【作者】 王俊普；

【导师】 文继舫； 李景和；

【作者基本信息】 中南大学 ， 基础医学， 2013， 博士

【摘要】 背景胃癌是人类常见的恶性肿瘤之一,国际癌症研究协会最新数据显示其发病率及死亡率高居恶性肿瘤前列。胃癌细胞增殖失控、侵袭、转移及耐药是导致其不良预后的重要原因。胃癌治疗的突破依赖于我们对胃癌发生发展复杂分子机制的深入探索。“非可控性炎症”在胃癌的发生发展进程中扮演着重要角色,在炎症与肿瘤动态网络的可调性与鲁棒性(robustness)辩证关系中,趋化因子受体与其配体是一类重要的非可控性炎症因素。最近有研究发现,趋化因子受体家族在数种肿瘤细胞中高表达,在肿瘤的发生、增殖、生长、侵袭、转移、血管新生及耐药等方面发挥重要的调控作用。CXCR1/2即CXCR1和CXCR2,二者具有75.8%的高度相似序列,属于趋化因子受体家族。最初研究表明CXCR1/2主要表达于中性粒细胞,被激活后调节信号通路下游的蛋白靶点(如NF-kB,AP-1, MMP9等),参与化学趋化、钙离子流动、细胞增殖、运动及血管新生的调节等。目前,有研究显示CXCR1/2在某些肿瘤进程中发挥一定的作用。而有关CXCR1/2在胃癌增殖生长、侵袭及转移中扮演何种角色?许多的研究表明MAPK/ERK和PI3K/AKT通路参与调控肿瘤的生物学行为,CXCR1/2是否与MAPK/ERK和PI3K/AKT通路在胃癌调控中有关联?哪些与胃癌细胞凋亡、增殖生长及侵袭转移相关的指标参与到CXCR1/2调控通路中?这些问题值得但尚未被深入研究。目的本课题着眼于一类重要的非可控性炎症因素--趋化因子受体CXCR1/2:1.研究CXCR1/2在胃癌中的表达及其与临床病理特征的关系。2.探究靶向阻断CXCR1/2和CXCR1基因沉默及过表达对胃癌细胞生物学行为的影响及其机制。3.为利用CXCR1/2设计靶向药物治疗胃癌提供有价值的实验基础。方法1.探讨CXCR/2表达与临床病理特征及与调控胃癌细胞生物学行为相关指标的相关性(1)收集胃癌及癌旁组织标本69例,采用免疫组织化学方法(IHC)检测,并分析CXCR1/2表达与临床病理特征之间的相关性；(2)分析CXCR1/2表达与ERK和AKT磷酸化、凋亡增殖生长(bcl-2, bax, CyclinD1, EGFR和ki-67)、血管新生(VEGF和CD34)及侵袭转移(MMP-9, MMP-2, TIMP-2和E-cadherin)等指标的相关性。2.探讨靶向阻断CXCR1/2对胃癌生物学行为的影响及其机制建立体内外Repertaxin(一种CXCR1/2小分子特异性抑制剂,能阻断CXCR1/2与其配体结合)靶向阻断胃癌细胞CXCR1/2体系：Repertaxin单独或联合化疗药物5-氟尿嘧啶(fluorouracil,5-FU)应用到培养的胃癌细胞MKN45和裸鼠成瘤模型中。(1)MTT实验、平板克隆形成实验、流式细胞周期检测及凋亡检测观察胃癌细胞增殖、细胞周期和凋亡情况变化；(2)划痕实验、迁移实验和侵袭实验比较细胞迁移及侵袭性；(3) real-time-PCR检测凋亡增殖生长(bcl-2, bax, CyclinD1, EGFR和ki-67)、血管新生(VEGF)及侵袭转移(MMP-9, MMP-2, TIMP-2和E-cadherin)指标的mRNA水平；(4) Western-blotting检测CXCR1/2、凋亡增殖生长(bc1-2, bax, CyclinD1, EGFR和ki-67)、血管新生(VEGF)及侵袭转移(MMP-9, MMP-2, TIMP-2和E-cadherin)指标的蛋白表达以及ERK和AKT磷酸化；(5)裸鼠成瘤实验：观察裸鼠体内移植瘤生长情况；切取移植瘤,制作切片,观察肿瘤细胞形态；TUNEL法检测细胞凋亡情况。3.探讨CXCR1沉默和过表达对胃癌细胞生物学行为的影响及机制(1) RT-PCR和Western-blotting检测CXCR1在胃黏膜正常细胞系GES-1及胃癌细胞系MKN28、MKN45、BGC823、SGC7901和AGS中的表达,筛选CXCR1低表达和高表达胃癌细胞株；(2)构建CXCR1干扰质粒载体(pYr-1.1-CXCR1-shRNA)、过表达质粒载体(pIRES2-ZsGreen1-CXCR1)及阴性对照质粒载体；建立CXCR1沉默及过表达的稳定转染细胞系；(3)为验证未出现脱靶效应(off-target effects):针对CXCR1干扰质粒载体(pYr-1.1-CXCR1-shRNA),设计构建干扰回复载体(pIRES2-ZsGreen1-CXCR1-Mut),双转染胃癌细胞,进行功能回复实验；(4)MTT实验、平板克隆形成实验和流式细胞周期检测观察癌细胞增殖、细胞周期和凋亡情况变化；(5)划痕实验、迁移实验和侵袭实验比较细胞迁移及侵袭性；(6) real-time-PCR检测凋亡增殖生长(bcl-2, bax, CyclinD1, EGFR和ki-67)、血管新生(VEGF)及侵袭转移(MMP-9, MMP-2, TIMP-2和E-cadherin)指标的mRNA水平；(7) Western-blotting检测CXCR1/2、凋亡增殖生长(bcl-2, bax, CyclinD1, EGFR和ki-67),血管新生(VEGF)及侵袭转移(MMP-9, MMP-2, TIMP-2和E-cadherin)指标的蛋白表达以及ERK和AKT磷酸化；(8)裸鼠成瘤实验：观察裸鼠体内移植瘤生长情况；切取移植瘤,制作HE切片,观察肿瘤细胞形态。结果1. CXCR1/2表达与胃癌的临床病理参数具有相关性(1)CXCR1/2在胃癌组织中的表达高于对应的癌旁组织,CXCR1/2的表达与年龄、性别和分化程度无关,而与胃癌的侵袭深度、淋巴结转移和TNM分期成正相关性。(2) CXCR1/2的表达与pAKT、pERK、Cyclin D1、EGFR、Bcl-2、微血管密度(MVD)、MMP-9和MMP-2的表达具有正相关性,但与AKT、ERK、Ki-67、Bax、VEGF、TIMP-2和E-cadherin的表达不具有相关性。2. Repertaxin靶向阻断CXCR1/2改变胃癌生物学行为并增敏5-FU(1) Repertaxin阻断CXCR1/2能诱导细胞周期阻滞和凋亡发生,阻止细胞增殖生长,抑制细胞迁移和侵袭；Repertaxin和5-FU联合应用效果更明显。(2) Repertaxin能降低ERK1/2和AKT磷酸化水平,Repertaxin和5-FU联合应用效果更明显。(3) Repertaxin能抑制bcl-2、cyclin D1、EGFR、VEGF、MMP2和MMP9的表达,而促进bax、TIMP-2和E-cadherin的表达；Repertaxin和5-FU联合应用效果更明显。3. CXCRl沉默和过表达能改变胃癌细胞生物学行为(1)CXCR1干扰未出现脱靶效应。(2)CXCR1沉默能诱导细胞周期阻滞和细胞凋亡,降低细胞增殖生长,抑制细胞迁移和侵袭；而CXCR1过表达则能促进细胞分裂、增殖生长、细胞迁移和侵袭,降低瘤细胞凋亡比例。(3)CXCR1沉默能降低ERK1/2和AKT磷酸化水平。(4)CXCR1沉默能抑制bcl-2、cyclin D1、EGFR、VEGF、MMP2和MMP9的表达,促进bax和E-cadherin的表达。结论：1.化学趋化因子受体CXCR1/2在胃癌组织和胃癌细胞系中高表达,发现CXCR1/2表达与侵袭、转移及TNM分期成正相关。2.在胃癌中CXCR1/2与其配体结合能激活MAPK/ERK和PI3K/AKT信号通路,通过调节与凋亡增殖生长(Bcl-2、Bax、 CyclinD1、EGFR和VEGF)、侵袭转移(MMP-9、MMP-2、TIMP-2和E-cadherin)相关指标中bcl-2、CyclinD1、EGFR、VEGF、MMP2和MMP9的上调以及bax、TIMP2和E-cadherin的下调,进而参与调控胃癌细胞的增殖生长、侵袭及转移,最终影响胃癌的发展。更多还原

【Abstract】 BackgroundGastric cancer is one of the most common causes of cancer-related deaths worldwide, and the data from International Agency for Research on Cancer show that the morbidity and mortality of gastric cancer ranges the forefront of the malignant tumors. Uncontrolled proliferation, invasion, metastasis and drug resistance are the major poor prognostic factors for gastric cancer. Therefore, improvement in the therapy of gastric cancer now depends on improving our knowledge of the complicated molecular mechanisms governing the progression and aggressiveness of the disease. Non-resolving inflammation plays a critical role in the development and process of gastric cancer. In the dialectical relationship between inflammation and tumor dynamic network scalability and robustness, chemokine receptors and their ligands are an important class of non-controlled inflammatory factors, and they express in some tumors and involve in tumor carcinogenesis, proliferation, growth, invasion, metastasis, and drug-resistance process.C-X-C chemokine receptor1/2(CXCR1/2), including CXCR1and CXCR2, belong to chemokine receptor families. Initial studies indicated that CXCR1/2express mainly on neutrophils and are originally characterized by their ability to regulate downstream protein targets (such as NF-kB, AP-1, MMP9, etc.) of signaling pathways and then induce chemotaxis of leukocytes, calcium ion flow, cell proliferation, movement, and regulation of angiogenesis. Recently, it was found that CXCR1/2are overexpressed in numerous solid tumors, and the studies revealed a close correlation with proliferation, angiogenesis, invasion, metastasis and drug resistance of the tumor. Although there have been some studies on CXCR1/2in several cancer types and there have been a few reports on expression of CXCR1/2in gastric carcinoma, the expression and functional roles of CXCR1/2in gastric adenocarcinoma progression remain controversial and unclear. ObjectiveThis study focused on an important class of non-resolving inflammation factors CXCR1/2to detect the expression of CXCR1/2in gastric cancer, explore its relationship with clinical and pathological features and study the changes of biological behavior of gastric cancer cells and their mechanisms after targeted blocking CXCR1/2and CXCR1gene silencing and over-expression. The project may provide some valuable experimental evidence for designing targeted drugs of CXCR1and/or CXCR2to treat gastric cancer.Methods1. Studying the correlation between the expression of CXCR1/2and clinicopathological factors, and and relevant indicators expression of regulating gastric cancer cell biological behaviors in patients with gastric carcinoma.(1)69primary and sporadic gastric adenocarcinoma tissue samples and their corresponding non-neoplastic mucosa specimens was retrieved, and we detected CXCR1/2expression by IHC and discussed its correlation with clinicopathological factors.(2) we analyzed the correlation between the expression of CXCR1/2and ERK1/2and AKT phosphorylation, and relevant indicators expression of the proliferation, growth and apoptosis (Bcl-2and Bax, CyclinDl, EGFR and Ki-67), angiogenesis(VEGF and CD34), invasion and metastasis (MMP-9, MMP-2, TIMP-2and E-cadherin), which are involved in the regulation of tumor proliferation, growth, angiogenesis, invasion and metastasis.2. Investigating the changes of gastric cancer cell biological behaviors and the mechanism of inhibiting CXCR1/2. The system of Repertaxin (a small molecule inhibitor of CXCR1/2activation) blocking CXCR1/2was established in vivo and in vitro. Repertaxin alone,5-FU alone, Repertaxin and5-FU in combination, and a control group were applied to gastric cancer cells and nude mice model.(1) Cell proliferation, cycle and apoptosis of gastric cancer cells were investigated by MTT assay, colony forming assay, cell cycle analysis and apoptosis assay. (2) Migration and invasion of gastric cancer cells were detected by wound-healing assay, cell migration and invasion assay.(3) It was detected that the mRNA level of the proliferation, growth, apoptosis(bcl-2, bax, CyclinDl, epidermal growth factor receptor EGFR and ki-67), angiogenesis(vascular endothelial growth factor VEGF), invasion and metastasis (matrix metallopeptidase MMP9, MMP2, tissue inhibitor of metalloproteinases TIMP2and E-cadherin) related indicators were by real-time-PCR.(4) It was assayed that the extracellular signal regulated protein kinase ERK1/2and AKT phosphorylation and expression of proliferation, growth, apoptosis(bcl-2, bax, CyclinDl, EGFR and ki-67), angiogenesis(VEGF), invasion and metastasis(MMP9, MMP2, TIMP2and E-cadherin) related indicators by Western-blotting.(5) Nude mice model:The growth of transplanted tumors was observated. After three weeks of the therapy, all mice were euthanized according to institutional guidelines, and local tumors were resected and analyzed. A part of every tumor tissue was fixed and processed for immunohistochemistry and TUNEL assay.3. Exploring the changes of gastric cancer cell biological behaviors and the mechanism of CXCR1gene silencing and over-expression.(1) The CXCR1expression was investigated by RT-PCR and Western-blotting in normal gastric mucosa cell line GES-1and gastric cancer cell lines(MKN28, MKN45, BGC823, SGC7901and AGS) to identify the cell lines of CXCR1over expression and that of CXCR1low expression.(2) Constructing interference vector (pYr-1.1-CXCR1-shRNA), over-expression plasmid vector (pIRES2-ZsGreenl-CXCR1) and negative control plasmid vector, and establishing the stable transfected cell lines of CXCR1silencing and over-expression.(3) Investigating off-target effects, for CXCR1interference plasmid vector, designing and constructing interference reverted plasmid vector (pIRES2-ZsGreenl-CXCR1-Mut), gastric cancer cells were doubly transfected to carry out functional rescue experiment.(4) Cell proliferation, cycle and apoptosis were investigated by MTT assay, colony forming assay, cell cycle analysis and apoptosis assay.(5) Migration and invasion of gastric cancer cells were detected by wound-healing assay, cell migration and invasion assay.(6) It was detected that the mRNA level of the proliferation, growth, apoptosis(bcl-2, bax, CyclinD1, EGFR and ki-67), angiogenesis (VEGF), invasion and metastasis(MMP9, MMP2, TIMP2and E-cadherin) related indicators were by real-time-PCR.(7) It was assayed that CXCR1, ERK1/2and AKT phosphorylation and expression of the proliferation, growth, apoptosis(bcl-2and bax, CyclinD1, EGFR and ki-67), angiogenesis(VEGF), invasion and metastasis(MMP9, MMP2, TIMP2and E-cadherin) related indicators by Western-blotting.(8) Nude mice model:The growth of transplanted tumors was observed. After three weeks of the therapy, all mice were euthanized according to institutional guidelines, and local tumors were resected and analyzed. A part of every tumor tissue was fixed and processed for HE and immunohistochemistry assay.Results4. CXCR1/2expression was associated with clinicopathological parameters of gastric cancer.(1) The CXCR1/2expression was significantly higher in gastric carcinoma compared to corresponding non-neoplastic mucosa tissues and was significantly associated with invasion, lymph node metastasis and TNM staging. However, no correlation was observed between CXCR1/2expression and gender, age and tumor differentiation.(2) Correlation analysis between CXCR1/2expression and the indicators expression of the phosphorylation(AKT, ERK, pAKT and pERK), proliferation, growth and apoptosis(Bcl-2, Bax, Cyclin Dl, EGFR and Ki-67), angiogenesis(VEGF and MVD), invasion and metastasis (MMP-9, MMP-2, TIMP-2and E-cadherin) by using the Spearman correlation test showed that CXCR1/2expression significantly correlated with pAKT, pERK, Cyclin D1, EGFR, Bcl-2, MVD, MMP-9and MMP-2, but CXCR1/2and AKT, ERK, Ki-67, Bax, VEGF, TIMP-2and E-cadherin expression had no significant correlation in gastric carcinoma.5. Repertaxin blocking CXCR1/2could change gastric cancer cell biological behaviors and enhance the effects of5-FU on gastric cancer in vitro and xenografts.(1) Repertaxin alone effectively induced gastric cancer cell cycle arrest, apoptosis, retarded proliferation and growth, and inhibited gastric cancer cell migration and invasion. The efficacy Repertaxin and5-FU in combination was much better than that of Repertaxin alone or5-FU alone.(2) Repertaxin repressed the phosphorylation of AKT and ERK1/2. The efficacy Repertaxin and5-FU in combination was much better than that of Repertaxin alone or5-FU alone.(3) Repertaxin decreased the expression of bcl-2, cyclin D1, EGFR, VEGF, MMP2and MMP9, and increased the level of bax, TIMP2and E-cadherin. The efficacy Repertaxin and5-FU in combination was much better than that of Repertaxin alone or5-FU alone.6. CXCR1silencing and over-expression could change gastric cancer cell biological behaviors.(1) The off-target effects didn’t appear in the CXCR1interference.(2) CXCR1silencing induced gastric cancer cell cycle arrest and apoptosis, retarded proliferation and growth, and inhibited gastric cancer cell migration and invasion. CXCR1over-expression promoted gastric cancer cell cycle, proliferation, growth, migration and invasion, and inhibited gastric cancer cell apoptosis.(3) CXCR1silencing decreased the phosphorylation level of AKT and ERK1/2.(4) CXCR1silencing inhibited the expression of bcl-2, CyclinDl, EGFR, VEGF, MMP2and MMP9, and promoted the expression of bax and E-cadherin.Conclusion:1. CXCR1/2was overexpressed in gastric cancer tissue and gastric cancer cells, and for the first time it was found that CXCR1/2expression was correlated positively with the proliferation, invasion, development and progression of gastric cancer.2. For the first time, it was found that CXCR1/2binding its ligands promoted the proliferation, invasion and metastasis of gastric cancer by inhibiting ERK1/2and AKT signaling pathway, and hereafter significantly decreasing the expression of bcl-2, cyclin D1, EGFR, VEGF, MMP2and MMP9, and increasing the level of bax, TIMP2and E-cadherin.更多还原