Identification of C/EBPβ Target Genes in ALK+ Anaplastic Large Cell Lymphoma (ALCL) by Gene Expression Profiling and Chromatin Immunoprecipitation

C/EBPβ (CCAAT enhancer binding protein) is a transcription factor that plays a crucial role in survival and transformation of ALK+ anaplastic large cell lymphoma (ALCL). The aim of this study was to identify the downstream targets of C/EBPβ responsible for ALK-mediated oncogenesis. C/EBPβ was knocked down in ALK+ ALCL cell lines with a C/EBPβ-shRNA, followed by gene expression profiling (GEP). GEP analysis revealed a reproducible signature of genes that were significantly regulated by C/EBPβ. Classification into biological categories revealed overrepresentation of genes involved in the immune response, apoptosis and cell proliferation. Transcriptional regulation by C/EBPβ was found in 6 of 11 (BCL2A1, G0S2, TRIB1, S100A9, DDX21 and DDIT4) genes investigated by chromatin immunoprecipitation. We demonstrated that BCL2A1, G0S2 and DDX21 play a crucial role in survival and proliferation of ALK+ ALCL cells. DDX21, a gene involved in rRNA biogenesis, was found differentially overexpressed in primary ALK+ ALCL cases. All three candidate genes were validated in primary ALCL cases by either immunohistochemistry or RT-qPCR. In conclusion, we identified and validated several key C/EBPβ-regulated genes with major impact on survival and cell growth in ALK+ ALCL, supporting the central role of C/EBPβ in ALK-mediated oncogenesis. Kijk and SUDHL1 cell lines transfected with shRNA for C/EBPbeta were compared to control cells (3 biological replicates per group) and untreated cells (1 biological replicate)

CCAAT增强子结合蛋白β（C/EBPβ，CCAAT enhancer binding protein）是一种转录因子，在间变性淋巴瘤激酶阳性间变性大细胞淋巴瘤（ALK+ anaplastic large cell lymphoma, ALCL）的存活与转化进程中发挥关键调控作用。本研究旨在明确介导ALK致癌作用的C/EBPβ下游靶基因。研究人员利用C/EBPβ短发夹RNA（C/EBPβ-shRNA）对ALK+ ALCL细胞系进行C/EBPβ敲低处理，随后开展基因表达谱（gene expression profiling, GEP）分析。GEP分析结果显示，受C/EBPβ显著调控的基因呈现出可重复的表达特征。对上述基因进行生物学功能分类后发现，参与免疫应答、细胞凋亡与细胞增殖的基因显著富集。通过染色质免疫沉淀（chromatin immunoprecipitation）实验对11个候选基因展开验证，其中6个基因（BCL2A1、G0S2、TRIB1、S100A9、DDX21及DDIT4）的转录调控确实受C/EBPβ介导。研究证实，BCL2A1、G0S2与DDX21在ALK+ ALCL细胞的存活与增殖过程中发挥关键作用。DDX21作为参与核糖体RNA生物发生的基因，在原发性ALK+ ALCL病例中呈现显著过表达。通过免疫组化或逆转录定量聚合酶链反应（RT-qPCR）对原发性ALCL病例中的这3个候选基因进行验证，结果均得到确认。综上，本研究鉴定并验证了多个受C/EBPβ调控的关键基因，这些基因对ALK+ ALCL的细胞存活与生长具有重要影响，进一步支持了C/EBPβ在ALK介导的致癌过程中的核心地位。将转染C/EBPβ短发夹RNA的Kijk与SUDHL1细胞系，与对照组细胞（每组设置3次生物学重复）及未处理细胞（1次生物学重复）进行对照分析。