Lifestyle intervention for gastroesophageal reflux disease: a national multicenter survey of lifestyle factor effects on gastroesophageal reflux disease in China.

BACKGROUND: Poor habits can worsen gastroesophageal reflux disease (GERD) and reduce treatment efficacy. Few large-scale studies have examined lifestyle influences, particularly eating habits, on GERD in China, and research related to eating quickly, hyperphagia, and eating hot foods is quite limited. The aim of this study was to evaluate the relationship between GERD pathogenesis and lifestyle factors to produce useful information for the development of a clinical reference guide through a national multicenter survey in China. METHODS: Symptom and lifestyle/habit questionnaires included 19 items were designed. The questionnaire results were subjected to correlation analysis relative to GERD symptom onset. A standard proton pump inhibitor (PPI) was advised to correct patients with unhealthful lifestyle habits. RESULTS: A total of 1518 subjects (832 GERD, 686 non-GERD) enrolled from six Chinese hospitals completed symptom and lifestyle/habit questionnaires. The top lifestyle factors related to GERD were fast eating, eating beyond fullness, and preference for spicy food. Univariate analysis showed that 21 factors, including male gender, a supra-normal body mass index (BMI), smoking, drinking alcohol, fast eating, eating beyond fullness, eating very hot foods, and drinking soup, among others, were associated with GERD (p < 0.05). Logistic multivariate regression analysis revealed the following risk factors for GERD [with odds ratios (ORs)]: fast eating (4.058), eating beyond fullness (2.849), wearing girdles or corsets (2.187), eating very hot foods (1.811), high BMI (1.805), lying down soon after eating (1.544), and smoking (1.521). Adjuvant lifestyle interventions improved outcomes over medication alone (z = -8.578, p < 0.001 Mann-Whitney rank sum test). CONCLUSIONS: Lifestyle interventions can improve medication efficacy in GERD patients. Numerous habits, including fast eating, eating beyond fullness, and eating very hot foods, were associated with GERD pathogenesis. The present results may be useful as a reference for preventive education and treatment.

[Deuterium depleted environment inhibits the growth of gastric cancer cells: in vitro study].

OBJECTIVE: To examine whether deuterium depleted environment may affect the biological features of human gastric cancer cells(SGC-7901)and explore the possible underlying mechanisms. METHODS: SGC-7901 cells were cultured in RPMI-1640 medium prepared with distilled water of different deuterium concentrations(experimental group:25ppm deuterium;control group:150ppm deuterium). Assays on cellular proliferation, cell cycle and apoptosis were conducted at different time points and comparison. The protein expression of proliferating cell nuclear antigen (PCNA) was measured using Western blot. RESULTS: In contrast to 150ppm group, the proliferation rate of SGC-7901 cells in 25ppm deuterium was decreased by 10% as indicated by the CCK-8 assay. Wound healing ability and the colony formation ability of these cells were also significantly suppressed (P<0.05). Flow cytometry analysis further revealed that exposure to 25ppm significantly increased the ratio of cancer cells at G1 phase (P<0.01) while decreased the ratio at S phase (P<0.05) compared to the 150ppm group. There was no significant difference in apoptosis between the two groups. Down-regulated expression of PCNA was also identified in cancer cells treated with 25ppm deuterium. CONCLUSIONS: Deuterium depleted environment inhibited the proliferation of gastric cancer cells, which may be attributed to the down-regulation of PCNA and cell cycle arrest at G1 phase.

[Effect of hypoxia on gastric cancer cell proliferation and LINE-1 endonuclease variant GCRG213 expression].

OBJECTIVE: To investigate the change of gastric cancer cell proliferation and the expression of gastric cancer related gene 213 (GCRG213), a long interspersed nuclear element-1 (LINE-1) endonuclease variant, during hypoxia. METHODS: Normal gastric mucosa cell GES-1 and gastric cancer cell BGC-823 were cultured in 20% or 3% oxygen concentrations, respectively. MTT test was used to analyze the proliferation of the GES-1 and BGC-823 cells. The change of GCRG213 mRNA and protein expression in GES-1 and BGC-823 cells was detected by using RT-PCR and Western blot analysis. Blast was used at the NCBI Blast server to identify GCRG213 sequence to any alignment in the GeneBank databases. RESULTS: Compared with 20% oxygen condition, 3% oxygen concentration could promote cell growth. Mean-while, the expression of GCRG213 at mRNA and protein levels was increased. GCRG213 sequence shared high homology with LINE-1 endonuclease sequence. CONCLUSION: GCRG213 is a variant of LINE-1 endonuclease. Hypoxia as in 3% oxygen condition can promote cell proliferation and lead to GCRG213 overexpression.

[Preparation and identification of monoclonal antibody against human GCRG213].

AIM: To prepare and characterize the monoclonal antibody against human GCRG213. METHODS: The HIS-GCRG213 fusion protein was expressed in E.coli. Mice were immunized with the purified HIS-GCRG213 protein. Hybridoma cell lines secreting monoclonal antibodies against GCRG213 were screened by regular cell fusion and subcloning approach. The titer and specificity of the antibody was characterized by ELISA and Western blot, respectively. The expression of GCRG213 was determined using immunohistochemistry technique on paraffin-embedded tissue sections from normal gastric mucosal tissues and advanced gastric cancer. RESULTS: The HIS-GCRG213 fusion protein with relative molecular mass of 20 800 was over expressed in E.coli. Two hybridoma cell lines which secreted monoclonal antibody specifically against human GCRG213 fusion protein were successfully obtained. The ascite titers of this monoclonal antibody reached 1:10(6);. Western blot analysis showed that the monoclonal antibody could bind to the recombinant HIS-GCRG213 protein specifically.The immunohistochemistry showed that GCRG213 were expressed higher in gastric cancer tissues than in normal ones. CONCLUSION: The monoclonal antibody against human GCRG213 with high titer and specificity has been successfully prepared, which could be utilized as a useful reagent for further studying the biological function of the GCRG213.

[Preparation, identification and preliminary application of the polyclonal antibody against gastric cancer-related protein GCRG224].

AIM: To prepare the polyclonal antibody against gastric cancer-related protein GCRG224. METHODS: The thioredoxin/GCRG224 fusion protein was expressed in E.coli. The polyclonal antibody against GCRG224 was obtained by immunizing a rabbit with the purified GCRG224 protein. The titer and specificity of the antibody were determined by ELISA and Western blot, respectively. The expression of GCRG224 in paraffin-embedded tissue sections from normal gastric mucosal tissues and advanced gastric cancer was determined using immunohistochemistry technique. RESULTS: The thioredoxin/GCRG224 fusion protein with relative molecular mass of 16.8 kDa was over-expressed in E.coli. The purity of the expressed product directly purified from a denaturing polyacrylamide gel was about 100%. The polyclonal antibody against GCRG224 was prepared. ELISA detection proved the titer of antiserum against GCRG224 was about 1:256,000. Western blot analysis showed that the antiserum could bind to the expressed fusion protein specifically. GCRG224 was found to have higher expression in gastric cancer tissues than in normal ones by immunohistochemistry. CONCLUSION: The successful preparation of the polyclonal antibody against GCRG224 lays a foundation for further study of the biological function and the possible role of GCRG224 in the development of gastric carcinoma.

LINE-1 family member GCRG123 gene is up-regulated in human gastric signet-ring cell carcinoma.

AIM: To analyze the expression profiles of a human gastric-cancer-related gene, GCRG123, in human gastric signet-ring cell carcinoma tissues, and to perform bioinformatics analysis on GCRG123. METHODS: In situ hybridization was used to explore the GCRG123 expression pattern in paraffin-embedded gastric tissues, including 15 cases of signet-ring cell carcinoma, 15 of intestinal-type adenocarcinoma, and 15 of normal gastric mucosa. Northern blotting was used to analyze the differences in GCRG123 expression between stomach signet-ring cell carcinoma and intestinal-type adenocarcinoma tissues. Online software, including BLAST, Multalin and BLAT, were applied for bioinformatics analysis. National Center for Biotechnology Information (NCBI) and the University of California Santa Cruz (UCSC) databases were used for the analyses. RESULTS: The in situ hybridization signal appeared as blue precipitates restricted to the cytoplasm. Ten out of 15 cases of gastric signet ring cell carcinoma, normal gastric mucosal epithelium and pyloric glands showed high GCRG123 expression. Low GCRG123 expression was observed in gastric intestinal-type adenocarcinoma and normal gastric glands. Northern blotting revealed that GCRG123 was up-regulated in signet-ring cell carcinoma tissue but down-regulated in intestinal-type adenocarcinoma tissue. BLAST and Multalin analyses revealed that the GCRG123 sequence had 92% similarity with the ORF2 sequence of human long interspersed nuclear element retrotransposons (LINE-1, L1). BLAT analysis indicated that GCRG123 mapped to all chromosomes. GCRG123 was found to integrate in the intron-17 and -23 of Rb, 5' flanking region of IL-2 and clotting factor IX genes. CONCLUSION: GCRG123, an active member of the L1 family, was up-regulated in human gastric signet-ring cell carcinoma.

[Gene expression profile of human normal gastrointestinal tract tissues: bioinformatic study].

AIM: To identify up-regulated genes specific to human normal gastrointestinal tissues. METHODS: Study was made on human normal tissue gene expression database open to the public. Tissue-specific genes were identified using one-tailed student T test. Online software including Ingenuity and KEGG were applied for physiological function analyses. Unsupervised two-way hierarchical clustering method was used to analyze the expression profile of stomach-specific genes in gastric cancer gene expression datasets. RESULTS: The analyses identified 196 stomach-specific genes, 203 ileum-specific genes and 224 colon-specific genes, respectively. The gene expression profiles reflect major organ-specific physiological functions on the molecular level. Some putative oncogenes and tumor suppressor genes were found in the tissue-specific gene list. Hierarchical clustering analysis revealed that the stomach-specific genes were up-regulated in normal stomach tissues but down-regulated in stomach cancer tissues. The normal tissues clustered together, so did the cancer tissues. At the meantime, clustering could also distinguish the moderate and severe differentiated stomach cancer. CONCLUSION: Human normal stomach, ileum and colon possess tissue-specific up-regulated genes, which are closely associated with physiological functions.

[Preparation and characterization of rabbit antibody against gastric cancer-related protein GCRG213].

AIM: To prepare the rabbit antibody against gastric cancer-related protein GCRG213. METHODS: The thioredoxin/GCRG213 fusion protein was expressed in E. coli. The rabbit antibody against GCRG213 was obtained by immunizing a rabbit with the purified GCRG213 protein. The titer and specificity of the antibody was determined by ELISA and Western-blot, respectively. RESULTS: The thioredoxin/GCRG213 fusion protein with relative molecular mass (Mr) of 29,400 was overexpressed in E. coli. The purity of expressed product directly purified from a denaturing polyacrylamide gel was about 100%. The rabbit antibody against GCRG213 was obtained. The ELISA titer of antiserum against GCRG213 was about 1:256,000. Western blot analysis showed that the antiserum could bind to the expressed fusion protein specifically. CONCLUSION: The rabbit antibody against GCRG213 has been successfully prepared, which lays the foundation for further studying the biological function and the possible role of the GCRG213 in the development of gastric carcinoma.

A gene encoding an apurinic/apyrimidinic endonuclease-like protein is up-regulated in human gastric cancer.

AIM: To identify the gene that may predispose to human gastric cancer and to analyze its expression in gastric cancer and non-tumorous gastric mucosa. METHODS: Cancer, para-tumor, and non-tumor gastric tissues were studied for gene expression profile using fluorescent differential display reverse transcription polymerase chain reaction (DDRT-PCR). The differentially expressed bands of interest were analyzed by cloning, Northern blotting, and sequencing. The sequencing results were compared with the GenBank database for homology and conserved domain analysis. In situ hybridization with DIG-labeled cRNA probes was used to detect the expression of gene in paraffin embedded gastric adenocarcinoma and non-cancerous tissues. RESULTS: A gene expressed higher in tumor and para-tumor tissues than in their non-tumor counterparts of all 7 tested gastric adenocarcinoma patients was identified by means of DDRT-PCR analysis. It was named GCRG213 (gastric cancer related gene 213). Northern blot confirmed the differential expression. GCRG213 (GenBank No. AY053451) consisted of 1094 base pairs with an open reading frame (ORF) which encoded 142 amino acids. The deduced amino acid sequence contained a putative conserved domain, apurinic/apyrimidinic endonuclease (APE). In situ hybridization analysis showed that GCRG213 was expressed higher in gastric cancer tissues than in their corresponding non-tumor ones. Precancerous leisions of gastric adenocarcinoma showed a high GCRG213 expression, too. No difference of the expression patterns was found between the early and advanced gastric cancer. CONCLUSION: A gene named GCRG213 was identified in human gastric adenocarcinoma. It encoded an APE-like protein which was probably a new member of the APE family. GCRG213 was over-expressed not only in gastric cancer, but also in its precancerous leisions. The role of GCRG213 expression in carcinogenesis needs further study.

[Cloning and tissue expression analysis of up-regulated cDNA fragment in human gastric cancer].

OBJECTIVE: To identify novel human gastric cancer-associated susceptibility gene for early diagnosis and treatment of gastric cancer. METHODS: A primer was designed for 3'-rapid amplification of cDNA end(RACE) and amplified fragments were cloned, then they were analyzed by sequencing. Compared with ESTs in Genbank, the EST fragment represented a novel gene. Combination of Northern blot and virtual Northern and multiple tissues Northern blot, expression of the cDNA in multiple normal and carcinoma tissues were analyzed. RESULTS: One of the important cDNA bands with poly(A) tail was cloned. This band was named W41. Sequence analysis showed that W41 consists of 533 bp. Basic local alignment search tool analysis revealed that W41 has low identity with any genes from GenBank. This sequence data was submitted to GenBank with accession No. AF 325202. Northern blot revealed that W41 presented higher expression in gastric cancer tissue than in normal tissue. Multiple tissue Northern blot revealed that W41 presented higher expression in multiple cancers than in normal tissues. Virtual Northern revealed that the cDNA presented higher expression in tumor series analysis of gene expression libraries than in normal. CONCLUSION: A novel human gastric cancer-associated cDNA fragment was identified.

[Expression of human anti-apoptotic gene survivin and its splice in normal human gastric tissue and gastric cancer].

OBJECTIVE: To analyze the expression of human anti-apoptotic gene survivin (SVV) in normal human gastric tissues and gastric cancer. METHODS: SVV cDNA clone was obtained from human gastric cancer tissues by virtue of RT-PCR, using Dig-marked cRNA probe in situ hybridization to analyze its expression in normal human gastric tissues and gastric cancer. RESULTS: Two SVV cDNA clones, SVV-S4A and SVV-S1B were obtained. The sequence of the former is identical to that of the well-known SVV cDNA; however, in the sequence of the latter, the third exon was missed, i.e., there are only two exons in SVV-S1B. In situ hybridization showed that SVV-S4A is mainly expressed in gastric cancer tissues whereas SVV-S1B is mostly expressed in normal gastric tissues. CONCLUSION: There is difference between SVV-S4A and SVV-S1B in respect to their characteristics of expression in gastric cancer and normal gastric tissues.

A novel gene, GCRG224, is differentially expressed in human gastric mucosa.

AIM: To clone genes that may predispose us to human gastric cancer and to analyze it's expression in gastric tissues. METHODS: Specimens of paired tumor, paratumor and normal gastric mucosa tissues collected from fifteen patients who suffered from stomach antrum adenocarcinoma were used for analysis. Seven out of the fifteen cases were first studied by fluorescent differential display reverse transcription polymerase chain reaction (DDTR-PCR) analysis. The differentially expressed bands of interest were cloned, analyzed by Northern blot, sequencing and RT-PCR. Through BLAST, the sequencing results were compared with GenBank database for homology analysis. In situ hybridization with DIG-labeled cRNA probes was used to analyze the expression of interesting cDNA bands in paraffin embedded paired normal gastric mucosa and cancer tissues isolated from 30 gastric adenocarcinoma patients. RESULTS: DDRT-PCR showed that one of the interesting cDNA bands, which was named W2, expressed much higher in all seven tested tumor and paratumor samples than in their normal counterparts, it was sub-cloned into a pGEM-T Easy vector. Two subclones were subsequently obtained. One of the subclone, GCRG224, was studied further. The sequencing result showed that GCRG224 consisted of 1 159 base pairs and had one open reading frame (ORF). It located at human chromosome 11q14. No homologue was found in GenBank database with GCRG224-ORF. This nucleotide sequence data were submitted to GenBank with accession No. AF438406. RT-PCR showed that GCRG224 expressed higher in 11/15 gastric cancer tissues than in non-tumor tissues. However, the result of Northern blot analysis showed a higher GCRG224 expression in the non-tumor tissue than in the tumor one. Human multiple tissue Northern blot analysis revealed that GCRG224 also expressed in human normal colon tissue, and peripheral blood leukocyte. In situ hybridization analysis showed that only 5/30 adenocarcinoma, 3/18 dysplasia and 6/18 intestinal metaplasia showed higher GCRG224 expression level than the normal gastric glands. However, GCRG224 was over-expressed predominantly in 26/30 cases of normal mucosal epithelium. CONCLUSION: A novel gene named GCRG224 was identified from human gastric mucosal tissue. It overexpressed in almost all gastric mucosal epithelium but only a small portion of cancer and precancerous leisions. The role of GCRG224 expression in gastric epithelium needs further study.