β3GnT8调控胃肠道肿瘤侵袭转移和化疗耐药的机制研究

发布时间：2018-04-21 02:04

本文选题：β3GnT8 + 胃癌　；参考：《苏州大学》2016年博士论文

【摘要】：目的胃癌和结肠癌是常见的消化道系统恶性肿瘤,发病率和死亡率较高。尽管临床上对胃肠道肿瘤采取了手术、放疗、化疗、生物治疗等综合治疗措施,但治疗效果差,仍有相当数量的患者最终死于癌灶的复发和转移,给社会和家庭带来了沉重的经济及精神负担。β1,3-N乙酰氨基葡萄糖基转移酶Ⅷ(β3Gn T8)以UDP-Glc NA为供体,将Glc NAc添加到三天线N-聚糖Galβ1-4Glc NAc的非还原性末端形成多聚乳糖胺型糖链。本课题从基因到糖链、从细胞内到细胞外等多个层次和水平上,通过对糖基转移酶β3Gn T8、多聚乳糖胺型N-糖链展开系统研究,以阐释β3Gn T8调控胃癌侵袭转移和结肠癌化疗耐药的分子机制。方法(1)研究β3Gn T8调控胃癌细胞侵袭转移的分子机制(1)运用胃癌组织芯片采用免疫组化染色观察β3Gn T8、多聚乳糖胺的表达,并将结果与患者临床病理参数进行统计分析,进而阐明β3Gn T8、多聚乳糖胺的表达水平与胃癌患者临床病理特征的关系。(2)培养不同来源的三种胃癌细胞株(AGS、SGC-7901、NCI-N87),通过Tanswell侵袭实验和划痕修复实验,比较各实验细胞的侵袭性和迁移性;采用荧光定量PCR和Western blot技术,检测β3Gn T8在各胃癌细胞中的表达高低;采用流式细胞术、Lectin blot、凝集素芯片技术,分析多聚乳糖胺在各胃癌细胞中的表达、结构和功能特点,进而阐明β3Gn T8、多聚乳糖胺的表达与胃癌细胞侵袭转移潜能的关系。(3)通过基因转染和RNA干扰技术靶向调控相应胃癌细胞中β3Gn T8的表达,采用流式细胞术和Lectin blot技术,检测多聚乳糖胺的表达;通过Transwell实验比较各实验细胞的侵袭能力;采用Western blot技术,检测糖蛋白CD147糖基化修饰改变;采用荧光定量PCR、Western blot,分析基质金属蛋白酶MMPs的表达变化,进而阐明β3Gn T8对胃癌细胞侵袭转移的调控机制。(4)采用免疫沉淀技术富集胃癌细胞中β3Gn T8的相互结合蛋白,通过SDSPAGE对免疫沉淀复合物进行分离。然后从胶中切取染色明显的蛋白条带,经过脱色、还原、烷基化、酶解等处理,进行质谱分析。搜索SWISS-PROT数据库,进而筛选和鉴定胃癌细胞中β3Gn T8的靶蛋白。(5)采用PNGase F酶解释放胃癌细胞中总糖蛋白N-糖链,对所得N-糖链用Sep-Pak C18柱纯化后进行完全甲基化衍生,质谱解析N-糖链的结构轮廓,进而阐明胃癌细胞中N-糖链变化规律。(2)研究β3Gn T8调控结肠癌细胞化疗耐药的分子机制(1)采用荧光定量PCR技术检测β3Gn T8在结肠癌5-氟尿嘧啶(5-FU)耐药细胞SW620/5-FU及其母细胞SW620中的表达水平;采用流式细胞术、Lectin blot检测多聚乳糖胺在SW620/5-FU及SW620中的表达特征,进而阐明β3Gn T8、多聚乳糖胺的表达与结肠癌细胞化疗耐药的关系。(2)通过RNA干扰技术抑制SW620/5-FU细胞中β3Gn T8的表达,采用流式细胞术、Lectin blot检测多聚乳糖胺的表达改变;采用MTT药物敏感性实验检测细胞耐药性状的改变;采用Annexin V-FITC/PI双染法检测细胞凋亡;采用PI单染检测细胞周期,进而阐明β3Gn T8对结肠癌细胞化疗耐药的调控机制。(3)通过多聚乳糖胺合成抑制剂3'-叠氮胸苷(AZT)干预SW620/5-FU细胞中多聚乳糖胺的合成,采用MTT药物敏感性实验检测细胞耐药性状的改变,进而阐明多聚乳糖胺对结肠癌细胞化疗耐药的调控机制。结果(1)β3Gn T8高表达通过诱导多聚乳糖胺的合成进而促进胃癌细胞侵袭转移(1)β3Gn T8阳性产物主要定位于胃癌组织细胞的胞核和胞浆中,呈棕黄色颗粒状分布。多聚乳糖胺阳性表达主要集中在胃癌组织细胞的胞浆,表现为胞浆内呈现棕黄色或者棕褐色颗粒,也有少量为胞膜中呈现棕黄色。胃癌患者中β3Gn T8的表达与年龄、性别、分化程度无明显相关性,但是与肿瘤的病理分级和临床分期正相关。胃癌患者中多聚乳糖胺的表达与肿瘤病理分级和临床分期存在正相关,而与年龄、性别、分化程度不具有相关性。β3Gn T8、多聚乳糖胺在胃癌组织中的表达强度呈正相关关系。(2)胃癌AGS细胞的侵袭转移能力最强,其次是SGC-7901细胞,而NCI-N87细胞的侵袭转移能力最弱。AGS细胞中β3Gn T8基因和蛋白表达最高,其次是SGC-7901细胞,而NCI-N87细胞中β3Gn T8基因和蛋白表达最低。AGS细胞中多聚乳糖胺含量最多,其次是SGC-7901细胞,而NCI-N87细胞中多聚乳糖胺含量最少。β3Gn T8、多聚乳糖胺在胃癌细胞中的表达水平存在一致性。(3)上调胃癌NCI-N87细胞中β3Gn T8的基因表达,多聚乳糖胺的含量上升,细胞侵袭能力增强,高糖基化(HG)-CD147表达升高,MMP-14的基因和蛋白表达增多。下调胃癌AGS细胞中β3Gn T8的基因表达,多聚乳糖胺的含量下降,细胞侵袭能力减弱,高糖基化(HG)-CD147表达降低,MMP-14的基因和蛋白表达受到抑制。(4)在胃癌AGS细胞中,共分离鉴定出45种可与β3Gn T8相互结合的蛋白;在NCI-N87细胞中,共分离鉴定出32种可与β3Gn T8相互结合的蛋白。通过对比分析,发现AGS和NCI-N87细胞中存在23种可与β3Gn T8结合的共有蛋白。针对这些蛋白进行功能研究,并结合免疫共沉淀技术,发现膜联蛋白A2(annexin A2,ANXA2)是β3Gn T8潜在靶蛋白。(5)胃癌AGS、SGC-7901、NCI-N87细胞中所含的N-糖链种类没有明显差别,但存在量上的差异。AGS细胞中岩藻糖和唾液酸的N-糖链含量均很少;SGC-7901细胞中岩藻糖的N-糖链所占比例明显高于其他两种细胞;NCI-N87细胞中唾液酸的N-糖链所占比例明显高于其他两种细胞。(2)β3Gn T8高表达通过诱导多聚乳糖胺的合成进而促进结肠癌细胞耐药(1)与结肠癌SW620细胞相比,耐药细胞SW620/5-FU中β3Gn T8的表达和多聚乳糖胺的含量均明显增加。(2)抑制SW620/5-FU细胞中β3Gn T8的基因表达,多聚乳糖胺的含量下降,对化疗药物5-FU的敏感性上升,细胞凋亡增多,G1期细胞减少,而S期和G2/M期细胞增加。(3)AZT可有效抑制SW620/5-FU细胞中多聚乳糖胺的合成,提高其对化疗药物5-FU的敏感性。结论(1)β3Gn T8、多聚乳糖胺在胃癌组织中高表达,且均与胃癌患者的病理分级和临床分期有关联,二者在胃癌组织的表达具有正相关性,提示其与胃癌的恶性进展有密切关系。随着胃癌细胞侵袭转移能力的增强,β3Gn T8的表达和多聚乳糖胺的含量也随之升高。β3Gn-T8通过催化CD147分子中多聚乳糖的合成调控MMP-14的表达,从而改变胃癌细胞侵袭转移能力,这可能需要ANXA2的协同作用。此外,N-糖链表达异常也与其胃癌细胞恶性表型有关。(2)β3Gn T8和多聚乳糖胺在结肠癌5-FU耐药细胞中高表达。抑制β3Gn T8的表达和多聚乳糖胺的生物合成,可增强结肠癌耐药细胞对5-FU的敏感性,实现其耐药性的逆转。总之,β3Gn T8及多聚乳糖胺在调控胃肠道肿瘤侵袭转移和化疗耐药过程中起着关键作用。这一研究为针对胃肠道肿瘤治疗相关分子药物设计、筛选提供新的靶点,为临床个体化治疗提供基础,具有重要的科学意义和实用价值。
[Abstract]:Objective gastric cancer and colon cancer are common malignant tumors of the digestive tract, with high morbidity and mortality. Despite the clinical treatment of gastrointestinal tumors, such as surgery, radiotherapy, chemotherapy and biological treatment, the curative effect is poor, and a considerable number of patients eventually die from the recurrence and metastasis of the cancer and bring to the society and family. The heavy economic and spiritual burden. The beta 1,3-N acetylglucosaminotransferase VIII (beta 3Gn T8), with UDP-Glc NA as the donor, added Glc NAc to the non reductive terminal of the three antenna N- Gal beta 1-4Glc NAc to form a polygalactamine chain. This subject has passed from genes to sugar chains, from intracellular to extracellular and other levels and levels. A systematic study of glycosyltransferase beta 3Gn T8 and poly lactamamine type N- chain expansion to explain the molecular mechanism of beta 3Gn T8 regulating invasion and metastasis of gastric cancer and chemotherapeutic resistance of colon cancer. Method (1) the molecular mechanism of the invasion and metastasis of gastric cancer cells by beta 3Gn T8 was studied (1) using immunohistochemical staining of gastric carcinoma tissue to observe the beta 3Gn T8 and polygalamines. The expression of the results and the clinicopathological parameters of the patients were analyzed, and the relationship between the expression level of beta 3Gn T8 and polygalamines and the clinicopathological features of gastric cancer patients. (2) to cultivate three kinds of gastric cancer cell lines (AGS, SGC-7901, NCI-N87) from different sources, through the Tanswell invasion experiment and the scratch repair experiment, to compare the experimental cells. The expression of beta 3Gn T8 in gastric cancer cells was detected by fluorescence quantitative PCR and Western blot. Flow cytometry, Lectin blot, and agglutinin chip technology were used to analyze the expression, structure and energy characteristics of polygalamine in gastric cancer cells, and then clarify the expression of beta 3Gn T8 and the expression of polygalamine. The relationship between the invasion and metastasis potential of gastric cancer cells. (3) the expression of beta 3Gn T8 in the corresponding gastric cancer cells was regulated by gene transfection and RNA interference technique. Flow cytometry and Lectin blot were used to detect the expression of polyamines; the invasion ability of the experimental cells was compared by Transwell experiment; and the Western blot technology was used to detect the sugar eggs. White CD147 glycosylation modification, fluorescence quantitative PCR, Western blot, analysis of the expression changes of matrix metalloproteinase MMPs, and further elucidate the regulation mechanism of the invasion and metastasis of gastric cancer cells by beta 3Gn T8. (4) enrichment of the mutual binding protein of beta 3Gn T8 in gastric cancer cells by immunoprecipitation technique, and the immunoprecipitation complex by SDSPAGE. Then, the obvious protein bands were cut from the glue, and the SWISS-PROT database was used to search and identify the target protein of the beta 3Gn T8 in gastric cancer cells. (5) the PNGase F enzyme was used to explain the sugar chain of the total glycoprotein N- in the gastric cancer cells and the Sep-Pak C18 in the N- chain. After purification, complete methylation was carried out, the structural profile of N- sugar chain was analyzed by mass spectrometry, and then the changes of N- sugar chain in gastric cancer cells were elucidated. (2) the molecular mechanism of chemotherapy resistance of colon cancer cells by beta 3Gn T8 was studied (1) the fluorescence quantitative PCR technique was used to detect the 3Gn T8 in the colon cancer 5- fluorouracil (5-FU) resistant cells SW620/5-FU and its mother The expression level in cell SW620; using flow cytometry, Lectin blot to detect the expression of polygalamines in SW620/5-FU and SW620, and further elucidate the relationship between the expression of beta 3Gn T8, the expression of polygalamines and chemotherapeutic resistance of colon cancer cells. (2) the expression of beta 3Gn T8 in SW620/ 5-FU cells was suppressed by RNA interference technique, and flow cytometry was used. In blot detected the changes in the expression of polygalactamine; detected the change of cell resistance by MTT drug sensitivity test; Annexin V-FITC/PI double staining was used to detect cell apoptosis; PI single staining was used to detect cell cycle, and then the regulation mechanism of beta 3Gn T8 to chemotherapy resistance of colon cancer cells was elucidated. (3) 3'synthesis of inhibitor 3' by polygalamines. - azidoside (AZT) interfered with the synthesis of polygalactamine in SW620/5-FU cells. The sensitivity test of MTT was used to detect the change of cell resistance characteristics and the regulation mechanism of polygalactamine on chemotherapy resistance of colon cancer cells. Results (1) the high expression of beta 3Gn T8 was induced by the synthesis of polygalamine to promote the invasion of gastric cancer cells. The positive products of transfer (1) beta 3Gn T8 were mainly located in the nucleus and cytoplasm of gastric carcinoma tissue cells, with brown and yellow granular distribution. The positive expression of polygalamines mainly concentrated in the cytoplasm of gastric cancer tissue cells, showing brown or brown brown granules in the cytoplasm, and a small amount of brown yellow in the membrane. The beta 3Gn T in gastric cancer patients. The expression of 8 was not associated with age, sex and differentiation, but it was positively correlated with the pathological grade and clinical stage of the tumor. There was a positive correlation between the expression of polygalactamine and the pathological grade and clinical stage of tumor, but not related to age, sex and differentiation. Beta 3Gn T8, polyamines were in gastric cancer tissue. The expression intensity was positive correlation. (2) the invasion and metastasis of gastric cancer AGS cells was the strongest, followed by SGC-7901 cells, while the weakest.AGS cells in the NCI-N87 cells were the weakest.AGS cells with the highest expression of the 3Gn T8 gene and protein, followed by the SGC-7901 cells, while the beta 3Gn T8 gene and protein expression in NCI-N87 cells expressed the lowest concentration of polygalactose in the.AGS cells. The content of amines is the most, followed by SGC-7901 cells, and the content of polygalamines in NCI-N87 cells is the least. The expression level of beta 3Gn T8 and polyamines in gastric cancer cells is consistent. (3) the gene expression of beta 3Gn T8 in gastric cancer NCI-N87 cells, the rise of polygalamines, the enhancement of cell invasiveness, and the -CD147 expression of high glycosylation (HG) in gastric cancer cells are up. The gene and protein expression of MMP-14 increased. The gene expression of beta 3Gn T8 in gastric cancer AGS cells decreased, the content of polygalamine decreased, the cell invasiveness decreased, the expression of high glycosylated (HG) -CD147 decreased, and the expression of MMP-14 gene and protein was inhibited. (4) 45 kinds of beta 3Gn T8 were identified in the gastric cancer AGS cells. In NCI-N87 cells, 32 proteins that could be combined with beta 3Gn T8 were isolated and identified. Through comparative analysis, 23 common proteins associated with beta 3Gn T8 were found in AGS and NCI-N87 cells. The functional study of these proteins and the combination of immunoprecipitation techniques showed that the membrane associated protein A2 (annexin A2, ANXA2) was beta 3G. N T8 potential target protein. (5) there is no significant difference in the N- sugar chain in AGS, SGC-7901, NCI-N87 cells of gastric cancer, but there is a significant difference in the N- sugar chain content of fucose and sialic acid in.AGS cells; the N- sugar chain of fucose in SGC-7901 cells is higher than the other two cells; the N- sugar of sialic acid in NCI-N87 cells The proportion of the chain was significantly higher than that of the other two cells. (2) the high expression of beta 3Gn T8 was induced by the synthesis of polyamines to promote the drug resistance of colon cancer cells (1), compared with the colon cancer SW620 cells, the expression of beta 3Gn T8 and the content of polylactiamines in the drug resistant cell SW620/5-FU increased significantly. (2) the inhibition of the gene of T8 in SW620/5-FU cells. It was expressed that the content of lactamines decreased, the sensitivity of the chemotherapeutic drug 5-FU increased, cell apoptosis increased, G1 cells decreased, and S and G2/M cells increased. (3) AZT could effectively inhibit the synthesis of polygalactamine in SW620/5-FU cells and improve its sensitivity to chemotherapy drug 5-FU. Conclusion (1) beta 3Gn T8 and polygalamines in gastric cancer tissue The expression of middle and high levels is associated with the pathological grade and clinical stage of gastric cancer patients. The expression of the two is positively related to the expression of gastric cancer, suggesting that it is closely related to the malignant progression of gastric cancer. With the enhancement of the invasion and metastasis of gastric cancer cells, the expression of beta 3Gn T8 and the content of polyamines also increase. Beta 3Gn-T8 through the catalytic C The synthesis of polygalactose in D147 molecules regulates the expression of MMP-14 and changes the invasion and metastasis of gastric cancer cells, which may require a synergistic effect of ANXA2. In addition, the abnormal expression of N- sugar chain is related to the malignant phenotype of gastric cancer cells. (2) the expression of beta 3Gn T8 and Polyamines in colon cancer 5-FU resistant cells is highly expressed and the expression of T8 in beta 3Gn is inhibited and the expression of beta 3Gn T8 is inhibited. The biosynthesis of polygalamines can enhance the sensitivity of colon cancer resistant cells to 5-FU and achieve a reversal of their resistance. In a word, beta 3Gn T8 and polyamines play a key role in regulating the invasion and metastasis of gastrointestinal tumors and chemotherapeutic resistance. This study is to screen for the design of molecular drugs for the treatment of gastrointestinal tumors. It is of great scientific significance and practical value to provide new targets for clinical individualized treatment.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735

【参考文献】