LncRNA-HOTAIR在ATRA诱导AML细胞分化中的作用及机制

发布时间：2018-06-18 21:02

本文选题：HOTAIR + 急性髓系白血病　；参考：《安徽医科大学》2017年硕士论文

【摘要】：研究背景与目的:急性髓系白血病(Acute myeloid leukemia,AML)是一类髓系造血干祖细胞的恶性克隆性疾病,其在各个年龄段均可发病,且病程进展迅速、临床预后差。近年来,随着靶向药物全反式维甲酸(All-transretinoic acid,ATRA)的应用,AML-M3即急性早幼粒白血病(Acute promyelocytic leukemia,APL)的治疗及预后取得了极大进展。然而其他类型的AML治疗效果仍不佳,因此,需要更加深入的发病机制研究来为临床治疗方案提供新的思路和理论依据。长链非编码RNA(long non-coding RNA,lnc RNA)是一类长度大于200bp、不具有或者很少具有编码蛋白质功能的RNA,其不仅参与了机体细胞正常增殖、分化、凋亡等生理过程,还参与了肿瘤发生发展等病理过程。HOTAIR为一长度为2158bp,位于12号染色体HOXC区域的lnc RNA,其作为促癌基因通过调控细胞分化、组蛋白修饰等多种方式参与了肿瘤的发生发展,但在AML中的作用及机制尚不明确。鉴于此,本研究旨在探讨HOTAIR在全反式维甲酸诱导AML细胞分化过程中的作用及机制,为AML的诊治提供新的思路及实验基础。方法:收集我院临床确诊AML患者45例,缺铁性贫血患者15例的骨髓标本;实时定量PCR(q PCR)检测HOTAIR在AML患者,缺铁性贫血患者骨髓组织以及AML细胞株(HL-60,NB4,U937)中的表达水平;全反式维甲酸诱导分化AML细胞株,q PCR检测和分析HOTAIR经ATRA分化干预前后HOTAIR表达变化,流式检测AML细胞分化及细胞周期;si RNA沉默HOTAIR基因,研究HOTAIR在AML细胞分化过程中的作用及机制;进一步Western Blot检测HOTAIR对细胞周期相关蛋白的水平变化;此外,采用Kaplan-meier法分析HOTAIR与患者临床预后的相关性。结果:我们研究发现:全反式维甲酸处理AML细胞后,细胞分化增多,CD11b及HOTAIR表达水平上调,HL-60细胞周期G1/G0期上升,S期下降;而沉默HOTAIR基因,细胞分化减少,HL-60细胞周期G1/G0期下降,S期上升,提示HOTAIR可部分逆转全反式维甲酸诱导分化作用。而且,全反式维甲酸可下调HL-60细胞CDK4,cylin D1蛋白水平,上调p21蛋白水平;沉默HOTAIR基因,HL-60细胞CDK4,cylin D1蛋白水平表达上升,p21蛋白表达下降。此外,临床骨髓标本的q PCR结果显示,HOTAIR表达水平在AML-M2型患者组中较缺铁性贫血患者组低,相应结果在AML细胞株也得到验证;进一步统计分析还发现HOTAIR与AML患者WBC高低相关,而与预后无明显相关。结论:1.HOTAIR在ATRA诱导AML细胞分化过程中,表达上调;2.HOTAIR通过调控p21,cylin D1及CDK4的表达,调控细胞周期,参与AML细胞分化;3.HOTAIR表达与AML患者高WBC数存在相关,HOTAIR可能为AML诊治提供新靶点。
[Abstract]:Background & objective: acute myeloid leukemia (myeloid) is a kind of malignant clonal disease of hematopoietic stem progenitor cells of myeloid system. In recent years, great progress has been made in the treatment and prognosis of the target drug All-Transretinoic Acid ATRAA (AML-M3). However, other types of AML still do not work well. Therefore, more in-depth research on pathogenesis is needed to provide new ideas and theoretical basis for clinical treatment. Long-chain noncoding RNAs (LNRNAs) are a class of RNAs with length larger than 200bpand with little or no function of encoding proteins, which not only participate in the physiological processes of normal cell proliferation, differentiation and apoptosis, but also participate in the physiological processes of cell proliferation, differentiation and apoptosis. HOTAIR is also involved in tumorigenesis and development. HOTAIR is a lnc RNAs located in the HOXC region of chromosome 12, which is involved in tumor development by regulating cell differentiation and histone modification. However, the role and mechanism in AML is unclear. In view of this, the purpose of this study was to explore the role and mechanism of HOTAIR in the differentiation of AML cells induced by all-trans retinoic acid, and to provide a new idea and experimental basis for the diagnosis and treatment of AML. Methods: bone marrow samples of 45 patients with AML and 15 patients with iron deficiency anemia were collected, and the expression of HOTAIR in bone marrow tissue of AML patients, patients with iron deficiency anemia and AML cell line HL-60NB4U937was detected by real-time quantitative PCRQ PCR. All trans retinoic acid (ATRA) induced differentiation AML cell line was detected and analyzed by qPCR. The expression of HOTAIR was detected before and after ATRA. Flow cytometry was used to detect the differentiation of AML cells and the silencing of HOTAIR gene by cell cycle siRNA. The role and mechanism of HOTAIR in the differentiation of AML cells were studied. In addition, Kaplan-meier method was used to analyze the correlation between HOTAIR and clinical prognosis. Results: in AML cells treated with all-trans retinoic acid, the expression of CD11b and HOTAIR increased and the expression of CD11b and HOTAIR upregulated and decreased in the G _ 1 / G _ 0 phase of HL-60 cell cycle, while the HOTAIR gene was silenced. The cell differentiation decreased and the G1 / G0 phase decreased and the S phase increased, suggesting that HOTAIR could partially reverse the differentiation induced by all-trans retinoic acid (ATRA). In addition, all trans retinoic acid could down-regulate the level of CDK4cylin D1 and up-regulate the level of p21 protein in HL-60 cells, while silencing the expression of CDK4 cylin D1 in HL-60 cells of HOTAIR gene could increase the expression of CDK4cylin D1 protein and decrease the expression of p21 protein. In addition, the Q PCR results of clinical bone marrow samples showed that the expression level of HOTAIR was lower in AML-M2 group than in iron deficiency anemia group, and the corresponding results were also verified in AML cell line. Further statistical analysis also found that HOTAIR was correlated with WBC level in AML patients. There was no significant correlation between prognosis and prognosis. Conclusion: 1. The expression of HOTAIR is up-regulated during ATRA-induced AML cell differentiation. Secondly, HOTAIR regulates the cell cycle by regulating the expression of p21 cylin D1 and CDK4. The expression of HOTAIR may provide a new target for the diagnosis and treatment of AML. 3. The expression of HOTAIR is related to the high WBC number of AML patients.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.71

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