Expression Of Tumor Suppressor Gene DBC2 In Gastric Cancer Infected By Helicobacter Pylori And Its Function

ObjectiveGastric cancer is now the most common malignant tumor of digestive tract in China, and it’s early diagnosis rate is low, prone to local and distant metastasis in patients with short survival, poor prognosis,poor life quality and so on. The incidence of gastric cancer and associated with abnormal expression of tumor related gene structural changes, and it has great significance for developing targeted drug to study on key genes related to gastric cancer. As a class I carcinogen, Helicobacter pylori infection and gastric cancer are closely related,specification of Helicobacter pylori eradication can significantly reduce the incidence of gastric cancer. In recent years it is always the focus of research the specific mechanism of gastric cancer caused by Helicobacter pylori infection, especially the relationship between Helicobacter pylori infection and anti oncogene. It can provide an important basis for the prevention and treatment of gastric cancer to clear the carcinogenic mechanism.Activation of oncogenes and (or) inactivation of tumor suppressor genes are the key links in the occurrence of tumors. Mutations in genes often alter signaling pathways in cells, ultimately leading to the generation of malignant tumors. The products of tumor suppressor genes inhibit tumor generation via cell cycle regulation and programmed cell death. However, malignant transformation and tumor formation can be accelerated if they are deleted or dysfunctional. Recent studies have found that breast cancer suppressor gene DBC2 (deletion in breast cancer 2) has a variety of biological functions, such as regulating the cell cycle, proliferation and apoptosis. DBC2 expression is closely related to the occurrence of breast cancer, gastric cancer and other tumors. Determining its function and mechanism could provide a new strategy for the treatment of cancer.Imbalance of cell apoptosis and proliferation related with the occurrence of gastric cancer.Apoptosis is one of the important ways to remove tumor cells of body under the pathological condition, abnormal expression of apoptosis regulating gene may promote the occurrence of tumors. Oncogenes and tumor suppressor genes play their functional eventually affect the cell cycle through different forms, the normal cells showed uncontrolled growth,and lead to the occurrence of malignant tumor.Invasion and metastasis of gastric cancer not only related to the biological characteristics of cancer cells,but also related to the interaction of tumor cells and the body microenvironment, which is involved in adhesion, chemotaxis and other mechanisms. The chemokine receptor CXCR4 is a cytokine receptor widely expressed in tissue, related to growth and invasion of gastric cancer and other malignant tumor. By virtue of chemokine receptor and its ligand binding ability tumor cells achieve distant metastasis outside of the primary. To explore the functional genes related to gastric cancer invasion and metastasis, and to obtain the predicted molecular markers and gastric cancer targeted therapy measures, it is the final goal of study. Therefore, it is necessary to study the effect of tumor suppressor gene on invasion and metastasis of gastric cancer.Although there are lots of researchs about DBC2 as an anti oncogene related with tumor, loss of DBC2 in Helicobacter pylori associated gastric carcinoma how to affect the development, invasion and metastasis of tumor that is not very clear. Can DBC2 play a role in the inhibition of gastric cancer? What’s the relationship between expression of DBC2 and Helicobacter pylori infection, invasion and metastasis of gastric cancer, and clinicopathological characteristics? These are the purpose of this research.In this study, we will approach the antitumor effect of DBC2 at the following aspects:1) The expression status of DBC2 in gastric cancer infected Helicobacter pylori and it’s clinical significance.2) Construction of recombinant vector of human DBC2.3) Overexpression of exogenous RhoBTB2 gene in gastric cancer cells infected Helicobacter pylori and the function study of DBC2.Methods1. Immunohistochemistry and immunocytochemistry method was used to detect the expression status of DBC2 in different human tissues and cells.2. The expression of DBC2mRNA was detected by RT-PCR, and was analyzed the relationship between Helicobacter pylori infection and clinical pathological features.3. Using cell transfection technique, the design of DBC2 over expression vector.4. Transfection of exogenous DBC2 gene in gastric cancer SGC7901 cells infected Helicobacter pylori, the expression of DBC2mRNA and protein was detected by RT-PCR method and Western Blot method.5. Using MTT method and flow cytometry to detected proliferation, apoptosis and cell cycle of normal gastric epithelial cells and SGC7901 cells before and after of DBC2 transfection.6. Using immunohistochemistry method and Western Blot method to detected the expression status of caspase-3 protein in SGC7901 cells infected Helicobacter pylori before and after of DBC2 transfection.7. Transwell assay to detect the invasion ability of SGC7901 cells before and after of DBC2 transfection.8. Using Western Blot method to detected the expression status of CXCR4 protein in SGC7901 cells infected Helicobacter pylori before and after of DBC2 transfection.Results1. In normal or inflamed tissues infected by Helicobacter pylori DBC2 showed higher levels of expression,however in gastric carcinoma tissues DBC2 mainly showed low expression or no expression (P<0.05). The expression status of DBC2 in GES-1-Hp cells was positive in high level, while the SGC7901-Hp group showed the expression status of DBC2 was low or deletion, the difference was significant (P<0.05). It is also found that the expression of DBC2 was no significant difference in the same group of cells infected Helicobacter pylori whatever 24h or 48 h.2. The expression of DBC2 mRNA in GES-1 cells infected by Helicobacter pylori showed high amplification, high expression, but in SGC7901 cells DBC2 mRNA amplification factor was lower and highly expressed at low levels, and the difference has statistical significance (P<0.05).3. Compared with the loss of DBC2 expression in gastric cancer that the depth of invasion was T1 or T2, the depth of invasion was T3 or T4 was more obvious (P<0.05). Compared with the loss of DBC2 expression in stage I or II gastric carcinoma, the loss of expression in stage III and IV gastric cancer tissue was more obvious (P<0.05). Moreover, closely related to the loss of DBC2 expression and tumor differentiation, the lower the degree of tumor differentiation and DBC2 deletion expression is more obvious. At the same time, there was no significant correlation between the loss of DBC2 expression and gender, age, tumor size or Borrmann type of patients with gastric cancer (P>0.05).4. Compared with the expression level of DBC2 in GES-1 cells, the expression in SGC7901 cells is significantly reduced, and the difference was statistically significant (P<0.05). Compared with the SGC7901 empty vector group, the expression level of DBC2mRNA in transfected SGC7901 cells at 24h after transfection was significantly increased (P<0.05). And 48h after the transfection DBC2mRNA amplification levels in SGC7901 cells was increased further and higher than the fold expansion in GES-1 cells, the difference was statistically significant (P<0.05).5. By DBC2/GAPDH absorbance ratio represents the expression level of DBC2 protein, SGC7901-DBC2 transfection group was the highest among groups. DBC2 gene protein expression level of GES-1 group and SGC7901-DBC2 transfection group was significantly higher levels than SGC7901 empty vector transfected group, the difference between the former two and the latter was statistically significant (P<0.05).It indicated that exogenous DBC2 gene in gastric cancer cell line SGC7901 has been relatively stable expression.6. Compared with GES-1 cells, SGC7901 cell proliferation activity was significantly enhanced (P<0.05). Compared with the SGC7901 group transfected with empty vector, the group transfected with DBC2 began to proliferate and slow down from second.If the time become long, the difference was more significant (P<0.05).7. Compared with non transfected SGC7901 cells, the ratio of S phase in the cell cycle in DBC2 transfected group was increased, accompanied by decreased G2 and G1 ratio. This shows that DBC2 implemented inhibition on gastric cancer SGC7901 cells growth was mediated by inducing cell cycle S (DNA copy) block and to prevent the cells into G2 phase.8. After 24h, Compared with GES-1 cells (3.55%±0.73%) and SGC7901 empty vector cells (6.17%±1.85%) group,the apoptosis rate of the group cells transfected with DBC2 (12.37%±1.67%) was significantly increased (P<0.05). After 48h, compared with GES-1 cells (3.50%±0.89%) and SGC7901 empty vector cells (6.18%±1.97%) group, the apoptosis rate of the group cells transfected with DBC2 (16.84%±3.62%) was also significantly increased (P<0.05).9. Compared with the negative control group (final score 2.18±0.06) and empty vector cells group (final score 2.21±0.11), Caspase3 activation (final points 9.87 ±1.65) in SGC7901-DBC2 transfection group was obvious and it’s expression ratio was significantly increased, the difference was statistically significant (P< 0.01).10. Compared with the negative control group (0.46±0.04),the Caspase3 protein expression level of SGC7901-DBC2 group (0.63±0.08) was increased significantly (P<0.01).11. At 24h this point, compared with the gastric cancer SGC7901-vector group and SGC7901-DBC2 transfected cells, the migration ability of gastric mucosal epithelial cells of GES-1 is very low, the difference was statistically significant (P<0.05). At the same time, compared with the SGC7901-empty vector group, the number of transmembrane cells transfected with SGC7901-DBC2 group was decreased significantly (P<0.05). At 48h the point, the number of transmembrane cells in SGC7901-DBC2 transfection group was still relatively decreased significantly compared with the SGC7901-empty vector group,the difference was statistically (P<0.05).12. At 24h the point, compared with GES-1 cells group, invasion transmembrane cell number of gastric cancer SGC7901-vector group and SGC7901-DBC2 transfection group were increased markedly,the difference was statistically significant (P<0.05). At the same time, compared with the SGC7901-empty vector group.the invasion transmembrane cell number of SGC7901-DBC2 transfection group was decreased significantly (P<0.05). At 48h the point, compared with the SGC7901-empty vector group, the invasion transmembrane cell number of SGC7901-DBC2 transfected group was decreased significantly, and the difference was also statistically significant (P<0.05).13. Compared with blank control group (0.15±0.01) and SGC7901-empty vector group (0.16±0.01), CXCR4 protein expression levels of SGC7901-DBC2 transfection group (0.08 0.01) was significantly lower (P<0.05).Conclusion1. DBC2 showed high expression in normal tissues and cells. At the level of transcription and protein levels DBC2 gene in gastric cancer tissues and gastric cancer cells showed a high frequency of loss of DBC2 expression deletion which closely related to Helicobacter pylori infection, clinical stage, invasion depth and differentiation.2. We have successfully completed the gastric cancer cell line SGC7901 DBC2 recombinant expression vector construction and transfection. The application of RT-PCR and Western-blot in transcription and protein levels to detected the DBC2 expression in SGC7901 cells before and after transfection. Compared with the control group, DBC2 level in the cells after transfection increased significantly.3. DBC2 can inhibit the proliferation of gastric cancer cells.It also can affect the cell cycle and promote tumor cell apoptosis, so as to achieve the role of tumor suppressor. The regulation of apoptosis may be achieved by influencing the expression of Caspase-3. DBC2 may promote cell apoptosis by enhancing the expression of Caspase-3 in cells, which leads to the inhibitory effect on cancer.4. DBC2 can inhibit the invasion and metastasis of gastric cancer cells and inhibit the expression of chemokine receptor CXCR4. DBC2 may inhibit the invasion and metastasis of tumor by inhibiting the expression of CXCR4.OriginalityCurrently the relationships between DBC2 and gastric carcinoma related Helicobacter pylori have been rarely reported. This study detected DBC2 expression deletion in gastric cancer associated Helicobacter pylori, relatively comprehensive study biological characteristics influence in the gastric cancer cell infected Helicobacter pylori caused by DBC2 intervention.The study provides the theoretical basic foundation for future clinical molecular target to the treatment of gastric cancer infected Helicobacter pylori.