基于定量蛋白质组学研究OLFM4在胃癌发生发展中的作用机制

发布时间：2018-05-10 14:11

本文选题：胃癌 + 蛋白质组学　；参考：《重庆医科大学》2016年硕士论文

【摘要】：目的:胃癌是危害人类健康的常见恶性肿瘤之一,名列全球第五大常见肿瘤。据最新统计,全球每年新发病例约95万人,死亡约72万人。胃癌病人的预后与肿瘤分期密切相关,而肿瘤的分期与肿瘤浸润深度、局部淋巴结和远端脏器是否转移相关。目前胃癌的发病及转移的机制尚不明确。因此深入研究胃癌的发生及转移机制显得尤为重要。近年来,蛋白质组学技术的快速发展为我们研究疾病的发病机制提供了强有力的工具。本研究旨在运用同位素标记的相对和绝对定量技术(iTRAQ)寻找与胃癌发病相关的蛋白,探讨其在胃癌发生发展中的作用机制。方法:本研究运用iTRAQ技术对胃癌组织和癌旁组织进行定量蛋白质组学研究,收集15例胃癌病人的胃癌组织和癌旁组织,通过i TRAQ筛选出胃癌组织和癌旁组织中差异表达的蛋白分子。从筛选出的蛋白分子中进一步选出几个蛋白,运用免疫印迹、实时定量PCR验证在蛋白水平、RNA水平差异蛋白的表达情况,并且用组织芯片进行免疫组化染色在组织水平验证差异蛋白的表达。然后运用胃癌细胞株模型,利用小干扰RNA技术特异性的下调蛋白的表达后观察其对胃癌细胞株生物学行为的影响。结果:通过iTRAQ技术以及质谱分析,共筛选出了134个差异表达的蛋白,其中51个蛋白上调,83个蛋白下调。通过免疫印迹和实时定量PCR验证了AZU1、CPVL、OLFM4、VIL1等差异蛋白的表达,发现AZU1、CPVL、OLFM4、VIL1在胃癌组织的表达明显高于癌旁组织,免疫组化检测也有同样的结果。结果与iTRAQ结果一致。从差异蛋白中筛选出OLFM4进行下一步的研究,在两株胃癌细胞AGS、MKN28中通过小干扰RNA技术下调胃癌细胞株中OLFM4的表达。结果发现胃癌细胞株中OLFM4的表达下调后,胃癌细胞的增殖生长能力,迁移和侵袭能力均明显降低。结论:本研究成功的运用了定量蛋白质组学方法筛选胃癌组织与癌旁组织中差异表达的蛋白,通过进一步的验证表明iTRAQ技术的结果是可靠的。同时也发现过表达的OLFM4在胃癌的发病和转移过程可能起了一定的促进作用,而降低OLFM4的表达可以抑制胃癌细胞的增殖生长能力、迁移侵袭能力。
[Abstract]:Objective: gastric cancer is one of the most common malignant tumors, which is the fifth most common tumor in the world. According to the latest statistics, the world's annual new cases of about 950000 people, about 720000 deaths. The prognosis of gastric cancer patients is closely related to the tumor stage, and the tumor stage is related to the depth of tumor invasion, the metastasis of local lymph nodes and distal organs. At present, the pathogenesis of gastric cancer and metastasis is not clear. Therefore, it is very important to study the pathogenesis and metastasis of gastric cancer. In recent years, the rapid development of proteomics technology provides a powerful tool for us to study the pathogenesis of diseases. The aim of this study was to explore the role of iTRAQ in the pathogenesis and progression of gastric cancer by using the relative and absolute quantitative techniques of isotopic labeling. Methods: the iTRAQ technique was used to study the quantitative proteomics of gastric cancer tissues and paracancerous tissues in 15 patients with gastric cancer. I TRAQ was used to screen differentially expressed protein molecules in gastric cancer tissues and paracancerous tissues. Several proteins were further selected from the selected protein molecules. The expression of differential proteins at the protein level was verified by Western blot and real-time quantitative PCR. Tissue microarray immunohistochemical staining was used to verify the differential protein expression at the tissue level. Then the biological behavior of gastric cancer cell line was observed by using a small interfering RNA technique to specifically down-regulate the expression of protein in gastric cancer cell line model. Results: 134 differentially expressed proteins were screened by iTRAQ and mass spectrometry, among which 51 proteins were up-regulated and 83 proteins were down-regulated. The differential protein expression of AZU1CPVLFL-OLFM4 / VIL1 was verified by Western blot and real-time PCR. It was found that the expression of AZU1CPVLFL-OLFM4 / VIL1 was significantly higher in gastric cancer tissues than that in adjacent tissues, and the same results were obtained by immunohistochemistry. The results were consistent with those of iTRAQ. OLFM4 was screened out from the differential protein for further study. The expression of OLFM4 was down-regulated by small interfering RNA in two gastric cancer cell lines AGSN MKN28. The results showed that the ability of proliferation, migration and invasion of gastric cancer cells decreased significantly after the expression of OLFM4 was down-regulated. Conclusion: quantitative proteomics was successfully used to screen differentially expressed proteins between gastric cancer tissues and paracancerous tissues, and the results of iTRAQ technique were proved to be reliable. It was also found that overexpression of OLFM4 might play a role in the pathogenesis and metastasis of gastric cancer, while the decrease of OLFM4 expression could inhibit the proliferation and invasion of gastric cancer cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.2

【参考文献】