【作者】 刘珊；

【导师】 徐进； 颜光玗；

【作者基本信息】 华南理工大学 ， 生物学， 2022， 硕士

【摘要】 背景:成体造血起源于早期胚胎发育,是由一群具有自我更新及分化能力的造血干细胞(Hematopoietic stem cell,HSC)分化而来的。当受到外界刺激后,HSC被激活并增殖分化成下游各个谱系。造血干细胞的发育过程受到多种转录因子及信号通路的精密调控,任何调控失衡都将导致严重的发育缺陷或者血液疾病。其中,转录因子RUNX1在造血发育过程中起着重要的作用。近些年临床研究发现在癌症疾病,特别是在人源白血病疾病中,相对于正常细胞,野生型RUNX1的表达量呈现不同程度地升高。近期有研究报道,使用化学药物抑制RUNX1表达会导致白血病细胞的生长阻滞,且过表达野生型RUNX1导致白血病细胞数量剧增。虽然研究发现白血病细胞异常高表达野生型RUNX1,但异常高表达的野生型RUNX1如何影响白血病细胞的潜在机制仍不清楚。目的:基于以上背景,本研究重点针对异常表达的野生型转录因子RUNX1对白血病细胞生长功能的影响展开研究,希望对RUNX1在白血病疾病中的作用进行进一步地阐述,且对临床疾病的用药有一定的提示作用。方法:本研究利用公共数据库中下载的RNA-seq、Ch IP-seq等数据进行生物信息学分析,对人源白血病细胞THP-1对于正常细胞CD34+的差异基因进行全面的生物学通路研究;进而借助载体构建、慢病毒转染等分子生物学手段,对正常细胞CD34+和白血病细胞THP-1进行RUNX1的敲降和过表达;通过对RUNX1异常表达导致正常细胞CD34+和白血病细胞THP-1中的表型进行分析,结合生物信息学分析得到的生物学通路进行验证;随后通过流式分析、克隆形成能力等细胞生物学手段对RUNX1影响白血病细胞的生物学途径进行进一步的探究;最后在富集出的通路中筛选出主要影响的基因,对RUNX1影响白血病细胞的潜在机制进行深入探索。结论:本研究发现RUNX1影响白血病细胞的生长主要通过以下两个途径:RUNX1促进细胞增殖能力和影响细胞分化潜能。通过筛选及验证发现CENPE基因是RUNX1直接潜在的调控基因。在拯救实验中,过表达CENPE可以拯救RUNX1缺失导致的细胞数量下降的表型。此外,CENPE通过促进细胞增殖能力来拯救白血病细胞生长数量。本研究发现RUNX1通过上调CENPE基因表达,从而促进白血病细胞的生长优势。更多还原

【Abstract】 Background: Adult hematopoiesis originates from hematopoietic stem cells(HSCs),which are a group of cells that contain self-renewal and differentiation ability.In response to external stimuli,activated HSCs proliferate and differentiate into downstream lineages.The developmental process of hematopoietic stem cell formation is precisely regulated by multiple transcription factors and signaling pathways.Dysregulation in this process could cause severe developmental defects or major hematological diseases.Transcription factor RUNX1 is a key transcription factor in hematopoietic development.In recent years,clinical studies have revealed that aberrantly expressed wild-type RUNX1 plays an important role in leukemogenesis.In cancer diseases,especially in leukemic diseases,wild-type RUNX1 expression showed varying degrees of elevation,relative to normal cells.In addition,recent studies have reported that inhibition of RUNX1 expression led to leukemic cell cycle arrest.Not only RUNX1 mutations could lead to leukemia diseases,but dysregulated wild-type RUNX1 also contributes to leukemia development.However,the underlying mechanism of dysregulated wild-type RUNX1 in leukemogenesis remains unclear.Objective: Hence,this study focused on the effect of the dysregulated wild-type RUNX1 on leukemia cell growth.We hope to clarify the roles of dysregulated wild-type RUNX1 in leukemia disease and provide a basis for clinical use.METHODS: In this study,we performed bioinformatics analysis to identified RUNX1-targeted biological functions in Acute myeloid leukemia(A ML)by examining publicly available transcriptome profiles and RUNX1 Ch IP-seq datasets of THP-1 Human leukemia and CD34+ hematopoietic stem/progenitor cells.Then,we overexpressed and knocked down RUNX1 expression in THP-1 and CD34+ cells to investigate the biological functions affected by dysregulated RUNX1.Finally,we identified CENPE,also known as Centrosome-associated protein E,as the direct target gene of RUNX1.Overexpressing CENPE can rescue the cell number depletion phenotype caused by RUNX1 knockdown in the THP-1 cell line.CONCLUSION: Our study suggested RUNX1 could induce leukemia cell growth by promoting cell proliferation,which was regulated by the RUNX1-CENPE axis.Our results investigate the role of RUNX1 in promoting the growth advantage of leukemia and pave the way for further exploration of the molecular mechanisms associated with dysregulated RUNX1 in leukemia.更多还原