Expressions Of Seven Gastric Cancer Associated Genes And Their Relevance With Wnt, NF-κB And Stat3 Signalings

Background and objective:Gastric cancer (GC) is one of the cancers with higher morbidity and mortality in China and the world. It has been demonstrated that the development of GC is a multi-step process including normal mucosa, precancerous lesions (chronic atrophic gastritis and intestinal metaplasia) and GC (intestinal GC and diffuse GC) and that many mechanisms involve in GC such as the activation of many oncogenes, inactivation of anti-oncogenes, aberrant expression of repairing genes and eating habits. But the exact mechanism of GC hasn't been elucidated. By cooperation with Cancer Research Center of University of Melbourne, researchers of the Cancer Research Center of Dalian medical university find some genes up-regulated commonly in GCs using gene chip to detect the spectra of genes correlated to GC. In present study, we classify genes and screen the representative genes in development of GC including c-Myc, cyclin D1, CD44, MMP7, VEGF and survivin which play a key role in cell survival and proliferation, regulation of cell cycle, cell migration, neovascularization and apoptosis escape. By detecting expressions of these genes in GC and precancerous lesions, we try to clarify the role of these genes, which will benefit the researches on the study of gene regulation and oncotherapy. It is suggested that these genes follow a likewise dynamic changes and have obvious homogenicity, as well as the transcription of them relates to the activation of Wnt signaling.A body of evidence has demonstrated that Wnt signaling pathway is involved in modulating cell proliferation, differentiation, transformation and many other functions, contains several important elements such as initiation of signals, transduction of molecules, transcriptional activation of target genes and is a complex system with the interactions of multiple molecules. In a variety of human tumors, the Wnt signaling is aberrantly activated and its functions rely on the successive molecular events,up-regulation of Wnt signals, and nuclear translocation ofβ-catenin, binding ofβ-catenin/TCF/LEF complex to the special DNA sequence of target genes, relief of the transcription repression and initiation of target gene transcription. Data from our study show the abnormalities of Wnt signaling including high frequency of Wnt expression, reduction of membrane E-cadherin, cytoplasm accumulation ofβ-catenin and nuclear translocation of TCF4 andβ-catenin. Forthcoming study usually defines the activation of Wnt signaling as the activation of a certain component of this pathway, the reports about the activation of total activation of it and its correlation with the expressions of GC associated genes. In present study, we detect the expression of key molecules Wnt2,β-catenin and TCF4 of Wnt signaling by paraffin-tissue array and immunohistochemisty, and for the first time investigate the role of Wnt signaling and the associativity of target genes expression in the full-scale view.The transcription of a certain gene like the component of Wnt pathway is regulated by many signalings. Previous researches show the target genes such as surviving, VEGF and c-Myc of Wnt signaling are also targets of NF-κB and Stat3 signaling, implying that different signaling pathways can interact and function synergistically by mediating the common downstream genes. Accordingly, the signaling pathway mediated by NF-κB and Stat3 may function in cooperative and supplementary mode and play a crucial role in certain steps of carcinogenesis of GCs.Surgical specimens and 4 GC cell lines were selected. The specimens were divided into two subtypes-intestinal GC and diffuse GC-according to the Lauren system. Precancerous lesions-chronic atrophic gastritis and intestinal metaplasia-were selected as the control. We detected the expression of GC associated genes and the status of Wnt, NF-κB and Stat3 signaling activation in the same specimens in order to explore the relevance of these pathways. As there have not been reports about the above issues and the relationship between the downstream genes, our present study will provide the basis and thoughts of mechanism, diagnosis, prevention and therapy and has its significance in biology and precaution of oncology. Materials and methods:GC and precancerous lesions were all selected from the Gastric Tissue Band of the Cancer Institute, Dalian Medical University and the GC cell lines AGS, HGC-27, MGC803 and BGC823 are from American Type Cell Culture and the Cancer research institute of Beijing. By the methods of paraffin embedded tissue array immunohistochemistry, immunofluorescence, RT-PCR and Western blotting, theexpression of GC associated genes and the activation of Wnt, NF-κB and Stat3 signaling were detected in different gastric tissues and cell lines. SPSS 12.0 software was adopted to analyze the results.Results:1. The expression and cellular distribution of seven genes in different gastric tissues1) The expressions of c-Myc, cyclin D1, CD44s, CD44v6, MMP-7, VEGF and survivin in GC are higher than that in precancerous lesions (p<0.05).2) The expressions of MMP-7 are higher in I-GC than in D-GC (p<0.01),whereas other genes express in the same level (p>0.05).3) Except cyclinD1 and survivin, the expressions of the rest genes are identical in intestinal metaplasia and chronic atrophic gastritis.2. The expressions of Wnt2,β-catenin and TCF4 in various gastric lesions1) Wnt2 detection rates were 90.3%(28/31)in GC, the expression were significant higher than that in precancerous lesions especially in 17 cases (p<0.01). The expressions of Wnt2 in two types GC were identical (p>0.05).2) There was difference in the nuclear translocation rate ofβ-catenin between the GC group and precancerous tissue (p<0.01) and no difference between two types of GC (p>0.05).3) All 31 GC cases displayed strong positive staining for TCF4 and expression elevated level of TCF4 in comparison to precancerous lesions (p<0.01).3. The activation status of Wnt signaling in various gastric tissues and the expressional relation of 7 target genes1) The Wnt signaling pathway was activated in 29/54 cases, most of them were GC (27/29), only two precancerous lesions with activated Wnt pathway. Among the 25 unactivated cases, most are precancerous lesions (84.0%). The distribution of Wnt+ and Wnt-are significant different between precancerous and GC lesions (p<0.001).2) Expressions of all 7 target genes in Wnt+ were higher than those in Wnt-.4. Expressions of NF-κB and Stat3 in gastric tissues1) NF-κB expresseed in 74.2% (23/31) GC and displayed higher expression than precancerous lesions (p<0.01), whereas no difference between two type of GC (p>0.05).2) Stat3 has a higher frequency in GC 96.8% (30/31) than precancerous group 30.4% (7/23) (p<0.01), and no difference between two type of GC (p>0.05).5. The relation of activations of Wnt, NF-κB and Stat3 pathway and the expressions of 7 target genes1) Among the 25 Wnt-cases, NF-κB activation were closely related to the expressions of survivin and VEGF (Z=2.437, p=0.027; Z=2.997, p=0.006) but not with that of c-Myc, CD44v6, cyclin D1 and especially CD44s and MMP-7 (p>0.05).2) Among the 17 cases with non-activation of both Wnt and NF-κB, 75% (3/4) of the cased (expressing VEGF) show activated Stat3. Rates of Stat3 activation accompanying these target genes are 66.7% (4/6) for c-Myc, 50.0% (4/8) for survivin, 50.0% (2/4) for MMP-7, 50.0% (1/2) for CD44v6, 44.4% (4/9) for cyclin D1 and 40.0% (2/5) for CD44s.6. The activation of Wnt, NF-κB and Stat3 signaling pathways and expression of7 target genes in GC cell linesWnt and Stat3 signaling were activated in MGC803 and BGC823 cell lines. The activation and nuclear translocation of NF-κB existed in MGC803 and AGS, whereas in HGC-27 cell line all these 3 pathways were inactivated. c-Myc,cyclin D1,VEGF,survivin,CD44s and CD44v6 express in all 4 cell lines, while MMP-7 express only in BGC823 and AGS and expressed weakly in HGC-27 cell line.Conclusion1. 7 oncogenes express in GC and precancerous lesions and display certain similarity, high frequency in GC and low frequency in precancerous lesions, suggesting their expression abide by certain dynamic change and display obvious homogeneity.2. Wnt2,β-catenin and TCF4, the key components of Wnt signaling are overexpress in GC. Wnt signaling pathway is activated in GC, whereas inactivated in precancerous lesions although a certain component expresses. It means that fully activation of Wnt signaling pathway play an important role in the development of GC.3. The expressions of target genes tightly relate to the activation of integrated Wnt signaling pathway, demonstrating full Wnt signaling pathway activation is the crucial regulator of expression of them especially in GC.4. NF-κB and Stat3 pathway are in an active situation in GC and relate to the expressions of some target genes of Wnt signaling pathway. At the early stage of GC, NF-κB and Stat3 can regulate the expression of GC associated genes by synergistic effect of Wnt signaling pathway.5. Crosstalk of at least 3 pathways-Wnt, NF-κB and Stat3-function in theformation and development of GC and synergistically regulate the expression of these oncogenes. The full study of signaling pathways is significant for the understanding the expression of many GC associated genes.In summary, by screening the genes associated with GC using gene chip hybridization and detecting the expression and relation to the activation of Wnt, NF-κB and Stat3 signaling, we demonstrate that the expressions of key genes in development of GC are regulated by many signaling pathways and the complex association exist in the development of GC.