结肠直肠癌分子标记物筛选研究

发布时间：2018-05-01 10:14

本文选题：结肠直肠癌 + 基因表达　；参考：《武汉大学》2016年博士论文

【摘要】：背景：结肠直肠癌是一种高度异质性疾病,具有多种分子表型和基因组变异特征,其基因的异常表达与癌细胞的发生和发展密切相关。然而,具有高度恶性的结肠直肠低分化腺癌组织的基因表达和基因组改变特征仍不十分清楚。目的：旨在深入了解Ⅱ期结肠直肠低分化腺癌组织中的基因改变及其恶性表型的相关性。方法：我们收集了四对Ⅱ期低分化结肠直肠腺癌组织和对应癌旁正常组织,利用包含有440个肿瘤相关基因的表达谱芯片和聚类分析获得显著差异表达基因；生物信息学Gene Ontology和Ingenuity,Pathway Analysis分析肿瘤组织中的差异表达基因参与的信号通路及相关的生物学功能；利用新型的生物信息学计算机模型预测了差异表达基因的转录调控机制；结合肿瘤基因组图谱数据(TCGA)中的195例结肠直肠癌组织高通量测序及基因表达数据,进行比对分析获得表达差异基因的基因组变异特征；通过文献检索获得与临床表型相关的50个基因芯片实验数据进行比对分析,并结合常见实体肿瘤的基因组图谱信息,进一步验证和筛选与结肠直肠癌进展高度相关的基因；利用免疫组织化学染色验证候选基因的蛋白表达情况与临床恶性表型的相关性。结果：在Ⅱ期低分化结肠直肠腺癌组织与其对应癌旁正常组织中,共获得93个差异表达基因(表达差异1.5倍以上,p小于0.05)；聚类分析结果显示,在肿瘤组织内包含显著表达上调基因群和显著表达下调基因群；系统生物信息学分析结果显示差异表达基因主要参与细胞凋亡、磷酸化、细胞周期调控等生物学过程,在癌组织中以NFKB、p53、AP1、STAT1、HSP90和CTNNB1信号通路紊乱最为显著；同时,依据生物信息学原理预测与差异表达基因相关的7个转录因子包括NFKB1、RELA、AP1、STAT3、p53和p63及其转录调控关系；与TCGA数据库比对分析,筛选出24个mRNA表达相一致的基因,其基因组变异频率在5%-37%；其中14个基因呈现拷贝数增加或减少,且mRNA表达量与其拷贝数变化密切相关；结合50个基因芯片实验数据共筛选出8个候选基因与肿瘤恶性表型具有潜在相关性,包括RPN2,HMGB1,AARS,IGFBP3,STAT1,YOU1,NQO1 and PEA15;在9种不同类型的实体肿瘤比较研究中,PRN2和HMGB1在结肠直肠癌中的变异频率最高；免疫组化实验结果显示在78例结肠直肠癌样本中,RPN2和HMGB1蛋白表达在癌组织中显著高表达,RPN2与组织分化程度和转移存在显著相关性。结论：本研究通过差异基因表达谱结合基因组数据库、不同的人群的基因芯片实验数据,共筛选出八个与肿瘤转移高度相关的候选基因,并揭示了RPN2和HMGB1在结肠直肠癌中作为早期肿瘤转移预测的分子标记物和RNAi药物靶标的潜在的临床应用价值。
[Abstract]:Background: colorectal cancer is a highly heterogeneous disease with a variety of molecular phenotypes and genomic variations. The abnormal expression of the gene is closely related to the occurrence and development of cancer cells. However, the gene expression and genomic changes of highly malignant colorectal adenocarcinoma tissue are still not very clear. The purpose of this study is to understand the correlation between gene change and its malignant phenotype in stage II colorectal carcinoma with low differentiation. Methods: We collected four pairs of low differentiated colorectal adenocarcinoma tissues and normal tissues adjacent to the carcinoma, and obtained significant differential expression by using expression profiles and cluster analysis containing 440 tumor related genes. Gene; bioinformatics Gene Ontology and Ingenuity, Pathway Analysis to analyze the signaling pathway and related biological functions of differentially expressed genes in tumor tissues; use a new bioinformatics computer model to predict the transcriptional regulation mechanism of differentially expressed genes; combined with the tumor genome map data (TCGA). High throughput sequencing and gene expression data of 195 cases of colorectal cancer were compared and analyzed to obtain genomic variation characteristics of differentially expressed genes. 50 gene chip experimental data related to clinical phenotype were compared and analyzed by literature retrieval, and combined with the genome map information of common solid tumors, it was further verified. And screening the genes associated with the progression of colorectal cancer, and using immunohistochemical staining to verify the correlation between the protein expression of the candidate gene and the clinical malignant phenotype. Results: 93 differentially expressed genes (1.5 times of the difference in expression) were obtained in stage II low differentiated colorectal adenocarcinoma and corresponding to the normal tissue adjacent to the carcinoma. P was less than 0.05), and the results of cluster analysis showed that the up-regulated gene group and down regulated gene group were significantly expressed in the tumor tissues. The system bioinformatics analysis showed that the differentially expressed genes were mainly involved in the biological process of cell apoptosis, phosphorylation, cell cycle regulation, and NFKB, p53, AP1, STAT1, HSP90 in the cancer tissues. At the same time, the 7 transcription factors related to differentially expressed genes were predicted by bioinformatics principles, including NFKB1, RELA, AP1, STAT3, p53 and p63, and their transcriptional regulation relationships. 24 mRNA tables were screened out with the same gene as TCGA database, and the frequency of genomic variation was in 5%-37%; 7 14 genes showed an increase or decrease in the number of copies, and the expression of mRNA was closely related to the change in the number of copies. A total of 8 candidate genes were screened with 50 gene chip data to be associated with the malignant phenotype of tumor, including RPN2, HMGB1, AARS, IGFBP3, STAT1, YOU1, NQO1 and PEA15, and 9 different types of solid tumor ratio. In the comparative study, PRN2 and HMGB1 had the highest frequency of variation in colorectal cancer; the immunohistochemical results showed that the expression of RPN2 and HMGB1 protein expressed significantly in the cancer tissues in 78 cases of colorectal cancer, and there was a significant correlation between RPN2 and the degree of tissue differentiation and metastasis. A total of eight candidate genes associated with tumor metastasis were screened from the group database and the gene chip experimental data of different populations, and the potential clinical application value of RPN2 and HMGB1 in colorectal cancer as a molecular marker and RNAi drug target for early tumor metastasis prediction was revealed.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.3

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