| Gastric cancer is one of the most common malignant tumor of gastrointestinal in the world, as the world’s leading cause of cancer-related death. In 2015, according to the American cancer society’s latest global cancer statistics data show that by the year 2012, the world has about 951,600 new cases of gastric cancer,723,100 deaths from cancer of the stomach. In East Asian countries, such as China, Japan and South Korea, has the high incidence of gastric cancer in the world area. In high economic level countries, such as Japan and Korea, because of the perfect medical services system, patients are found mostly at early stage, and the prognosis is relatively good; However, about 40% of gastric cancer in China, due to the poor economic conditions and backward medical, when a lot of patients with gastric cancer diagnosed has been in the advanced stage, so the stomach cancer mortality is significantly higher than the former. The latest cancer statistics show that China has an estimated 4.292 million new cancer cases and 2.814 million cases of cancer death in 2015. Tumor etiology incidence rate of gastric cancer in male and female ranks the second and third places, respectively. And it also among the forefront in the causes of death due to malignant tumors. Although with the progress of numerous clinical and basic research and development of science and technology, diagnosis and treatment of gastric cancer has made great progress, but its prognosis conditions are still not optimistic. Metastasis (especially the peritoneal metastasis) is still one of the main factors which affects the prognosis, accounting for 31% of all transfers cases, and the average survival time is less than 6 months. Peritoneal metastasis prevents the tumor from radical resection and it often complicated with intestinal obstruction, massive ascites and other symptoms, these factors often lead to poor quality of life and short of death. Therefore, we should actively look for the key molecules associated with peritoneal metastases in the development of gastric cancer, research the specific molecular mechanisms, and provide the basis for individual treatment of patients with gastric cancer. Only in this way will it be possible to do early detection, diagnosis and treatment, improve the prognosis, and lay a solid foundation for the development of diagnosis and treatment of gastric cancer.In order to better study the development and peritoneal metastasis-related molecular mechanisms of gastric cancer, we first have to understand the theory about tumor metastasis. Development of tumor is determined by genetic factors, environmental factors and lifestyle factors, and is a multi-stage, multiple-step, and participated by multiple signaling pathways. Currently accepted theories of gastrointestinal tumor metastasis has four kinds, including direct invasion, lymph node metastasis, hematogenous spread and peritoneal cavity metastasis. Lymphatic metastasis is the most common tranfer model in gastric cancer, it is mainly tumor cells transfered into the lymph nodes around the stomach through the lymphatic vessel. In addition, it can also tranfer to distant lymph nodes by jumping metastasis, lymph node dissection around the stomach plays an important role in the gastric cancer postoperative pathologic staging, follow-up treatment and prognosis assessment. Implantation metastasis is the main cause of gastric cancer peritoneal metastasis, it originates from the mucosa, and tumor cells shed into the abdominal cavity leading to peritoneal metastasis after breaking the serosal layer of gastric cancer. A large sum of previous studies showed that normal tissue, primary tumors and tumor metastases there are many differences between the expressed genes, precisely because of these variation, leading to a significant change in the growth of tumor cell metabolism, adhesion ability and athletic ability, and resulting in significantly enhance its capability of tumor cell invasion and metastasis.In recent years, with the development of proteomics technique, it becomes one of the most effective methods of looking for molecular markers and drug targets. In addition, proteomics also have very attractive prospects in clinical diagnosis and treatment of human diseases, such as cancer. Protein is the performer of the physiological function, and also the direct manifestation of the phenomenon, so the study of protein structure and function will directly state the life changing mechanism which in physiological or pathological condition. The form of the protein itself and the rule of the activity, such as post-translational modification, protein-protein interactions, protein conformation and other issues, still depend directly on the protein research to solve, for complex post-translational modification of proteins, protein subcellular localization or migration, protein-protein interaction, almost could hardly judging from mRNA level. The core technologies of proteome research is two-dimensional gel electrophoresis-mass spectrometry (Two-dimensional polyacry-lamide gel electrophoresis-mass spec-trometry,2-DE-MS), which is separated by two-dimensional gel electrophoresis of proteins, then using mass spectrometry to identify protein one by one. Because the two-dimensional gel electrophoresis has some disadvantages, such as tedious, instability and low sensitivity, the fluorescent difference gel electrophoresis (difference gel electrophoresis, DIGE) technology gradually becomes one of the most popular technology of proteomics research due to the advantage of high throughput, sensitivity and precision.Based on the clinical, to the challenges of recurrence of gastric cancer metastasis, the aim is to look for the biomarker and the biological therapeutic targets which associated with tumor invasion and metastasis. In the earlier stage, we analysised the differences protein group on primary tumor, peritoneal transfer stove and corresponding normal stomach epithelial tissue from 6 cases who have advanced gastric cancer (Laparoscopic palliative resection for gastric carcinoma) used the proteomic technology. We finally identified 35 abnormal expression of protein. By further analysis and verification, we found that muscle globulin heavy chain (Myosin, heavy chain 9, non-muscle, MYH9) and the human red membrane integration protein sample protein 2 (Stomatin-like protein 2,SLP-2) is associated with the invasion and metastasis, TNM stage and the prognosis of gastric carcinoma. According to reports, MYH9 is involved in cell polarity forming processes, cell division, contraction and relocation, and it is also associated with many diseases, such as hereditary thrombocytopenia, herpes virus infections and embryonic development and so on. In many solid tumors MYH9 is abnormal expression, it promote the occurrence and development of gastric cancer, colorectal cancer, esophageal cancer and breast cancer, but plays a suppressor role in head and neck squamous cell carcinoma, and positively correlated with prognosis. As a "molecular motor" in cell, abnormal MYH9 how to effect the invasion and metastasis of gastric cancer cell cause our interests. In earlier stage, we construct the MYH9 gene knockdown cell lines using Lentiviral interference, and conduct relevant experiment using the most effective and stable monoclonal strains. wo found that the interference group of gastric cancer cells infected with the virus morphogenesis changes obviously, loose connection between cells, and cell shrinkage ability weakened when pancreatic enzyme digestion; In the same view under a fluorescent microscope, the infected and uninfected cells differ significantly. At the same time, after the MYH9 gene knockdown, its migration ability enhancement and invasion ability weakened. On the basis of relative literatures, the inconformity of migration and invasion considers that MYH9 as a skeleton protein, mainly involved in process of cell anchored and adhesion, so the decrease of MYH9 reduces the cell adhesion and enhances the random motion in levels.Generating a MYH9 knockouts in MGC803 cells through Lentiviral interference technology has some limitations, such as 1. after the success of building of MYH9 gene knockout cell lines, with the increase of cell culture and batches, we cannot construct a stable knockdown cell line due to the cell increase of not transfection into virus.2. Although Lentiviral has transfected into cells, but they do not play a relevant role. In order to further verify related biological function after building a gastric cancer cell line with MYH9 gene knockdown through Lentiviral interference, at the same time as potential therapeutic targets and prognostic indicators, MYH9 how to influence the molecular mechanisms of gastric cancer cell biology behavior is also our subsequent research priorities. Therefore, to obtain reliable knockout cells model is a powerful guarantee for further study of specific gene function. With the continuous development of molecular biology technology, gene editing technology constantly emerging and improvement, so in this study we chooses the Transcription Activator-Like Effector Nuclease technique to generate MYH9 gene silence in MGC803 by TALEN, which will provide models for the gene function study. It is also good for further exploration of molecular mechanism of MYH9, and laying a foundation to study the relationship between gastric cancer and peritoneal metastasis.This project consists of two parts, the details are as follows:Chapter I:Generate MYH9 knockouts in MGC803 cells by TALENObjective To construct a MYH9 gene knockout model in MGC803 cell line through a novel gene editing technology — transcription activator-like effector nucleases(TALEN).Methods According to FastTALETM TALEN Kit, we designed TALEN pairs and constructed the plasmids targeting to MYH9 gene. After detecting their activity in MGC803 cells by plasmid transfection, gene sequencing, we successfully selected the monoclonal cells.Results we successfully generate a MYH9 gene knockdown model in MGC803 cell line. Because of the cells deadly after MYH9 knockout, we cannot get MYH9 gene knockout cell line completely.Chapter II:Verification of MYH9 gene knockdown model and its influences on the proliferation and apoptosis of MGC803 cellObjective To validate the MYH9 gene knockout model in MGC803 cell line, test related cell biology function, and provide reliable model and potential research directions for exploring the molecular mechanisms of MYH9.Methods:To detect the relative expressive of MYH9 gene at mRNA and protein levels using qRT-PCR and Western blot methods, and research the cell proliferation and apoptosis function.Results 1. We found that the MYH9 mRNA and protein levels were significantly lower than those of wild-type controls in selected monoclonal cell.2. After MYH9 gene was knockdowned in MGC803 cell line, its cell proliferation restrained.3. Compared with wild-type controls, since the MYH9 gene was knockdowned in MGC803 cell line, its cell proliferation restrained, cell cycle arrested at G2/M phase (P<0.05) and the cell number undergoing apoptosis increased significantly(P<0.01).Through the above research research, we can come to the following conclusion:1. We successfully generate a MYH9 gene knockout model in MGC803 cell line by TALEN. Through its verification and related experiments on cell function, we obtained reliable MYH9 gene knockdown model which was good for the MYH9 function study. In addition, we also found that TALEN was an effective and stable gene editing technology.2.We can’t get the MYH9 gene knockout cell line completely except MYH9 gene knockdown model. It also reflects the importance of the MYH9 gene.3.Since the MYH9 gene was knockdowned in MGC803 cell line, its cell proliferation restrained, cell cycle arrested at G2/M phase (P<0.05) and the cell undergoing early apoptosis increased signif!cantly(P<0.01). This phenomenon could not be explained by the MYH9 gene as a skeleton for the increasing of cell early apoptosis. Whether MYH9 affects the development and progression of gastric cancer through other mechanisms? This research provides a potential research directions to further study the molecular mechanism of the MYH9 gene.The innovation of this research:We first generate a MYH9 gene knockdown model in MGC803 cell line through a novel gene editing technology — transcription activator-like effector nucleases(TALEN). |
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