| Cancer is a leading cause of death in the world. According to GLOBOCAN 2012, the most commonly diagnosed cancers worldwide were those of the lung, breast and colorectum, and the most common causes of cancer death were cancers of the lung, liver and stomach. The report also shows that cancer incidence and mortality of China is the number one in the world. According to cancer statistics in 2014 from The National Central Cancer Registry of China, the most commonly diagnosed cancers nationwide of top three were the lung, stomach and liver cancer, and the most common causes of cancer death of top three were the lung, liver and stomach cancer. Thus it can be seen that cancer, even gastric cancer constitutes an enormous burden on human health in China, so it is of great importance to prevention, diagnosis and treatment for cancer.Gastric cancer is a complex and multi-factorial process disease, resulting from the interaction between genetic and environmental factors that potentially deregulate cell oncogenic signaling pathways to promote gastric cancer development. Common methods for treating gastric cancer patient are surgery, chemotherapy and radiotherapy. But early gastric cancer is difficult to diagnose due to the histological and genetic heterogeneity, and most patients are asymptomatic in the initial stages, making it difficult to control the malignancy rate, so gastric cancer patients are often diagnosed after the disease has progressed to the advanced stage where the long term outlook is very poor(5-year survival rate of 10%-30%). In recent years, the development of molecular targeted therapy has led to a revolutionary breakthrough in clinical therapy and become the hope of cancer treatment. But targeted therapy still exist many obstacles, especially cancer driving factors, the key signaling pathways and ideal targets of cancer cells were unclear, so effective drug were so rare. Therefore, it is very important to effectively solve early diagnostic markers and clinical treatment of targeted drugs for gastric cancer. Thus, a better revelation of gastric carcinogenesis development can provide important novel biomarkers for gastric cancer, and also can provide candidate molecular targets and new strategy for clinical treatment.In this study, our objective was to investigate gastric cancer-related differential proteins in the proteome of pairs of patients’ cancer tissues and adjacent normal tissues to find any potential molecular and signaling networks, to reveal potential carcinogenic mechanisms, and to evaluate any specific biomarkers for gastric cancer diagnosis and treatment by a coupled label-free MS approach, in silico analysis and experimental verification. In addition, we investigate that molecular mechanism of heterogeneous nuclear ribonucleoprotein D(hnRNPD) in the process of gastric cancer at cellular level by immunofluorescence, gene transfection, flow cytometry, MTT, Transwell chamber, Quantitative Real-time PCR(qRT-PCR), immune coprecipitation and western blot, as to provide a potential target for gastric cancer.We obtained the following results and conclusions through a series of experimental research.First, a total of 3100-3700 proteins were identified in pairs of gastric cancer and adjacent normal tissues, respectively. While 146 dysregulated proteins were reliably quantified after limited selection in these proteins by a coupled label-free MS approach. Of 146 proteins, 81 proteins were downregulated and 65 proteins were upregulated in gastric cancer tissues.Second, 146 differentially expressed proteins were closely related to cancer and nucleic acid binding proteins of 146 differentially expressed proteins comprised the largest group by in silico analysis. Molecular functions of these proteins were mostly concerned with nucleic acid binding, catalytic, structural molecule and transporter activity. Also these proteins could interact with each other and formed strong networks. Comprehensive analysis by in silico analysis revealed that many enriched biological process and signal pathway take part in the development of gastric cancer, for example metabolic and energy pathways, oxidative phosphorylation, gene expression, nucleotide synthesis, RNA splicing and processing, and transcriptional regulation.Third, a significant up-regulation of hnRNPA2B1, hnRNPD, hnRNPL and YBX-1 transcription and translation in the most of the ten gastric cancer tissues compared with adjacent tissues by qRT-PCR, western blot and immunohistochemistry(IHC). IHC also revealed nucleo-cytoplasmic shuttling of hnRNPA2B1, hnRNPD and YBX-1, that is hnRNPA2B1, hnRNPD and YBX-1 antibodies displayed a weak to moderate glandular epithelium cell cytoplasm and nucleus staining in adjacent tissues, whereas strong nucleus staining in tumor tissues. HnRNPD and hnRNPL were first reported that high expression in gastric cancer tissues.Finally, in order to explore the relationship between hnRNPD and the development of gastric cancer and its possible molecular mechanisms, we elaborated the following questions. HnRNPD expression was distributed in the nucleus of gastric cancer cells by immunofluorescence again. HnRNPD can promote gastric cancer cell proliferation and migration, regulate cell cycle, inhibit cell apoptosis by MTT, Transwell chamber and flow cytometry. Also hnRNPD can combine with a series of cell cycle regulatory factors and proto-oncogenes mRNA to mediated its expression level, and very likely take part in YBX-1-PI3K/AKT signaling pathways, involving in gastric cancer cell proliferation, to affect the gastric cancer development by RNA-IP, qRT-PCR and western blot. |
PDF Full Text Request