DNMT3a在结直肠癌组织中的表达及其对HCT116细胞株生物学功能的影响
发布时间:2018-06-21 02:44
本文选题:结直肠癌 + DNMT3a ; 参考:《河北医科大学》2017年硕士论文
【摘要】:目的:通过Real time-q PCR技术检测DNA甲基化转移酶3a(DNMT3a)mRNA在结直肠癌(colorenctal cancer,CRC)组织及相应切缘正常粘膜组织中的表达水平,分析DNMT3a表达水平与结直肠癌患者病理和临床特征之间的关系。同时进一步应用Cell Counting Kit-8(CCK-8)法、流式细胞技术(FCM)及Transwell迁移实验等方法检测DNMT3a对结肠癌细胞株HCT116的增殖、凋亡以及迁移等生物学功能的影响。探讨DNMT3a作为潜在肿瘤标志物对结直肠癌早期诊断及预后评估的价值,进一步探究DNMT3a在肿瘤发生发展过程中的作用机制,以便为结直肠癌的早期筛查、诊断、药物治疗提供新的理论和实验依据。方法:1收集2012年5月-2015年3月河北医科大学第四医院以及河北医科大学第一医院共计25例结直肠癌组织标本,每例标本均取自结直肠原发癌组织及其两侧切缘正常黏膜组织(术后病理证实切缘无癌细胞侵犯)。新鲜标本离体后迅速置于液氮中,于-80℃冰箱保存备用。25例患者均为第一次手术,且术前均未接受放疗或化疗。2应用实时荧光定量PCR(RT-q-PCR)技术检测DNA甲基化转移酶3a(DNMT3a)mRNA在结直肠癌(colorenctal cancer,CRC)组织及相应切缘正常粘膜组织中的表达水平,分析DNMT3a表达水平与结直肠癌患者病理和临床特征之间的关系。选取结肠癌细胞株HCT116进行培养,应用Lipofectamine 2000将DNMT3a小干扰RNA(siRNA)转入HCT116细胞,设为实验组,同时设立对照组(转染阴性对照siRNA),转染后24小时检测两组细胞中DNMT3a mRNA的表达情况。3转染DNMT3a siRNA后1d、2 d、3 d、4 d、5d分别应用CCK-8法检测5个时段的OD值,绘制细胞生长曲线以观察细胞增殖改变情况。4应用流式细胞技术检测转染前后细胞凋亡的变化情况。5应用Transwell技术检测转染前后肿瘤细胞迁移能力的变化情况。6应用spss13.0软件对数据进行统计分析,数据以中位数(四分位数间距)或均数±标准差表示,两组间配对样本采用Wilcoxon检验或t检验,两组间独立样本采用Mann-Whitney检验,多组间比较采用单因素方差分析,均以P0.05为差异有统计学意义。结果:1 DNMT3a mRNA在25例结直肠癌组织及其对应切缘正常粘膜组织中的表达水平分别为2.332(1.3685,4.653)和0.9073(0.2708,1.5185),结直肠癌组织中的表达较正常黏膜组织显著上调(P0.0001)。2 DNMT3a的表达水平与结直肠癌患者的部分临床病理特征密切相关,即T分期为T3、T4的患者明显高于T1、T2期的患者(P=0.0239),肿瘤直径5cm的患者明显高于≤5cm的患者(P=0.0043),而与性别、年龄、病理类型、分化程度和有无淋巴结转移无关(均P0.05)。3转染24h后,实验组细胞中DNMT3a mRNA的表达水平明显低于阴性对照组。4转染DNMT3a siRNA实验组在转染后1d、2d、3 d、4 d、5d,5个时段OD值均低于对阴性照组,差异具有统计学意义(均P0.05)。5 HCT116细胞中,实验组细胞凋亡率(3.35±0.451)%,相较于对照组(2.733±0.459)%呈升高趋势,但差异无统计学意义(P=0.4016)。6转染24小时后对细胞进行Transwell实验,实验组细胞迁移数目为(140±12)个,阴性对照组细胞迁移数目为(231±14)个,实验组的迁移能力明显弱于阴性对照组,(P=0.0395)。结论:1 DNMT3a可能作为一种促癌因子在结直肠癌发生过程中发挥作用;2 DNMT3a可能通过沉默多种抑癌基因的表达而促进了直肠癌的发生及肿瘤恶性程度的进展,并可以为肿瘤预后的判断提供参考;3 DNMT3a可作为潜在的新型肿瘤标记物和治疗靶点,为结直肠癌患者的诊断及肿瘤药物的研发提供新的思路。
[Abstract]:Objective: to detect the expression level of DNA methyltransferase 3A (DNMT3a) mRNA in colorectal cancer (colorenctal cancer, CRC) tissue and the normal mucosal tissue by Real time-q PCR, and to analyze the relationship between DNMT3a expression level and the pathological and clinical characteristics of colorectal cancer patients. 8) method, flow cytometry (FCM) and Transwell migration test to detect the effects of DNMT3a on the proliferation, apoptosis and migration of colon cancer cell line HCT116, and explore the value of DNMT3a as a potential tumor marker for early diagnosis and prognosis of colorectal cancer, and further explore DNMT3a in the process of tumor development. The mechanism of action is provided to provide new theoretical and experimental basis for early screening, diagnosis and treatment of colorectal cancer. Methods: 1 a total of 25 specimens of colorectal cancer were collected from the fourth hospital of Hebei Medical University in March -2015 and the first hospital of Hebei Medical University in May 2012. Each sample was taken from the primary colorectal carcinoma and two The normal mucosa tissue of the lateral incisor (confirmed by postoperative pathology confirmed that there was no cancer cell invasion in the cutting edge). Fresh specimens were quickly placed in liquid nitrogen in vitro, and.25 patients were first operated at -80 C fridge for the first time, and no radiotherapy or chemotherapy.2 was used to detect 3A (DNMT3a) mRNA DNA methyltransferase 3A (DNMT3a) mRNA before operation. The expression level in the colorenctal cancer (CRC) tissue and the normal mucosal tissue of the corresponding cutting edge was analyzed. The relationship between the level of DNMT3a expression and the pathological and clinical features of colorectal cancer patients was analyzed. The colon cancer cell line HCT116 was cultured and Lipofectamine 2000 was used to transfer DNMT3a small interference RNA (siRNA) into HCT116 cells. In the experimental group, the control group was set up (transfected negative control siRNA), and the expression of DNMT3a mRNA in the two groups of cells was detected 24 hours after transfection..3 transfected to DNMT3a siRNA 1D, 2 D, 3 D, 4 D, 5D applied CCK-8 method to detect the 5 time periods respectively, and plotted the cell growth curve to observe the cell proliferation changes. Changes of cell apoptosis before and after dyeing.5 Transwell technique was used to detect the change of migration ability of tumor cells before and after transfection.6 applied SPSS13.0 software to analyze the data, the data were expressed with median (four quantile spacing) or mean standard deviation, and two pairs of paired samples were examined by Wilcoxon test or t test, and the two groups were independent. The samples were compared with single factor analysis of variance with Mann-Whitney test. The results showed that the expression level of 1 DNMT3a mRNA was 2.332 (1.3685,4.653) and 0.9073 (0.2708,1.5185) in the normal mucosa of 25 cases of colorectal cancer and its corresponding cutting edge, and the expression in colorectal cancer tissues was compared. The expression level of normal mucosal tissue (P0.0001).2 DNMT3a is closely related to some clinicopathological features of colorectal cancer patients, that is, T staging is T3, T4 patients are significantly higher than T1, T2 stage patients (P=0.0239), tumor diameter 5cm patients are significantly higher than those of 5cm (P=0.0043), and sex, age, pathological type, differentiation degree. After transfection of 24h with or without lymph node metastasis (P0.05), the expression level of DNMT3a mRNA in the experimental group was significantly lower than that of the negative control group.4 transfected DNMT3a siRNA experimental group, 1D, 2D, 3 D, 4 D, and the 5 periods were lower than the negative group, the difference was statistically significant. The rate of death (3.35 + 0.451)%, compared with the control group (2.733 + 0.459)%, was higher, but the difference was not statistically significant (P=0.4016).6 transfection for 24 hours after the Transwell experiment, the number of cell migration in the experimental group was (140 + 12), and the number of cell migration numbers in the negative control group was (231 + 14). The migration ability of the experimental group was significantly weaker than the negative control. Group, (P=0.0395). Conclusion: 1 DNMT3a may play a role in the carcinogenesis of colorectal cancer; 2 DNMT3a may promote the progression of rectal cancer and the malignancy of cancer by silencing the expression of a variety of tumor suppressor genes, and can provide reference for the prognosis of cancer; 3 DNMT3a may be a potential new type of swelling. Tumor markers and therapeutic targets provide new ideas for the diagnosis of colorectal cancer and the development of tumor drugs.
【学位授予单位】:河北医科大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:R735.34
【参考文献】
相关期刊论文 前2条
1 Yan Jia;Mingzhou Guo;;Epigenetic changes in colorectal cancer[J];Chinese Journal of Cancer;2013年01期
2 关志宇;戴冬秋;;胃癌TIMP3基因启动子甲基化及其蛋白表达的研究[J];世界华人消化杂志;2006年02期
,本文编号:2046834
本文链接:https://www.wllwen.com/yixuelunwen/zlx/2046834.html