RASSF6基因在弥漫大B细胞淋巴瘤中的甲基化状态、mRNA表达及其临床意义的研究

发布时间：2018-08-03 18:56

【摘要】：目的:非霍奇金淋巴瘤是血液系统恶性肿瘤之一,其中弥漫大B细胞淋巴瘤(Diffuse large B-cell lymphoma,DLBCL)是非霍奇金淋巴瘤中最常见的一种类型,约占成人淋巴瘤的一半,是一组具有高度异质性的侵袭性B细胞淋巴瘤,治疗方法主要以化疗为主。美罗华联合化疗方案显著提高了弥漫大B细胞淋巴瘤的疗效,R-CHOP方案被认为是标准治疗方案。然而,即便是应用标准方案,最终仍有将近40%的患者会复发进展。因此,弥漫大B细胞淋巴瘤病因学研究、基因组学分析为早期诊断、靶向治疗及预后评估提供了新的方向。弥漫大B细胞淋巴瘤的发生发展是多基因、多因素共同作用的结果,因此明确肿瘤相关基因是早期诊断及后期治疗的关键。研究表明,肿瘤的发生发展至少包含遗传和表观遗传两个层面的调控,包括癌基因激活、抑癌基因失活等,且表观遗传似乎起着更加重要的作用。近年来,表观遗传现象在多种肿瘤的发病机制中被明确,包括DNA甲基化、组蛋白修饰、染色质重塑等。研究证实RAS相关区域家族6基因(RASSF6)是一种抑癌基因,RASSF6的表达可以通过与K-Ras结合,协同抑制肿瘤细胞的增殖,且可通过激活JNK信号通路组织细胞周期进展;另外可通过激活caspase通路和其他通路诱导肿瘤细胞凋亡。有研究显示在儿童白血病、胃癌、乳腺癌等肿瘤中出现RASSF6基因启动子区高甲基化现象,甲基化是导致RASSF6基因失活的重要原因之一。而RASSF6基因在弥漫大B细胞淋巴瘤中的相关研究尚未见报道。本研究将通过应用甲基化特异性聚合酶链反应(Methylation-Specific PCR,MSP)和实时荧光定量逆转录-聚合酶链反应(real-time Quantitative Reverse Transcriptase-PCR,QRT-PCR)的方法验证RASSF6在弥漫大B细胞淋巴瘤中启动子区甲基化状态及表达情况,分析其与样本资料的关系,为弥漫大B细胞淋巴瘤的发病机制、临床诊断及治疗提供一定的理论依据。1应用MSP方法检测弥漫大B细胞淋巴瘤患者和健康人外周血RASSF6基因甲基化状态,并分析其与临床资料之间的关系。2应用QRT-PCR方法检测对比弥漫大B细胞淋巴瘤患者和健康人外周血中的RASSF6基因mRNA的相对表达量,并分析其与临床资料之间的关系。3应用统计软件SPSS19.0对数据进行统计学分析。结果:1 RASSF6基因启动子区在弥漫大B细胞淋巴瘤患者和健康人外周血中的甲基化率分别为48.6%(17/35)和14.3%(5/35)。弥漫大B细胞淋巴瘤患者RASSF6基因启动子区的甲基化率显著高于健康人,差异有统计学意义(P0.05)。2分组分析发现,在弥漫大B细胞淋巴瘤患者中,RASSF6甲基化与Ki-67、LDH、临床分期相关(P0.05);与患者的性别、年龄、结外累及、A/B症状、生发中心(Germinal center origin,GCB)或非生发中心(Nongerminal center origin,Non-GCB)、β2微球蛋白、骨髓累及、IPI均无关(P0.05)。3弥漫大B细胞淋巴瘤患者RASSF6基因的mRNA表达量为0.0415±0.0287,显著低于健康人mRNA的表达量0.0634±0.0427,差异有统计学意义(P0.05)。4分组分析发现,弥漫大B细胞淋巴瘤患者RASSF6基因mRNA的表达量与患者的IPI、Ki-67、结外累及相关(P0.05);与患者的性别、年龄、LDH、A/B症状、GCB或Non-GCB、β2微球蛋白、骨髓累及、临床分期均无关(P0.05)。5弥漫大B细胞淋巴瘤外周血中RASSF6基因启动子甲基化阳性的mRNA表达量为0.0306±0.0256,甲基化阴性的mRNA相对表达量平均为0.0518±0.0283,差异有统计学意义(P0.05)。结论:1弥漫大B细胞淋巴瘤患者RASSF6基因启动子甲基化率显著高于健康人,这表明RASSF6基因启动子甲基化可能是弥漫大B细胞淋巴瘤的发生机制之一。2弥漫大B细胞淋巴瘤患者RASSF6基因mRNA的表达显著低于健方法:康人,这表明RASSF6基因可能在弥漫大B细胞淋巴瘤中起抑癌基因的作用。3弥漫大B细胞淋巴瘤患者RASSF6基因启动子区甲基化率与其mRNA表达呈负相关,提示RASSF6基因启动子区甲基化可能是导致弥漫大B细胞淋巴瘤患者mRNA表达减低的原因之一。4弥漫大B细胞淋巴瘤患者RASSF6基因启动子区甲基化与患者的Ki-67、LDH、临床分期相关,这说明RASSF6基因启动子甲基化状态可能与弥漫大B细胞淋巴瘤的侵袭性及预后相关。
[Abstract]:Objective: non Hodgkin's lymphoma is one of the malignant tumors of the blood system. The diffuse large B cell lymphoma (Diffuse large B-cell lymphoma, DLBCL) is the most common type of non Hodgkin's lymphoma, which accounts for about half of the adult lymphoma, and is a group of highly heterogeneous invasive B cell lymphoma. The treatment is mainly by chemotherapy. Mainly. The combined chemotherapy regimen has significantly improved the efficacy of diffuse large B cell lymphoma, and the R-CHOP scheme is considered as a standard treatment. However, even with the application standard scheme, nearly 40% of the patients will continue to recur. Therefore, the etiological study of the diffuse large B cell lymphoma, the genomics analysis as an early diagnosis, and the targeted treatment. The development of the treatment and prognosis provides a new direction. The development of the diffuse large B cell lymphoma is the result of multiple genes and multiple factors. Therefore, it is clear that the tumor related genes are the key to early diagnosis and later treatment. The research shows that the development of the tumor includes at least two levels of genetic and epigenetic regulation, including oncogenes. Activation, inactivation of tumor suppressor genes, and epigenetics seem to play a more important role. In recent years, epigenetic phenomena have been identified in the pathogenesis of various tumors, including DNA methylation, histone modification, chromatin remodeling and so on. The study confirmed that the family 6 gene (RASSF6) of the RAS related region is a tumor suppressor gene, and the expression of RASSF6 can be expressed. Combined with K-Ras, the proliferation of tumor cells can be inhibited, and cell cycle progression can be organized by activating the JNK signaling pathway, and apoptosis of tumor cells can be induced by activating the caspase pathway and other pathways. It is one of the important reasons for the inactivation of RASSF6 gene, and the related research of RASSF6 gene in diffuse large B cell lymphoma has not been reported. This study will be based on the application of methylation specific polymerase chain reaction (Methylation-Specific PCR, MSP) and real-time fluorescent quantitative reverse transcriptase polymerase chain reaction (real-time Quantitative Reve). RSE Transcriptase-PCR, QRT-PCR) method verifies the methylation status and expression of RASSF6 in the promoter region of diffuse large B cell lymphoma, and analyzes its relationship with the sample data. It provides a certain theoretical basis for the pathogenesis, clinical diagnosis and treatment of diffuse large B cell lymphoma, and the.1 application MSP method for the detection of diffuse large B cell lymphoma. The methylation status of RASSF6 gene in peripheral blood of patients and healthy people and analysis of the relationship between them and clinical data.2 application QRT-PCR method to detect the relative expression of RASSF6 gene mRNA in diffuse large B cell lymphoma and healthy human peripheral blood, and to analyze the relationship between the clinical data and the.3 Application Statistics Software SPSS19.0 logarithm. Results: the methylation rates of 1 RASSF6 gene promoter region in diffuse large B cell lymphoma and healthy human peripheral blood were 48.6% (17/35) and 14.3% (5/35) respectively. The methylation rate of RASSF6 gene promoter region of diffuse large B cell lymphoma was significantly higher than that of healthy people, and the difference was statistically significant (P0.05).2 grouping. It was found that in patients with diffuse large B cell lymphoma, RASSF6 methylation was associated with Ki-67, LDH, clinical staging (P0.05), and the sex, age, extranodal involvement, A/B symptoms, Germinal center origin, GCB, or non germinal centers (Nongerminal Center), beta 2 microglobulin, and bone marrow involvement in the patients. The mRNA expression of RASSF6 gene in the patients with diffuse large B cell lymphoma was 0.0415 + 0.0287, which was significantly lower than that of healthy mRNA (0.0634 + 0.0427). The difference was statistically significant (P0.05).4 group analysis found that the expression of RASSF6 gene mRNA in patients with diffuse large B cell lymphoma was associated with the patient's IPI, Ki-67, and extranodal involvement (P0.05). The sex, age, LDH, A/B symptoms, GCB or Non-GCB, beta 2 microglobulin, bone marrow involvement, and clinical stages were not related to (P0.05).5 diffuse large B cell lymphoma in the peripheral blood of RASSF6 gene promoter methylation positive mRNA expression of 0.0306 + 0.0256, methylation negative mRNA relative expression of 0.0518 + 0.0283, the difference was statistically significant (P0). .05) conclusion: the methylation rate of RASSF6 gene promoter of RASSF6 gene in patients with diffuse large B cell lymphoma is significantly higher than that of healthy people. This indicates that the methylation of RASSF6 promoter may be one of the mechanisms of diffuse large B cell lymphoma. The expression of RASSF6 gene mRNA in patients with diffuse large B cell lymphoma is significantly lower than that of the healthy method: Kang people, which indicates the RASSF6 base. The methylation rate of the RASSF6 gene promoter region of the diffuse large B cell lymphoma patients with diffuse large B cell lymphoma is negatively correlated with the mRNA expression, suggesting that the methylation of the RASSF6 gene promoter region may be one of the reasons for the decrease of mRNA expression in patients with diffuse large B cell lymphoma, one of the.4 diffuse B cells. The methylation of the promoter region of the RASSF6 gene in the patients with the tumor is associated with the patient's Ki-67, LDH, and clinical staging. This suggests that the methylation status of the RASSF6 gene promoter may be associated with the invasiveness and prognosis of diffuse large B cell lymphoma.
【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】：R733.1

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