本文關(guān)鍵詞：在急性髓細(xì)胞白血病中Ⅰ類組蛋白去乙�；刚{(diào)控BRCA1、CHK1和RAD51表達(dá)的研究　出處：《吉林大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 急性髓細(xì)胞白血病 組蛋白去乙�；� BRCA1 CHK1 RAD51 DNA損傷應(yīng)答 細(xì)胞周期檢驗(yàn)點(diǎn)

【摘要】：急性髓細(xì)胞白血病(Acute myeloid leukemia，AML)是最常見的急性白血病。阿糖胞苷（Cytosine arabinoside, ara-C）和柔紅霉素（Daunorubicin，DNR）是目前在AML治療中應(yīng)用最廣泛和最有效的兩種常規(guī)化療藥物，對二者的抗藥性是導(dǎo)致AML治療失敗的主要原因。因此，引入抗癌新藥來增強(qiáng)常規(guī)化療藥物的活性或降低其毒副作用,進(jìn)而提高AML的治愈率,具有特殊的重要性和迫切性。 在眾多的抗癌新藥中，組蛋白去乙�；敢种苿�(Histonedeacetylase inhibitor, HDACI)類藥物尤其引人關(guān)注。目前，，至少有十余種HDACI處在早期的臨床試驗(yàn)階段。其中，Vorinostat、Depsipeptide、Belinostat和Panobinostat已經(jīng)獲得美國食品及藥品管理局(Food and Drug Administration, the United States of America, USFDA)的批準(zhǔn)用于治療與血液相關(guān)的腫瘤。針對AML的相關(guān)研究表明，HDACI能抑制AML細(xì)胞的增殖并能誘導(dǎo)細(xì)胞的凋亡，但對正常組織細(xì)胞的作用卻有限。因此，HDACI是一類非常有前途的治療AML的靶向新藥。然而臨床實(shí)驗(yàn)顯示，作為單藥治療癌癥，HDACI的療效有限。但是，HDACI與其它化療或靶向性藥物聯(lián)合應(yīng)用時卻能顯著提高這些藥物的抗腫瘤活性。因此，將HDACI與化療藥物或其它靶向性藥物聯(lián)合治療癌癥已成為一個新的研究熱點(diǎn)。 我們課題組在之前的研究中發(fā)現(xiàn)，廣譜HDAC抑制劑panobinostat通過下調(diào)BRCA1、CHK1和RAD51蛋白（在DNA損傷應(yīng)答網(wǎng)絡(luò)中起關(guān)鍵作用）和mRNA的表達(dá)來部分消除常規(guī)化療藥物阿糖胞苷和柔紅霉素引起的S和/或G2/M細(xì)胞周期檢驗(yàn)點(diǎn)的激活，并促進(jìn)阿糖胞苷和柔紅霉素引起的DNA損傷，從而顯著增強(qiáng)二者引 起的AML細(xì)胞的凋亡。然而，究竟哪些HDAC調(diào)控BRCA1、CHK1和RAD51的表達(dá)尚不清楚。這是本論文要解決的核心問題。 首先，我們應(yīng)用不同類別的選擇性HDACI來篩查究竟哪些大類的HDAC調(diào)控BRCA1、CHK1和RAD51的表達(dá)。我們發(fā)現(xiàn)，在AML細(xì)胞中，只有I類HDAC選擇性抑制劑MGCD0103（選擇性抑制HDAC1、HDAC2和HDAC3）對BRCA1、CHK1和RAD51蛋白和mRNA的表達(dá)產(chǎn)生了下調(diào)作用，并增加了阿糖胞苷和柔紅霉素所誘導(dǎo)的DNA損傷和細(xì)胞凋亡，MGCD0103還部分消除了阿糖胞苷所誘導(dǎo)的S和G2/M細(xì)胞周期阻滯，也部分消除了柔紅霉素所誘導(dǎo)的G2/M細(xì)胞周期阻滯，這與應(yīng)用panobinostat得到的結(jié)果一致。然而，IIa類HDAC抑制劑MC1568和HDAC6選擇性抑制劑Tubastatin A對AML細(xì)胞則沒有這方面的影響。上述結(jié)果表明，MGCD0103通過選擇性抑制HDAC1、HDAC2和HDAC3來下調(diào)AML細(xì)胞中BRCA1、CHK1和RAD51的表達(dá)，至少部分消除了阿糖胞苷和柔紅霉素所誘導(dǎo)的細(xì)胞周期阻滯，并促進(jìn)阿糖胞苷和柔紅霉素所引起的DNA損傷，從而使大量AML細(xì)胞在帶有DNA損傷的情況下進(jìn)行細(xì)胞周期運(yùn)行而誘發(fā)細(xì)胞凋亡。 隨后，我們應(yīng)用免疫沉淀和HDAC酶活分析法進(jìn)一步證明了HDAC1、HDAC2和/或HDAC3對上述基因的調(diào)控起關(guān)鍵作用，但與HDAC8無關(guān)。對HDAC1、HDAC2和HDAC3調(diào)控BRCA1、CHK1和RAD51表達(dá)的分子機(jī)制的初步探索發(fā)現(xiàn)，HDAC1、HDAC2和/或HDAC3對BRCA1、CHK1和RAD51表達(dá)的下調(diào)作用可能是通過下調(diào)轉(zhuǎn)錄因子E2F1來實(shí)現(xiàn)的。 為進(jìn)一步研究HDAC1、HDAC2和HDAC3中究竟哪些調(diào)控BRCA1、CHk1和RAD51的表達(dá)，我們應(yīng)用慢病毒shRNA在AML細(xì)胞株中分別沉默了HDAC1、HDAC2和HDAC3，結(jié)果表明單獨(dú)沉默HDAC1、HDAC2和HDAC3中的任何一個對BRCA1、CHK1和RAD51的表達(dá)沒有影響。另外，分別在HDAC1和HDAC2沉默的AML細(xì)胞中加入HDAC3選擇性抑制劑RGFP966后都不能降低上述基因的表達(dá)水平，預(yù)示至少HDAC1和HDAC2協(xié)作調(diào)控上述基因的表達(dá)。如果在后續(xù)的實(shí)驗(yàn)中同時抑制HDAC1和HDAC2也不能對BRCA1、CHK1和RAD51的表達(dá)產(chǎn)生影響，那么則說明HDAC1、HDAC2和HDAC3都參與了對BRCA1、CHK1和RAD51表達(dá)的調(diào)控。 綜上所述，本論文的研究明確了同時抑制HDAC1、HDAC2和HDAC3能在AML細(xì)胞中下調(diào)BRCA1、CHK1和RAD51的表達(dá)，并能部分消除阿糖胞苷和柔紅霉素引起的S和G2/M細(xì)胞周期阻滯，促進(jìn)二者引起的DNA損傷和細(xì)胞凋亡。進(jìn)一步的研究預(yù)示，HDAC1和HDAC2是調(diào)控BRCA1、CHK1和RAD51不可缺少的因素，是否需要HDAC3還需要進(jìn)一步的實(shí)驗(yàn)來確定。本課題的完成對研發(fā)選擇性更強(qiáng)的HDACI具有理論指導(dǎo)意義，為AML的臨床治療提供新思路。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R733.71

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本文編號：1377949