【作者】 张德才；

【导师】 沈守荣；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 研究背景与目的：结直肠癌是世界上第三大常见的恶性肿瘤,全球每年新发病例约120万例,死亡人数达60万。在我国,结直肠癌位居恶性肿瘤死亡率第四位。近年来,我国结直肠癌的发病和死亡呈明显上升趋势。结直肠癌的发生、发展是一个涉及多基因、多步骤、多阶段的复杂过程。80%以上的结直肠癌是从腺瘤癌变而来,而从腺瘤的发生至癌变的发生、发展,主要涉及癌基因、抑癌基因、错配修复基因等三类基因的变化。其中抑癌基因的异常在结直肠癌的发生发展中起关键作用,其失活或缺失可导致细胞去调节性生长,克隆性扩张。因此,在结直肠癌病因学研究中,有必要寻找与其发生、发展相关的抑癌基因,探讨其在疾病发生发展中的作用,进一步从分子水平阐明结直肠癌的发病机制,为疾病的早发现、早诊断和早治疗提供重要的理论基础。本课题组前期研究采用荧光差异双向电泳技术构建了不同临床分期结直肠癌蛋白质差异表达谱,发现琥珀酸脱氢酶(succinate dehydrogenase, SDH)是结直肠癌发病过程中一个差异表达蛋白质分子,可能具有潜在的抑癌功能。SDH也称为线粒体复合物Ⅱ,定位于线粒体内膜,是由A、B、C、D四个亚基组成的异源四聚体,分别由相应的核基因编码。SDH是三羧酸循环和线粒体呼吸链的组成部分,其在细胞能量代谢中起到重要的作用。SDH作为一个抑癌基因,其基因缺陷或表达异常与多种疾病的发生有关,如副神经节瘤、嗜铬细胞瘤、肾癌、胃肠道间质瘤、Leigh综合征等。前期研究中我们利用免疫组化技术检测了包含103例病例的结直肠组织芯片中SDH的表达情况,发现SDHB蛋白在结直肠癌组织的表达较正常结直肠粘膜明显下调,且SDHB在低分化结直肠癌组织中表达较中、高分化癌组织明显减弱。目前对SDH基因在结直肠癌发生发展中的作用及机制国内外尚未见报道。本课题在前期工作的基础上,扩大样本量检测SDHB在低分化结肠癌组织中的表达以进一步证实SDHB的表达与结肠癌分化程度的关系。为了进一步深入研究SDHB基因在结直肠癌发生发展中的作用,我们构建了SDHB过表达稳定转染细胞系以明确SDHB基因对结肠癌细胞SW620生物学行为的影响。同时研究了SDHB基因对SW620细胞基因表达谱的影响。此外,我们从SDHB基因突变和启动子甲基化两个方面对SDHB在结直肠癌中表达下调的机制进行了探讨。方法：本研究在包含32个点阵的低分化结肠癌组织芯片中应用免疫组化技术检测了SDHB蛋白的表达。而后采用真核表达载体pIRESneo3构建了SDHB基因稳定表达载体pIRES-SDHB,利用脂质体转染的方法将其导入SW620细胞,建立了SDHB稳定转染结肠癌细胞系。通过细胞生长曲线、平板集落形成、裸鼠移植瘤形成试验等明确SDHB对结肠癌细胞生长与增殖的作用。通过流式细胞仪检测SDHB对SW620细胞周期的影响,同时采用Western blot及免疫细胞化学法分别检测细胞周期相关分子cyclin D1和PCNA的表达。通过构建人全基因组表达谱芯片探究了SDHB基因过表达对SW620细胞基因表达谱的影响。此外,提取了40对结直肠癌及配对的癌旁正常结直肠粘膜组织gDNA,一方面采用PCR扩增了SDHB基因各外显子片段,产物纯化后进行双向测序检测各外显子突变情况；另一方面将gDNA经亚硫酸氢盐修饰后,采用甲基化特异性PCR检测SDHB基因启动子甲基化状态。结果：(1) SDHB在结直肠癌组织中的表达①免疫组织化学检测SDHB蛋白在结直肠癌组织芯片中的表达,发现SDHB在结直肠癌组织的表达较正常结直肠粘膜明显下调,且其在低分化结直肠癌组织中表达较中、高分化明显减弱。②Western blot检测SDHB蛋白在结直肠癌组织中的表达,发现SDHB在结直肠癌组织中表达正常粘膜组织明显减弱。(2) SDHB基因转染对结肠癌SW620细胞生物学功能的影响①细胞生长曲线显示转染SDHB基因显著抑制了SW620细胞的生长。②平板集落形成试验显示转染SDHB基因显著抑制SW620细胞的集落形成能力。③裸鼠移植瘤形成实验表明SDHB抑制结肠癌SW620细胞致瘤性。④流式细胞仪检测发现稳定转染SDHB基因导致SW620细胞周期发生G1-S期阻滞。⑤Western blot及免疫细胞化学法检测发现SDHB下调SW620细胞中cyclin D1及PCNA的表达。(3) SDHB对结肠癌细胞基因表达谱的影响①采用Illumina公司的人全基因组表达谱芯片(Human HT-12v4)共筛选出受SDHB基因调控结肠癌SW620细胞中差异表达的基因共1653个,其中上调基因1382个,下调基因271个。②采用real-time PCR验证了部分差异表达基因,结果与芯片检测结果一致,说明芯片数据可靠。③GO分析发现差异表达基因主要参与细胞周期及其调控、细胞增殖调控、转录调控、细胞信号传导等过程。④KEGG数据库分析发现差异表达基因主要参与p53、MAPK、 Toll样受体、ErbB、Jak-STAT、T细胞受体、Notch、Wnt、TGF-β、 VEGF、细胞因子相互作用等信号传导途径。(4) SDHB在结直肠癌组织中表达下调的机制①PCR扩增SDHB基因各外显子片段,测序后发现在40例结直肠癌和与配对的正常结直肠粘膜组织标本中SDHB基因第一外显子第18个碱基处存在一个SNP位点,碱基由C颠换成A,致第6位密码子由GCC变为GCA,两个密码子均编码丙氨酸,为一同义突变；未发现致病性突变。结果提示SDHB基因外显子突变可能不是单纯性结直肠癌的致病原因。②MSP法未能在上述40对组织中检测出SDHB基因启动子区CpG岛甲基化,推测SDHB在结肠癌组织中表达下调可能不是由启动子区甲基化引起的。结论：(1) SDHB在结直肠癌组织中表达下调,且表达水平与结直肠癌组织分化程度相关,在低分化癌组织中表达较中、高分化癌组织明显减弱。(2)SDHB可抑制体外SW620细胞的生长与增殖,诱导细胞G1-S期阻滞；抑制SW620细胞裸鼠皮下移植瘤的形成。(3) SDHB基因外显子点突变和启动子区甲基化可能不是SDHB在结直肠癌组织中表达下调的机制。更多还原

【Abstract】 Background and objective:Colorectal cancer (CRC) is the third most prevalent cancer in the world, accounting for approximately1.2million new cases and more than600,000cancer-related deaths each year worldwide. In China, CRC ranks fourth in cancer mortality, and in recent years, the morbidity and mortality of CRC in China shows an obvious upward trend. The occurrence and development of CRC is a complex process involving multi-gene, multi-step, and multi-stage. More than80%of CRC begins with adenomatous polyp, further advances to adenoma with high grade dysplasia and finally progresses to an invasive cancer. This process mainly involves three types of genetic changes in oncogenes, tumor suppressor genes, and mismatch repair genes. The defect of tumor suppressor genes plays a key role in the development of CRC, which could cause cells to grow out of control and tumor progression. Therefore, it is necessary to find the tumor suppressor genes involved in the development of CRC, to explore their roles in tumor development, and to further clarify the pathogenesis at the molecular level in the etiology study, which could provide an important theoretical basis for early detection, diagnosis, and treatment of CRC.In previous study, protein dynamic expression patterns in different stages of CRC were established using two-dimensional gel electrophoresis. The results showed that SDH exhibited potential tumor suppressor function in the pathogenesis of CRC. SDH (also known as mitochondrial complex Ⅱ) is a heterotetrameric protein consists of A, B, C and D subunits encoded by corresponding nuclear genes, localized on the inner mitochondrial membrane which catalyse the oxidative dehydrogenation of succinate and, as part of complex Ⅱ of the electron transport chain, couple this to the reduction of ubiquinone (succinate-ubiquinone oxidoreductase). As a tumor suppressor gene, genetic defects or abnormal expression in SDH are associated with a diverse collection of diseases, including paragangliomas (PGL), pheochromocytoma, renal cancer, gastrointestinal stromal cell tumor, and Leigh syndrome. Initially, the SDH expression was detected by immunohistochemistry using tissue microarray (TMA) sections which containing103cases of CRC tissue specimens. Expression of SDHB protein was found to be significantly lower in CRC tissues compared to the normal mucosa. And the expression of SDHB was negatively correlated with the differentiation status of CRC tissues.The role of SDH in CRC development has not been reported previously. Based on preliminary work, we detect SDHB expression in poorly differentiated CRC tissues with expanded sample to further confirm the correlation between SDHB expression and differentiation status of CRC in the present study. In order to further investigate the potential SDH tumor suppressor functions in CRC, we stably expressed SDHB protein in human colon cancer cells to determine the effects of SDHB expression on tumor cell growth and tumorigenesis. And differential expression of genes induced by ectopic SDHB expression in cancer cells was screened by microarray analysis. Moreover, we investigated the mechanism for downregulated expression of SDHB in CRC in genetic and epigenetic aspects, such as gene mutations and DNA methylation.Methods:In this study, we examined SDHB protein expression in a TMA containing32cases of poorly differentiated colon cancer tissues by immunohistochemistry. Then SDHB was transfected into colon cancer cell line (SW620) by lipofectin and a stable cell line of pIRES-SDHB overexpressing SDHB gene was established. The effect of SDHB on malignant behavior of SW620was assessed by growth curves of cells, colony formation, and tumor formation in nude mice. The effect of SDHB on cell cycle progression in SW620was investigated by flow cytometry.The expression of cell cycle related proteins cyclin D1and PCNA was detected by western blot and immunocytochemistry. In order to identify genes modulated by SDHB in CRC, the expression profile of SDHB expressing colon cancer cells and controls were investigated. Moreover, genomic DNA was extracted in forty CRC and paired normal mucosa tissues. On the one hand, SDHB gene exons were amplified by PCR and all products were purified and sequenced from both directions to detect gene mutations. On the other hand, genomic DNA was treated by sodium bisulfite modification and then amplified by methylation-specific PCR to assess the SDHB promoter methylation status.Results:(1) Protein expression of SDHB in CRC①Immunohistochemical results showed that expression of SDHB protein was found to be significantly lower in CRC tissues compared to the normal mucosa. And the expression of SDHB in poorly differentiated CRC was significantly lower compared to that in moderately and well differentiated CRC.②Western blot showed the same results which were consistent with that of immunohistochemistry.(2) Effects of SDHB expression on tumor cell growth and tumorigenesis①Cell growth curves showed the restoration of SDHB significantly inhibited the growth of SW620cells.②Colony formation showed the ectopic expression of SDHB substantially inhibited tumor cell colony formation.③Xenograft tumor formation assay showed that SDHB inhibited tumorigenicity in SW620cells.④Flow cytometry results of the cell cycle distribution showed that SDHB induced cell cycle arrest at the G1phase.⑤Western blot and immunocytochemistry showed that SDHB downregulated expression of cyclin D1and PCNA.(3) Effects of SDHB expression on the gene expression profile of colon cancer cells①cDNA microarray analysis showed total of1,653differentially expressed genes with more than a twofold change (p<0.01), of which1,382gene expressions were upregulated and271gene expressions were downregulated in SW620-SDHB cells compared with vector control cells.②The differentially expressed genes were validated by real-time RT-PCR.③Most of the differentially expressed genes were grouped in GO with regard to "cell cycle","cellular process","transcriptional regulation" or "cell signaling".④The KEGG database analysis showed that the differentially expressed genes were mainly involved in the p53, MAPK, Toll-like receptors, ErbB, Jak-STAT, T cell receptor, Notch, Wnt, TGF-β, VEGF, cytokines interaction signal transduction pathway.(4) Mechanism for downregulated expression of SDHB in CRC①Forty cases of CRC and paired normal mucosa tissues contained a single nueleotide polymorphism in SDHB exonl which located in the18th base pair, C substituted by A, making the6th codon changed from GCC to GCA. However, both GCC and GCA are encode for alamine. And no pathogenic mutation was found. The results suggested that the exon mutation in SDHB gene may not be the cause of CRC.②Methylation-specific PCR failed to detect SDHB promoter methylation in all these CRC and paired normal mucosa tissues, which suggested that the mechanism for downregulated expression of SDHB in CRC may not be caused by promoter methylation.Conclusion:(1) Expression of SDHB protein was significantly lower in CRC tissues compared to the normal mucosa, and its expression was negatively correlated with the differentiation status of CRC tissues.(2) Restoration of SDHB could inhibit tumor cell growth, colony formation, tumorigenicity, and induce cell cycle arrest at the G1phase.(3) The mechanism for downregulated expression of SDHB in CRC may not be due to exon mutation or promoter methylation.更多还原