【作者】 孟会敏；

【导师】 李建明；

【作者基本信息】 南方医科大学 ， 病理学与病理生理学， 2010， 硕士

【摘要】 结直肠癌是常见的恶性肿瘤之一,近十年来,结直肠癌在我国的发病率有逐年上升的趋势。转移是影响患者治疗效果和导致患者死亡的主要原因。阐明结直肠癌转移相关的分子机制及寻找预防和治疗的有效途径是目前结直肠癌研究的重要课题。锌指转录因子Snail属于转录抑制因子Snail超家族。Snail是一个重要的转录因子,在肿瘤上皮间叶转化(epithelial-mesenchymal transition,EMT)过程中起重要作用。研究发现,Snail可通过调节E-cadherin的表达参与EMT的发生。这一结论已经在多种肿瘤中如结肠癌、乳腺癌、胰腺癌、恶性黑色素瘤及口腔鳞癌等获得证实。我们课题组前期生物信息学分析表明,Snail的结合位点存在于PRL-3基因的启动子区域。作为肿瘤转移发生过程中重要的调节因子,Snail参与PRL-3基因表达及其转录调控的关系并不清楚。阐明Snail与PRL-3基因启动子之间的作用机制有重要作用。胚胎干细胞(ESCs)是指当受精卵分裂发育成囊胚时内细胞团的细胞,它具有体外培养无限增殖、自我更新和多向分化的特性。胚胎干细胞(ES细胞)是一种高度未分化细胞,它具有发育的全能性,能分化出成体动物的所有组织和器官,包括生殖细胞。研究和利用ES细胞是当前生物工程领域的核心问题之一。诱导性胚胎干细胞(Induced pluripotent stem cells, iPS cells),是通过特定的基因组合与转染可以将已分化的体细胞诱导重新编程使细胞回归到胚胎干细胞的状态,具有和胚胎干细胞类似的功能,能分化生成各种组织细胞。例如在成人体细胞中导入4种基因(OCT4、Nanog、SOX2、LIN28或OCT3/4、SOX2. C-MYC、KLF4),通过基因重新编程,使分化的细胞被逆转恢复到全能分化的状态。有文献报道这些胚胎干细胞基因在一些恶性肿瘤中均有表达,但它们在肿瘤的发生、进展与转移中的作用并不清楚。因此阐明这些诱导性胚胎干细胞基因与恶性肿瘤发生发展的关系有重要的作用。在此实验中我们研究的是Snail转录抑制因子与结直肠癌中诱导性胚胎干细胞基因(Nanog)之间的联系及Nanog对结直肠癌发生发展的影响。研究方法：1、Snail对结直肠癌细胞生物学特性的影响课题组前期工作已经证明了锌指转录因子Snail与结直肠癌发生、演进及转移相关。进一步我们观察Snail的上调或下调对结直肠癌细胞生物学特性的影响。鉴定Snail干扰载体及过表达载体,转染结直肠癌SW480细胞及Lovo细胞,通过MTT、粘附实验、Transwell小室及平板克隆实验来检测Snail基因上调或下调后对结直肠癌细胞生物学特性的影响。2、Snail参与结直肠癌细胞中PRL-3基因的表达调控在课题组前期确定PRL-3基因启动子区域的基础上,将PRL-3基因启动子相关片段(-642bp至-383bp及-754bp至455bp)与Snail干扰载体及过表达载体共同转染至结直肠癌细胞系SW480及Lovo细胞,然后检测PRL-3基因启动子片段(-642bp至-383bp及-754bp至455bp)萤光素酶的活性；同时应用Western blotting检测Snail上调或下调对结直肠癌细胞PRL-3蛋白表达的影响。3、Snail参与调控结直肠癌中的Nanog基因提取新鲜结直肠癌组织蛋白,Western blotting检测结直肠癌组织中Snail与Nanog的表达,初步确定Snail与Nanog在结直肠癌组织中表达的关系。然后Western blotting检测Snail上调、下调或TGF-β1刺激后对结直肠癌细胞SW480中Nanog蛋白表达的影响。应用Nanog慢病毒过表达系统,建立Nanog过表达细胞株,荧光定量PCR检测Nanog上调后α-catenin、β-catenin、γ-catenin、E-cadheri、vimentin、Snail、Slug及CK的表达。4、Nanog在结直肠癌细胞中的作用应用Western blotting检测结直肠癌组织及其正常对照Nanog表达的差异。选择175例结直肠癌样本作为研究对象,运用免疫组织化学方法分析Nanog在这些组织中的表达情况。MTT、平板克隆、流式细胞术、Transwell小室及划痕实验检测Nanog上调后对结直肠癌SW480细胞增殖、细胞周期、侵袭及迁移能力的影响。结果：1、Snail对结直肠癌细胞生物学特性的影响应用MTT法,我们检测了Snail上调或下调对Lovo-mock和Lovo shRNA-Snail2细胞体外增殖能力的影响,与对照组比,Lovo shRNA-Snail2细胞的增殖能力下降,具有显著性差异(F=9030.72,P=0.000)；另外还检测了Snail上调或下调对SW480-mock和SW480 GFP-Snail WT细胞体外增殖能力的影响,与对照组比,SW480GFP-SnailWT细胞的增殖能力增高,具有显著性差异(F=2837.717,P=0.000)。平板克隆形成实验显示Lovo shRNA-Snail2细胞增殖能力下降,具有显著性差异(t=3.332,P=0.029)；SW480GFP-Snail WT细胞的增殖能力明显增高,具有显著性差异(t=-10.328,P=0.000)。粘附实验结果表明,与Lovo-mock细胞相比,Lovo shRNA-Snail2细胞的粘附能力下降,具有显著性差异(t=5.258,P=0.006)；与SW480-mock相比,SW480GFP-Snail WT细胞的粘附能力明显增高,具有显著性差异(t=-11.023,P=0.000)。Transwells小室实验证明,与Lovo-mock细胞相比,Lovo shRNA-Snail2细胞的迁移能力下降,具有显著性差异(t=21.822,P=0.000)；与SW480-mock相比,SW480GFP-Snail WT细胞的迁移能力明显增高,具有显著性差异(t=-25.509,P=0.000)。2、Snail参与结直肠癌细胞中PRL-3基因的表达调控Snail下调后,PRL-3基因启动子片段(-642bp至-383bp及-754bp至455bp)的萤光素酶活性明显下降,PRL-3启动子的活性下降；Snail上调后,PRL-3基因启动子片段(-642bp至-383bp)的萤光素酶活性明显增高,PRL-3启动子的活性增高。与PRL-3基因启动子活性改变相一致,Snail下调后,结直肠癌Lovo细胞中PRL-3蛋白表达明显降低；Snail上调后,结直肠癌SW480细胞中PRL-3蛋白表达明显增高。3、Snail参与调控结直肠癌中的Nanog基因初步确定结直肠癌组织中Snail与Nanog基因的相关性；Snail上调、下调或TGF-β1刺激后,Nanog的表达增高；荧光定量PCR检测Nanog上调后,结直肠癌SW480细胞中γ-catenin、slug及Snail的表达增高。4、Nanog在结直肠癌细胞中的作用结直肠癌组织中Nanog蛋白的表达比正常结直肠组织中的表达要高；免疫组化结果分析表明：Nanog的表达水平与淋巴结状态(P=0.024)及Dukes分期(P=0.049)有关,具有显著性差异；肿瘤的分化(P=0.034)、Dukes分期(P=0.002)及Nanog蛋白的表达(P=0.003)都可作为结直肠癌病人潜在的独立的预后因素,具有显著性差异。应用MTT法,我们检测了Nanog上调对SW480/Mock.SW480/Nanog clonel、SW480/Nanog clone2及SW480/Nanog clone3细胞体外增殖能力的影响,与对照组SW480/Mock比,SW480/Nanog clonel、SW480/Nanog clone2及SW480/Nanog clone3细胞的增殖能力增高,具有显著性差异(F=222.994,P=0.000)。平板克隆形成实验显示,与对照组SW480/Mock细胞相比,SW480/Nanog clone1、SW480/Nanog clone2及SW480/Nanog clone3细胞的增殖能力明显增高,具有显著性差异(F=201.740,P=0.000)。划痕实验结果表明,与对照组SW480/Mock细胞相比,SW480/Nanog clone1、SW480/Nanog clone2及SW480/Nanog clone3细胞的迁移能力增强,具有显著性差异(F=67.023,P=0.000)。运动小室实验证明,与SW480/Mock细胞相比,SW480/Nanog clone1、SW480/Nanog clone2及SW480/Nanog clone3细胞的迁移能力增高,具有显著性差异(F=245.267,P=0.000)。细胞周期实验证明,与SW480/Mock细胞相比,SW480/Nanog clone1、SW480/Nanog clone2及SW480/Nanog clone3细胞的S期增殖指数明显增高(F=52.399,P=0.000),具有显著性差异,说明Nanog可促进细胞DNA的合成。结论：1、Snail可促进结直肠癌细胞的增殖、粘附、侵袭与迁移。2、Snail通过调控结直肠癌组织中PRL-3启动子的活性调节PRL-3蛋白的表达,这是Snail影响结直肠癌细胞的增殖、粘附与迁移的重要机制之一。3、Snail参与调控结直肠癌组织中Nanog基因的表达,这是影响结直肠癌的增殖、粘附与迁移的重要机制之一4、Nanog可促进结直肠癌细胞的增殖、粘附、侵袭与迁移,其中Nanog还可促进结直肠癌细胞DNA的合成。5、在结直肠癌发生发展过程中,Nanog作为诱因和EMT信号传导受体发挥着作用；Nanog蛋白的表达水平与淋巴结状态和Dukes分期有关；Nanog蛋白的表达可作为结直肠癌病人一个潜在的独立预后因素。更多还原

【Abstract】 Colorectal cancer (CRC) is a common malignant tumor leading to death in the world. The incidence rate of CRC in china is increasing fast during the past decades. Metastasis is the main cause affecting the therapeutic efficacy and leading to the death of cancer patients. It is urgent to elucidate molecular mechanisms of metastasis and find out the preventive and therapeutic strategies.Snail is an important transcription factor which plays an important role in the process of epithelial mesenchymal transition (EMT) in the tumor. It is studied that Snail is involved in the occurrence of EMT through regulating the expression of E-cadherin. This has been confirmed in several types of tumors such as colon cancer, breast cancer, pancreatic cancer, malignant melanoma, oral squamous cell carcinoma and et al.Our previous work by bioinformatic analysis showed that Snail binding sites were present in the PRL-3 gene promoter region. As an important regulatory factor involved in the process of tumor metastasis, the regulatory role of Snail involved in PRL-3 gene expression is still unknown. Therefore, it is necessary to clarify the relationship between Snail and PRL-3 gene.Embryonic stem cells (ESCs) are the cells of inner cell mass when the fertilized egg develops into the blastula, which has the characteristics of unlimited proliferation, self-renewal and multi-potentiall differentiation. ESCs are a kind of highly undifferentiated cells, which possess developmental totipotency and can differentiate into all tissues and organs of animals, including reproductive cells. Research about ES cells is one of most excited filelds of bio-engineering. Induced embryonic stem cells (iPS cells) are cells which were reprogrammed to embryonic stem cell state from somatic cells through forced induction of a kind of specific combinations of genes. They possess similar functions of embryonic stem cells (ES cells) and can generate many kinds of tissue cells. For example, when we imported the four kinds of genes (OCT4, Nanog, SOX2, LIN28, or OCT3/4, SOX2, C-MYC, KLF4) into human somatic cells, differentiated cells are reversed to the embryonic stem cell state through genetic reprogramming. It has been reported that these embryonic stem cell genes were also expressed in a number of malignant tumors. However, their roles in development, progress and metastasis of cancer are still not very clear. In this study, we preliminary uncover the relationship between Snail transcriptional repressors and induced embryonic stem cell gene (Nanog) in colorectal cancer and the effects of Nanog on the development of colorectal cancer.Methods1. The effects of Snail gene on the biological behaviors of colorectal cancer (CRC) cells.In order to know the effects of Snail gene on the biological behaviors of colorectal cancer (CRC) cells, we knocked down Snail expression in Lovo cells and upreguted Snail expression in SW480 cells. Proliferation, adhesion and migration capacities of CRC cells using MTT, adhesion experiments, Transwell chamber and plate colony formation assay were analyzed.2. The effects of Snail gene on the luciferase activities of the PRL-3 gene promoter and PRL-3 protein expression in CRC cells.The luciferase activities of the PRL-3 gene promoter fragments (-642bp to-383bp and-754bp to 455bp) was studied after expression of Snail was upregulated in SW480 cells or downregulated in Lovo cells. Meanwhile, PRL-3 protein expression was detected using Western blotting after expression of Snail was upregulated in SW480 cells or downregulated in Lovo cells.3. Snail was involved in the regulation of Nanog gene in CRC.Protein expressions of Snail and Nanog in fresh CRC tissues were detected using Western blotting to preliminarily determine the possible correlation between Snail and Nanog genes. Nanog protein expression was detected using Western blotting after Snail was upregulated or down-regulated in CRC cells. The effects of TGF-β1 on Nanog protein expression in CRC cells were also determined. After Nanog was upregulated, mRNA expressions of a-catenin,β-catenin,γ-catenin, E-cadherin, vimentin, snail, slug, and CK were studied by fluorescence quantitative PCR assay.4. Immunohistochemical evaluation of Nanog protein expression in CRCImmunohistochemical staining of Nanog protein expression in 175 human tissue samples of CRC was done using a Dako EnVision System. Rabbit anti-human Nanog polyclonal antibody from Biosynthesis Biotechnology Co., LTD (Beijing, China) was used for immunohistochemical staining of Nanog protein expression in human tissue samples of CRC. The specificity of this antibody was determined by western blot and compared with that of mouse anti-Nanog monoclonal antibody from Abnova (Taiwan). The cellular localization of Nanog was studied by indirect immunofluorescence and confocal microscopy in SW480 cells. The staining intensity was scored on a scale of 0 to 3 as negative (0), weak (1), medium (2) or strong (3). The extent of the staining, defined as the percentage of positive staining areas of tumor cells in relation to the whole tumor area, was scored on a scale of 0 to 4:0 (0%),1 (1-25%),2 (26-50%),3 (51-75%) and 4 (76-100%). An overall protein expression score (overall score range,0-12) was calculated by multiplying the intensity and positivity scores. For statistical analysis, a final staining score of≥4 was considered to be high expression of Nanog protein.5. The effects of Nanog gene on the biological behaviors of colorectal cancer (CRC) cells.Proliferation, adhesion and migration capacities of CRC cells using MTT, adhesion experiments, Transwell chamber and plate colony formation assay were analyzed in the stable nanog-overexpression SW480 cells.Results1. Effect of Snail gene on the biological behaviors of CRC cells.A significantly increased proliferation was found after Snail expression in SW480 cells was upregulated by in vitro MTT assay. A significantly decreased proliferation was found after Snail expression in Lovo cells was knocked down by in vitro MTT assay.Snail overexpression had a significant enhanced ability to form colonies in plates in SW480, and Snail downregulation had a significant inhibtory ability to form colonies in plates in Lovo cells.Using Transwells migration assay, we observed that Snail overexpression had a significant enhanced migratory ability in SW480, and Snail downregulation had a significant inhibtory migration ability in Lovo cells.Results of adhesion assay showed Snail promoted adhesion of human CRC cells.2. PRL-3 was regulated by Snail in CRC cells.Luciferase activity of PRL-3 promoter fragments (-642bp to-383bp and-754bp to 455bp) decreased significantly after Snail expression was knocked down. Luciferase activity of PRL-3 promoter fragments (-642bp to-383bp) increased significantly after Snail expression was up-regulated. Consistent with the changes of PRL-3 promoter activity, we found that PRL-3 protein expression was significantly reduced after after Snail expression was knocked down in Lovo cells and PRL-3 protein expression was significantly increased after Snail expression was up-regulated in SW480 cells.3. Snail was involved in regulation of Nanog gene expression in CRCWe observed that expression of Nanog protein was significantly induced after TGFβ1 stimulation in HT-29 and colo205 cells. Knockdown of Snail, a key gene involved in EMT process, led to inhibitory expression of Nanog and overexpression of Snail resulted in increasing expression of Nanog in SW480 cells. The results suggest that Nanog may participate in EMT process initiated by TGFβ1 and also can be regulated by Snail.Interestingly, semi-quantitative real-time PCR analyses demonstrated that Nanog significantly induced the transcription of Snail, Slug andγ-catenin in at least two Nanog-overexpression clones.4. Relationship between clinicopathologic features and nanog expression in CRCWestern blots results showed that Nanog protein expression in CRC tissues was higher than that in normal colorectal tissues. Nanog protein was localized mainly in the cytoplasm of cancer cells. Nuclear accumulation of Nanog was only observed in a small fraction of cancer cells Cytoplasmic and perinuclear distribution of Nanog protein was found in SW480 cells by means of cytoimmunofluorescent labeling and confocal microscopy. No significant associations were found between Nanog expression and age, gender, tumor size, tumor site, differentiation and invasion of CRC patients (p>0.05). Interestingly, we observed that Nanog expression was positively correlated with lymph node status (p=0.024) and Dukes classification (p= 0.049) of patients. Using Kaplan-Meier analysis method, we found that the protein expression of Nanog in CRC was significantly correlated with overall survival (p= 0.000) of CRC patients. Using a public microarray expression profiling data,11 we also found the expression level of Nanog mRNA was significantly correlated with recurrence-free survival of stage II CRC (p=0.038). These results indicated that the high expression of Nanog was correlated with a shorter survival or recurrence-free survival.To determine whether the expression of Nanog was an independent prognostic factor of outcomes, multivariate survival analysis including invasion, differentiation, Dukes classification and Nanog expression, was done. Results showed that the expression of Nanog protein was a potential independent prognostic factor of outcomes of CRC patients.5. Nanog promotes proliferation, invasion and migration of human CRC cells.A significantly increased proliferation was found in SW480/Nanog clone2, SW480/Nanog clone3 compared with that in Mock cells by in vitro MTT assay. Nanog overexpression had a significant enhanced ability to form colonies in plates. Concordant with these results, Nanog-overexpression clones especially in clone 2 and clone 3 showed a significantly increased number of S phase (PI) by cell cycle analysis of flow cytometry.We also observed that three stable Nanog-overexpression clones displayed significant increase in invasive ability compared with Mock cells). In vitro wound healing migration assay indicated that cells in three stable Nanog-overexpression clones filled in from 50% to 80% of the scratched area after 60 h, whereas Mock cells filled in only about 20% of the scratched area after 60 h.Conclusions1. Snail promotes proliferation, adhesion, invasion and migration of CRC cells.2. Snail is involved in progression of CRC by regulating PRL-3 expression.3. Nanog gene is a potential new target of Snail in colorectal cancer.4. Nanog promotes proliferation, adhesion, invasion and migration of CRC cells.5. Nanog functions as an inducer and also a recipient of EMT in tumor progression of CRC. Nanog expression level is correlated with lymph node status and Dukes stage. Nanog protein expression in CRC patients can be considered as a potential independent prognostic factor.更多还原