressed in 6 out of 9 HCC cell lines. Loss of BCL6B expression was associated with promoter hypermethylation. Ectopic expression of BCL6B in HepG2 and Huh7 cell lines inhibited colony formation (P <0.05), cell viability (P <0.01), and tumorigenicity in nude mice (P <0.05). BCL6B expression also induced apoptosis (P <0.05), an effect associated with activation of the caspase cascade and cleavage of PARP. Stable expression of BCL6B in MHCC97L cells suppressed cell migration (P <0.05) and invasion (P <0.05), and significantly reduced the incidence and severity of lung metastasis in an orthotopic HCC mouse model. The anti-metastatic effect of BCL6B was mediated by up-regulation of cell adhes...
expression of cytoskeletal markers of quiescent stellate cells. Gene-expression profiling identif... more expression of cytoskeletal markers of quiescent stellate cells. Gene-expression profiling identified IL-8 signalling and the wnt-β-catenin pathway as the key canonical pathways affected. Organotypic co-culture of PSCs and cancer cells demonstrated an indirect effect of treated PSCs on cancer cell morphology, proliferation (decrease) and apoptosis (increase) and confirmed the engagement of the wnt-β-catenin signalling pathway. There was less βcatenin signalling (TOPFlash reporter assays) in cancer cells co-cultured with treated quiescent PSCs, which was mediated by an increased expression of sFRP4 (secreted frizzledrelated protein 4) resulting in decreased invasion of cancer cells. Additionally, in contrast to vehicle treated orgnaotypic cultures, PSCs exposed to ATRA did not invade into the extracellular matrix gel but formed a "wall" at the cancer-matrix junction, blocking cancer cell invasion. Conclusion: Targeting the desmoplastic stroma with agents such as ATRA interferes with the, for tumour progression important, tumour-stroma cross-talk and offers exciting opportunities for combinatorial therapy for pancreatic cancer.
In flowering plants, tapetum degeneration is proposed to be triggered by a programmed cell death ... more In flowering plants, tapetum degeneration is proposed to be triggered by a programmed cell death (PCD) process during late stages of pollen development; the PCD is thought to provide cellular contents supporting pollen wall formation and to allow the subsequent pollen release. However, the molecular basis regulating tapetum PCD in plants remains poorly understood. We report the isolation and characterization of a rice (Oryza sativa) male sterile mutant tapetum degeneration retardation (tdr), which exhibits degeneration retardation of the tapetum and middle layer as well as collapse of microspores. The TDR gene is preferentially expressed in the tapetum and encodes a putative basic helix-loop-helix protein, which is likely localized to the nucleus. More importantly, two genes, Os CP1 and Os c6, encoding a Cys protease and a protease inhibitor, respectively, were shown to be the likely direct targets of TDR through chromatin immunoprecipitation analyses and the electrophoretic mobility shift assay. These results indicate that TDR is a key component of the molecular network regulating rice tapetum development and degeneration.
Recent 16S ribosomal RNA gene (rRNA) molecular profiling of the stomach mucosa revealed a surpris... more Recent 16S ribosomal RNA gene (rRNA) molecular profiling of the stomach mucosa revealed a surprising complexity of microbiota. Helicobacter pylori infection and non-steroidal anti-inflammatory drug (NSAID) use are two main contributors to gastritis and peptic ulcer. However, little is known about the association between other members of the stomach microbiota and gastric diseases. In this study, cloning and sequencing of the 16S rRNA was used to profile the stomach microbiota from normal and gastritis patients. One hundred and thirty three phylotypes from eight bacterial phyla were identified. The stomach microbiota was found to be closely adhered to the mucosa. Eleven Streptococcus phylotypes were successfully cultivated from the biopsies. One to two genera represented a majority of clones within any of the identified phyla. We further developed two real-time quantitative PCR assays to quantify the relative abundance of the Firmicutes phylum and the Streptococcus genus. Significantly higher abundance of the Firmicutes phylum and the Streptococcus genus within the Firmicutes phylum was observed in patients with antral gastritis, compared with normal controls. This study suggests that the genus taxon level can largely represent much higher taxa such as the phylum. The clinical relevance and the mechanism underlying the altered microbiota composition in gastritis require further functional studies.
The basic/helix-loop-helix (bHLH) transcription factors and their homologs form a large family in... more The basic/helix-loop-helix (bHLH) transcription factors and their homologs form a large family in plant and animal genomes. They are known to play important roles in the specification of tissue types in animals. On the other hand, few plant bHLH proteins have been studied functionally. Recent completion of whole genome sequences of model plants Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) allows genome-wide analysis and comparison of the bHLH family in flowering plants. We have identified 167 bHLH genes in the rice genome, and their phylogenetic analysis indicates that they form wellsupported clades, which are defined as subfamilies. In addition, sequence analysis of potential DNA-binding activity, the sequence motifs outside the bHLH domain, and the conservation of intron/exon structural patterns further support the evolutionary relationships among these proteins. The genome distribution of rice bHLH genes strongly supports the hypothesis that genome-wide and tandem duplication contributed to the expansion of the bHLH gene family, consistent with the birth-and-death theory of gene family evolution. Bioinformatics analysis suggests that rice bHLH proteins can potentially participate in a variety of combinatorial interactions, endowing them with the capacity to regulate a multitude of transcriptional programs. In addition, similar expression patterns suggest functional conservation between some rice bHLH genes and their close Arabidopsis homologs. Figure 3. NJ phylogenetic tree of the OsbHLH members. This tree indicates the predicted DNA-binding activities, the intron distribution pattern, and the conservative sequence out of the bHLH domain. The unrooted tree, constructed using MEGA 3.0, summarizes the evolutionary relationships among the 167 members of the OsbHLH protein family. The proteins are named according to OsbHLH numbers (see Supplemental Fig. 1; ). The colorful dots on the nodes indicate the bootstrap values of the tree, which is built by the maximum parsimony method. The variation rates across the amino acid positions were shown by the length of the branch. The tree shows the 22 phylogenetic subfamilies (A-V) with high predictive value. The bootstrap values lower than 50 are not shown in the phylogenetic tree. The markers in front of the OsbHLH numbers indicate the predicted DNAbinding activity of each protein, i.e. the roundish marker indicates putative G-box binders, the square marker indicates putative non-G-box but E-box binders, the triangle marker indicates putative non-E-box binders (i.e. possible DNA-binding capacity but no predicted recognition of an E box), and the upside-down triangle marker indicates putative non-DNA binders (see for categories). The colors of these markers indicate the numbers and positions of the introns localized in the bHLH domain of each (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 15: 63-78 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403-410 Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389-3402 Atchley WR, Fitch WM (1997) A natural classification of the basic helixloop-helix class of transcription factors. Proc Natl Acad Sci USA 94: 5172-5176 Atchley WR, Terhalle W, Dress A (1999) Positional dependence, cliques, and predictive motifs in the bHLH protein domain. J Mol Evol 48: 501-516
Using microRNA (miRNA) expression array, we identified that miR-7 was deregulated in colorectal c... more Using microRNA (miRNA) expression array, we identified that miR-7 was deregulated in colorectal cancer (CRC). We studied the biological role and molecular target of miR-7 in CRC. miR-7 was downregulated in six out of seven colon cancer cell lines. Ectopic expression of miR-7 suppressed colon cancer cell proliferation (P&lt;0.05), induced apoptosis (P&lt;0.05) and caused cell-cycle arrest in G1 phase (P&lt;0.05). The tumor suppressive function of miR-7 was further confirmed in nude mice (P&lt;0.05). The 3&#39;-untranslated region (3&#39;UTR) of Yin Yang 1 (YY1) mRNA contains an evolutionarily conserved miR-7 binding site using in silico searches, luciferase reporter assay and western blot analysis confirmed that miR-7 directly bound to YY1 3&#39;UTR to negatively regulate the protein expression of YY1 in colon cancer cell lines HCT116 and LOVO. Intriguingly, knock-down of YY1 in three colon cancer cell lines (HCT116, LOVO and DLD1) consistently suppressed cell proliferation (P&lt;0.01) and induced apoptosis (P&lt;0.01), indicating the opposite functions of miR-7 and YY1 in CRC. Consistent with these data, ectopic expression of YY1 promoted cell growth by increasing proliferation (P&lt;0.01) and suppressing apoptosis (P&lt;0.001). The tumorigenic ability of YY1 was further confirmed in vivo in xenograft-nude mouse model (P&lt;0.01). In addition, pathway analyses revealed that the oncogenic effect by YY1 was associated with inhibiting p53 and modulating its downstream effectors p15, caspase cascades and C-Jun, and activating Wnt signaling pathway through activating β-catenin, anti-apoptotic survivin and fibroblast growth factor 4. Furthermore, multivariate analysis revealed that patients with YY1 protein high expression had a significant decrease in overall survival, and Kaplan-Meier survival curves showed that these patients had significantly shorter survival than others (P&lt;0.0001). In conclusion, MiR-7 is a novel miRNA with tumor suppressive function in colon cancer by targeting oncogenic YY1. YY1 promotes colon cancer growth through inhibiting p53 and promoting Wnt signaling pathways and serves as an independent prognostic biomarker for CRC patients.
Using genome-wide promoter methylation analysis, we identified a disintegrin-like and metalloprot... more Using genome-wide promoter methylation analysis, we identified a disintegrin-like and metalloprotease with thrombospondin type 1 motif 9 (ADAMTS9) is methylated in cancer. We aim to clarify its epigenetic inactivation, biological function and clinical implication in gastric cancer. ADAMTS9 was silenced in 6 out of 8 gastric cancer cell lines. The loss of ADAMTS9 expression was regulated by promoter hypermethylation and could be restored by demethylation agent. Ectopic expression of ADAMTS9 in gastric cancer cell lines (AGS, BGC823) inhibited cell growth curve in both the cell lines (Po0.0001), suppressed colony formation (Po0.01) and induced apoptosis (Po0.001 in AGS, Po0.01 in BGC823). Moreover, conditioned culture medium from ADAMTS9-transfected cell lines significantly disrupted the human umbilical vein endothelial cell tube formation capacity on Matrigel (Po0.01 in AGS, Po0.001 in BGC823). The in vivo growth of ADAMTS9 cells in nude mice was also markedly diminished after stable expression of ADAMTS9 (Po0.001). On the other hand, ADAMTS9 knockdown promoted cell proliferation (Po0.001). We further revealed that ADAMTS9 inhibited tumor growth by blocking activation of Akt and its downstream target the mammalian target of rapamycin (mTOR). ADAMTS9 also reduced phosphorylation of mTOR downstream targets p70 ribosomal S6 kinase, eIF4E-binding protein and downregulated hypoxia-inducible factor-1a. Therefore, this is the first demonstration that ADAMTS9 is a critical tumor suppressor of gastric cancer progression at least in part through suppression of oncogenic AKT/mTOR signaling. Moreover, promoter methylation of ADAMTS9 was detected in 29.2% (21/72) of primary gastric tumors. Multivariate analysis showed that patients with ADAMTS9 methylation had a poorer overall survival (relative risk (RR) ¼ 2.788; 95% confidence interval, 1.474-5.274; P ¼ 0.002). Kaplan-Meier survival curves showed that ADAMTS9 methylation was significantly associated with shortened survival in gastric cancer patients (P ¼ 0.001, log-rank test). In conclusion, ADAMTS9 acts as a functional tumor suppressor in gastric cancer through inhibiting oncogenic AKT/mTOR signaling pathway. Methylation of ADAMTS9 is an independent prognostic factor of gastric cancer.
Aberrant expression and altered function of transcription factors (TFs) have vital roles in many ... more Aberrant expression and altered function of transcription factors (TFs) have vital roles in many aspects of tumor development and progression. In this study, we investigated the functional significance of a TF, Yin Yang1 (YY1) in tumorigenesis of endometrioid endometrial carcinoma (EEC). We demonstrated that YY1 is upregulated in EEC cell lines and primary tumors; and its expression is associated with tumor stages. Depletion of YY1 inhibits EEC cell proliferation and migration both in vitro and in vivo, whereas overexpression of YY1 promotes EEC cell growth. These results suggest that YY1 functions as an oncogenic factor in EEC. Transcriptome analysis revealed a significant effect of YY1 on critical aspects of EEC tumorigenesis through inhibition of APC expression. Further mechanistic investigation uncovered a new epigenetic silencing mode of APC by YY1 through recruitment of EZH2 and trimethylation of histone 3 lysine 27 on its promoter region. Moreover, YY1 overexpression was found to be a consequence of miR-193a-5p downregulation through direct miR-193a-5p-YY1 interplay. Our results therefore establish a novel miR-193a-5p-YY1-APC axis, which contributes to EEC development, and may serve as future intervention target.
expression of cytoskeletal markers of quiescent stellate cells. Gene-expression profiling identif... more expression of cytoskeletal markers of quiescent stellate cells. Gene-expression profiling identified IL-8 signalling and the wnt-β-catenin pathway as the key canonical pathways affected. Organotypic co-culture of PSCs and cancer cells demonstrated an indirect effect of treated PSCs on cancer cell morphology, proliferation (decrease) and apoptosis (increase) and confirmed the engagement of the wnt-β-catenin signalling pathway. There was less βcatenin signalling (TOPFlash reporter assays) in cancer cells co-cultured with treated quiescent PSCs, which was mediated by an increased expression of sFRP4 (secreted frizzledrelated protein 4) resulting in decreased invasion of cancer cells. Additionally, in contrast to vehicle treated orgnaotypic cultures, PSCs exposed to ATRA did not invade into the extracellular matrix gel but formed a "wall" at the cancer-matrix junction, blocking cancer cell invasion. Conclusion: Targeting the desmoplastic stroma with agents such as ATRA interferes with the, for tumour progression important, tumour-stroma cross-talk and offers exciting opportunities for combinatorial therapy for pancreatic cancer.
Using genome-wide promoter methylation assay, B cell CLL/lymphoma 6 member B (BCL6B) was found to... more Using genome-wide promoter methylation assay, B cell CLL/lymphoma 6 member B (BCL6B) was found to be preferentially methylated in cancer. A study was undertaken to examine the epigenetic regulation, biological function and clinical significance of BCL6B in gastric cancer (GC). BCL6B promoter methylation was evaluated by combined bisulfite restriction analysis and sequencing. The biological functions of BCL6B were determined by cell viability, colony formation, flow cytometry and in vivo tumorigenicity assays. The molecular targets of BCL6B were identified by cDNA expression array. BCL6B was silenced or downregulated in all nine GC cell lines and readily expressed in normal gastric tissues. Loss of BCL6B expression was regulated by promoter hypermethylation. Re-expression of BCL6B in GC cell lines inhibited colony formation, suppressed cell viability, induced apoptosis and restrained the tumorigenecity in nude mice. These effects were associated with upregulation of the pro-apoptosis genes tumour necrosis factor receptor superfamily member 1A, caspase-8, caspase-9, caspase-3 and caspase-7 and nuclear enzyme poly (ADP-ribose) polymerase, downregulation of the pro-proliferation genes S100 calcium binding protein A4 and vascular endothelial growth factor A, and induction of the tumour suppressor genes ataxia telangiectasia mutated homologue and p53. BCL6B hypermethylation was detected in 49.0% (102/208) and 66.3% (67/101) of two independent cohorts of patients with GC, respectively. BCL6B methylation was an independent factor for the survival of patients with GC (p=0.001 for cohort I, p=0.02 for cohort II). BCL6B plays a pivotal role as a potential tumour suppressor in GC. Detection of methylated BCL6B may serve as an independent biomarker for the prognosis of GC.
pathogenecity of disease-specific genes of H. pylori. Results; 14 TOF spots were significantly el... more pathogenecity of disease-specific genes of H. pylori. Results; 14 TOF spots were significantly elevated in H. pylori from duodenal ulcer compared to strains from gastric ulcer; bII 5012, ATP synthase, EF-Tu, acetyl Co-A transferase, PGK, etc, whereas 26 spots were significantly in strains from gastric ulcer, including trigger factors, Hsp, proline peptidase, pilt like protein, etc. Interestingly, 30 spots were significantly increased in strain from gastric cancer compared to gastric ulcer, including EF.Tu, adenylate cyclase, pyruvate ferrodoxin oxidoreductase, gln A MCP, zinc protease, ASPS etc. Biomarker validations performed through immunoblotting with proteomes from H. pylori and sera from patients include Zinc protease and Hsp. Conclusion: In summary, putative biomarker might come from the authentic proteomes of H. pylori, of which we could identify zinc protease and Hsp could be candidates for potential biomarker to discriminate pathogenecity of H. pylori.
are unknown. METHODS: Using a genetic approach, we have assessed the impact of Tff2 deficiency in... more are unknown. METHODS: Using a genetic approach, we have assessed the impact of Tff2 deficiency in the gp130 Y757F/Y757F mouse model of gastric tumourigenesis. To investigate TFF2 transcriptional silencing in human cancer, gene expression and promoter methylation analysis was conducted upon ninety-one gastric mucosal specimens encompassing different stages of gastric cancer development: H. pylori-positive gastritis, pre-neoplastic adjacent to cancer showing intestinal metaplasia (IM), cancer and normal controls. RESULTS: Antral tumour size in gp130 Y757F/Y757F /Tff2 -/compound mutant mice was increased 54% and 33% at 6 and 12 weeks respectively compared to gp130 Y757F/Y757F single mutant littermates. Increased tumour load was positively correlated with epithelial cell proliferation and negatively correlated with transcription of gastric tumour suppressor genes, Tff1 and Gastrokines (Gkn)1 and Gkn2. In addition to effects on tumourigenesis, the gp130 Y757F/Y757F /Tff2 -/fundus displayed pervasive glandular atrophy, characterised by loss of oxyntic and zymogenic lineages, expansion of the mucous neck cell layer, and increased type-1 T-helper (Th1) cytokine release, recapitulating the pre-neoplastic atrophy induced by H. pylori in humans. In translational studies we present novel data showing that the TFF2 promoter is frequently and progressively methylated during human gastric tumourigenesis. TFF2 methylation is positively correlated with transcriptional silencing, and discriminates normal, H. pyloriinfected/IM and cancer tissue by hierarchical cluster analysis. Genome demethylation restored TFF2 expression in gastric cancer cell lines confirming that TFF2 silencing is methylation dependent. CONCLUSIONS: TFF2 is a novel tumour suppressor gene that regulates gastric differentiation and exerts anti-proliferative effects in gastric tumourigenesis. Promoter methylation is a major TFF2 silencing mechanism that operates during pre-neoplastic progression with potential therapeutic value in early detection and/or prognostic outcome.
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