Mutational landscape of TP53 and CDH1 in gastric cancer

In this editorial we comment on an article published in a recent issue of the World J Gastrointest Surg.A common gene mutation in gastric cancer(GC)is the TP53 mutation.As a tumor suppressor gene,TP53 is implicated in more than half of all tumor occurrences.TP53 gene mutations in GC tissue may be related with clinical pathological aspects.The TP53 mutation arose late in the progression of GC and aided in the final switch to malignancy.CDH1 encodes E-cadherin,which is involved in cell-to-cell adhesion,epithelial structure maintenance,cell polarity,differentiation,and intracellular signaling pathway modulation.CDH1 mutations and functional loss can result in diffuse GC,and CDH1 mutations can serve as independent prognostic indicators for poor prognosis.GC patients can benefit from genetic counseling and testing for CDH1 mutations.Demethylation therapy may assist to postpone the onset and progression of GC.The investigation of TP53 and CDH1 gene mutations in GC allows for the investigation of the relationship between these two gene mutations,as well as providing some basis for evaluating the prognosis of GC patients.

Anti-gastric cancer active immunity induced by FasL/B7-1 gene-modified tumor cells

AIM: To study the activation of cytotoxic T lymphocytes (CTLs) against gastric cancer cells induced by FasL/B7-1 (FB-11) gene-modified tumor cells, and to explore whether co-expression of FasL and B7-1 in SGC-7901 tumor cells could initiate synergistic antitumor effect. METHODS: FasL and B7-1 genes were transfected into human SGC-7901 gastric cancer cells with adenovirus vectors. The positive clones were selected by G418. FasL and B7-1 genes were detected by flow cytometry and RT-PCR. Abdominal infiltrating lymphocytes and sensitized spleen cells were obtained from mice that were immunized with SGC-7901/FB-11 or wild type SGC-7901 cells intraperitoneally, and cytotoxicity of these CTLs against tumor cells was determined by MTT assay. RESULTS: Flow cytometry and RT-PCR showed that FasL and B7-1 genes were highly expressed. FasL and B7-1 transfected cancer cells had a high apoptosis index. DNA laddering suggested that FasL and B7-1 genes induced gastric cancer cell apoptosis. FasL+/B7-1+SGC-7901 cells (SGC-7901/FB-11) were inoculated subcutaneously in the dorsal skin of C57BL/6 mice and then decreased their tumorigenicity greatly (z = 2.15-46.10, P＜0.01). SGC- 7901/FB-11 cell-sensitized mice obtained protective immune activity against the rechallenge of wild type SGC 7901 cells (z = 2.06-44.30, P＜0.05). The cytotoxicity of CTLs induced by SGC-7901/FB-11 cells against SGC-7901 was significantly higher than that of CTLs activated by wild-type SGC-7901 cells (84.1±2.4% vs30.5±2.3%,P＜0.05).CONCLUSION: FasL and B7-1 genes can effectively promote the activity of CTLs against gastric cancer cells. FasL/B7-1 molecules play an important role in CTL cytotoxicity.

Rationales for expression and altered expression of apoptotic protease activating factor-1 gene in gastric cancer

AIM: To elucidate the relationship between apoptotic protease activating factor-1 (Apaf-1) gene and gastric cancer. METHODS: Thirty-five postoperative cancer and adjacent normal tissue samples were collected in the present study. Expression of the Apaf-1 gene in these samples was analyzed by semi-quantitative RT-PCR. Loss of heterozygosity (LOH) was used to determine whether there was loss of Apaf-1 gene in domain of 12q22-23 in the samples. Promoter methylation of Apaf-1 gene in the samples was analyzed by methylation specific (MSP) PCR. RESULTS: The expression of Apaf-1 mRNA in gastric cancer tissue samples was 51%. The LOH frequency of D12S346, D12S1706, D12S327, D12S1657 and D12S393 was 33%, 8%, 58%, 12% and 42%, respectively. Fifty percent LOH was found at two sites and 17% LOH at three sites. Apaf-1 mRNA expression decreased significantly in 13 cases (rs = 0.487, P = 0.003). The rate of Apaf-1 promoter methylation was 49% in gastric cancer tissue samples and 23% in para-cancerous tissue samples. Promoter methylation occurred significantly in 16 of 18 gastric cancer tissue samples with decreased expression of Apaf-1 mRNA rs = 0.886, P = 10-6). CONCLUSION: The expression of Apaf-1 gene is low in gastric cancer tissues. Methylation of Apaf-1 gene promoter and LOH in domain of 12q22-23 are the main reasons for the expression and altered expression of Apaf-1 gene.

Association between endogenous gene expression and growth regulation induced by TGF-β1 in human gastric cancer cells

AIM: To investigate the association between endogenous gene expression and growth regulation including proliferation and apoptosis induced by transforming growth factor-pi (TGF-βl) in human gastric cancer (GC) cells. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the main components of the TGF-β1/Smads signal pathway in human poorly differentiated GC cell line BGC-823. Localization of Smad proteins was also determined using immunofluorescence. Then, the BGC-823 cells were cultured in the presence or absence of TGF-β1 (10 ng/mL) for 24 and 48 h, and the effects of TGF-β1 on proliferation and apoptosis were measured by cell growth curve and flow cytometry (FCM) analysis. The ultrastructural features of BGC-823 cells with or without TGF-β1 treatment were observed under transmission electron microscope. The apoptotic cells were visualized by means of the terminal deoxynucleotidyl transferase (TdT)-mediated dTUP in situ nick end-labeling (TUNEL) method. Meanwhile, the expression levels of endogenous p15, p21 and Smad7 mRNA and the corresponding proteins in the cells were detected at 1, 2 and 3 h after culture in the presence or absence of TGF-β1 (10 ng/mL) by semi-quantitative RT-PCR and Western blot, respectively. RESULTS: The TGF-β1/Smad signaling was found to be intact and functional in BGC-823 cells. The growth curve revealed the most evident inhibition of cell proliferation by TGF-β1 at 48 h, and FCM assay showed G1 arrest accompanied with apoptosis induced by TGF-β1. The typical morphological changes of apoptosis were observed in cells exposed to TGF-β1. The apoptosis index (AI) in TGF-β1-treated cells was significantly higher than that in the untreated controls (10.7±1.3% vs 0.32±0.06%, P＜0.01). The levels of p15, p21 and Smad7mRNA and corresponding proteins in cells were significantly up-regulated at 1 h, but gradually returned to basal levels at 3 h following TGF-βl (10 ng/mL) treatment. CONCLUSION: TGF-β1 affects both proliferation and apoptosis of GC cells through the regulation of p15 and p21, and induces transient expression of Smad 7 as a negative feedback modulation of TGF-β1 signal. Our results suggest a novel functional role of p21 as an accelerant of TGF-β1-mediated apoptosis in GC cells.

Silence of HIN-1 expression through methylation of its gene promoter in gastric cancer

AIM: To clarify the role of high in normal-1 (HIN-1) gene promoter methylation during gastric cancer development. METHODS: Gastric cancer cell lines and tissue specimens were analyzed for expression of HIN-1 mRNA and protein using the semi-quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The methylation of the HIN-1 gene promoter was detected in gastric carcinoma cells and tissues using methylation-specific polymerase chain reaction. The 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell viability assay and flow cytometry were used to assess the changes in behaviors of gastric cancer cells with or without 5-aza-2’-deoxycytidine treatment. RESULTS: HIN-1 was not expressed in 4 of 5 gastric cancer cell lines. The demethylation reagent 5-aza-2’-deoxycytidine was able to induce or upregulate HIN-1 expression in gastric cancer cell lines, which is associated with reduction of tumor cell viability. Furthermore, methylation of the HIN-1 gene promoter was shown in 57.8% (26/45) of the primary gastric cancer and 42.1% (17/38) of adjacent tissue samples, but was not shown in normal gastric mucosa (0/10). From the clinicopathological data of the patients, methylation of the HIN-1 gene promoter was found to be associated with tumor differentiation (P = 0.000). CONCLUSION: High methylation of HIN-1 gene promoter results in silence of HIN-1 expression in gastric cancer. 5-aza-2’-deoxycytidine reverses HIN-1 methylation and reduces viability of gastric cancer cells.

Suppression of gastric cancer growth by baculovirus vector-mediated transfer of normal epithelial cell specific-1 gene

AIM: To study the inhibitory effect of baculovirus- mediated normal epithelial cell specific-1 (NES1) gene therapy on gastric cancer (GC) in vitro and in vivo. METHODS: We first constructed recombinant baculovirus vectors and then transfected them into gastric cancer cells (SGC-7901). Efficiency of the baculovirus for gene transfer into SGC-7901 cells and cell growth curves were detected by fluorescence microscopy, Western blot and 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in vitro, respectively. The therapeutic effect of this gene therapy on GC was confirmed in xenografted nude mice. Tumor growth was determined by tumor volume, and expression of NES1 in tumor was analyzed by immunohistochemistry. RESULTS: Baculovirus vectors were successfully transfected into SGC-7901 cells. SGC-7901 cells transfected with the NES1 gene inhibited cell growth. In the Bac-NES1 treated group, tumor growth was significantly reduced with a high level of NES1 expression CONCLUSION: Baculovirus-mediated NES1 gene can be used in gene therapy for GC.

Genetic instability of BRCA1 gene at locus D17S855 is related to clinicopathological behaviors of gastric cancer from Chinese population

AIM： To investigate genetic instability of gene BRCA1 at locus D17S855, and their relationship with clinicopathological characteristics of gastric cancer in Chinese population. METHODS： Microsatellite instability （MSI） and loss of heterozygosity （LOH） of gene BRCA1 at locus D17S855 were compared between 37 samples of gastric cancer and corresponding non-cancerous gastric tissue. RESULTS： MSI at locus D17S855 was positive in 7 of 37 samples of gastric cancer （18.95%）. MSI had a close relationship with TNM staging but no relation with lymph node metastasis, histological type or tumor differentiation. MSI positive frequency in TNM Ⅰ ＋ Ⅱ （31.58%, 6/19） was much higher than that in TNM Ⅲ＋ Ⅳ （5.56%, 1/18）, （P 〈 0.05）. LOH positive rate was 18.92% （7/37）. LOH had no relationship to histological type, tumor differentiation or lymph node metastasis, but LOH positive rate in TNM Ⅲ＋ Ⅳ was 33.33% （6/18）, much higher than that in TNM Ⅰ ＋ Ⅱ （ 5.26%, 1/19）, （P 〈 0.05）. BRCA1 protein was expressed in 14 of 37 samples of gastric cancer. The positive rates of BRCA1 protein in TNM Ⅰ ＋ Ⅱ and TNM Ⅲ＋ Ⅳ were 57.89% and 16.67%, respectively, （P 〈 0.05）. The positive rate of BRCA1 protein was 77.78% in high differentiation samples, 30.77% in middle differentiation and 12.50% in lower differentiation samples, （P 〈 0.05）. CONCLUSION： MSI of BRCA1 gene could be used as a molecular marker in early phases of sporadic gastric cancer in Chinese population. LOH occurs at later period of gastric cancer, therefore, it could be used as prognostic factor.

XRCC1 genetic polymorphism Arg399Gln and gastric cancer risk:A meta-analysis

AIM： To evaluate the association between X-ray crosscomplementing gene 1 （XRCCl） genetic polymorphism Arg399Gln and gastric cancer risk by means of metaanalysis. METHODS： We searched PubMed and NCBI up to June 1, 2008. A total of 16 clinical trials and reports were identified, but only 8 trials qualified under our selection criteria. Statistical analysis was performed with the software program Review Manage, version 4.2.8. RESULTS： Of the 8 case-control studies selected for this meta-analysis, a total of 1334 gastric cancer cases and 2194 controls were included. For Arg399GIn, the Gin/Gin genotype carriers did not have a decreased cancer risk compared with those individuals with the Arg/Arg genotype （OR = 0.92, 95% CI, 0.71-1.19; P = 0.51）. Similarly, no associations were found in the recessive and dominant modeling （Gin/Gin vs Arg/GIn ＋ Arg/Arg： OR = 0.96; 95% CI, 0.77-1.19; P = 0.70 and Gin/Gin ＋ Arg/GIn vs Arg/Arg： OR = 0.90, 95% CI, 0.77-1.05; P = 0.18）. CONCLUSION： No association is found between the XRCC1 polymorphism Arg399GIn and gastric cancer risk.

Prognostic significance of X-ray cross-complementing gene 1 expression in gastric cancer

Objective： The aim of this study is to identify the prognostic significance of X-ray cross-complementing gene 1 （XRCCI） in patients with gastric cancer undergoing surgery and platinum-based adjuvant chemotherapy. Methods： Immunohistochemistry （IHC） was used to evaluate XRCCI protein expression profiles on surgical specimens of 612 gastric cancer patients. The relationship between XRCC1 expression and existing prognostic factors, platinum-based adjuvant chemotherapy, disease-free survival （DFS） and overall survival （OS） were analyzed. Results： Among 612 patients staged II/III in our study, 182 （29.74%） were evaluated as XRCC1 IHC positive. XRCC1 expression was not significantly related to OS （P=0.347） or DFS （P=0.297）. Compared with surgery only, platinum-based adjuvant chemotherapy significantly improved the OS （P=0.031）. And the patients with negative XRCC1 expression benefited more from platinum-based adjuvant chemotherapy （P=0.049）. Multivariate analysis demonstrated that tumor size, T category, N category, vascular or nerve invasion and platinum-based chemotherapy were good prognostic factors for OS （P〈0.05）. Though XRCCI plays an important role in DNA repair pathways, no significant relationship is found in XRCCI expression and OS among gastric cancer in our study. Conclusions： XRCC1 might be an alternative prognostic marker for the patients of gastric cancer after radical resection. The patients with negative XRCC1 expression can benefit more from platinum-based adjuvant chemotherapy.

Association of NOD1 and NOD2 genes polymorphisms with Helicobacter pylori related gastric cancer in a Chinese population

AIM:To investigate the association between the tag single nucleotide polymorphisms(TagSNPs) of NOD1 and NOD2 and the risk of developing gastric cancer.METHODS:We conducted a hospital-based case-control study including 296 incident gastric cancer patients and 160 gastritis controls.Eight TagSNPs in the NOD1 and NOD2 genes were selected from the Hapmap database using the haploview software and genotyped by the Sequenom MassArray system.The serum levels of anti-Helicobacter pylori(H.pylori) IgG were measured by enzyme-linked immunosorbent assay to indicate H.pylori infection.The odds ratios(OR) and 95% confidence intervals(CI) were calculated by unconditional logistic regression,including sex and age as confounding factors.RESULTS:The NOD1 rs2907749 GG genotype showed a decreased risk for gastric cancer(OR 0.50,95% CI:0.26-0.95,P = 0.04) while the rs7789045 TT genotype showed an increased risk(OR 2.14,95% CI:1.20-3.82,P = 0.01).An elevated susceptibility to gastric cancer was observed in the subjects with H.pylori infection and the NaOD1 rs7789045 TT genotype(OR 2.05,95% CI:1.07-3.94,P = 0.03) or the NOD2 rs7205423 GC genotype(OR 2.52,95% CI:1.05-6.04,P = 0.04).Haplotype analysis suggested that the distribution of AGT(rs2907749,rs2075820 and rs7789045) in NOD1 between the cases and control groups was significantly different(P corrected:0.04),and the diplotype AGT/AGT was associated with an elevated gastric cancer risk(OR 1.98,95% CI:1.04-3.79,P = 0.04).The association of the NOD1 rs7789045 TT genotype and the diplotype AGT/AGT was significant with H.pylori-related diffuse-type gastric cancer(OR 3.00,95% CI:1.38-6.53,P = 0.01;OR 4.02,95% CI:1.61-10.05,P < 0.01,respectively).CONCLUSION:Genetic polymorphisms in NOD1 and NOD2 may interact with H.pylori infection and may play important roles in promoting the development of gastric cancer in the Chinese population.

Piwi like RNA-mediated gene silencing 1 gene as a possible major player in gastric cancer

AIM To establish a permanent piwi like RNA-mediated genesilencing 1(PIWIL1) gene knockout in AGP01 gastric cancer cell line using CRISPR-Cas9 system and analyze phenotypic modifications as well as gene expression alterations.METHODS CRISPR-Cas9 system used was purchased from Dharmacon GE Life Sciences(Lafayette, CO, United States) and permanent knockout was performed according to manufacturer's recommendations. Woundhealing assay was performed to investigate the effect of PIWIL1 knockout on migration capability of cells and Boyden chamber invasion assay was performed to investigate the effect on invasion capability. For the gene expression analysis, a one-color microarray-based gene expression analysis kit(Agilent Technologies, Santa Clara, CA, United States) was used according to the protocol provided by the manufacturer. RESULTS PIWIL1 gene knockout caused a significant decrease in AGP01 migration capacity as well as a significant decrease in cell invasiveness. Moreover, functional analysis based on grouping of all differentially expressed m RNAs identified a total of 35 genes(5 up-regulated and 30 down-regulated) encoding proteins involved in cellular invasion and migration. According to current literature, 9 of these 35 genes(DOCK2, ZNF503, PDE4 D, ABL1, ABL2, LPAR1, SMAD2, WASF3 and DACH1) are possibly related to the mechanisms used by PIWIL1 to promote carcinogenic effects related to migration and invasion, since their functions are consistent with the changes observed(being up-or down-regulated after knockout). CONCLUSION Taken together, these data reinforce the idea that PIWIL1 plays a crucial role in the signaling pathway of gastric cancer, regulating several genes involved in migration and invasion processes; therefore, its use as a therapeutic target may generate promising results in the treatment of gastric cancer.

Inhibitory effect of Polo-like kinase 1 depletion on mitosis and apoptosis of gastric cancer cells

AIM： Polo-like kinase 1 （PLK1） serine/threonine kinase plays a vital role in multiple phases of mitosis in gastric cancer cells. To investigate the effect of PLK1 depletion on mitosis and apoptosis of gastric cancer cells. METHODS： PLK1 expression was blocked by small RNA interference（siRNA）. The expression levels of PLK1, cdc2, cyclin B and caspase 3 were detected by Western blotting. Then, PLK1 depletion, cdc2 activity, cell proliferation, cell cycle phase distribution, mitotic spindle structure, and the rate of apoptosis of the PLK1 knockdown cells were observed. RESULTS： PLK1 gene knockdown was associated with increased cyclin B expression, increased cdc2 activity （but not with the expression levels）, accumulation of gastric cancer cells at G2/M, improper mitotic spindle formation, delayed chromosome separation and delayed or arrested cytokinesis. Moreover, PLK1 depletion in gastric cancer cells was associated with decreased proliferation, attenuated pro-caspase 3 levels and increased apoptosis. CONCLUSION： Blockage of to decreased mitosis or even PLK1 expression may lead apoptosis in gastric cancer cells, indicating that PLK1 may be a valuable therapeutic target for gastric cancer.

Frequent loss of heterozygosity at 8p22 chromosomal region in diffuse type of gastric cancer

AIM: To study the loss of heterozygosity (LOH) at 8p21-23 locus in diffuse gastric cancer.METHODS: To evaluate the involvement of this region in gastric cancer, we used eight microsatellite markers covering two Mb of mentioned region, to perform a high-resolution analysis of allele loss in 42 cases of late diffuse gastric adenocarcinoma.RESULTS: Six of these STS makers: D8S1149, D8S1645, D8S1643, D8S1508, D8S1591, and D8S1145 showed 36%, 28%, 37%, 41%, 44% and 53% LOH, respectively.CONCLUSION: A critical region of loss, close to the NAT2 locus and relatively far from FEZ1 gene currently postulated as tumor suppressor gene in this region.

ERCC1 polymorphism, expression and clinical outcome of oxaliplatin-based adjuvant chemotherapy in gastric cancer

AIM： TO determine the influence of excision repair cross complementing group 1 （ERCC1） codon 118 polymorphism and mRNA level on the clinical outcome of gastric cancer patients treated with oxaliplatin-based adjuvant chemotherapy. METHODS： Eighty-nine gastric cancer patients treated with oxalipatin-based adjuvant chemotherapy were included in this study. ERCC1 codon 118 C/T polymorphism was tested by polymerase chain reaction-ligation detection reaction （PCR-LDR） method in peripheral blood lymphocytes of those patients; and the intratumoral ERCC1 mRNA expression was measured using reverse transcription PCR in 62 patients whose tumor tissue specimens were available. RESULTS： No significant relationship was found between ERCC1 codon 118 polymorphism and ERCC1 mRNA level. The median relapse-free and overall survival period was 20.1 mo and 28.4 too, respectively. The relapse-free and overall survivals in patients with lOW levels of ERCC1 mRNA were significantly longer than those in patients with high levels （P 〈 0.05）, while there was no significant association found between ERCC1 118 genotypes and the disease prognosis. Multivariate analysis also showed that ERCC1 mRNA level was a potential predictor for relapse and survival in gastric cancer patients treated with oxaliplatin-based adjuvant chemotherapy （P 〈 0.05）. CONCLUSION： ERCC1 codon 118 polymorphisrn has no significant impact on ERCC1 rnRNA expression, and the intraturnoral ERCC1 rnRNA level but not codon 118 polymorphisrn may be a useful predictive parameter for the relapse and survival of gastric cancer patients receiving oxaliplatin-based adjuvant chemotherapy.

Long noncoding RNA NALT1-induced gastric cancer invasion and metastasis via NOTCH signaling pathway

BACKGROUNDLong noncoding RNAs (lncRNAs) are aberrant and play critical roles in gastriccancer (GC) progression and metastasis. Searching for coexpressed lncRNAclusters or representative biomarkers related to malignant phenotypes of GC mayhelp to elucidate the mechanism of tumor development and predict the prognosisof GC.AIMTo investigate the prognostic value of NOTCH1 associated with lncRNA in T cellacute lymphoblastic leukemia 1 (NALT1) in GC and the mechanism of itsinvolvement in GC invasion and metastasis.METHODSRNA sequencing and corresponding clinical data were downloaded from TheCancer Genome Atlas database. The significance module was studied byweighted gene coexpression network analysis. A total of 336 clinical sampleswere included in the study. Gene silencing, reverse transcription polymerasechain reaction, western blotting, scrape motility assay, and Transwell migrationassay were used to assess the function of hub-lncRNAs.RESULTSAt the transcriptome level, 3339 differentially expressed lncRNAs were obtained.weighted gene coexpression network analysis was used to obtain 15 lncRNAclusters and observe their coexpression. Pearson’s correlation showed that blue module was correlated with tumor grade and survival. NALT1 was the hublncRNAof blue module and was an independent risk factor for GC prognosis.NALT1 was overexpressed in GC and its expression was closely related toinvasion and metastasis. The mechanism may involve NALT1 regulation ofNOTCH1, which is associated with lncRNA in T cell acute lymphoblasticleukemia, through cis regulation, thereby affecting the expression of the NOTCHsignaling pathway.CONCLUSIONNALT1 is overexpressed and promotes invasion and metastasis of GC. Themechanism may be related to regulation of NOTCH1 by NALT1 and its effect onNOTCH signaling pathway expression.

The role of endoscopy in the management of hereditary diffuse gastric cancer syndrome

Hereditary diffuse gastric cancer(HDGC) syndrome is an inherited cancer risk syndrome associated with path-ogenic germline CDH1 variants. Given the high risk for developing diffuse gastric cancer, CDH1 carriers are recommended to undergo prophylactic total gastrectomy for cancer risk reduction. Current guidelines recommend upper endoscopy in CDH1 carriers prior to surgery and then annually for individuals deferring prophylactic total gastrectomy.Management of individuals from HDGC families without CDH1 pathogenic variants remains less clear, and management of families with CDH1 pathogenic variants in the absence of a family history of gastric cancer is particularly problematic at present. Despite adherence to surveillance protocols, endoscopic detection of cancer foci in HDGC is suboptimal and imperfect for facilitating decision-making. Alternative endoscopic modalities, such as chromoendoscopy,endoscopic ultrasound, and other non-white light methods have been utilized,but are of limited utility to further improve cancer detection and risk stratification in HDGC. Herein, we review what is known and what remains unclear about endoscopic surveillance for HDGC, among individuals with and without germline CDH1 pathogenic variants. Ultimately, the use of endoscopy in the management of HDGC remains a challenging arena, but one in which further research to improve surveillance is crucial.

Up-regulation of T-lymphoma and metastasis gene 1 in gastric cancer and its involvement in cell invasion and migration

Background T-lymphoma and metastasis gene 1 （Tiaml） produces a guanine nucleotide exchange factor （GNEF） that regulates guanosine triphosphatase, which transforms guanosine diphosphate to guanosine triphosphate. Recently published data indicate that Tiaml was associated with gastric cancer. The aim of this study was to investigate biological effects and potential mechanisms of Tiara1 in gastric carcinoma. Methods We analyzed the expression of Tiaml in 114 pair-matched gastric neoplastic and adjacent non-neoplastic tissues by quantitative real-time PCR. We investigated Tiaml expression and its prognostic value for gastric cancer. Furthermore, the functions of Tiaml over-expression were analyzed with stable-expression Tiara1 plasmid in human gastric cancer cell lines. Results Tiaml expression was significantly associated with cell differentiation and lymphatic metastasis; expression of Tiaml mRNA was up-regulated in gastric cancer compared to pair-matched adjacent non-tumor tissues. Analyses of surgical tissue samples and 5-year survival of gastric cancer patients showed that those with strong Tiaml expression had significantly shorter overall survival time than those with negative Tiaml expression. Ectopic expression of Tiaml promoted cell growth, migration and invasion of gastric cancer cells in vitro. Conclusions In gastric cancer cells, Tiaml affects multiple properties associated with acquisition of the metastatic phenotype, and may be a marker of gastric cancer progression and metastasis in a subset of cancer.

Insulin gene enhancer protein 1 mediates glycolysis and tumorigenesis of gastric cancer through regulating glucose transporter 4

Background:Insulin gene enhancer protein 1,(ISL1),a LIM-homeodomain transcription factor,is involved in multiple tumors and is associated with insulin secretion and metabolic phenotypes.However,the role of ISL1 in stimulating glycolysis to promote tumorigenesis in gastric cancer(GC)is unclear.In this study,we aimed to characterize the expression pattern of ISL1 in GC patients and explore its molecular biological mechanism in glycolysis and tumorigenesis.Methods:We analyzed the expression and clinical significance of ISL1 in GC using immunohistochemistry and real-time polymerase chain reaction(PCR).Flow cytometry and IncuCyte assays were used to measure cell proliferation after ISL1 knockdown.RNA-sequencing was performed to identify differentially expressed genes,followed by Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis and Gene Set Enrichment Analysis(GSEA)to reveal key signaling pathways likely regulated by ISL1 in GC.Alteration of the glycolytic ability of GC cells with ISL1 knockdown was validated by measuring the extracellular acidification rate(ECAR)and oxygen consumption rate(OCR)and by detecting glucose consumption and lactate production.The expression of glucose transporter 4(GLUT4)and ISL1 was assessed by Western blotting,immunohistochemistry,and immunofluorescent microscopy.The luciferase reporter activity and chromatin immunoprecipitation assays were performed to determine the transcriptional regulation of ISL1 on GLUT4.Results:High levels of ISL1 and GLUT4 expression was associated with short survival of GC patients.ISL1 knockdown inhibited cell proliferation both in vitro and in vivo.KEGG analysis and GSEA for RNA-sequencing data indicated impairment of the glycolysis pathway in GC cells with ISL1 knockdown,which was validated by reduced glucose uptake and lactate production,decreased ECAR,and increased OCR.Mechanistic investigation indicated that ISL1 transcriptionally regulated GLUT4 through binding to its promoter.Conclusion:ISL1 facilitates glycolysis and tumorigenesis in GC via the transcriptional regulation of GLUT4.

Effect of NHE1 antisense gene transfection on the biological behavior of SGC-7901 human gastric carcinoma cells

AIM: To study the effect of type 1 Na+/H+ exchanger (NHE1 ) antisense human gene transfection on the biological behavior of gastric carcinoma cell line SGC-7901. METHODS: Antisense NHE1 eukaryotic expression on vector pcDNA3.1 was constructed by recombinant DNA technique and transfected into gastric carcinoma cell line SGC-7901 with DOTAP liposome transfection method. Morphological changes of cells were observed with optic and electron microscopes. Changes in cell proliferative capacity, apoptosis, intracellular pH (pHi), cell cycle, clone formation in two-layer soft agar, and tumorigenicity in nude mice were examined. RESULTS: Antisense eukaryotic expressing vectors were successfully constructed and transfected into SGC-7901. The transfectant obtained named 7901 -antisense (7901-AS) stablely produced antisense NHE1. There was a significant difference between the pHi of 7901-AS cells (6.77 ± 0.05) and that of 7901-zeo cells and SGC-7901 cells (7.24 ± 0.03 and 7.26 ± 0.03, P < 0.01). Compared with SGC-7901 and 7901-zeo cells, 7901-AS cells mostly showed cell proliferation inhibition, G1/G0 phase arrest, increased cell apoptotic rate, recovery of contact inhibition, and density contact. The tumorigenicity in nude mice and cloning efficiency in the two-layer soft agar were clearly inhibited. CONCLUSION: NHE1 antisense gene significantly restrains the malignant behavior of human gastric carcinoma cells, suppresses cell growth and induces cell apoptosis, and partially reverses the malignant phenotypes of SGC-7901 . These results suggest a potential role for human tumor gene therapy.