【作者】 房雪峰；

【导师】 郑树； 林标扬；

【作者基本信息】 浙江大学 ， 肿瘤学， 2010， 博士

【摘要】 SOX (SRY-like HMG box)基因家族是一类具有HMG (high mobility group)特征性结构域的转录因子家族。HMG是DNA结合区域,其基因序列在真核细胞内高度保守。目前,共发现20种SOX基因,分为10个亚类。SOX2是SOX家族组中一个主要成员,表达与胚胎干细胞,在胚胎的各个发育过程中起重要作用。近年来,发现SOX2是诱导人和鼠成体细胞细胞为pluripotency细胞的一个关键和必需的分子。干细胞和肿瘤都具有自我更新等共同特性,在干细胞起关键作用的基因在肿瘤的发生和发展中也起一定作用。SOX2已被发现在多种肿瘤中高表达,如肺癌、胰腺癌、乳腺癌和神经内分泌癌等；在中枢神经系统发育的早期,SOX2表达于全部的神经上皮,但神经系统发育成熟之后,其表达仅局限于胚基的神经胶质干细胞,而当神经胶质细胞恶变后,在脑胶质瘤中被激活而重新表达,表达显著高于正常组织,并且在RNA和蛋白水平上得到了证实。文献报道在结肠癌中SOX2高表达并且是判断预后的候选生物标记,并且SOX2的表达与干细胞标记CD133以及对化疗药物抵抗有相关性。然而SOX2结肠癌中的作用及其分子机理目前尚无研究。本研究中,我们在结肠癌细胞中应用基因干涉技术,观察SOX2基因对细胞生物学行为的影响,发现SOX2基因干涉后其增殖、克隆形成以及动物体内致瘤能力明显降低。我们利用了基因组,转录组以及蛋白质组学技术：基因表达芯片,Chip-Seq (chromatin immunoprecipitation-sequencin)和Co-IP-MS-MS (Immunoprecipitation coupled with mass spectrometry),应用系统生物学理念,探讨该基因对肿瘤发生的分子机理。1.免疫组织化学检测SOX2在结肠癌中的表达用免疫组织化学检测30例结肠癌组织和配对正常结肠组织SOX2蛋白的表达,结果表明SOX2在癌组织细胞核中的表达显著高于正常组织,差异有显著性。2.基因干涉技术对SOX2在肿瘤细胞中生物学功能的研究利用慢病毒介导的基因干涉技术,病毒感染结肠癌细胞后SOX2的表达显著下调,体外功能实验显示,SOX2基因基因干涉后细胞增殖能力和克隆形成能力显著减低,细胞周期呈现S和G2／M期阻滞；动物体内致瘤能力也显著降低。3.SOX2基因干涉后细胞基因表达谱的改变及生物信息分析SOX2基因干涉后,通过基因芯片表达谱分析,发现1205个基因的表达因SOX2基因干涉而下调；Go Ontology分析,这些下调的基因参与了数条重要生物过程,如：cell surface receptor linked signal transduction (GO:0007166), BMP signaling pathway (GO:0030509), response to retinoic acid (GO:0032526)等,说明SOX2可能通过这些基因表达水平的调节而调控这些生物过程。4. Chip-seq SOX2蛋白分子基因组结合位点分析我们利用Chip-seq技术确定了SOX2在基因组上的结合位点,并与表达谱芯片整合分析,进一步确定了SOX2在转录水平上可能直接调节的基因；并将SOX2在肿瘤中的结合位点与在胚胎干细胞中的结合位点进行比较,两者共有372个结合位点,说明Sox2在肿瘤中和在干细胞中对基因的调节具有一定的保守性。在结肠癌细胞中SOX2在基因组DNA上的结合位点富含AT序列,并且共有一共同motif:wwTGywTT;将SOX2的结合区域向两侧延伸,以发现SOX2结合区域内是否含有其他已知转录因子的结合motif,结果发现了已知的与SOX2有协同作用的OCT家族蛋白的结合motif,并且发现新的转录因子的结合motif如：Fox和HNF等家族蛋白的结合motif,这说明这些蛋白可能与SOX2共同参与对基因表达的调节。5. Co-IP-MS-MS SOX2相互作用蛋白的鉴定和分析Co-IP-MS/MS分析共鉴定到与SOX2蛋白相互作用形成复合物的蛋白144个,每个蛋白的可信度都大于0.9,并且每个蛋白在质谱中出现的肽段都至少有两个不同唯一肽段出现。随机选取了4个蛋白进行western-blot验证；我们发现在鉴定到的蛋白中,有很多蛋白为HnRNP家族蛋白,并与以往鉴定到的HnRNP复合物组分相比较,说明SOX2可能是HnRNP复合物的一个组分,SOX2与这蛋白相互作用参与了基因的表达各个过程的调节。6.整合分析整合基因表达谱改变、Sox2 DNA结合位点和Sox2相互作用蛋白,我们发现在Sox2基因组的结合区域存在CEBPB转录因子结合motif,在相互作用的蛋白中存在CEBPB蛋白,这说明Sox2可能与和CEBPB共同结合一个的DNA区域内调节基因的表达,这有待于经典的生物学进一步验证。结论：SOX2在结肠癌组织的细胞核中的表达显著高于正常组织。SOX2基因干涉降低了结肠癌细胞的体外增殖能力和体内致瘤能力,细胞周期呈现S和G2／M期阻滞。SOX2基因干涉后数条与结肠癌发生相关的生物过程中的基因表达下调。确定了SOX2在基因组上的结合位点和结合特征；SOX2在肿瘤细胞中和人胚胎干细胞中共有372个共同的基因组结合位点,说明Sox2在肿瘤中和在胚胎中对基因的调节具有一定的保守性；基因芯片数据和Chip-seq数据整合分析确定了SOX2可能直接调节94个基因的转录,其中53个基因受SOX2的正调节,41个受SOX2的负调节。蛋白相互作用分析显示SOX2与HNRNP家族蛋白相互结合,这种相互结合可能共同参与了基因表达的调节。因此,本研究为我们深入了解SOX2基因在肿瘤中作用的分子机理提供研究基础和线索。更多还原

【Abstract】 SOX (SRY-like HMG box) gene family represents a family of transcriptional factors characterized by the presence of a homologous sequence called the HMG (high mobility group) box in their genes. HMG box is a DNA binding domain that is highly conserved throughout eukaryotic species. So far, twenty SOX genes have been identified in humans and mice, and they can be divided into 10 subgroups on the basis of sequence similarity and genomic organization. SOX genes bind to the minor groove in DNA to control diverse developmental processes. SOX2, one of the key members of the SOX family gene, is highly expressed in embryonic stem cells. Recent research showed that SOX2 is a key transcription factor whose over expression can induce pluripotency in both mouse and human somatic cells. These suggest that SOX2 is the key gene in conferring sternness of cells. The sternness program can also play an important role in cancer because self-renewal is a hallmark of for both stem cells and cancer cells.Recent research showed that SOX2 are over expressed in malignant glioma, but display minimal expression in normal tissues. They further confirmed by RT-PCR and immunohistochemistry staining that SOX2 mRNA and protein expression were increased in GBM compared to normal brain tissues. We previously completed massively parallel signature sequencing (MPSS) and identified SOX2 as significantly over expressed in GBM tissues compared to normal brain tissues. SOX2 has also been implicated in several cancers including gastric cancer, breast cancer, pancreatic cancer, pulmonary non-small cell and neuroendocrine carcinomas. In addition, SOX2 was identified to be a prognostic marker for human esophageal squamous cell carcinoma and colorectal cancer.Therefore, SOX2 may be a gene that is predominantly expressed in embryonic and adult stems cells including neural progenitor cells and re-activates in cancers including brain tumors. However, up to the present, systems-wide investigation of the functions of SOX2 has concentrated on embryonic development in mouse embryonic stem cells. To understand how SOX2 exerts its function in a system-wide (genome wide and proteome wide) manner in brain tumors and colorectal cancers, we took advantages of recent advances in genomic and proteomic technologies and conducted a study combining two novel technologies--ChIP-seq analysis and Immunoprecipitation (IP) coupled with mass spectrometry analysis. We report here our results of the genome wide targets of SOX2 in cancer cells identified by ChIP-seq analysis. In addition, we report the components of the SOX2 interactome identified by IP coupled with mass spectrometry analysis. Our integrated analysis provides us with a comprehensive picture for better understanding of the SOX2’s role in brain tumors and colorectal cancers.1. immunohistochemical staining analysis of SOX2 protein in 30 colon cancer tissue.To analyze its protein expression, we performed Immunohistochemical staining in 30 colorectal cancer tissues and 30 normal adjacent tissues. We observed positive immunoreactivities in colorectal cancer with nucleus staining pattern in 46%(14/30) of cancer tissues, compared to only 7% in the adjacent mucosa tissue cells. Statistical analysis using Pearson Chi-Square (df=1, two-sided) indicates that the difference in SOX2 expression between cancer and adjacent tissues is significant (P<0.01).2. SOX2 knockdown decreased cell colony formation on soft agar and in vivo tumorigenesis potential.We knocked down the expression of SOX2 in colorectal cancer cell and gliomas cell, and found that reduced expression of SOX2 correlates with decreased growth rates, colony formation and migration abilities of cancer cells. In addition, knocking down of SOX2 resulted in an accumulation of cells in the G0/G1 phases and a decrease of cells in S and G2/M phases. Using in vivo mouse models, we showed that SOX2 knockdown cells had reduced tumor sizes compared with the MOCK known down cells. Taking together, these data suggested functional important roles of SOX2 in colorectal carcinogenesis.3. Identification of SOX2 regulated genes by microarrays.We used Affymetrix array U133 plus 2 to identify genes regulated by SOX2 by comparing expression profiles of parental SW620 cells and SOX2-knockdown SW620 cells. We found that the expression of 1,205 genes was decreased by 2 fold or more in the SOX2 knockdown SW620 cells comparing with the mock control cells.Gene Ontology analysis of down regulated genes by SOX2 knockdown revealed that the GO terms cell surface receptor linked signal transduction (GO:0007166), BMP signaling pathway (GO:0030509). However, there were no significantly (FDR＜0.05) enriched GO terms for the genes that were up regulated by SOX2 knowndown.4. Identification of DNA binding consensus and SOX2 collaborating TFs in the SOX2 binding regions.To see whether the human SOX2 binding regions in GBM cells and colon cancer cells have their own unique and enriched binding motif, we used the MotifSampler program to identify binding consensus sequences enriched in the SOX2 binding regions. We found a consensus sequence wwTGywTT and wwTGnwTw in colon cancer cells and glioma cells. We then wondered whether known TFs could bind to the SOX2 binding regions and act as SOX2 cooperators for the regulation of gene expression. In order to systematically search for potential bindings of other transcription factors, we used the MotifScanner program and scanned all TF motif matrices using the human transcription factor subset of the Transfac professional 7.0. Matched matrices with likelihood (LR) ratios of 500 or higher were tabulated and frequencies calculated. Among the top known TF matrices identified with SOX2 in the SOX2-binding region include the known OCT family and some new unknown families such as the FOX family, the HNF family, the GATA family and several others.5. IP-MS/MS analysis of SOX2 interacting proteins revealed that SOX2 interacts with heterogeneous nuclear ribonucleoproteins.We carried out IP-MS/MS analysis to determine the interacting protein partners of SOX2 in cancer cells. We identified SOX2 as a protein in the HnRNP complex because it interacts with many heterogeneous nuclear ribonucleoproteins. Comparing the SOX2 interactome and the HNRPNK interactome, we identified 16 proteins that are common interacting proteins for SOX2 and HNRNPK. suggesting that SOX2 is part of the HNRNP complex, interacting with other HNRNP proteins. Protein-protein interaction analysis revealed that HNRNPK interacts with multiple proteins involved in multiple steps in the gene expression including chromatin remodeling, transcription, RNA processing and translation. Gene Ontology analysis of the SOX2 interacting proteins suggested that SOX2 also interacts with proteins involved in multiple steps in regulating gene expression including chromatin remodeling, transcription, RNA processing and translation. This is not surprising in light of SOX2’s essential role in development and in conferring sternness property of cells.更多还原