在急性髓细胞白血病中Ⅰ类组蛋白去乙酰化酶调控BRCA1、CHK1和RAD51表达的研究

发布时间：2018-01-04 09:20

本文关键词：在急性髓细胞白血病中Ⅰ类组蛋白去乙酰化酶调控BRCA1、CHK1和RAD51表达的研究　出处：《吉林大学》2015年硕士论文　论文类型：学位论文

【摘要】：急性髓细胞白血病(Acute myeloid leukemia，AML)是最常见的急性白血病。阿糖胞苷（Cytosine arabinoside, ara-C）和柔红霉素（Daunorubicin，DNR）是目前在AML治疗中应用最广泛和最有效的两种常规化疗药物，对二者的抗药性是导致AML治疗失败的主要原因。因此，引入抗癌新药来增强常规化疗药物的活性或降低其毒副作用,进而提高AML的治愈率,具有特殊的重要性和迫切性。在众多的抗癌新药中，组蛋白去乙酰化酶抑制剂(Histonedeacetylase inhibitor, HDACI)类药物尤其引人关注。目前，，至少有十余种HDACI处在早期的临床试验阶段。其中，Vorinostat、Depsipeptide、Belinostat和Panobinostat已经获得美国食品及药品管理局(Food and Drug Administration, the United States of America, USFDA)的批准用于治疗与血液相关的肿瘤。针对AML的相关研究表明，HDACI能抑制AML细胞的增殖并能诱导细胞的凋亡，但对正常组织细胞的作用却有限。因此，HDACI是一类非常有前途的治疗AML的靶向新药。然而临床实验显示，作为单药治疗癌症，HDACI的疗效有限。但是，HDACI与其它化疗或靶向性药物联合应用时却能显著提高这些药物的抗肿瘤活性。因此，将HDACI与化疗药物或其它靶向性药物联合治疗癌症已成为一个新的研究热点。我们课题组在之前的研究中发现，广谱HDAC抑制剂panobinostat通过下调BRCA1、CHK1和RAD51蛋白（在DNA损伤应答网络中起关键作用）和mRNA的表达来部分消除常规化疗药物阿糖胞苷和柔红霉素引起的S和/或G2/M细胞周期检验点的激活，并促进阿糖胞苷和柔红霉素引起的DNA损伤，从而显著增强二者引起的AML细胞的凋亡。然而，究竟哪些HDAC调控BRCA1、CHK1和RAD51的表达尚不清楚。这是本论文要解决的核心问题。首先，我们应用不同类别的选择性HDACI来筛查究竟哪些大类的HDAC调控BRCA1、CHK1和RAD51的表达。我们发现，在AML细胞中，只有I类HDAC选择性抑制剂MGCD0103（选择性抑制HDAC1、HDAC2和HDAC3）对BRCA1、CHK1和RAD51蛋白和mRNA的表达产生了下调作用，并增加了阿糖胞苷和柔红霉素所诱导的DNA损伤和细胞凋亡，MGCD0103还部分消除了阿糖胞苷所诱导的S和G2/M细胞周期阻滞，也部分消除了柔红霉素所诱导的G2/M细胞周期阻滞，这与应用panobinostat得到的结果一致。然而，IIa类HDAC抑制剂MC1568和HDAC6选择性抑制剂Tubastatin A对AML细胞则没有这方面的影响。上述结果表明，MGCD0103通过选择性抑制HDAC1、HDAC2和HDAC3来下调AML细胞中BRCA1、CHK1和RAD51的表达，至少部分消除了阿糖胞苷和柔红霉素所诱导的细胞周期阻滞，并促进阿糖胞苷和柔红霉素所引起的DNA损伤，从而使大量AML细胞在带有DNA损伤的情况下进行细胞周期运行而诱发细胞凋亡。随后，我们应用免疫沉淀和HDAC酶活分析法进一步证明了HDAC1、HDAC2和/或HDAC3对上述基因的调控起关键作用，但与HDAC8无关。对HDAC1、HDAC2和HDAC3调控BRCA1、CHK1和RAD51表达的分子机制的初步探索发现，HDAC1、HDAC2和/或HDAC3对BRCA1、CHK1和RAD51表达的下调作用可能是通过下调转录因子E2F1来实现的。为进一步研究HDAC1、HDAC2和HDAC3中究竟哪些调控BRCA1、CHk1和RAD51的表达，我们应用慢病毒shRNA在AML细胞株中分别沉默了HDAC1、HDAC2和HDAC3，结果表明单独沉默HDAC1、HDAC2和HDAC3中的任何一个对BRCA1、CHK1和RAD51的表达没有影响。另外，分别在HDAC1和HDAC2沉默的AML细胞中加入HDAC3选择性抑制剂RGFP966后都不能降低上述基因的表达水平，预示至少HDAC1和HDAC2协作调控上述基因的表达。如果在后续的实验中同时抑制HDAC1和HDAC2也不能对BRCA1、CHK1和RAD51的表达产生影响，那么则说明HDAC1、HDAC2和HDAC3都参与了对BRCA1、CHK1和RAD51表达的调控。综上所述，本论文的研究明确了同时抑制HDAC1、HDAC2和HDAC3能在AML细胞中下调BRCA1、CHK1和RAD51的表达，并能部分消除阿糖胞苷和柔红霉素引起的S和G2/M细胞周期阻滞，促进二者引起的DNA损伤和细胞凋亡。进一步的研究预示，HDAC1和HDAC2是调控BRCA1、CHK1和RAD51不可缺少的因素，是否需要HDAC3还需要进一步的实验来确定。本课题的完成对研发选择性更强的HDACI具有理论指导意义，为AML的临床治疗提供新思路。
[Abstract]:Acute myeloid leukemia (Acute myeloid, leukemia, AML) is the most common acute leukemia. Cytarabine (Cytosine arabinoside, ara-C) and daunorubicin (Daunorubicin, DNR) are two kinds of conventional chemotherapy drugs in the treatment of AML is currently the most widely used and most effective, the two resistance is the main cause AML treatment failure. Therefore, the introduction of new anticancer drug to enhance chemotherapy activity or reduce the toxicity and improve the cure rate of AML, is of special importance and urgency.
In many of the new anticancer drug, histone deacetylase inhibitors (Histonedeacetylase, inhibitor, HDACI) drugs of particular concern. At present, there are at least a dozen HDACI in early phase clinical trials. Among them, Vorinostat, Depsipeptide, Belinostat and Panobinostat has received U.S. Food and Drug Administration (Food and Drug Administration. The United States of America, USFDA) approval for the treatment of blood related tumors. The AML study showed that apoptosis of HDACI can inhibit the proliferation of AML cells and induce cells, but in normal tissues is limited. Therefore, HDACI is a very promising therapeutic target of AML to the new drug. However, clinical trials showed that as monotherapy for the treatment of cancer, the efficacy of HDACI. However, HDACI and other chemotherapy or targeted drug combination should be used when they can significantly improve Therefore, the combined treatment of HDACI with chemotherapeutic drugs or other targeted drugs has become a new research hotspot.
Found in previous studies of our research group, a broad-spectrum HDAC inhibitor panobinostat through downregulation of BRCA1, CHK1 and RAD51 (protein plays a key role in DNA damage response network) and the expression of mRNA to eliminate the activation of S and / or G2/M cell cycle checkpoint conventional chemotherapeutic cytarabine and daunorubicin induced, and to promote the DNA damage of cytarabine and daunorubicin induced, thereby significantly increasing the two lead
However, it is not clear which HDAC regulates the expression of BRCA1, CHK1 and RAD51, which is the core problem to be solved in this paper.
First, we used different types of selective HDACI screening what categories of HDAC regulation of BRCA1, the expression of CHK1 and RAD51. We found that in AML cells, only the I class HDAC selective inhibitor MGCD0103 (selective inhibition of HDAC1, HDAC2 and HDAC3) on the expression of BRCA1, CHK1 and RAD51 protein and mRNA produced down-regulation effect, and increase the DNA damage and apoptosis induced by cytarabine and daunorubicin, MGCD0103 also eliminates the induced by cytarabine S and G2/M cell cycle arrest, but also eliminates the G2/M cell cycle arrest induced by daunorubicin, which is consistent with the result and application of panobinostat. However, II a HDAC inhibitor MC1568 and HDAC6 inhibitor Tubastatin A has no effect on the AML cells. These results indicate that MGCD0103 selectively inhibits HDAC1, HDAC2 and HDAC3 in B cells to down AML RCA1, the expression of CHK1 and RAD51, at least in part, to eliminate the cell cycle arrest induced by cytarabine and daunorubicin, and promote the DNA damage of cytarabine and daunorubicin induced, so that a large number of AML cells and induce cell apoptosis and cell cycle in a case of DNA damage.
Then, we used immunoprecipitation and HDAC enzyme activity analysis further proved that HDAC1, HDAC2 and / or HDAC3 regulation of the gene plays a key role, but has nothing to do with the HDAC8. The HDAC1, HDAC2 and HDAC3 regulate BRCA1, explore the molecular mechanism of the expression of CHK1 and RAD51 found that HDAC1, HDAC2 and / or HDAC3 for BRCA1, the expression of CHK1 and RAD51 down-regulation may down regulate the expression of transcription factor E2F1.
For the further study of HDAC1, HDAC2 and HDAC3 in which the regulation of BRCA1, the expression of CHk1 and RAD51, we used lentivirus shRNA in AML cell line were silent HDAC1, HDAC2 and HDAC3, the results showed that silencing of HDAC1, HDAC2 and HDAC3 in any one of BRCA1, did not affect the expression of CHK1 and RAD51. In addition, with selective HDAC3 inhibitor RGFP966 in HDAC1 and HDAC2 respectively after silencing AML cells can reduce the expression level of the gene expression of HDAC1 and HDAC2, indicate that at least the cooperating control genes. If at the same time in subsequent experiments on inhibition of HDAC1 and HDAC2 can influence the expression of BRCA1, CHK1 and RAD51. It indicates that HDAC1, HDAC2 and HDAC3 are involved in the regulation of BRCA1 expression of CHK1 and RAD51.
In summary, this study clearly the same inhibition of HDAC1, HDAC2 and HDAC3 can downregulate BRCA1 in AML cells, the expression of CHK1 and RAD51, and can eliminate cytarabine and daunorubicin induced S and G2/M cell cycle arrest, DNA damage and apoptosis promotes the two cause. Further study shows. HDAC1 and HDAC2 are indispensable factors in regulation of BRCA1, CHK1 and RAD51, HDAC3 also need further experiments are needed to determine. The completion of this project for the development of more selective HDACI has important significance in theory, provide new ideas for clinical treatment of AML.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R733.71

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