非小细胞肺癌侵袭转移中Wnt通路差异基因表达研究

发布时间：2018-09-12 15:06

【摘要】：第一部分全基因表达谱技术筛选与Wnt通路相关的NSCLC转移相关性基因目的：本研究旨在运用全基因表达谱技术初步筛选NSCLC转移差异表达基因,寻找与Wnt通路相关的转移基因并分析其基因功能,为非小细胞肺癌的浸润转移机制研究及治疗位点提供新思路。方法：收集2014年4月至2014年10月山东大学齐鲁医院胸外科手术切取的肺癌组织,离体后立即放入冻存管中,并于10分钟内置于液氮罐中冷冻2-3小时,后转入-80℃冰箱中冻存。同时收集留取标本肺癌患者的临床资料。所选病例均未行术前放疗和化疗,且经病理证实为原发性非小细胞肺癌。从中选取淋巴结转移组6例,为实验组,无淋巴结转移组6例,为对照组。Trizol法抽提RNA,使用NanoDrop ND-1000评估RNA量和质量,RNA完整性通过标准变性凝胶电泳进行评估。采用Agilent人类4×44K基因表达微阵列v2,将实验组和对照组进行全基因组表达谱实验。使用Agilent Feature Extraction软件获得芯片图,得到原始数据。使用GeneSpring GX v12.1软件对原始数据进行Quantile标准化,并进行基因本体论(Gene Ontology, GO)分析和Pathway分析,筛选与Wnt通路相关的转移差异基因并分析其可能参与的生物学过程。结果：1、RNA质量控制：基因芯片实验样本的OD值显示A260/A280均在1.8-2.1之间,琼脂糖凝胶电泳结果显示28SrRNA和18SrRNA条带清晰,RNA纯度和完整性较好,样品合格。2、聚类分析：聚类分析显示转移组和非转移组组间差异显著,同一类样品能通过聚类出现在同一个簇(cluster)中,聚在同一个簇的样品具有相似的生物学性质,提示芯片试验具有一定的重复性,结果可靠。3、差异基因：根据倍数法筛选出差异基因11975个,转移上调基因5812个,下调基因6163个。4、GO分析：结果显示差异基因主要涉及多细胞生物过程、信号转导、G蛋白偶联受体活性、细胞外基质等功能集团。5、Pathway分析：转移相关差异基因主要涉及MAPK信号通路、细胞周期、PI3K-Akt信号通路、Wnt通路、p53信号通路等。6、筛选Wnt通路差异基因：筛选出与Wnt通路相关的NSCLC转移差异基因共34个,其中上调基因18个,下调基因16个,涉及细胞周期素基因、蛋白激酶C基因、卷曲家族受体基因、转录因子基因等,GO分析显示上述基因参与蛋白结合、细胞质膜、细胞内信号转导、卷曲蛋白结合、细胞增殖正性调节、特定序列DNA结合转录因子活性等生物学过程。结论：1、NSCLC淋巴结转移与未转移之间存在大量差异表达基因,涉及多种细胞生物过程、细胞外基质等功能集团和MAPK、Wnt、p53等多条信号转导通路。2、与Wnt通路相关的差异基因主要参与特定序列DNA结合转录因子活性、细胞内信号转导、细胞增殖正性调节等生物学过程,但尚需进一步验证。第二部分与Wnt通路相关的非小细胞肺癌转移相关基因的验证目的：由于芯片结果存在一定的假阳性率,本研究采用实时荧光定量PCR(real-time quantitative polymerase chain reaction, RT-qPCR)技术对目的基因进行相对定量检测与分析,旨在验证表达谱芯片的可靠性,指导下一步研究方向。方法：从上述筛选出的34个差异基因中挑选出与肿瘤发生发展有密切关系的基因CCND2, PRKCA, NFATC1作为目的基因,继续应用上述12例肺癌冻存组织,淋巴结转移组6例,为实验组,无淋巴结转移组6例,为对照组。RNA抽提方法及质量检测方法同上,分别对实验组和对照组的目的基因及管家基因进行实时定量PCR技术,并绘制浓度稀释DNA标准曲线,计算目的基因相对含量,采用配对t检验检测实时定量PCR结果与基因芯片结果是否具有一致性。结果：所选取的差异基因CCND2, PRKCA, NFATC1在淋巴结转移组中较无淋巴结转移组均呈表达上调,且实时定量PCR与表达谱芯片的上调倍数对比,二者差别无统计学意义(t=0.4539,P0.05)。结论：1、CCND2, PRKCA, NFATC1实时定量PCR结果与表达谱芯片结果一致,全基因表达谱芯片技术能够高通量筛选出具有研究意义的差异表达基因。2、CCND2, PRKCA, NFATC1基因与人类肿瘤发生发展密切相关,在肺癌转移中表达水平上调,表明其在肺癌侵袭转移中具有重要研究价值。
[Abstract]:Part I Screening of NSCLC metastasis-related genes associated with Wnt pathway by whole gene expression profiling: The purpose of this study was to screen differentially expressed genes for NSCLC metastasis by using whole gene expression profiling technology, search for Wnt pathway-related metastasis genes and analyze their gene functions, and study the invasion and metastasis mechanism of non-small cell lung cancer. Methods: Lung cancer tissues were collected from the thoracic surgery of Qilu Hospital of Shandong University from April 2014 to October 2014. The tissues were put into cryopreservation tube immediately after being detached and frozen in liquid nitrogen tank for 2-3 hours within 10 minutes. Materials. None of the patients underwent preoperative radiotherapy or chemotherapy, and were confirmed to be primary non-small cell lung cancer by pathology. Six patients with lymph node metastasis were selected as experimental group, six patients without lymph node metastasis as control group. RNA was extracted by Trizol method, and RNA quantity and quality were evaluated by NanoDrop ND-1000. RNA integrity was evaluated by standard denaturing gel electrophoresis. Using Agilent human 4 *44K gene expression microarray v2, the whole genome expression profiles of the experimental group and the control group were tested. The chips were obtained by using Agilent Feature Extraction software, and the original data were obtained. The original data were standardized by using GeneSpring GX v12.1 software, and the gene ontology (GO) was performed. Results: 1. RNA quality control: OD values of DNA chip samples showed that A260/A280 were between 1.8 and 2.1. Agarose gel electrophoresis showed that 28SrRNA and 18SrRNA bands were clear, RNA purity and integrity were good. Cluster analysis: Cluster analysis showed that there was a significant difference between metastatic group and non-metastatic group. Samples of the same group could appear in the same cluster by clustering. Samples clustered in the same cluster had similar biological properties, suggesting that the chip test had a certain repeatability and the results were reliable. Methods 11975 differentially expressed genes, 5812 up-regulated genes and 6163 down-regulated genes were screened. GO analysis showed that the differentially expressed genes were mainly involved in multicellular biological processes, signal transduction, G protein-coupled receptor activity, extracellular matrix and other functional groups. Cycle, PI3K-Akt signaling pathway, Wnt pathway, p53 signaling pathway and so on. 6. Screening of Wnt pathway differential genes: Screening of Wnt pathway related to NSCLC transfer differential genes a total of 34, including 18 up-regulated genes, 16 down-regulated genes, involving cyclin genes, protein kinase C genes, convolution family receptor genes, transcription factor genes, GO analysis was obvious. These genes are involved in protein binding, cytoplasmic membrane, intracellular signal transduction, convolution protein binding, positive regulation of cell proliferation, and activity of specific DNA binding transcription factors. Functional groups, MAPK, Wnt, p53 and other signal transduction pathways. 2. Differential genes related to Wnt pathway are involved in specific sequence DNA binding transcription factor activity, intracellular signal transduction, positive regulation of cell proliferation and other biological processes, but need to be further verified. The second part is related to metastasis of non-small cell lung cancer associated with Wnt pathway. Objective: In order to verify the reliability of the microarray and guide the next research direction, real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect and analyze the target gene. CCND2, PRKCA and NFATC1 were selected as the target genes from the 34 differentially expressed genes. The 12 cases of lung cancer cryopreserved tissues, 6 cases of lymph node metastasis group, 6 cases of experimental group and 6 cases of non-lymph node metastasis group were used as control group. RNA extraction method and quality detection method were the same. Real-time quantitative PCR was performed on the target genes and housekeeping genes of experimental group and control group respectively, and the concentration dilution DNA standard curve was drawn to calculate the relative content of the target genes. There was no significant difference between the two groups (t = 0.4539, P 0.05). Conclusion: 1. The results of CCND2, PRKCA, NFATC1 real-time quantitative PCR were consistent with those of expression microarray, and the whole gene expression microarray technology could achieve high throughput. The differentially expressed genes. 2, CCND2, PRKCA and NFATC1 are closely related to the occurrence and development of human tumors. They are up-regulated in the metastasis of lung cancer, indicating that they have important research value in the invasion and metastasis of lung cancer.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2

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