| BackgroundCompared with all kinds of malignant digestive tumor, gastric cancer is the most common one. The occurrence of gastric cancer is related to several mirco factors, such as genes and proteins. The incidence of gastric cancer is high in our country, has been in the first place for several years among all kinds of digestive tumor. A large number of new cases takes place in China every year. At the same time, the incidence and mortality of gastric cancer happened in China lead the third place in the worldwide. In China, gastric cancer ranks as the first leading cause of cancer death in rural areas, and gastric cancer ranks as the second leading cause of cancer death in urban areas. However, the mortality of gastric cancer accounts for first place among malignant tumor. China has a large number of rural people, compared with urban people, the level of education, health condition and economy is quite low. The gap between rural and urban lead to a unique result, which means rural people is suffering a threat of higher incidence and mortality than the people live in city. Recently, there is a rising trend that more and more adenocarcinoma happened in gastroesophageal junction(GCA). The malignant degree of gastric cancer is very high. Though there are many study about the mechanism of gastric cancer on molecular level, the details of etiology is still stay unknown. More effort should be put in the study of gastric cancer.Autophagy is a dynamic process by which cytosolic materials are sequestered into membrane vesicles called autophagosomes, and then delivered to the lysosome for degradation. It’s a lysosomal-mediated cytoplasmic degradation process in the case of hunger, oxidative stress and damage, lack of energy metabolism and accept metabolic pressure from inside and outside cells, which is relative rare in normal circumstances. First all inside or outside stimulation act on cells, induct the cup shape structure of double membrane form the preautophagosome, leading to a significant rise in autophagy level. Autophagy can promote cell material recycling and autophagy body membrane will fall off and recycle. Autophagy has very important biological significance, which is a major contributor to cell metabolism, autophagy can provide endogenous nutrition when lack of external nutrients. And autophagy also plays an important role in cell updates, tissue metabolism and other normal developmental processes. Besides, autophagy also has a relationship with the development of many diseases, such as neurodegenerative diseases, Danon myopathy, etc. Autophagy is a common biological phenomenon in all eukaryotic cell’s metabolic activity, and its also involved in tumor cells. The role of process about autophagy in the occurrence and development of tumor is very complex, we cannot simply think the function of autophagy is promotion or inhibition effect in carcinogenesis. Autophagy can help the mitochondria to form a separation membrane to prevent pro-apoptotic factors from diffusion, then help cells to escape apoptosis; autophagy suppression can reduce the protein degradation and increase anabolism, resulting in the sustained growth of precancerous cells. Currently, many studies have confirmed that autophagy plays an important role in the development of gastric cancer, but the mechanisms and the clinical significance of autophagy in gastric cancer is still unknown.Beclin-1 is the first gene to be found related to autophagy, it is a key factor related with the location of the autophagic protein, and it is also a special gene involved in the formation of the mammalian autophagy regulation system. Autophagy is closely related to the occurrence of a wide variety of tumor development. Beclin-1 can not only promotes the cell autophagy level, but also a kind of specific tumor suppressor factor. Studies have shown that the mutations and mutations of Beclin-1 allele is closely related to the development of several worldwide common malignant diseases, such as liver cancer, lung cancer, lymphoma, breast cancer, etc, which also have relative high fatality rate. But whether the inner mechanism of disease is directly related to autophagy this process remains to be further discussed. Beclin-1 gene can regulate the autophagy activity through forming a new complex with PI3K(phosphatidylinositol 3, kinase), the key sites in the process of autophagy structure, than regulate other ATG protein activation, change the relative space position of ATG protein in the autophagy precursor structure, finally complete the genetic effect on the effective regulation of autophagy. Another important molecular structure of the autophagy is LC3(micro-tubule-associated protein1, light chain 3). The genes of LC3 is associated with autophagy in yeast Atg8 are analogues of the same source. LC3-Ⅱ is one of the existence of LC3 form, its distribution area is quite different from the other form of LC3, which is called LC3-Ⅰ. LC3-Ⅱ mainly exists in the double membrane structure of autophagy, is a very important marker for autophagy research, can pass for LC3- Ⅱ detection and observation, determine whether autophagy and the level of autophagy.PurposeThis article elaborated from several aspects to explore the expression and clinical significance of autophagy related gene expression of Beclin-1 in different staging of gastric cancer tissues. To explore the Beclin-1’s action mechanism in the process of occurrence and development of gastric cancer. Explore the function mechanism of IFN-γ on the occurrence and development of gastric cancer. To provide theoretical basis for finding new therapeutic targets of gastric cancer during the clinical treatment.Method1. In this study, specimens of gastric cancer tissue in Department of General Surgery, the China Japan Union Hospital of Jilin University were collected, from June 2013 to September 2014,among which 16 belonged to stage 1, 40 stage 2, 19 stage 3. Then using technique of immunohistochemistry, RT-PCR and Western Blot test, to detect the m RNA expression and protein level of Beclin-1 and IFN-γ in gastric cancer tissue samples. Flow cytometry is used to analyze the percentage of immune cells, which secrete IFN-γ. 2. Western Blot to detect the expression level of Beclin-1 in BGC – 823, MKN-28 and GES-1 cell lines. Semi-quantitative RT-PCR to detect the expression level of Beclin-1’s m RNA; CCK 8 testing the influence of IFN-γ on BGC- 823 cell line proliferation. Western Blot and semi-quantitative RT-PCR to detect the influence of IFN-γ on BGC- 823 cell line, detecting m RNA expression and protein level of autophagy marker LC3, Beclin-1. Analysis of Beclin-1 in different expression level of apoptosis regulation mechanism of gastric cancer, to detect the m RNA expression and protein level of apoptosis related proteins the Bcl-2, Casepase3. PCR and WB were used to detect the m RNA and protein expression of Beclin-1 an important protein, which relative to cell autophagy. 3. Finally, by building a rodent species tumor transplantation model(conditions for breeding, free food and drink) to detect in vivo induced autophagy occurs whether can inhibit the growth of xenograft tumor. In vitro studies showed that after stimulation of IFN-γ,autophagy-related proteins, Beclin-1 and LC3 were activated. Moreover, activation of autophagy inhibits the xenograft growth in nude mice.Result1. Immunohistochemistry, RT-PCR and Western Blot results show that the Beclin-1 of Ⅰstage and Ⅱstage gastric cancer clinical tissue specimens have higher expression level, the expression level in Ⅲ period gastric cancer clinical tissue samples is lower than the other two stage, and the difference has statistical significance(p < 0.05). Flow cytometry instrument IFN-γ inflammatory markers detection results show that the inflammation factor IFN-γ in phase Ⅰ and Ⅱ also have high level expression in gastric cancer clinical tissue specimens, and the percentage of positive cells Ⅰ and Ⅱ period was associated with a significantly higher. While the result in perid Ⅲ is low. 2. The expression of Beclin-1 is highest in MNK-28, is higher in GES-1 than BGC-823, and a significant difference between GES-1 and BGC-823(P < 0.05). After treatment of IFN-γ in BGC- 823 cell lines, the expression of Beclin-1 and LC3 significantly increased, loss of cell proliferation, apoptosis related proteins expression level has been changed. 3. The in vitro experiment results show that: after IFN-γ stimulus, autophagy related protein expression and LC3 Beclin-1 the quantity will increase. The use of nude mice to establish the xenograft model results show that accept autophagy induced cells inhibits the growth of xenograft tumor in nude mice in vivo, nude mice weight and higher survival rate than the control group obviously.Conclusion1. There is a close relationship between autophagy and the pathological staging of gastric cancer. Beclin-1, INF-γ play an important role in the occurrence and development of gastric cancer. There is a close relationship between Beclin-1 and the pathological features of gastric cancer. Beclin-1 with high expression level in periodⅠ and periodⅡ in gastric cancer tissue samples, and expressed low in Ⅲ stage gastric cancer tissue samples compared with the previous period. 2. IFN-γ also closely related to the occurrence and development of gastric cancer. After IFN-γ stimulate autophagy related genes in gastric cancer tissue Beclin 1 and LC3 expression quantity increase. 3. Autophagy related gene Beclin-1 is closely related to gastric cancer infiltration depth and degree of differentiation, while whether there is any relationship with lymph node metastasis is not clear. 4. INF-γ induce the activation of autophagy, which can inhibit tumor cell proliferation. Promote apoptosis and inhibit the growth of tumor, can obviously reduce the tumor volume and prolong survival time of mice a tumor-burdened. |
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