【作者】 罗敏；

【导师】 霍继荣；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 第一章人结肠癌细胞中PCDH8表达及启动子区域甲基化目的：结直肠癌是全球范围内发病率高和死亡率高的恶性肿瘤之一,在我国,结直肠癌居恶性肿瘤发病率的第3位,死亡率的第4位。DNA甲基化是结直肠癌研究的热点,DNA异常甲基化被认为是抑癌基因失活的一个重要原因,因此,其在结直肠癌的发生、发展及转移中起着关键的作用。在膀胱癌、胃癌、乳腺癌、鼻咽癌等恶性肿瘤的研究中,PCDH8基因被认为是一种可能的候补抑癌基因,但是在结直肠癌中尚无报道。该章研究的主要目的是比较人正常结肠上皮细胞株和人结肠癌细胞株中PCDH8mRNA、PCDH8蛋白水平及其启动子区域甲基化差异。方法：1、用RT-PCR检测人结肠正常上皮细胞株(NCM460)和人结肠癌细胞株(HT29、HCT116、SW480、SW620)中PCDH8mRNA的表达；2、用Werstem Blotting方法检测人结肠正常上皮细胞株(NCM460)和人结肠癌细胞株(HT29、HCT116、SW480、SW620)中PCDH8蛋白的表达；3、用甲基化特异性PCR (MSP)检测人结肠正常上皮细胞株(NCM460)和人结肠癌细胞株(HT2、HCT116、 SW480、SW620)中PCDH8基因启动子区域的甲基化。结果：1、PCDH8mRNA在人正常结肠细胞株NCM460中稳定表达,在人结肠癌细胞株HT29和HCT116中表达缺失,在人结肠癌细胞株SW480和SW620中表达下调；2、人正常结肠细胞株NCM460中PCDH8蛋白稳定表达,人结肠癌细胞株HT29、HCT116、SW480和SW620中,PCDH8蛋白表达下调；3、人正常结肠细胞株NCM460中,PCDH8基因启动子区域甲基化阴性；人结肠癌细胞株SW620、SW480、HCT116和HT29中,PCDH8基因启动子区域甲基化阳性。结论：PCDH8基因在人结肠癌细胞株SW620、SW480、HCT116和]HT29中表达下调,可能与PCDH8基因启动子区域甲基化有关,提示为结直肠癌发生发展的因素之一。第二章人结直肠癌组织中PCDH8蛋白表达水平及其启动子区域甲基化目的：探讨人结直肠癌组织、远离结直肠癌的正常组织和人结直肠腺瘤组织中PCDH8蛋白表达水平及启动子区域甲基化方法：1、用免疫组织化学染色检测结直肠癌组织、远离结直肠癌的正常组织和结直肠腺瘤组织中PCDH8蛋白表达水平；2、用甲基化特异性PCR (MSP)检测结直肠癌组织、远离结直肠癌的正常组织和结直肠腺瘤组织中PCDH8基因启动子区域的甲基化变化；3、应用SPSS13.0软件,采用卡方检验统计学方法,分析PCDH8蛋白表达水平与结直肠癌患者临床病理参数的相关性；4、应用SPSS13.0软件,采用卡方检验统计学方法,分析PCDH8基因启动子区域的甲基化变化与结直肠癌患者临床病理参数的相关性。结果：1、结直肠癌组织中,16/68(30.77%)PCDH8蛋白阳性；远离结直肠癌的正常组织中,32/34(94.12%)PCDH8蛋白阳性；结直肠腺瘤组织中,16/20(80%)PCDH8蛋白阳性。远离结直肠癌的正常组织和结直肠腺瘤组织中PCDH8蛋白阳性率均高于结直肠癌组织(P<0.01),远离结直肠癌正常组织和结直肠腺瘤组织中PCDH8蛋白阳性率无显著差异(P=0.321)。2、结直肠癌组织中,56/68(82.35%)发生了PCDH8基因启动子区域的甲基化；远离结直肠癌的正常组织中,仅2/34(5.88%)发生PCDH8基因启动子区域的甲基化；结直肠腺瘤组织中,14/20(70%)发生PCDH8基因启动子区域的甲基化。结直肠癌和结直肠腺瘤组织中PCDH8基因启动子区域甲基化阳性率均高于远离结直肠癌的正常组织(P<0.01),结直肠癌组织和结直肠腺瘤组织中PCDH8基因启动子区域甲基化阳性率无显著差异(P=0.402)。3、PCDH8蛋白的表达水平与结直肠癌患者的性别、诊断时的年龄、肿瘤大小、TNM分期、分化程度、淋巴结转移无明显相关(P>0.05)。4、PCDH8基因启动子区域甲基化与结直肠癌肿瘤大小有明显的相关性(P<0.01),但与患者的性别、诊断时的年龄、TNM分期、分化程度、淋巴结转移无明显相关(P>0.05)。结论：PCDH8基因启动子区域甲基化在人结直肠癌组织和人结直肠腺瘤组织中频繁发生,可能是结直肠癌发生的早期事件,是结直肠癌发生发展的原因之一。更多还原

【Abstract】 Chapter1:PCDH8mRNA, PCDH8promoter methylation and PCDH8protein in colorectal cancer cell linesPurpose:Colorectal cancer is one of the world-wide malignancy leading to highest morbidity and mortality. In China, colorectal cancer is the3rd frequentest malignancy, and the4th cause of death among all of the malignancy. The study of DNA methylation in colorectal cancer has become a hotspot research in recent years, and aberrant methylation has been considered as an alternative mechanism, which is responsible for inactive tumor suppressor gene. Therefore, DNA methylation is likely to play an very important role in the development and progression of colorectal cancer. Low expression and methylation of the promoter in protocadherin8(PCDH8) gene have been detected in gastric cancer, breast cancer and bladder cancer, in which PCDH8is confirmed a novo tumor suppressor gene. However, none reserch has been done in colorectal cancer about PCDH8. In the part, we studied the expression of PCDH8in the cell lines of colorectal cancer.Methods:1.The expression of PCDH8mRNA was detected by RT-PCR in the human normal colonic epithelial cell line (NCM460) and four human colorectal cancer cell lines;2. PCDH8protien was detected by Werstern Blotting in human normal colonic epithelial cell line and four human colorectal cancer cell lines;3.PCDH8methylation was evaluated by methylation-specific PCR in human normal colonic epithelial cell line and four human colorectal cancer cell lines.Results:1. PCDH8mRNA was stablely expressed in the human normal colonic epithelial cell line (NCM460), PCDH8mRNA was nearly not detected in the human colorectal cancer cell lines HT29and HCT116, PCDH8mRNA was lowly expressed in he human colorectal cancer cell lines SW480and SW620.2. PCDH8protein was lowly expressed in all of the four human colorectal cancer cell lines,while it was high expressed in the human normal colonic epithelial cell line.3. PCDH8promoter methylation was showed in all of the four human colorectal cancer cell lines, while none PCDH8promoter methylation was detected in the human normal colonic epithelial cell line.Conclusions:PCDH8gene is lowly expressed in all of the four human colorectal cancer cell lines, which is likely to be associated with the PCDH8promoter methylation. Maybe, it is the one of the reason for the development and progression of colorectal cancer. Chapter2:PCDH8protein and PCDH8promoter methylation in colorectal cancer tissuesPurpose:To explore the PCDH8protein and PCDH8promoter methylation in the human colorectal cancer tissues, human colonic adenoma tissues and adjacent non-tumor tissues;.Methods:1.PCDH8protien was detected by Immunohistochemical staining in human colorectal cancer tissues,human colonic adenoma tissues and adjacent non-tumor tissues;2. PCDH8promoter methylation was evaluated by methylation-specific PCR (MSP) in human colorectal cancer tissues,human colonic adenoma tissues and adjacent non-tumor tissues;3. Applicate SPSS13.0software and chi-square test statistics method to explore the possible correlation between PCDH8protein and clinicopathologic charters of colorectal cancer;4. Applicate SPSS13.0software and chi-square test statistics method to explore the possible correlation between PCDH8promoter methylation and clinicopathologic charters of colorectal cancer.Results:1. Immunohistochemical staining showed that PCDH8protein was frequently detected in adjacent non-tumor tissues (94.12%,32/34), human colonic adenoma tissues (80%,16/120), and human colorectal cancer tissues (30.77%,16/68). Statistically, there was no difference in expression of the PCDH8protein between adjacent non-tumor tissues and human colonic adenoma tissues(P=0.321). However, there were statistically significant differences between colorectal cancer tissues and adjacent non-tumor tissues (P<0.01), and between human colorectal cancer tissues and human colonic adenoma tissues (P<0.01).2. MSP analysis showed that PCDH8gene promoter methylation was frequently detected in human colorectal cancer tissues (82.35%,56/68) and human colonic adenoma tissues (70%,14/20), but in adjacent non-tumor tissues only2of32(5.88%) were showed PCDH8gene promoter methylation. Statistically, there was no difference in methylation of the PCDH8gene promoter between human colorectal cancer tissues and human colonic adenoma tissues(P=0.402). However, there were statistically significant differences between colorectal cancer tissues and adjacent non-tumor tissues (P<0.01), and between human colonic adenoma tissues and adjacent non-tumor tissues (P<0.01).3. PCDH8protein expression was not significantly correlated with sex, age of diagnose, size of tumor, TNM stage, tumor differentiation, lymph node metastasis in colorectal cancer(P>0.05).4. PCDH8promoter methylation was statistical significantly correlated with size of tumor, in colorectal cancer(P<0.01),while not significantly correlated with sex, age of diagnose, TNM stage, tumor differentiation, lymph node metastasis in colorectal cancer(P>0.05). ConcIusions:PCDH8gene promoter methylation was frequently detected in human colorectal cancer tissues and human colonic adenoma tissues, which is likely to be an early stage in colorectal cancer. Maybe, it is the one of the reason for the development and progression of colorectal cancer.更多还原