WWP1在胃癌发生发展中的作用及其初步的分子调控机制

发布时间：2017-12-28 00:15

本文关键词：WWP1在胃癌发生发展中的作用及其初步的分子调控机制　出处：《郑州大学》2015年博士论文　论文类型：学位论文

更多相关文章： WWP1 胃癌 siRNA 细胞增殖 预后

【摘要】：背景和目的胃癌(gastric cancer, GC)是最常见的消化道恶性肿瘤,在肿瘤相关性死亡中居第二位。尽管胃癌的诊断策略和分子标记的研究取得了很大的进展,但是晚期胃癌的预后和生存率仍不理想,胃癌病人的中位存活时间仅为6-10个月。因此,寻找和鉴定新的诊断、预防或治疗胃癌病人的分子标记具有十分重要的意义。含有WW结构域的E3泛素蛋白连接酶1 (WW domain containing E3 ubiquitin protein ligasel, WWP1)定位于人染色体8q21.3区域。人WWP1包含922个氨基酸残基,分子量大约为110 kDa,由1个C2结构域,4个WW结构域和1个HECT结构域组成。WWPl能与多种不同的信号途径相互作用,如Notch信号途径、转化生长因子β(transforming growth factor β, TGFβ)信号途径、丝裂原活化蛋白激酶(mitogen-activated protein kinases, MAPKs)信号途径和表皮生长因子(Epithelial growth factor, EGF)信号途径等,提示其功能上的多样性和复杂性。越来越多的研究表明,WWP1牵涉到多种不同疾病的发生、发展和进展,如感染性疾病、神经性疾病、衰老,特别是肿瘤的发生发展过程。在前列腺癌和乳腺癌中,WWP1 mRNA和蛋白表达上调,在口腔癌和肝癌中表达也显著增加。这些研究表明WWP1可能在这些肿瘤的发生发展及进展中发挥重要的作用。然而,迄今为止,国内外尚未见WWP1在胃癌发生、发展中的作用的研究报道。本研究采用原位杂交、免疫组织化学、实时荧光定量PCR (Real-time qPCR)以及Western blotting等技术检测胃癌组织和正常胃粘膜组织中WWP1蛋白和mRNA的表达,探讨胃癌临床病理学参数与其蛋白和mRNA的表达之间的关系,并通过病人资料随访,采用Kaplan-Meier存活曲线研究WWP1的表达与胃癌病人预后之间的关系,进一步采用Cox比例风险模型分析胃癌病人独立的预后因子；采用Real-time qPCR和Western blotting技术检测多种胃癌细胞系中WWP1mRNA和蛋白的表达；采用RNA干扰技术研究WWP1表达下调对胃癌细胞增殖、细胞周期和凋亡的影响,并应用生物信息学手段分析WWP1可能相互作用的蛋白,利用Western blotting技术验证其可能的相互调控作用；最后建立胃癌的裸鼠移植瘤模型,并采用该模型研究WWP1表达下调对胃癌裸鼠移植瘤生长的影响,并通过Kaplan-Meier存活曲线分析其与裸鼠生存率之间的关系。第1章 WWP1在胃癌组织中的表达及其与胃癌病人预后之间的关系目的分析WWP1在胃癌组织中的表达,初步探讨其在胃癌发生发展中的可能作用。方法(1)采用原位杂交和免疫组化技术分别检测131例胃癌组织和相应的正常胃黏膜组织中WWP1 mRNA和蛋白的表达：采用Real-time qPCR和Western blotting技术检测胃癌组织和相对应的正常胃粘膜组织中WWP1 mRNA和蛋白的表达。(2)采用Kaplan-Meier存活曲线分析WWP1 mRNA和蛋白表达与胃癌病人预后之间的关系,并采用Cox比例风险模型分析胃癌病人独立的预后因子。(3)统计学分析：采用统计学软件SPSS17.0对数据进行分析处理,数据表示为x±s,原位杂交和免疫组化的结果采用X2进行评估,Real-time qPCR和Western blotting结果采用单因素方差分析(One-way ANOVA), WWP1 mRNA和蛋白的表达与胃癌病人预后之间的关系采用Kaplan-Meier存活曲线分析,Cox比例风险模型用来分析胃癌病人的独立预后因子,P＜0.05表示差异具有统计学意义。结果(1)在131例胃癌组织中,WWP1 mRNA和蛋白的阳性表达率分别为66.41%和61.83%,而在131例正常胃黏膜组织中,WWP1 mRNA和蛋白的阳性表达率分别为12.21%和9.92%。WWP1 mRNA和蛋白在胃癌组织和正常胃黏膜组织中的表达水平均具有统计学差异(X2=80.646和76.715,均P=0.000)。(2) Real-time qPCR和Western blotting结果表明,随机选择的10例胃癌组织中WWP1 mRNA和蛋白的相对表达水平均显著高于正常胃黏膜组织,且差异具有统计学意义(P0.05)。(3) WWP1 mRNA和蛋白表达均与胃癌病人肿瘤分化程度、TNM分期、浸润深度和淋巴结转移密切相关(所有P0.05),而与胃癌病人的年龄、性别和肿瘤大小无关(所有P0.05)。(4) Log-rank检验(Mantel-Cox)结果表明,WWP1 mRNA和蛋白低表达的胃癌患者的存活时间均显著长于WWP1 mRNA和蛋白高表达的胃癌患者,且差异具有统计学意义(P分别为0.0134和0.0098)。(5)Cox比例风险模型结果表明,TNM分期、淋巴结转移、WWP1 mRNA和蛋白均可作为胃癌病人独立的预后因子。第2章 WWP1表达下调对胃癌细胞增殖、细胞周期和细胞凋亡的影响及可能的分子调控机制目的探讨WWP1表达下调对胃癌细胞增殖、凋亡和细胞周期的影响及其调控机制。方法(1)采用Real-time qPCR和Western blotting技术检测不同胃癌细胞系和正常胃黏膜上皮细胞GES-1中WWP1 mRNA和蛋白的表达。(2)利用脂质体2000将WWP1 siRNA和对照siRNA分别转染胃癌MKN-45细胞和胃癌AGS细胞。(3)采用Western blotting技术检测转染前后胃癌细胞中WWP1、cyclin D1、 CDK4、Bcl-2和Bax蛋白的表达以及PTEN-Akt信号途径中关键蛋白PTEN、p-Akt和总Akt蛋白表达的变化；采用Caspase-3比色分析试剂盒检测不同方法处理的胃癌细胞中Caspase-3活性的变化。(4)采用CCK-8增殖实验检测WWP1表达下调对胃癌MKN-45和AGS细胞增殖的影响；采用流式细胞术检测WWP1表达下调对胃癌MKN-45和AGS细胞周期和凋亡的影响。(5)采用STRING 9.1在线软件对WWP1的结构及其相互作用的蛋白进行预测分析。(6)统计学处理：采用统计学软件SPSS17.0分析所有的实验数据,数据表示为x±S,采用单因素方差分析(One way ANOVA)比较多个样本的均数,以P0.05表示具有统计学差异。结果(1)所检测的胃癌细胞中WWP1 mRNA和蛋白的相对表达水平均显著高于正常胃粘膜上皮细胞GES-1中WWP1 mRNA和蛋白的相对表达水平,且差异具有统计学意义(P0.05)。此外,KN-45和AGS细胞中WWP1 mRNA和蛋白的相对表达水平显著高于其它各个胃癌细胞,且差异具有统计学意义(P0.05)。(2) WWP1 siRNA组中WWP1蛋白的表达水平均显著低于未处理的MKN-45和AGS以及对照siRNA,且差异具有统计学意义(P0.05)。(3)与未处理组和对照siRNA组相比,转染WWP1 siRNA组中胃癌MKN-45和AGS细胞的增殖均显著受到抑制,且差异具有统计学意义(P0.05)。(4) WWP1 siRNA组中MKN-45和AGS细胞在G0/G1期的比率(64.87±1.48%和64.88±1.34%)分别显著高于未处理组(50.19±1.46%和50.25±3.02%)和对照siRNA组(50.93±1.39%和51.60±2.09%),且差异具有统计学意义(F=98.515和38.528,均P=0.000),而未处理组和对照siRNA组中MKN-45和AGS细胞在G0/G1期的比率无差异(P＞0.05)。此外,WWP1 siRNA组中MKN-45和AGS细胞在S期的比率(20.32±1.25%和23.47±2.26%)分别显著低于未处理组(29.57±1.25%和30.30±0.96%)和对照siRNA组(30.36±1.17%和30.23±1.34%),差异具有统计学意义(F=62.386和17.692,P=0.000和0.003),而未处理组和对照siRNA组中MKN-45和AGS细胞在S期的比率无差异(P＞0.05)。(5)流式细胞术结果表明,WWP1 siRNA组中MKN-45和AGS的早期凋亡率分别为20.56±0.92%和28.99±1.71%,显著高于未处理组(6.95±0.69%和9.5±1.18%)和对照siRNA组(8.35±1.87%和10.0±1.46%),且差异具有统计学意义(F=104.561和171.636,均P=0.000),而未处理组和对照siRNA组之间MKN-45和AGS细胞的早期凋亡率无差异(P0.05)。WWP1 siRNA组中MKN-45和AGS的总凋亡率分别为22.95±1.92%和36.2±4.36%,显著高于未处理组(8.38±1.03%和11.49±0.48%)和对照siRNA组(9.9±1.29%和11.48±0.88%),且差异具有统计学意义(F=90.441和91.844,均P=0.000),而未处理组和对照siRNA组之间MKN-45和AGS细胞的总凋亡率无差异(P＞0.05)。(6) WWP1 siRNA组中MKN-45和AGS的CDK4、cyclin D1和Bcl-2蛋白表达水平显著低于对照siRNA组和未处理组,而Bax蛋白的表达和Caspase-3的活性均显著高于对照siRNA组和未处理组,差异具有统计学意义(P0.05)。(7) STRING 9.1软件预测结果表明,WWP1与很多蛋白可能具有潜在的相互作用,如PTE、TGFβ1、STAT3和SMAD7等。(8)与未处理组和对照siRNA组相比,WWP1 siRNA组中MKN-45和AGS细胞中PTEN蛋白的表达显著上调,而p-Akt蛋白表达显著下调,但不改变总Akt蛋白表达。第3章 WWP1在胃癌MKN-45和AGS细胞裸鼠移植瘤中的作用目的分析WWP1下调在胃癌裸鼠移植瘤中的作用。方法(1)实验动物分组：42只裸鼠随机分为MKN-45细胞移植组和AGS细胞移植组,每组21只,进行致瘤实验。每组的裸鼠均随机分为三小组,每组7只,分为未处理组、对照siRNA组、WWP1 siRNA组。(2)MKN-45细胞和AGS细胞的皮下接种及治疗：在裸鼠右侧背部皮下分别接种2×106 MKN-45细胞/只和3×106 AGS细胞/只。当肿瘤体积生长至55-80mm3时,未处理组不进行任何处理,其余两小组,每3天分别注射一次WWP1siRNA和对照siRNA(100ng/只)。每隔3天采用游标卡尺测量肿瘤的体积,当测量全部结束后,剥离肿块,称量瘤体重量,并采用Kaplan-Meier存活曲线分析WWP1表达与裸鼠生存率之间的关系。(3)采用Real-time qPCR和Western blotting分别检测胃癌细胞MKN-45和AGS裸鼠移植瘤中WWP1 mRNA和蛋白的表达。(4)统计学处理：采用SPSS17.0统计学软件进行所有实验数据的处理,数据表示为x±s,多个样本的均数比较采用单因素方差分析(One way ANOVA), WWP1表达与裸鼠存活之间的关系采用Kaplan-Meier存活曲线进行分析,P0.05表示差异具有统计学意义。结果(1) Real-time qPCR和Western blotting结果表明,与未处理组(MKN-45或AGS)和对照siRNA组相比,WWP1 siRNA组中的MKN-45或AGS裸鼠移植瘤中WWP1 mRNA和蛋白的表达均显著降低,且差异具有统计学意义(P0.05),而未处理组和对照siRNA组中WWP1 mRNA和蛋白的相对表达水平无差异(P＞0.05)。(2)与未处理的MKN-45或AGS和对照siRNA组相比,WWP1 siRNA组中MKN-45或AGS接种的裸鼠肿瘤的生长显著被抑制,且差异具有统计学意义(P0.05)。(3) WWP1 siRNA组中MKN-45和AGS裸鼠移植瘤的平均重量分别为0.467±0.111和0.457±0.118,显著低于未处理组(1.823±0.102和1.838±0.095)和对照siRNA组(1.714±0.119和1.950±0.153),且差异具有统计学意义(F=323.932和312.691,均P=0.000),而未处理组和对照siRNA组的MKN-45和AGS的裸鼠移植瘤的重量无差异(P0.05)。(4) Kaplan-Meier存活曲线分析结果显示,与未处理的MKN-45和对照siRNA组相比,WWP1 siRNA组中裸鼠的生存时间显著延长(P0.05)。同样的结果也在AGS裸鼠移植瘤中发现。结论(1)WWP1在胃癌组织中的高表达与胃癌病人肿瘤分化程度、TNM分期、浸润深度、淋巴结转移以及预后关系密切,并且WWP1可作为胃癌病人独立的预后因子,提示WWP1在胃癌发生发展中具有十分重要的作用。(2)WWP1表达下调诱导的胃癌细胞增殖抑制、细胞周期分布改变和细胞凋亡,这可能与cyclin D1、CDK4和Bcl-2表达下调以及Bax表达上调和Caspase-3活性的升高密切相关。(3)WWP1表达下调介导的胃癌生物学行为的变化可能通过调控PTEN-Akt信号途径发挥作用,因而联合靶向二者可能成为胃癌治疗的新途径。(4)WWP1表达下调在胃癌裸鼠移植瘤中的抑制作用可能为开辟胃癌治疗新靶点提供实验依据。
[Abstract]:Background and objective gastric cancer (GC) is the most common malignant tumor of the digestive tract and is the second largest in tumor related death. Although great progress has been made in the research of gastric cancer diagnosis strategy and molecular marker, the prognosis and survival rate of advanced gastric cancer is still not ideal. The median survival time of gastric cancer patients is only 6-10 months. Therefore, it is of great significance to find and identify the new molecular markers for the diagnosis, prevention, or treatment of gastric cancer patients. The E3 ubiquitin protein ligase 1 (WW domain containing E3 ubiquitin protein ligasel, WWP1), which contains the domain of the WW, is located in the 8q21.3 region of the human chromosome. Human WWP1 contains 922 amino acid residues, with a molecular weight of about 110 kDa, consisting of 1 C2 domains, 4 WW domains and 1 HECT domains. WWPl signaling pathway and various interactions, such as the Notch signaling pathway, transforming growth factor beta (transforming beta growth factor, TGF) signal pathway, mitogen activated protein kinase (mitogen-activated protein, kinases, MAPKs) signal pathway and epidermal growth factor (Epithelial growth, factor, EGF) signal pathway, suggesting its function the diversity and complexity of. More and more studies show that WWP1 is involved in the occurrence, development and progression of various diseases, such as infectious diseases, neurologic diseases and senescence, especially the occurrence and development of tumors. In prostate and breast cancer, the expression of WWP1 mRNA and protein is up-regulated, and the expression in oral and hepatocellular carcinoma is also significantly increased. These studies suggest that WWP1 may play an important role in the development and progression of these tumors. However, up to now, there has been no research report on the role of WWP1 in the occurrence and development of gastric cancer at home and abroad. This research adopts immunohistochemistry in situ hybridization, chemical, real time fluorescence quantitative PCR (Real-time qPCR) expression of WWP1 and mRNA and Western blotting detection of gastric cancer and normal gastric mucosa tissues, to explore the relationship between clinicopathological parameters and the expression of protein and mRNA, and the relationship between the survival of patients followed up. Study on WWP1 curve expression and prognosis of gastric cancer patients by Kaplan-Meier, further using Cox proportional hazards model analysis of prognostic factors in patients with gastric cancer independently; the expression of WWP1mRNA and protein detection in gastric cancer cell lines by Real-time qPCR and Western blotting using RNA WWP1 technology; research on jamming effect of down-regulation on cell cycle and apoptosis of gastric cancer cell proliferation the analysis of WWP1 protein interaction and possible application of bioinformatics method, using Western blot Ting technology to verify the possible effect of mutual regulation; finally to establish nude mouse model of gastric cancer, and using the model to study the effect of down-regulation of WWP1 expression on the growth of gastric cancer xenografts in nude mice, and through its relationship with the survival of Kaplan-Meier nude mice survival curve analysis. The first chapter is the expression of WWP1 in gastric cancer and its relationship with the prognosis of gastric cancer. Objective to analyze the expression of WWP1 in gastric cancer and explore its possible role in the development of gastric cancer. Methods (1) by in situ hybridization and immunohistochemistry techniques were used to detect the expression of WWP1 and mRNA protein in 131 cases of gastric cancer tissues and corresponding normal gastric mucosa tissues: the expression of WWP1 and mRNA protein in gastric cancer was detected by Real-time qPCR and Western Blotting Technology and the corresponding normal gastric tissues. (2) Kaplan-Meier survival curve was used to analyze the relationship between WWP1 mRNA and protein expression and prognosis of gastric cancer patients. Cox independent risk factor analysis was used to analyze the independent prognostic factors of gastric cancer patients. (3) statistical analysis: using statistical software SPSS17.0 to analyze the data, the data were expressed as x + s, in situ hybridization and immunohistochemical results were evaluated by X2, Real-time qPCR and Western blotting results using single factor analysis of variance (One-way ANOVA), the relationship between the expression and prognosis of gastric cancer patients of WWP1 mRNA and protein the Kaplan-Meier survival curve analysis, independent prognostic factor in Cox proportional hazards model for analysis of gastric cancer patients, P < 0.05 said the difference was statistically significant. Results (1) in 131 gastric cancer tissues, the positive expression rates of WWP1 mRNA and protein were 66.41% and 61.83%, respectively. In 131 normal gastric mucosa tissues, the positive expression rates of WWP1 mRNA and protein were 12.21% and 9.92%, respectively. The expression levels of WWP1 mRNA and protein in gastric cancer tissues and normal gastric mucosa were statistically different (X2=80.646 and 76.715, all P=0.000). (2) Real-time qPCR and Western blotting results showed that the relative expression levels of WWP1 mRNA and protein in 10 gastric cancer tissues were significantly higher than those in normal gastric mucosa tissues, and the difference was statistically significant (P0.05). (3) WWP1 mRNA and protein expression were closely related to tumor differentiation, TNM stage, depth of invasion and lymph node metastasis in all gastric cancer patients (all P0.05), but not related to age, sex and tumor size of gastric cancer patients (all P0.05). (4) Log-rank test (Mantel-Cox) showed that the survival time of gastric cancer patients with low expression of WWP1 mRNA and protein was significantly longer than those of patients with high expression of WWP1 mRNA and protein, and the difference was statistically significant (P 0.0134 and 0.0098 respectively). (5) the Cox proportional risk model showed that TNM staging, lymph node metastasis, WWP1 mRNA and protein could be an independent prognostic factor for gastric cancer patients. The second chapter is about the effect of downregulation of WWP1 expression on gastric cancer cell proliferation, cell cycle and apoptosis, and the possible molecular regulation mechanism. Objective to investigate the effect of downregulation of WWP1 expression on gastric cancer cell proliferation, apoptosis and cell cycle and its regulatory mechanism. Methods (1) Real-time qPCR and Western blotting were used to detect WWP1 mRNA and eggs in GES-1 of different gastric cancer cell lines and normal gastric mucosa epithelial cells.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.2

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