| Gastric cancer is the most common malignant tumor of alimentary tract, despite improved prognosis as result of early diagnosis, radical operation and the development of adjuvant therapy, the mortality rate is still ranked among the top of malignant tumor mortality. There are many factors that can affect the prognosis of gastric cancer, in which the peritoneal metastasis are common in recurrence and metastasis of gastric cancer. Peritoneal dissemination usually leads to the terminal stage of advanced GC and diminishes the quality of life by refractory ascites and intestinal obstruction. As a result, how to effectively forecast and control of peritoneal metastasis is one of the key issues on improving the curative effect of advanced gastric cancer currently.The current clinical method for early diagnosis of peritoneal metastasis from gastric cancer including detection of peritoneal lavage cytology and molecular marker. Peritoneal lavage cytology detection is the most commonly used method and the gold standard in early prediction of peritoneal subclinical metastasis from gastric cancer, but it lacks high sensitivity, since not all the advanced gastric cancer that soaked serous could be found the exfoliated cancer cells. Currently the molecular markers for the detection are CEA, CA125, E-cadherin, β1-integrin, MMPs and VEGF. However, basic researchers at home and abroad have not yet found an accepted molecular marker for early detection of peritoneal metastasis from advanced gastric cancer. At present, most of the research associated with metastasis so far has been focused on the genetic changes of related molecules or single or few proteins without systematicstudy. But little is known about the key factors to trigger tumorigenic cells to initiatefurther invasion and metastasis facing with so many regulators up to now.Proteomics along with its related technologies, an emerging branch of molecular biology in recent years, focuses on studies of the proteome encoded by genes, and makes complete analysis of the composition, expression levels and modification state of the intracellular proteins with dynamic changes, and understands the interactions and associations among proteins, thus revealing protein function and its relationship with laws of the cellular activities as well as regulation mechanisms of gene expression as a new research field. Its core technology is protein separation techniques based on two-dimensional polyacrylamide gel electrophoresis (2-DE) and protein identification technology based on mass spectrometry (MS) and bioinformatics. At present there are many novel differential proteomics analysis methods, among which the difference gel electrophoresis (difference gel electrophoresis, DIGE) technology becomes one of the most popular one, because it’s not only have the high-resolution feature, inherited from two-dimensional gel electrophoresis(2-DE), but also have high reproducibility, high sensitivity, high throughput and high dynamic range. DIGE is a method which can label protein samples with different fluorescent dyes before2-D electrophoresis, and can separate up to three different protein samples at the same time in one two-dimensional gel. The application of the internal standard could further increase the credibility of the experiment, and ensure the results could reflect the real biological differences, while avoid influence of systematic errors on experimental results. To date,2D-DIGE technologies have been used to identify novel candidate biomarkers associated with early diagnosis, differential diagnosis, prognosis, and response to chemotherapy in various diseases.Above all, this topic mostly focused on screening and identificating peritoneal metastasis related proteins of gastric cancer, and then selecting one of interesting protein for further functional verification and mechanism of a preliminary study. In the first part of experiment, two-dimensional fluorescence difference gel electropho-resis (2D-DIGE) was employed together with MALDI TOF/TOF mass spectrometry to screen and identify differentially expressed proteins between gastric cancer and corresponding peritoneal metastasis tissues.27differential proteins were identified,23proteins were significantly up-regulated and4were down-regulated in peritoneal metastatic foci. This result laid the foundation for future research. After analysis with Uniprot, Myosin-9was predicted to play important roles in the bioprocesses of actin cytoskeleton reorganization, actin filament-based movement, angiogenesis, cell-cell adhesion, cytokinesis, protein transport and regulation of cell shape. Above of all, we speculated that Myosin-9might be involved in the process of invasion and metastasis of gastric cancer. Hence the second and third parts were carried on to further study. The results showed that RNAi Myosin-9could inhibit the migration and invasion of gastric cancer cell, promote the adhesion of gastric cancer cell, and notably decrease the number of metastatic nodules in the peritoneal cavity of nude mice. Myosin-9promotes the invasion and metastasis of gastric cancer probably by facilitating the EMT process of gastric cancer.Although the present study demonstrated that Myosin-9expression impacted invasion and metastasis ability of the gastric carcinoma cells, but the development and progression of malignant tumors were the results of interactions between a variety of genes and proteins and it was difficult to fully explain the complex tumor biological behavior with a single gene. In the future research, we will analyze other differential proteins and contrast the relationships between these proteins, and eventually clarify the potential mechanism of peritoneal metastasis from gastric cancer. Besides, the study will be not only on tissues and cells but also on bold and body fluids of patients with peritoneal metastasis. We are looking forward to find an effective molecular marker to predict the peritoneal metastasis from gastric cancer.The specific content of this topic is as follows:Part1Isolation, identification and bioinformatics analysis of peritoneal metastasis related proteins of gastric cancer.Purpose:To separate proteins in gastric cancer and corresponding peritoneal metastasis tissues, find and identify the differences; combined the technology of bioinformatics analysis, to search for the predictive indicators and ulteriorly provide a theoretical basis for tumor mechanism study. In addition, through this experiment the practical value of2D-DIGE technology in the gastric carcinoma study will be verified.Methods:gastric cancer and corresponding peritoneal metastasis tissues served as the subjects,2D-DIGE method was applied to the purified protein for protein separation, According to the analysis of differential protein spots combining different maps of DIGE and Decyder software, we set these which was up-regulated or down-regulated2.0times as differential protein spots. Protein spots of interest were excised by automatic Spot picker, and then the picked spots were carried out with in-gel digestion, followed by using ABI4800MALDI TOF/TOF mass spectrometry. For understanding the relationship among proteins, we use the "String" protein interaction databases.Results:(1).According to the analysis of differential protein spots combining different maps of DIGE and Decyder software,32differential protein spots were found. Retrieving SWISSPROT database through the mascot software, getting rid of the same protein within appraisal result and unidentified protein spots,27differential proteins were identified.23proteins were significantly up-regulated and4were down-regulated in peritoneal metastatic foci.(2) After analysis with Uniprot, myosin-9was predicted to play important roles in the bioprocesses of actin cytoskeleton reorganization, actin filament-based movement, angiogenesis, cell-cell adhesion, cytokinesis, protein transport and regulation of cell shape. Furthermore, single protein analysis by the String9.0program indicated that myosin-9was a key functional partner with ACE, RHOB, RHOA, ACTB, SVIL, CDK5, ACTG1, ROCK2, CALD1, and S100A4.Based on the above results, we selected Myosin-9for subsequent function research.(3) Western blot and immunocytochemistry were used to further verify Myosin-9in order to ensure the reliability of the proteome results. The western blot and immunocytochemistry results showed that Myosin-9was higher in peritoneal metastasis tissues, compared to that in gastric cancer tissues. Evidently, western blot results were consistent with proteome analysis.Part2Studies on function of Myosin-9in the invasion and metastasis of gastric cancer.Purpose:To explore the effects of Myosin-9on invasive and metastatic ability of gastric cancer cells.Methods:The expression of Myosin-9mRNA was detected in gastric cancer cells by quantitate real-time PCR. The level of Myosin-9protein was detected by western blot. We transfected the lentivirus RNAi vectors into the gastric cancer cells to silence Myosin-9. Cell adhesion assay was used to analyze the effect of decreased Myosin-9on cell adhesion ability. Transwell assay was used to assess the effect of decreased Myosin-9on cell migration and invasion ability. The animal model with gastric cancer metastasized to peritoneal cavity was used to assess the effect of decreased Myosin-9on peritoneal metastasis ability of gastric cancer.Results:(1) Compared with GES-1cells (gastric epithelial cells), enhanced signals were found in AGS and BGC823cells (derived from the primary lesion), especially in SGC7901cells (derived from the metastatic lymph node).Western blot analysis confirmed high Myosin-9protein expression in AGS, BGC823, and SGC7901cells. We choose the AGS and SGC7901for subsequent experiments.(2)After several preliminary experiments, we determined the best optimization conditions:cell inoculation density=50%, MOI=10, Polygrene=4μg/ml. Western blot showed that the lenti-virus sequence1had the best interference effect.(3) RNAi Myosin-9could inhibit the migration and invasion of gastric cancer cell, and promote the adhesion of gastric cancer cell. The distinction between the groups was significantly different (P<0.01).(4) RNAi Myosin-9could notably decrease the number of metastatic nodules in the peritoneal cavity of nude mice. The distinction between the groups was significantly different (P<0.01).Part3The preliminary research on the mechanism of Myosin-9in invasion and metastasis of gastric cancer.Purpose:To explore to how that Myosin-9was involved in the invasion and metastasis of gastric cancer.Methods:The expression of EMT related markers E-cadherin, N-cadherin, vimentin and β-catenin were detected by western blot between interference and negative control groups.Results:RNAi Myosin-9could up-regulate the expression of E-cadherin, and down-regulate the expression of N-cadherin, vimentin and3-catenin.After all, we have used a technology platform based on difference gel electrophoresis, MALDI TOF/TOF mass spectrometry and bioinformatics analysis to find and identify the proteins associated with peritoneal metastasis from gastric cancer. And then we conducted the initial functional verification and mechanism study of a differential protein.After the research, four conclusions are drawn as follows:1. RNAi Myosin-9could inhibit the migration and invasion of gastric cancer cell, promote the adhesion of gastric cancer cell, and notably decrease the number of metastatic nodules in the peritoneal cavity of nude mice. These results suggest that Myosin-9can promote the invasion and metastasis of gastric cancer.2. Myosin-9promotes the invasion and metastasis of gastric cancer probably by facilitating the EMT process of gastric cancer, the exact mechanisms still needs further study.The innovation of this study:1. Establishment of peritoneal metastasis of gastric cancer tissue protein finger prints.2. Confirmed Myosin-9’s role in invasion and metastasis of gastric cancer, and found that inhibition of Myosin-9could restrain the EMT process of gastric cancer. |
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