儿童急性白血病RIZ1基因表达及启动子甲基化状态的实验研究

发布时间：2018-06-24 21:39

本文选题：RIZ1 + 儿童急性白血病　；参考：《华中科技大学》2012年博士论文

【摘要】：急性白血病(acute leukemia, AL)是儿童时期最常见的恶性肿瘤性疾病,由于联合化疗和支持治疗的不断完善,白血病患儿的长期生存率已得到显著提高,然而复发仍是儿童白血病治疗失败的最重要原因,因此白血病的发病机制亟待进一步阐明。视网膜母细胞瘤蛋白结合锌指结构基因1 (retinoblastoma protein-interacting zinc finger gene 1, RIZ1)是近来新发现的抑癌基因(tumor suppressor genes, TSGs),在多种肿瘤中表达下调甚至沉默,但在血液系统恶性疾病中的研究有限。DNA甲基化(DNA methylation)作为表观遗传学(epigenetics)的代表,在不改变DNA序列的前提下,通过改变染色质的构象导致抑癌基因表达关闭,参与肿瘤发生的过程,因而成为肿瘤发病机制的研究热点。DNA甲基化转移酶(DNA methyltransferases, DNMTs)是维持和催化甲基化的关键酶。DNMTs在多种肿瘤中表达上升,在肿瘤形成的过程中发挥关键作用。本研究检测急性白血病患儿在初诊、缓解及复发阶段RIZ1基因的表达情况,RIZ1启动子区域CpG岛的甲基化状态,结合患者临床特征,探讨抑癌基因RIZ1在儿童白血病发生中的作用,DNA甲基化对RIZ1表达的影响；还通过检测DNMTs的表达水平,分析DNMTs与RIZ1表达变化的关系,探讨DNMTs对急性白血病发生的影响。第一部分RIZ1基因在儿童急性白血病的表达改变目的：探讨RIZ1基因在儿童急性白血病患者中的表达情况及临床意义,以初步了解RIZ1基因在急性白血病中的作用。方法：应用实时定量PCR (qRT-PCR)技术检测92例ALL和64例AML患者骨髓中RIZ1基因的mRNA表达水平,其中男性77例,女性79例,平均年龄6.43岁。分析RIZ1表达与临床特征的关系。采集13例非恶性血液疾病患儿骨髓作为对照组。结果：按照CCLG-ALL2008和中华医学会儿科学分会血液学组《儿童急性髓细胞白血病诊疗建议》进行危险度分型,ALL患儿分为标危组(SR)59例、中危组(MR)17例以及高危组(HR)16例,AML患者分为低危组(LR)32例、中危组(MR)15例和高危组(HR)17例。ALL患者中,初诊41例、缓解46例、复发6例；AML病人中,初诊29例、缓解29例、复发5例及未缓解1例。与对照组相比,ALL和AML患儿中RIZ1基因表达下调的病人各占92.4%和96.9%。ALL患者在缓解、初诊和复发阶段,RIZ1表达水平依次降低(P0.01)；T-ALL表达水平低于B-ALL,但没有统计学差异；SR、MR和HR各组的mRNA表达水平逐渐降低(P0.001)。AML病人中,缓解、初诊和复发阶段RIZ1表达水平依次降低(P0.01),但初诊和复发病人表达差异无统计学意义；LR、MR和HR的mRNA表达水平依次降低(P0.001)。33例ALL和22例AML患者化疗前后自身对照分析,达到完全缓解时RIZ1表达均明显升高(P=0.005和P0.001)。AML患者mRNA整体表达水平和在初诊、缓解、复发阶段表达水平均低于ALL病人,且具有统计学差异。结论：RIZ1基因在儿童ALL和AML中表达下调,RIZ1表达下降可能是白血病的不良预后因素。第二部分急性白血病RIZ1基因启动子区域甲基化状态目的：探讨儿童白血病患者RIZ1基因启动子区域CpG岛甲基化状态与RIZ1表达下调的关系,以及甲基化在白血病发生过程中的作用。方法：应用甲基化特异性PCR (methylation-specific PCR, MSP)和亚硫酸盐测序(bisulfite sequencing)的方法检测64例ALL和32例AML患儿RIZ1基因启动子区域CpG岛甲基化状态,其中男性患者46例,女性50例,平均年龄6.43岁。分析DNA甲基化与RIZ1基因表达下调的相关性,以及对临床预后的影响。采集9例非恶性血液疾病患儿骨髓作为对照组。结果：按照前述标准进行危险分级,ALL患儿分为SR 40例、MR 11例、HR 12例；AML患者分为LR 12例、MR 7例和HR 13例。ALL患者中,初诊32例、缓解26例、复发6例；AML病人中,初诊16例、缓解10例、复发5例、未缓解1例。27例急性白血病患儿的RIZ1基因启动子CpG岛出现甲基化,甲基化率为28.1%,其中ALL为23.4%、AML为37.5%；而9例对照组病人均为非甲基化状态。ALL患者中,复发、初诊和完全缓解病人的甲基化率依次降低(P=0.006); B-ALL和T-ALL患者的甲基化率无明显差别(P=0.565)；随着危险度增加SR、MR、HR甲基化率逐渐升高,且有统计学差异(P=0.003)。AML病人中,复发、初诊和完全缓解患儿甲基化率依次降低,但不具有统计学差异(P=0.303)；12例LR组患者均未检出甲基化,LR、MR、HR组甲基化率依次增加(P=0.002)。甲基化患者的RIZ1表达水平明显低于非甲基化者(P0.001)。结论：RIZ1基因甲基化现象在儿童白血病尤其是ALL中并不常见,但仍是RIZ1表达降低的重要原因之一,RIZ1基因启动子区域DNA甲基化与儿童急性白血病的发生发展相关,并且可能提示预后不良。第三部分DNA甲基转移酶活性变化与RIZ1表达相关研究目的：研究DNA甲基转移酶(DNMTs)在ALL和AML患儿中的表达情况,探讨其与RIZ1基因表达下调以及CpG岛甲基化的相关性。方法：通过实时定量PCR (qRT-PCR)技术检测DNMTs三种亚型(DNMT1、DNMT3a和DNMT3b)在41例ALL和29例AML初诊患儿骨髓中的表达情况。男性和女性均为35例；平均年龄6.25岁。收集13例非恶性血液疾病患儿骨髓作为对照组。结果：与对照组相比,ALL患者的DNMT1基因表达水平上调3.86倍,DNMT3a上升4.44倍,DNMT3b升高8.21倍,；AML患者的DNMT1基因表达水平升高4.35倍,DNMT3a上升4.57倍,DNMT3b上调9.08倍,差异均具有统计学意义(P0.001)。ALL患儿中,HR组的DNMT1基因表达显著高于SR组(P=0.046),但DNMT3a和DNMT3b的表达均无明显差异。同样的结果见于AML的LR组和HR组患者(DNMT1:P=0.05)。在ALL患者中,甲基化患儿的DNMT1、DNMT3a和DNMT3b表达均高于非甲基化病人(DNMT1:P=0.015; DNMT3a:P=0.038; DNMT3b:P=0.003)。AML患者中,甲基化患儿的DNMT1和DNMT3b表达高于非甲基化病人(DNMT1: P=0.007; DNMT3b:P0.001),而DNMT3a表达无明显差异(P=0.131)。结论：儿童急性白血病患者的DNMTs表达水平显著升高,甲基化患儿表达明显上调,提示DNMTs表达上调可能是RIZ1基因启动子甲基化的原因,参与白血病的发生过程。
[Abstract]:Acute leukemia (AL) is the most common malignant tumor in childhood. Due to the continuous improvement of combined chemotherapy and support therapy, the long-term survival rate of children with leukemia has been significantly improved. However, the recurrence is still the most important reason for the failure of leukemia treatment in children. Therefore, the pathogenesis of leukemia needs to be further improved. Clarify.
Retinoblastoma protein combined with zinc finger structure gene 1 (retinoblastoma protein-interacting zinc finger gene 1, RIZ1) is a newly discovered tumor suppressor gene (tumor suppressor genes, TSGs), down regulation and even silence in a variety of tumors, but the study of limited.DNA methylation in hematological malignancies (DNA) As the representative of epigenetics (epigenetics), on the premise of not changing the DNA sequence, the expression of the tumor suppressor gene is closed by changing the conformation of chromatin, and it is involved in the process of cancer. Therefore, the.DNA methyltransferase (DNA methyltransferases, DNMTs) is the hot spot in the pathogenesis of cancer, which is the key to maintain and catalyze the methylation. The expression of.DNMTs is increased in many tumors and plays a key role in the process of tumor formation.
This study examined the expression of RIZ1 gene in the early diagnosis, remission and recurrence of acute leukemia, the methylation status of CpG island in the RIZ1 promoter region, and combined with the clinical features of the patients to explore the role of the tumor suppressor gene RIZ1 in children's leukemia, the effect of DNA methylation on the expression of RIZ1, and the level of DNMTs expression by detecting the expression level of DNMTs. Objective to analyze the relationship between DNMTs and RIZ1 expression and explore the effect of DNMTs on the occurrence of acute leukemia.
Part one expression of RIZ1 gene in children with acute leukemia
Objective: To investigate the expression and clinical significance of RIZ1 gene in children with acute leukemia, so as to preliminarily understand the role of RIZ1 gene in acute leukemia.
Methods: the mRNA expression level of RIZ1 gene in bone marrow of 92 ALL and 64 AML patients was detected by real-time quantitative PCR (qRT-PCR) technique, including 77 males and 79 females with an average age of 6.43 years. The relationship between the expression of RIZ1 and the clinical characteristics was analyzed. 13 cases of non malignant blood disease were collected as the control group.
Results: according to the risk classification of children's acute myelocytic leukemia in the hematology group, CCLG-ALL2008 and Chinese Medical Association, ALL children were divided into 59 cases of risk group (SR), 17 cases of middle risk group (MR) and 16 cases of high risk group (HR), AML patients were divided into 32 cases of low risk group (LR), 15 cases of middle risk group (MR) and 17.ALL patients in high risk group (HR). In 41 cases of initial diagnosis, 46 cases were relieved and 6 cases recurred. In AML patients, 29 cases were first diagnosed, 29 cases were remission, 5 cases relapsed and 1 cases were not relieved. Compared with the control group, the patients with RIZ1 gene expression decreased in 92.4% and 96.9%.ALL were relieved, and the level of RIZ1 expression decreased in the first diagnosis and relapse stage (P0.01); T-ALL expression level was lower than B-AL. L, but there was no statistical difference. The level of mRNA expression in SR, MR and HR gradually decreased (P0.001) in.AML patients. The level of RIZ1 expression in the initial and recurrent stages decreased in turn (P0.01), but there was no significant difference in the expression of primary and recurrent patients; LR, MR, and HR were reduced in turn and 22 cases were reduced. Before and after treatment, the expression of RIZ1 was significantly increased (P=0.005 and P0.001), and the expression level of mRNA in.AML patients was significantly higher than that of ALL patients, and the expression level in the recurrent stage was lower than that of the ALL patients, and the difference was statistically significant.
Conclusion: the expression of RIZ1 gene is down regulated in children's ALL and AML, and the decrease of RIZ1 expression may be a poor prognostic factor of leukemia.
The second part is the methylation status of RIZ1 gene promoter region in acute leukemia.
Objective: To investigate the relationship between the methylation status of CpG island in the promoter region of RIZ1 gene and the down regulation of RIZ1 expression in the promoter region of children with leukemia and the role of methylation in the process of leukemia.
Methods: the methylation specific PCR (methylation-specific PCR, MSP) and sulfite sequencing (bisulfite sequencing) were used to detect the CpG island methylation status of the RIZ1 gene promoter region of 64 children with ALL and 32 cases of AML, including 46 male patients, 50 women and 6.43 years of age. The DNA methylation and down regulation of RIZ1 gene expression were analyzed. 9 patients with non malignant hematological diseases were selected as control group.
Results: according to the criteria for risk classification, ALL children were divided into SR 40 cases, MR 11 cases, HR 12 cases, AML patients were divided into LR 12 cases, MR 7 cases and HR 13 cases.ALL patients, 32 cases, 26 cases, 6 cases, 16 cases, 10, recurrence 5, RIZ1 gene promoter of.27 cases of acute leukemia were not alleviated. Methylation, methylation rate was 28.1%, of which ALL was 23.4%, AML was 37.5%, and 9 cases of control patients were in non methylation state.ALL patients, relapse, first diagnosis and complete remission patients' methylation rate decreased in turn (P=0.006); B-ALL and T-ALL patients had no significant difference in methylation rate (P=0.565); SR, MR, HR methyl were increased with the risk degree. In the patients with statistical difference (P=0.003), there was a statistical difference (P=0.003) in patients with.AML. The rate of methylated in the patients with primary and complete remission decreased in turn, but there was no statistical difference (P=0.303). No methylation was detected in group LR, LR, MR, and the methylation rate in group HR was increased in turn (P=0.002). The level of RIZ1 expression in the methylation patients was significantly lower than that of non methylated patients. P0.001.
Conclusion: the methylation of RIZ1 gene is not common in children with leukemia, especially in ALL, but it is still one of the important reasons for the decrease of RIZ1 expression. The DNA methylation in the promoter region of the RIZ1 gene is associated with the development of acute leukemia in children, and may indicate poor prognosis.
The third part is about the correlation between the activity of DNA methyltransferase and the expression of RIZ1.
Objective: To investigate the expression of DNA methyltransferase (DNMTs) in children with ALL and AML, and to explore the correlation between RIZ1 expression and CpG island methylation.
Methods: the expression of DNMTs three subtypes (DNMT1, DNMT3a and DNMT3b) in 41 cases of ALL and 29 cases of AML newly diagnosed children was detected by real-time quantitative PCR (qRT-PCR). Both male and female were 35 cases, the average age was 6.25 years old. 13 cases of non malignant blood disease were collected as the control group.
Results: compared with the control group, the expression of DNMT1 gene expression in ALL patients was up to 3.86 times, DNMT3a increased 4.44 times, DNMT3b increased by 8.21 times, the DNMT1 gene expression level of AML patients increased 4.35 times, DNMT3a increased 4.57 times, DNMT3b increased 9.08 times, the difference was statistically significant (P0.001).ALL children, HR group DNMT1 gene expression was significantly higher than those R group (P=0.046), but there was no significant difference in the expression of DNMT3a and DNMT3b. The same results were found in group LR and HR in AML (DNMT1:P=0.05). In patients with ALL, DNMT1, DNMT3a, and DNMT3b expressions were higher than those of patients with non methylation. The expression of T3b was higher than that of non methylation patients (DNMT1: P=0.007; DNMT3b:P0.001), but DNMT3a expression was not significantly different (P=0.131).
Conclusion: the expression level of DNMTs in children with acute leukemia is significantly elevated, and the expression of methylation children is obviously up-regulated, suggesting that the up regulation of DNMTs expression may be the cause of the methylation of RIZ1 gene promoter and participate in the process of leukaemia.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R733.7

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