The Research Of Effect And Regulation Of BAMBI Gene Silencing On Growth Of Gastric Cancer And Nude Mouse Transplantation Tumor

BackgroundIn worldwide, the incidence of gastric cancer is the fourth after lung cancer, breast cancer, colorectal cancer, and gastric cancer mortality is still among the world’s second cancer mortality, it has become one of the main diseases of harm human health. Clinical symptoms and signs of early gastric cancer have no specificity, it is easy to be ignored. The most diagnosis of gastric cancer has belongs to the advanced stage of disease, those patients have lost effect a radical cure operation opportunity. Therefore, the molecular mechanisms of gastric cancer occurrence and development will be further understood, and the biological indicator associated with malignant biological function characteristics and prognosis of gastric cancer will be looked.It can help to find the early diagnosis of gastric cancer, and to provide new ideas for stomach cancer prevention and provide new targets for molecular targeted therapy of gastric cancer.Now the etiology and pathogenesis of gastric cancer has not been clarified, and its mechanism may be related to cellular signal transduction pathway is closely related to the abnormal, and signaling pathway of relevant factors is also involved in the regulation of the proliferation, differentiation, invasion and metastasis process of gastric cancer cell. In the study of tumor molecular pathogenic mechanism, TGF-β and Wnt signaling pathways in various tissues and organs of tumor and the occurrence of diseases and evolution play an important role, is one of the more and more attention by researchers in the world.Bone morphogenetic protein and activation of transmembrane inhibitors (BAMBI) is the receptors of TGF-β, and the expression of BAMBI is inducted by β-catenin which is the key components of Wnt signal transduction pathways.In this study, we researched the effect of BAMBI on the gastric cancer in vivo and in vitro, in order to find a new target for the treatment of gastric cancer by the regulation mechanism of TGF-β, Wnt and β-catenin signaling pathway.Part Ⅰ:The expression and significance of BAMBI in human gastric cancer tissueObjectiveTo detect the expression of BAMBI in human gastric cancer tissue samples of 60 cases and the corresponding tissue adjacent to carcinoma specimens of 60 cases by immunohistochemical methods, and to discuss the relationship between clinical pathological features and BAMBI development of gastric cancer, and the role of the mechanism, for theoretical basis of the next experiment.MethodsFrom October 2012 to October 2014 in Shandong Tumor hospital department of postoperative,60 cases of gastric cancer were collected, including cancers of the stomach cancer and corresponding adjacent tissues,60 cases each. The expression of BAMBI in human gastric cancer tissue samples of 60 cases and the corresponding tissue adjacent to carcinoma specimens of 60 cases were detected by immunohistochemical methods, and the relationship of BAMBI and the clinical pathological factors associated with gastric cancer were analyzed.ResultsThe positive expression of BAMBI was tan grains or mass targeting in cancer cells. The positive expression rate of BAMBI in gastric cancer and tissue adjacent to carcinoma were 66.7% and 20.0% respectively, and the positive expression rate of BAMBI in gastric cancer tissues was obviously higher than that of tissue adjacent to carcinoma (^=8.164, P<0.05).There were no significantly relationship between the expression of BAMBI in gastric cancer tissue and the patients’ age, gender, degree of differentiation, tumor size (P>0.05),and there significantly relationship between the expression of BAMBI and TNM staging, lymph node metastasis, whether infiltration serous. The positive expression rate of BAMBI in patients with lymph node metastasis (91.9%) were significantly higher than those patients without lymph node metastasis (26.1%) (P< 0.05). The positive expression rate of BAMBI of patients with stage Ⅲ+Ⅳ in lymph node metastasis(88.6%) were significantly higher than those in patients with stage Ⅰ+ Ⅱ (36.0%) (P< 0.05). The positive expression rate of BAMBI in patients with infiltration serous(87.9%) were significantly higher than those without infiltration serous (40.7%) (P< 0.05).ConclusionThe expression of BAMBI in gastric cancer tissue is high.It is to said that BAMBI is involved in the occurrence of gastric cancer process, and it suggest that BAMBI is possible the potential molecular targets of clinical drug development of gastric cancer.Part Ⅱ:The effects of BAMBI gene silencing mediated by lentivirus on biological behavior of human gastric cancer cellsObjectiveTo discuss the effects of BAMBI gene silencing mediated by lentivirus on biological behavior of human gastric cancer cells and expression changes of BAMBI protein and mRNA for providing new molecular targets for gene therapy of cancer of the stomach and experimental basis.MethodsShRNA-BAMBI was designed and synthesised, then it were transfected into human gastric cancer cells MGC-803 which were cultured in vitro. RT-PCR and Western blot analysis were used to detect the BAMBI mRNA and protein expression level of MGC-803 cells. MTT assay was used to detect the cell growth inhibition. And Transwell experiment was used to analyze the migration and invasion of MGC-803 cells.ResultsAfter shRNA-BAMBI transfecting into MGC-803 cells,BAMBI protein and mRNA expressions of the experimental group were all significantly decreased compared with negative control group,blank control group(P<0.05),but there was no significant difference between negative control group and blank control group (P>0.05)After shRNA-BAMBI transfecting into MGC-803 cells, by the curve according to the cell growth inhibition rate by MTT, we found that the proliferation,migration and invasion of cells in experimental group was significantly suppressed, the cell inhibition rate of experimental group significantly higher than the blank control group and the negative control group (P< 0.05), but there was no significant difference between negative control group and blank control group (P>0.05).ConclusionBAMBI-shRNA can inhibit BAMBI mRNA and protein expression of MGC-803 cells, inhibit proliferation and reduce the invasion and migration ability of MGC-803 cells.Part III:The influence and mechanism of BAMBI gene silencing mediated by lentivirus on proliferation and apoptosis of gastric cancer orthotopic transplantation tumor of nude mouseObjectiveThe gastric cancer orthotopic transplantation tumor of nude mouse model was established,and to discuss the influence and mechanism of BAMBI gene silencing mediated by lentivirus on proliferation and apoptosis of gastric cancer orthotopic transplantation tumor of nude mouse.MethodsShRNA-BAMBI was designed and synthesised,then to intervene the orthotopic transplantation tumor tissue of gastric cancer in nude mouse, in accordance with the mice were executed at observation point in time (the third day,sixth day,ninth day and twelfth day) to observe the effects of BAMBI gene silencing mediated by lentivirus on proliferation and apoptosis of gastric cancer orthotopic transplantation tumor of nude mouse and the expression of BAMBI,β-catenin and TGF-β transplanted tumor tissue were detected by RT-PCR, Western blot and immunohistochemistry methods.ResultsMGC-803 gastric cancer cells were injected into nude mice after 3 weeks, subcutaneous tumor rate of nude mice were 100%, and subcutaneous form tumors was about 1.0 cm in diameter. After six successive batches in situ tumor tissue block after transplantation, and transplantation tumor rate in situ could be improved by the improvement of medical adhesive stick method.Early in orthotopic transplantation, there was no obvious change in weight and activity of nude mice, after 4 to 5 weeks a hard nodules could touch on the abdomen. With nodules gradually increasing, nude mice were thin, activity decreased gradually, such as slow performance. All vaccinated animals could be a tumor and be survival.Anatomical all nude mice, round or oval bump is formed on the stomach of visible nude mice, wring transplanted tumor tissue, aspect as the gray, solid, fish. Wring in transplanted tumor tissue found in tumors in the process of injection BAMBI-shRNA slow virus particles experimental nude mice, transplanted tumors had smaller, and the surrounding tissue boundaries clear, no obvious adhesion phenomenon. Within the tumors had injected with normal saline and irrelevant sequence slow virus particles of nude mouse transplantation tumor volume is larger, and around the liver, spleen and other organs have different degree of adhesion. Orthotopic transplantation tumor tissue after HE staining, light microscope under visible large nuclear hyperchromatism of tumor cells, nuclear loading ratio are increased, the nuclear atypia, obviously sick rational fission, visible adenoid structure, confirm for gastric cancer.The inhibitory rate of tumor weight in experimental group was (47.44+ 1.64)%.According to comparison among tumor weight of three groups, the result showed: the weight of the experimental group (1.21+0.03 g) and the negative control group (2.48 +0.18 g) and blank control group (2.52+0.17 g), the weight of the experimental group had significantly lighter than those in blank control group and negative control group (P < 0.05), the difference was statistically significant (P<0.05), while there was no significant difference between negative control group and blank control group (P>0.05).Before gene silencing,there were no significant difference of tumor volume of the three teams(0.89±0.35,0.92±0.26,0.87±0.18 respectively). But after 3,6,9,12 day of gene silencing,there were significant different during three teams (According to comparison among inhibition rate of three groups:experimental team (1.02±0.29%1.84±0.21、 2.34±0.37、2.34±0.37、2.89±0.95 and3.14±0.96),blank team (2.12±0.19±3.94±0.28. 4.38±0.84、4.96±1.02 and 5.16±1.18) and negative control team (2.18±0.17、3.86±0.27、 4.32±0.86、4.95±1.05 and 5.19±1.12) respectively.The results showed that the inhibition rate of proliferation in experimental group was significantly higher than that in the blank control group and negative control group(24hr,48hr and 72hr).The experimental team were 9.27±2.05,19.24±3.54, 28.17±2.94,the blank team were 0.92±0.09,0.86±0.19,1.42±0.18 and the negative control team were 0.85±0.06,0.95±0.13,1.76±0.18.The results showed that the apoptosis rate(AI) in experimental group was significantly higher than that in the blank control group and negative control group(24hr,48hr and 72hr), experimental team were 25.18±1.25、 30.74±4.29,39.48±5.26,blank team were 5.02±1.38、7.45±2.35、9.64±2.89,negative control team were 4.52±1.34、7.19±2.13、8.97±2.74 respectively (P< 0.05), while there was no significant difference between negative control group and blank control group (P>0.05)The expression of BAMBI,β-catenin and TGF-β in experimental group were tested. The protein expression of experimental group in TGF-β in were significantly more than those of blank control group and negative control group (P< 0.05),but the BAMBI and β-catenin were significantly less than those of blank control group and negative control group (P< 0.05), while there was no significant difference between blank control group and negative control group (P>0.05)TGF-β mRNA expressions of the experimental group were all significantly increased compared with blank control group, negative control group (P<0.05),but BAMBI and P-catenin were significantly decreased with blank control group, negative control group (P<0.05),.There was no significant difference between negative control group and blank control group (P>0.05)Conclusion1. The study successfully built gastric cancer orthotopic transplantation tumor of nude mouse model, and slow virus shRNA-BAMBI was injected into tumor tissue to be transfected.It inhibited the growth of gastric cancer transplantation tumor and induced apoptosis of gastric carcinoma, and it proved that BAMBI was a potential molecular targets of clinical drug for gastric cancer.2. Slow virus shRNA-BAMBI was injected into tumor tissue successfully. The TGF-β mRNA and protein expression were promoted in the orthotopic transplantation tumor tissue.It inhibited the development of gastric cancer.3. Slow virus shRNA-BAMBI was injected into tumor tissue successfully, the β-catenin and BAMBI mRNA and protein expression were prohibited, The Wnt/β-catenin signaling pathway was suppressed for slowing the proliferation of cancer cell.It achieved the goal of treatment for gastric cancer.