miR-320c在急性髓系白血病中的临床意义及其机制研究

发布时间：2018-06-01 09:55

本文选题：急性髓系白血病 + microRNA　；参考：《浙江大学》2017年博士论文

【摘要】：背景:急性髓系白血病(acutemyeloid leukemia,AML)是目前成人最常见的血液系统恶性肿瘤之一,在细胞形态学、分子生物学、细胞遗传学、免疫分型、临床表现等方面具有高度异质性。AML的发病机制十分复杂,到目前为止,科学家们还不能完全将之阐述清楚。现在公认对疾病发生发展有重要启动作用的包括染色体核型异常和可重现性的基因突变。通过对这两个因素的分类,可以对大部分病人进行危险分层,根据病人不同的危险分层,采用相应的治疗方案,使得AML患者的五年生存率有显著的提高。但是,部分患者预后仍然不佳,提示临床医生需要进一步探索AML新的发病机制,完善现有的预后评估系统。因此,迫切需要发现新的预后因子并探讨这些预后因子在AML中发挥的功能,从而完善现有的评估系统。近年来,随着科学家们对表观遗传学认识的不断深入,越来越多的证据表明其在AML发生发展中发挥着重要的作用。microRNAs(miRNAs)作为表观遗传学中重要的一员,在AML中的作用正越来越受到重视起来。MiRNAs是一类长度为18-25个核苷酸,具有在转录后调控基因表达水平的非编码小RNA,可通过特异性结合靶基因的3'非翻译区(3'UTR)上的互补序列,从而下调靶基因的表达水平。目前已有很多文献表明miRNAs的表达异常和AML的发生发展有着密切的联系。目的:收集AML患者和正常对照的标本,利用荧光定量PCR检测miR-320c的表达情况,分析miR-320c的表达和AML患者预后之间的关系,探索miR-320c表达下调的调控机制,采用获得性功能实验观察miR-320c在AML细胞株中的生物学功能,利用生物信息学网站和荧光素酶双报告系统寻找miR-320c下游直接作用的靶基因,并进一步分析靶基因对AML患者预后的影响、对AML细胞株生物学表型的调节以及对下游信号通路的调控。方法:(1)利用定量PCR手段检测304例AML患者及正常对照中miR-320c的表达水平,统计学分析miR-320c表达和AML患者预后之间的关系;(2)探讨miR-320c低表达是否和其启动子区CpG岛高度甲基化相关;通过转染miR-320c模拟体(mimic)外源性过表达miR-320c来进行获得性功能实验,通过MTS、集落形成实验、流式检测细胞凋亡和细胞周期、WfesternBlot检测相关蛋白的改变等来探索miR-320c在AML中的生物学功能;利用生物信息学网站对miR-320c的靶基因进行预测,通过荧光定量PCR和WesternBlot检测靶基因RNA及蛋白水平的改变,荧光素酶双报告实验验证miR-320c和靶基因之间的关系;(3)检测304例AML患者及正常对照中靶基因的表达水平,分析靶基因表达和AML患者预后之间的关系;干扰靶基因表达,模拟miR-320c过表达在AML中发挥的作用,探索靶基因的生物学功能,明确靶向调控关系及其下游调控机制。结果:(1)和正常对照相比miR-320c在AML患者中表达显著下调,miR-320c低表达患者的OS较差,miR-320c高表达水平是AML患者的预后良好的独立因素;(2)去甲基化药物地西他滨处理AML细胞株THP-1、MV4-11和AML-OCI3后,可诱导miR-320c的表达上调;(3)过表达miR-320c可以通过PI3K/AKT及凋亡信号通路抑制AML的细胞活性、增殖及克隆形成能力,诱导细胞凋亡;(4)过表达miR-320c可以增加AML细胞对阿糖胞苷的敏感性;(5)利用生物信息学网站预测Musashi2(MSI2)是miR-320c的靶基因,并通过荧光定量PCR、WeesternBlot实验和荧光素酶双报告系统证实MSI2是miR-320c的下游靶基因;(6)和正常对照相比,MSI2在AML患者中表达上调,MSI2高表达患者年龄较大,NPM1和FLT3-ITD的突变率较高,CR率较低,OS和EFS较差;(7)慢病毒干扰MSI2的表达可以抑制AML细胞株的增殖,诱导细胞凋亡,并且可以下调MAPK及WNT通路。结论:(1)miR-320c在AML患者中显著低表达,miR-320c高表达水平是AML患者的预后良好的独立因素,低表达患者预后不良;(2)miR-320C低表达和其启动子区CpG岛高甲基化相关;(3)过表达miR-320c可以抑制AML细胞活性、阻碍细胞增殖、降低克隆形成能力,诱导细胞凋亡,下调PI3K/AKT通路活性,增加对阿糖胞苷的敏感性;(4)MSI2是miR-320c的直接靶点,MSI2在AML患者中高表达,MSI2过表达是AML患者的预后不良因素;(5)下调MSI2可以抑制AML细胞的增殖,诱导细胞凋亡,下调细胞内MAPK和WNT信号通路活性。
[Abstract]:Background: acutemyeloid leukemia (AML) is one of the most common hematological malignancies in adults. The pathogenesis of highly heterogeneous.AML in cellular morphology, molecular biology, cytogenetics, immunophenotype and clinical manifestations is very complicated. So far, scientists can not completely It is clear. It is now recognized that the genetic abnormalities and reproducibility of chromosomes are important for the development of disease. By classifying these two factors, the risk stratification of most patients can be stratified, according to the different risk stratification of the patients, and the corresponding treatment scheme is adopted to make the AML patients five years old. There is a significant improvement in the survival rate. However, the prognosis of some patients is still poor, suggesting that clinicians need to further explore the new pathogenesis of AML and improve the existing prognostic evaluation system. Therefore, there is an urgent need to discover new prognostic factors and explore the function of these prognostic factors in AML so as to improve the existing evaluation system. As scientists continue to understand epigenetics, more and more evidence suggests that.MicroRNAs plays an important role in the development of AML (miRNAs) as an important member of epigenetics. The role in AML is becoming more and more important, and.MiRNAs is a class of 18-25 nucleotides, which is in transcription. The non coding small RNA of the post regulatory gene expression level can be regulated by a complementary sequence on the 3'non translation region (3'UTR) that specifically combines the target gene, so that the expression level of the target gene is down regulated. Many documents have shown that there is a close relationship between the abnormal expression of miRNAs and the development of AML. Objective: to collect the specimens of the AML patients and the normal controls. The expression of miR-320c was detected by fluorescence quantitative PCR, the relationship between the expression of miR-320c and the prognosis of AML patients was analyzed. The regulation mechanism of the down regulation of miR-320c expression was explored. The biological function of miR-320c in the AML cell line was observed by the acquired functional experiment, and the bioinformatics website and the luciferase double report system were used to find miR-320. The direct target gene of C downstream, and further analysis of the effect of target gene on the prognosis of AML patients, the regulation of the biological phenotype of the AML cell line and the regulation of the downstream signal pathway. Methods: (1) the expression level of miR-320c in 304 cases of AML patients and normal controls was detected by quantitative PCR, and miR-320c expression and AML patients were statistically analyzed. The relationship between the prognosis; (2) to investigate whether the low expression of miR-320c is related to the high methylation of CpG island in the promoter region; through transfection of miR-320c analogue (mimic) exogenous overexpression of miR-320c for acquired functional experiments, MTS, colony formation experiments, flow detection of apoptosis and cell cycle, and the modification of WfesternBlot detection related proteins To explore the biological function of miR-320c in AML, to predict the target gene of miR-320c by bioinformatics website, to detect the change of target gene RNA and protein level by fluorescence quantitative PCR and WesternBlot, and to verify the relationship between miR-320c and the target gene by the double report of luciferase reporter; (3) to detect 304 cases of AML patients and normal pairs. According to the expression level of the target gene, the relationship between the target gene expression and the prognosis of AML patients was analyzed, the target gene expression, the role of miR-320c overexpression in AML were simulated, the biological function of the target gene, the target regulation relationship and the downstream regulation mechanism were explored. Results: (1) and the normal contrast miR-320c was expressed in AML patients. Significant downregulation, low expression of OS in patients with miR-320c low expression, high expression of miR-320c is an independent factor for good prognosis in AML patients; (2) the expression of miR-320c can be induced after AML cell line THP-1, MV4-11 and AML-OCI3 of demethylation drug, and (3) miR-320c can inhibit AML by PI3K/AKT and apoptotic signaling pathways. Cell activity, proliferation and clone formation ability, inducing cell apoptosis; (4) overexpression of miR-320c can increase the sensitivity of AML cells to cytarabine; (5) using bioinformatics website to predict Musashi2 (MSI2) is the target gene of miR-320c, and through fluorescence quantitative PCR, WeesternBlot experiment and luciferase double report system confirmed MSI2 is miR-320c (6) compared with normal control, the expression of MSI2 in AML patients was up-regulated, MSI2 high expression was older, NPM1 and FLT3-ITD had higher mutation rate, CR rate was lower, OS and EFS were poor; (7) the expression of lentivirus interfering MSI2 could inhibit the proliferation of AML cell lines, induce apoptosis and down regulation of MAPK and WNT pathways. Conclusion: (1) IR-320c was significantly lower expression in AML patients. High expression of miR-320c was an independent factor in the prognosis of AML patients, and low expression of patients had poor prognosis; (2) low expression of miR-320C was associated with CpG island Gao Jiaji in promoter region; (3) overexpression of miR-320c could inhibit the viability of AML cells, impede cell proliferation, and reduce the ability to clone and induce cells to induce cells. Apoptosis, down-regulation of PI3K/AKT pathway activity, increase the sensitivity of cytarabine; (4) MSI2 is a direct target of miR-320c, MSI2 is highly expressed in AML patients. MSI2 overexpression is a prognostic factor for AML patients; (5) down regulation of MSI2 can inhibit the proliferation of AML cells, induce apoptosis, and down regulate the activity of MAPK and WNT signaling pathway in cells.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R733.71

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