發(fā)布時間：2018-06-24 21:39

  本文選題：RIZ1 + 兒童急性白血病�。� 參考：《華中科技大學(xué)》2012年博士論文

【摘要】：急性白血病(acute leukemia, AL)是兒童時期最常見的惡性腫瘤性疾病,由于聯(lián)合化療和支持治療的不斷完善,白血病患兒的長期生存率已得到顯著提高,然而復(fù)發(fā)仍是兒童白血病治療失敗的最重要原因,因此白血病的發(fā)病機制亟待進一步闡明。 視網(wǎng)膜母細胞瘤蛋白結(jié)合鋅指結(jié)構(gòu)基因1 (retinoblastoma protein-interacting zinc finger gene 1, RIZ1)是近來新發(fā)現(xiàn)的抑癌基因(tumor suppressor genes, TSGs),在多種腫瘤中表達下調(diào)甚至沉默,但在血液系統(tǒng)惡性疾病中的研究有限。DNA甲基化(DNA methylation)作為表觀遺傳學(xué)(epigenetics)的代表,在不改變DNA序列的前提下,通過改變?nèi)旧|(zhì)的構(gòu)象導(dǎo)致抑癌基因表達關(guān)閉,參與腫瘤發(fā)生的過程,因而成為腫瘤發(fā)病機制的研究熱點。DNA甲基化轉(zhuǎn)移酶(DNA methyltransferases, DNMTs)是維持和催化甲基化的關(guān)鍵酶。DNMTs在多種腫瘤中表達上升,在腫瘤形成的過程中發(fā)揮關(guān)鍵作用。 本研究檢測急性白血病患兒在初診、緩解及復(fù)發(fā)階段RIZ1基因的表達情況,RIZ1啟動子區(qū)域CpG島的甲基化狀態(tài),結(jié)合患者臨床特征,探討抑癌基因RIZ1在兒童白血病發(fā)生中的作用,DNA甲基化對RIZ1表達的影響；還通過檢測DNMTs的表達水平,分析DNMTs與RIZ1表達變化的關(guān)系,探討DNMTs對急性白血病發(fā)生的影響。 第一部分RIZ1基因在兒童急性白血病的表達改變 目的：探討RIZ1基因在兒童急性白血病患者中的表達情況及臨床意義,以初步了解RIZ1基因在急性白血病中的作用。 方法：應(yīng)用實時定量PCR (qRT-PCR)技術(shù)檢測92例ALL和64例AML患者骨髓中RIZ1基因的mRNA表達水平,其中男性77例,女性79例,平均年齡6.43歲。分析RIZ1表達與臨床特征的關(guān)系。采集13例非惡性血液疾病患兒骨髓作為對照組。 結(jié)果：按照CCLG-ALL2008和中華醫(yī)學(xué)會兒科學(xué)分會血液學(xué)組《兒童急性髓細胞白血病診療建議》進行危險度分型,ALL患兒分為標(biāo)危組(SR)59例、中危組(MR)17例以及高危組(HR)16例,AML患者分為低危組(LR)32例、中危組(MR)15例和高危組(HR)17例。ALL患者中,初診41例、緩解46例、復(fù)發(fā)6例；AML病人中,初診29例、緩解29例、復(fù)發(fā)5例及未緩解1例。與對照組相比,ALL和AML患兒中RIZ1基因表達下調(diào)的病人各占92.4%和96.9%。ALL患者在緩解、初診和復(fù)發(fā)階段,RIZ1表達水平依次降低(P0.01)；T-ALL表達水平低于B-ALL,但沒有統(tǒng)計學(xué)差異；SR、MR和HR各組的mRNA表達水平逐漸降低(P0.001)。AML病人中,緩解、初診和復(fù)發(fā)階段RIZ1表達水平依次降低(P0.01),但初診和復(fù)發(fā)病人表達差異無統(tǒng)計學(xué)意義；LR、MR和HR的mRNA表達水平依次降低(P0.001)。33例ALL和22例AML患者化療前后自身對照分析,達到完全緩解時RIZ1表達均明顯升高(P=0.005和P0.001)。AML患者mRNA整體表達水平和在初診、緩解、復(fù)發(fā)階段表達水平均低于ALL病人,且具有統(tǒng)計學(xué)差異。 結(jié)論：RIZ1基因在兒童ALL和AML中表達下調(diào),RIZ1表達下降可能是白血病的不良預(yù)后因素。 第二部分急性白血病RIZ1基因啟動子區(qū)域甲基化狀態(tài) 目的：探討兒童白血病患者RIZ1基因啟動子區(qū)域CpG島甲基化狀態(tài)與RIZ1表達下調(diào)的關(guān)系,以及甲基化在白血病發(fā)生過程中的作用。 方法：應(yīng)用甲基化特異性PCR (methylation-specific PCR, MSP)和亞硫酸鹽測序(bisulfite sequencing)的方法檢測64例ALL和32例AML患兒RIZ1基因啟動子區(qū)域CpG島甲基化狀態(tài),其中男性患者46例,女性50例,平均年齡6.43歲。分析DNA甲基化與RIZ1基因表達下調(diào)的相關(guān)性,以及對臨床預(yù)后的影響。采集9例非惡性血液疾病患兒骨髓作為對照組。 結(jié)果：按照前述標(biāo)準(zhǔn)進行危險分級,ALL患兒分為SR 40例、MR 11例、HR 12例；AML患者分為LR 12例、MR 7例和HR 13例。ALL患者中,初診32例、緩解26例、復(fù)發(fā)6例；AML病人中,初診16例、緩解10例、復(fù)發(fā)5例、未緩解1例。27例急性白血病患兒的RIZ1基因啟動子CpG島出現(xiàn)甲基化,甲基化率為28.1%,其中ALL為23.4%、AML為37.5%；而9例對照組病人均為非甲基化狀態(tài)。ALL患者中,復(fù)發(fā)、初診和完全緩解病人的甲基化率依次降低(P=0.006); B-ALL和T-ALL患者的甲基化率無明顯差別(P=0.565)；隨著危險度增加SR、MR、HR甲基化率逐漸升高,且有統(tǒng)計學(xué)差異(P=0.003)。AML病人中,復(fù)發(fā)、初診和完全緩解患兒甲基化率依次降低,但不具有統(tǒng)計學(xué)差異(P=0.303)；12例LR組患者均未檢出甲基化,LR、MR、HR組甲基化率依次增加(P=0.002)。甲基化患者的RIZ1表達水平明顯低于非甲基化者(P0.001)。 結(jié)論：RIZ1基因甲基化現(xiàn)象在兒童白血病尤其是ALL中并不常見,但仍是RIZ1表達降低的重要原因之一,RIZ1基因啟動子區(qū)域DNA甲基化與兒童急性白血病的發(fā)生發(fā)展相關(guān),并且可能提示預(yù)后不良。 第三部分DNA甲基轉(zhuǎn)移酶活性變化與RIZ1表達相關(guān)研究 目的：研究DNA甲基轉(zhuǎn)移酶(DNMTs)在ALL和AML患兒中的表達情況,探討其與RIZ1基因表達下調(diào)以及CpG島甲基化的相關(guān)性。 方法：通過實時定量PCR (qRT-PCR)技術(shù)檢測DNMTs三種亞型(DNMT1、DNMT3a和DNMT3b)在41例ALL和29例AML初診患兒骨髓中的表達情況。男性和女性均為35例；平均年齡6.25歲。收集13例非惡性血液疾病患兒骨髓作為對照組。 結(jié)果：與對照組相比,ALL患者的DNMT1基因表達水平上調(diào)3.86倍,DNMT3a上升4.44倍,DNMT3b升高8.21倍,；AML患者的DNMT1基因表達水平升高4.35倍,DNMT3a上升4.57倍,DNMT3b上調(diào)9.08倍,差異均具有統(tǒng)計學(xué)意義(P0.001)。ALL患兒中,HR組的DNMT1基因表達顯著高于SR組(P=0.046),但DNMT3a和DNMT3b的表達均無明顯差異。同樣的結(jié)果見于AML的LR組和HR組患者(DNMT1:P=0.05)。在ALL患者中,甲基化患兒的DNMT1、DNMT3a和DNMT3b表達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,甲基化患兒的DNMT1和DNMT3b表達高于非甲基化病人(DNMT1: P=0.007; DNMT3b:P0.001),而DNMT3a表達無明顯差異(P=0.131)。 結(jié)論：兒童急性白血病患者的DNMTs表達水平顯著升高,甲基化患兒表達明顯上調(diào),提示DNMTs表達上調(diào)可能是RIZ1基因啟動子甲基化的原因,參與白血病的發(fā)生過程。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R733.7

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相關(guān)期刊論文 前1條

1 胡洪玻;胡群;;T系和B系急性淋巴細胞白血病ID4甲基化狀態(tài)分析[J];中國當(dāng)代兒科雜志;2010年12期

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本文編號：2063078