Detection and location of Helicobacter pylori in human gastric carcinomas.

AIM: To define the infection status of Helicobacter pylori in 109 patients with gastric cancers and H pylori localization in gastric carcinoma tissues in South China. METHODS: The incidence of H pylori infection in gastric carcinomas was estimated by polymerase chain reaction (PCR), simultaneously; both morphological features and the localization of H pylori in gastric carcinomas were demonstrated by Warthin-Starry (WS) staining. The relationships between H pylori infection and the clinical-pathologic factors of gastric carcinomas were analyzed by software SPSS10.0. RESULTS: H pylori was found in 42 (39.03%) and 58 (53.21%) cases of 109 patients with gastric carcinomas by PCR and WS, respectively. H pylori infection rate detected in gastric carcinomas by WS was higher than that by PCR (chi2 = 9.735, P < 0.005 < 0.01). WS stain showed that H pylori existed in the gastric antrum mucus, mucosal gland of normal tissues adjacent to gastric carcinomas and the gland, mucus pool of cancer tissues. The positive rate of H pylori in normal tissues adjacent to carcinomas was higher than that in cancer tissues (chi2 = 15.750, P < 0.005 < 0.01). No significant differences in age, sex, site, histological types and lymph node metastasis were found between H pylori-positive gastric carcinomas and H pylori-negative cases by both methods, but there were statistically significant differences of H pylori positive rate between early and advanced stage of gastric carcinomas (chi2 = 4.548 or 5.922, P = 0.033 or 0.015 < 0.05). CONCLUSION: These results suggested that H pylori infection might play a certain role in the early stage of carcinogenesis of human gastric mucosa epithelia.

Construction of hu-PBL/SCID chimeras and development of EBV-related lymphomas.

OBJECTIVE: To construct hu-PBL/SCID chimeras and to investigate the development of lymphoma and oncogenicity of the Epstein-Barr virus (EBV). METHODS: Human peripheral blood lymphocytes (PBLs) were isolated from healthy adult donors and transplanted intraperitoneally into severe combined immunodeficient (SCID) mice. Mice with hu-PBL engraftment from healthy EBV seronegative donors were injected intraperitoneally with EBV-containing supernatant from suspension culture of B95-8 cell line (active infection), whereas mice receiving lymphocytes from healthy EBV seropositive donors were not re-infected with B95-8 derived EBV (latent infection). Pathological examination and molecular analysis were performed on experimental animals and induced neoplasms. RESULTS: In the early stage of this experiment, 12 mice died of acute graft-versus-host disease, mortality was 34.3% (12/35 mice) with an average life span of 17.5 days. In 19 survival hu-PBL/SCID chimeric recipients from 12 healthy donors, tumor incidence was 84.2% (16/19 mice). The average survival time of tumor-bearing mice was 65.5 days. EBV-related neoplasms in SCID mice were nodular tumors with aggressive and fatal features. Histological morphology of tumors exhibited diffuse large cell lymphomas. Immunohistochemistry revealed that LCA (CD45) and L26 (CD20) were positive, but both PS1 (CD3) and UCHL-1 (CD45RO) were negative, and EBV products ZEBRA, LMP1, and EBNA2 were expressed in a small number of tumor cells. EB virus particles were seen in the nuclei of some tumor cells by electron microscopy, and EBV DNA could be amplified in the tumor tissues by PCR. In situ hybridization indicated that the nuclei of tumor cells contained human-specific Alu sequence. CONCLUSIONS: EBV-induced tumors were human B-cell malignant lymphomas. We obtained direct causative evidence dealing with EBV-associated tumor deriving from normal human cells.

[Molecular pathological characteristics of human B-cell lymphomas induced by Epstein-Barr virus].

To identify molecular features of neoplasms associated with EB virus, human peripheral blood lymphocytes (huPBL) were isolated from healthy volunteer donors and were transplanted intraperitoneally into SCID mice, and then huPBL/SCID mice were infected with EB virus. Serum levels of human IgG were measured by unidirectional immunodiffusion assay. Human Alu sequence and EBER-1 in tumor tissues were detected with PCR and in situ hybridization. Immunohistochemical staining was used to examine leukocyte differentiation antigens (LCA, L26, UCHL1, PS1), viral gene products (LMP1, EBNA2, BZLF1) and cellular oncoproteins (p53, C-myc, Bcl-2 and Bax). The experiments showed that tumors developed in 24 of 34 surviving huPBL/SCID mice by EBV infection. Histopathological and immunohistochemical observations demonstrated that all of the induced tumors in SCID mice were malignant lymphomas derived from human B-lymphocytes. In situ hybridization showed that tumor cells had EBV-encoded small RNA-1 (i.e. EBER-1). Alu sequence could be amplified by PCR from human genome of tumor tissues. Immunohistochemistry detected positive staining of BZLF1-encoded protein in a small population of tumor cells of almost all cases, and positive staining of LMP1 and EBNA2 only in small number of tumor cells. Human IgG could be found in the serum of 12 SCID mice on the 15th day after huPBL engraftment, and then increased with time and with the development of induced tumors in 6 mice. Positive rates of p53, C-myc, Bcl-2 and Bax expression were 83.33%, 100%, 95.83%, 91.67%, respectively, in 24 cases of the EBV-induced lymphomas. The results indicate that molecular lesions associated with the induced B-cell lymphoma involved EBV infection, expression of oncogenic viral genes, and abnormal expression of cellular oncogenes in human xenografts. Human IgG level in the serum of huPBL/SCID mice can be considered as a useful index for tumor development.

MiR-374b-5p suppresses RECK expression and promotes gastric cancer cell invasion and metastasis.

AIM: To profile expression of microRNAs (miRNAs) in gastric cancer cells and investigate the effect of miR-374b-5p on gastric cancer cell invasion and metastasis. METHODS: An miRNA microarray assay was performed to identify miRNAs differentially expressed in gastric cancer cell lines (MGC-803 and SGC-7901) compared with a normal gastric epithelial cell line. Upregulation of miR-374b-5p was newly identified and confirmed via quantitative real-time reverse transcription-PCR (qRT-PCR). MGC-803 cells were transfected with a synthesized anti-miR-374b-5p sequence or a control vector using Lipofectamine reagent, or treated with transfection reagent alone or phosphate-buffered saline as controls. Rate of transfection was verified after 48 h by qRT-PCR. Cells were then subjected to transwell migration, wound scratch and cell counting kit-8 assays. A bioinformatic analysis to identify miR-374b-5p target genes was performed using miRanda, PicTar and TargetScan software. A dual luciferase reporter assay was performed to evaluate the influence of miR-374b-5p on target gene activation, and qRT-PCR and Western blot were used to evaluate the levels of target mRNA and protein following transfection with miR-374b-5p antisense oligonucleotides. RESULTS: The microarray profiling revealed downregulation of 14 (fold change < 0.667; P < 0.05) and upregulation of 12 (fold change > 1.50; P < 0.05) miRNAs in MGC-803 and SGC-7901 cells compared with GES-1 controls. The upregulation of miR-374b-5p (fold change = 1.75 and 1.64 in MGC-803 and SGC-7901, respectively; P < 0.05) was confirmed by qRT-PCR. Compared with the control groups, the restoration of miR-374b-5p expression with anti-miR-374b-5p significantly suppressed the metastasis, invasion and proliferation of MGC-803 cells. The bioinformatic analysis predicted that the 3' untranslated region (UTR) of reversion-inducing cysteine-rich protein with Kazal motif (RECK) contains three miR-374b-5p target sequences. RECK was verified as a target gene in a dual luciferase reporter assay showing that activation of RECK 3'UTR-pmirGLO was increased by co-transfection with miR-374b-5p. Finally, transfection of miR-374b-5p antisense oligonucleotides increased mRNA and protein levels of RECK in MGC-803 cells (P < 0.05). CONCLUSION: These findings indicate that upregulation of miR-374b-5p contributes to gastric cancer cell metastasis and invasion through inhibition of RECK expression.

Differential gene and protein expression in primary gastric carcinomas and their lymph node metastases as revealed by combined cDNA microarray and tissue microarray analysis.

OBJECTIVE: To gain insight into the molecular events of lymph node metastasis of human gastric carcinoma. METHODS: The gene expression profile of five matched primary gastric carcinomas and their lymph node metastases was analyzed by complementary DNA (cDNA) microarray. Differential genes were identified in the metastatic and corresponding primary tumor pairs. Among the differentially expressed genes, carbonic anhydrase II (CAII) and insulin-like growth factor binding protein 4 (IGFBP 4) genes were detected by RT-PCR. CTTN protein expression was examined by tissue microarray. RESULTS: There was a high expression (over twofold) of 44 genes and a low expression (under twofold) of 32 genes in lymph node metastasis compared with primary gastric carcinoma, respectively. CAII mRNA was downregulated and IGFBP 4 mRNA was upregulated in paired lymph node metastases of gastric carcinomas. The overexpression of CTTN protein was related to the lymph node metastasis and the clinical stage of gastric carcinomas. CONCLUSION: This study showed that there is a low expression of genes relative to growth signal and immune response in lymph node metastases, and a high expression of genes relative to growth factor, cell cycle, cell motility and adhesion in lymph node metastases compared with primary gastric carcinomas. The expression of CTTN was related to the invasion and metastasis of gastric cancer.

[Refined mapping of the loss of heterozygosity on chromosome 1q21 in gastric carcinoma].

OBJECTIVE: To obtain allelic loss mapping and define the minimal lost region on chromosome 1q21 in gastric carcinomas, and explore role of 1q21 loss of heterozygosity (LOH) in the development and progression of gastric carcinogenesis. METHODS: Using 7 high-density microsatellite markers and PCR method, lq21 LOH was analyzed in 30 paired specimens of fresh gastric carcinoma, and the relation between 1q21 LOH and the clinicopathological features of the malignancy was tested. RESULTS: The LOH frequency on chromosome 1q21 from these gastric carcinoma tissues reached 60% (18/30). The LOH frequencies of the microsatellite markers D1S514, D1S2696, D1S498, D1S305, D1S2624, D1S2635 and D1S2702 were 13.3%, 10%, 20%, 23.3%, 33.3%, 40% and 23.3%, respectively. The minimal lost region on 1q21 LOH in the gastric carcinoma tissues was located in the region between D1S2624 and D1S2707, in the vicinity of D1S2635. No significant relations of 1q21 LOH to the patients' age, gender, location of the primary foci, clinical staging, or the tumor differentiation were noted (P>0.05), but 1q21 LOH was correlated to lymph node metastases of the malignancy (P<0.05). CONCLUSION: Higher frequency of 1q21 LOH occurs in gastric carcinoma cells, suggesting the presence of potential tumor suppressor genes closely associated with gastric carcinogenesis near the region of D1S2635.