【作者】 杨琳；

【导师】 陈春燕；

【作者基本信息】 山东大学 ， 内科学（血液病方向）， 2019， 硕士

【摘要】 研究目的:急性髓系白血病(Acute myeloid leukemia,AML)是最常见的成人急性白血病类型,近年来发病率呈现上升趋势,患者病情危重,预后凶险。尽管联合化疗的进步使AML治疗取得进展,但AML是一类高度异质性的疾病,不同亚型、不同染色体核型、不同基因表达及突变类型的患者预后不同。根据AML患者细胞遗传学和分子遗传学特征,结合分子异常与核型异常建立综合分层体系评估患者预后,从而制定个体化治疗方案成为AML治疗的发展趋势。对没有染色体核型改变同时也缺乏分子遗传学异常的AML患者,其危险度划分也有待细化。因此探寻新的AML预后相关分子,进一步完善AML诊断分层和预后评估体系,对于优化治疗方案提高AML患者生存率具有重要意义。本研究目的在于:通过搜索数据库中AML相关信息,挖掘与AML诊断分层与预后相关的候选分子,然后通过临床标本检测和细胞系实验初步验证候选分子在AML中的作用,期望发现急性髓系白血病诊断分层和预后评估新的候选标志分子。研究方法与结果:1.TCGA数据库生存分析利用TCGA数据库中AML患者预后数据信息,整合患者基因表达数据,使用R语言包作Kaplan-Meier生存分析,并进行Log-Rank检验。得到一组与TCGA数据库中AML患者生存相关的基因。按P值从小到大排列,选取与预后最具统计学相关性的30个分子作为候选分子。2.数据库筛选AML患者与正常人差异表达基因(Differentially Expressed Genes,DEGs)分析在GEO数据库里选取同时具有AML各亚型患者以及正常人基因表达信息的数据集,进行统计学分析,比较AML患者与对照组全基因组表达水平,得到一系列差异表达基因。检索1中得出的30个候选分子表达差异水平,选取表达差异最具统计学意义的生存相关分子——腺苷酸琥珀酸合成酶(Adenylosuccinate synthetase,ADSS)进行研究。在独立数据集中比较ADSS在AML患者骨髓中与正常人骨髓中表达差异。结果显示AML患者骨髓中ADSS表达受抑。3.探究ADSS与AML疾病诊断、分型、风险评估相关临床特征间相关性将ADSS列为AML诊断指标,做受试者曲线,探究ADSS与疾病相关性。根据ADSS基因表达量高低进行分组,统计各组的临床特征,进行卡方检验,探究发现ADSS与FAD分型,细胞生物学分层、分子遗传学分层具有相关性。4.比较高风险组AML患者与低风险组患者ADSS表达量比较LSC+亚群和LSC-亚群中ADSS表达差异。根据细胞生物学危险度分层将AML患者分为高危、中危和低危组后,比较ADSS在患者骨髓内表达差异。结果显示危险度越高的AML患者ADSS表达量越低。5.验证ADSS表达差异与预后相关性将TCGA和GEO数据库中患者根据ADSS表达量进行分组,做生存曲线,比较高表达组和低表达组预后情况差异。结果显示ADSS低表达组患者预后较高表达组差。6.差异表达基因的多因素COX风险模型分析将TCGA数据库中的各临床特征如性别、年龄等作为单一因素,分别检验其与生存的相关性。选取对生存有影响的临床特征为变量,结合候选分子的表达进行多因素COX回归分析,证实ADSS表达水平是AML预后的独立影响因素。7.蛋白互作网络分析在STRING蛋白数据库中对目的基因ADSS构建相关蛋白互作网络,并对结果进行富集分析,观察这些蛋白与哪些信号通路相关联。将AML患者依据候选分子表达量由大到小的顺序进行排序,选取前25%作为高表达组,后25%作为低表达组,用R语言分析高低表达组患者的全基因组表达水平,得到差异基因,对差异性最具统计学意义的分子进行如上富集分析。结果显示ADSS与腺苷酸代谢途径和相关蛋白可以相互作用,可能通过该途径影响AML的发生与发展。8.临床标本检测检测AML患者和正常对照组骨髓样本,比较正常对照组、AML初诊组、AML-CR组以及AML-NR组ADSS的表达情况。结果显示AML患者较正常对照组低表达ADSS。同一 AML患者ADSS在不同时期的表达量与疾病进程呈负相关。9.细胞系诱导分化实验在AML细胞系中,分别用PMA,Hemin,DMSO诱导巨噬细胞系、红系和粒系分化,检测诱导分化后的细胞系中ADSS表达情况,初步验证ADSS与AML分化相关性。研究结论:1.本实验基于生物信息学分析发现腺苷酸琥珀酸合成酶ADSS与AML预后紧密相关,ADSS的低表达与多种AML患者的临床和生物学特征相关,提示ADSS可能是AML患者新的诊断分层及预后评估潜在标志分子。2.通过对临床标本检测证实ADSS与AML疾病进程呈负相关,提示ADSS可能作为AML发生发展过程中的潜在抑癌基因。3.细胞系诱导分化实验结果证实ADSS能够诱导白血病细胞分化,虽然具体分子作用机制有待进一步研究,但初步提示ADSS可能成为AML的潜在干预靶点。更多还原

【Abstract】 Purposes:Acute myeloid leukemia(Acute myeloid leukemia,AML)is the most common type of Acute adult leukemia,and the incidence has increased in recent years.Patients are usually in critical condition and have a poor prognosis.Although advances in combination chemotherapy have advanced AML treatment,AML is a highly heterogeneous disease with different prognoses for patients with different subtypes,different karyotypes,different gene expression and mutation types.Based on the cytogenetic and molecular genetics characteristics of AML patients,a comprehensive stratified system was established to evaluate the prognosis of patients by combining molecular abnormalities and karyotype abnormalities,so that the development of individualized treatment plans became the trend of AML treatment.For patients without karyotype or molecular abnormalities,the risk classification needs to be refined.Therefore,it is of great significance to explore new prognostic molecules of AML and further improve the diagnostic stratification and prognostic evaluation system of AML to select the most appropriate treatment scheme and improve the survival rate of AML patients.The purpose of this study is to search for candidate molecules related to AML diagnostic stratification and prognosis by searching AML related biological information in the database,and then verify the role of candidate molecules in AML through clinical specimen detection and cell line experiments,and hope to find acute myeloids leukemia diagnostic stratification and prognosis assessment of new candidate marker molecules.Methods and results:1.TCGA database survival analysis Using the prognostic data of AML patients in the TCGA database,the patient gene expression data was integrated,and the R language package was used for Kaplan-Meier survival analysis and Log-Rank test.A set of genes related to the survival of AML patients in the TCGA database was obtained.According to the P value from small to large,30 molecules with the most statistically relevant prognosis were selected as candidate molecules.2.Database screening of differentially expressed genes(DEGs)in patients with AML and the control groupIn the GEO database,a data set containing information on the gene expression of patients with subtypes of AML and normal or non-leukemia people was selected for statistical analysis.The expression levels of whole genomes of AML patients and the control group were compared,and a series of differentially expressed genes were obtained.The level of expression difference of 30 candidate molecules mentioned above was searched,and the survival-related molecules Adenylosuccinate synthetase(ADSS)which has the most statistically significant differences were selected for further research.The expression of ADSS in the bone marrow of AML patients and in control group was compared in an independent data set.The results showed that ADSS expression was suppressed in the bone marrow of AML patients.3.To explore the correlation between clinical features related to the diagnosis,classification and risk stratification of ADSS and AML diseases.ADSS was listed as a diagnostic indicator for AML,and the receiver operating characteristic curve(ROC curve)was used to explore the correlation between the expression of ADSS and disease diagnosis.According to the level of ADSS gene expression,the clinical characteristics of each group were statistically analyzed.Chi-square test was performed to find out the correlation between ADSS and FAD classification,cell biology stratification and molecular genetic stratification.4.Comparison of ADSS expression in patients with high-risk AML and low-risk patientsThe differences in ADSS expression between LSC+ subpopulations and LSC-subpopulations were compared.According to the risk of stratification of cell biology,AML patients were divided into high-risk,intermediate-risk,low-risk groups,and the expression of ADSS in patients’ bone marrow was compared.The results showed that the higher the risk,the lower the expression level of ADSS in AML patients.5.Verify the difference between ADSS expression and prognosisPatients in the TCGA and GEO databases were grouped according to the median expression of ADSS,and the survival curve was compared.The results showed that patients with low expression of ADSS had a poor prognosis.6.Multi-factor cox regression model analysis of differentially expressed genesThe clinical characteristics of the TCGA database,such as gender and age,were used as a single factor to test their correlation with survival.The clinical features that have an impact on survival were selected as variables,and multi-factor cox regression analysis was performed in combination with the expression of candidate molecules,confirming that ADSS expression level is an independent factor of AML prognosis.7.Protein interaction network analysisA network of related proteins was constructed in the STRING protein database for the target gene ADSS,and the results were subjected to enrichment analysis to observe which biological functional pathways and disease profiles these proteins can be annotated.Patients with AML were ranked according to the order of candidate molecule expression.The top 25%was selected as the high expression group and the latter 25%was used as the low expression group.The whole genome expression level of the high and low expression groups was analyzed by R language.Genes were subjected to the above enrichment analysis for the most statistically significant differences.The results show that ADSS can interact with adenylate metabolic pathways and related proteins,which may affect the occurrence and development of AML.8.Clinical specimen testingThe clinical bone marrow samples collected by the laboratory were compared,and the expressions of ADSS in the normal control group,AML initial diagnosis group,AML-CR group and AML-NR group were compared.The results showed that AML patients had lower expression of ADSS than the normal control group.The expression levels of ADSS in different AML patients at different stages were negatively correlated with disease progression.9.Cell line induced differentiation experimentIn the AML cell line,PMA,hemin,DMSO were used to induce macrophage,erythroid,and granulocyte differentiation,and the expression of ADSS in the cell line after differentiation was detected,and the correlation between ADSS and AML differentiation was preliminarily verified.Conclusion:1.Based on bioinformatics analysis,this study found that Adenylosuccinate synthetase(ADSS)is closely related to the prognosis of AML,and the low expression of ADSS is related to the clinical and biological characteristics of a variety of AML patients,suggesting that ADSS may be a new diagnostic stratification and a potential marker for prognosis evaluation of AML patients.2.The negative correlation between ADSS and the progression of AML was confirmed by the detection of clinical specimens,suggesting that ADSS may be a potential tumor suppressor gene in the development of AML.3.The results of cell line induced differentiation experiments confirmed that ADSS could induce differentiation of leukemia cells.Although the specific molecular mechanism remains to be further studied,it preliminarily suggests that ADSS may be a potential intervention target for AML.更多还原