转座酶可及性染色质测序法联合RNA测序探究解旋酶样转录因子基因的缺失对肝细胞癌的影响  

Assay for transposase-accessible chromatin using sequencing combined with RNA sequencing to explore the effect of helicase like transcription factor deletion on hepatocellular carcinoma

作　　者：张艺旋 马亚锐 王小兵 焦宇辰 Zhang Yixuan;Ma Yarui;Wang Xiaobing;Jiao Yuchen(Department of Cell Biology and Molecular Biology,National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital,Chinese Academy of Medical Science and Peking Union Medical College,Beijing 100021,China)

机构地区：[1]国家癌症中心/国家肿瘤临床医学研究中心/中国医学科学院北京协和医学院肿瘤医院细胞生物学及分子生物学研究室,北京100021

出　　处：《肝癌电子杂志》2023年第2期7-15,共9页Electronic Journal of Liver Tumor

基　　金：国家自然科学基金面上项目(82172988)。

摘　　要：目的:探究解旋酶样转录因子(helicase like transcription factor,HLTF)基因在肝细胞癌(hepatocellular carcinoma,HCC)中潜在的调控机制。方法:构建HLTF基因稳定敲除的HCC细胞株。应用RNA测序法(RNA sequencing,RNA-seq)检测并分析肝癌细胞HLTF基因敲除前后的差异表达基因。应用转座酶可及性染色质测序法(assay for transposase-accessible chromatin using sequencing,ATAC-seq)检测HLTF基因敲除前后肝癌细胞染色质可及性的改变。采用RNA-seq和ATAC-seq多组学数据进行联合分析,寻找HLTF基因潜在的下游调控通路和关键基因。结果:癌症基因组图谱(the cancer genome atlas,TCGA)数据库分析表明HLTF基因在HCC组织中的表达水平升高,且其高表达与HCC预后不良有关联;RNA-seq分析结果显示,与野生型肝癌细胞相比,HLTF基因敲除细胞中有563个基因的表达水平上调,656个基因的表达水平下调;ATAC-seq分析结果显示,共27818个区域在HLTF基因缺失时染色质可及性发生显著改变,其中14225个区域染色质可及性增强,13593个区域染色质可及性减弱;对染色质可及性改变的区域进行motif富集分析,数据显示在染色质可及性增强的区域,Atf3、Fra1和BATF等被富集,在染色质可及性减弱的区域,Fra1、Fra2和JunB等被富集;RNA-seq和ATAC-seq多组学数据联合分析表明,重叠基因主要富集在花生四烯酸代谢通路、Wnt信号通路、钙离子信号通路和转化生长因子β(transforming growth factorβ,TGF-β)信号通路等。HLTF基因的缺失使核RNA输出因子3(nuclear RNA export factor 3,NXF3)基因表达水平升高。NXF3基因高表达的HCC患者的预后较NXF3基因低表达的HCC患者好。结论:在HCC中,HLTF基因可能参与调控花生四烯酸代谢通路、Wnt信号通路和TGF-β信号通路等,NXF3基因可能是HLTF基因的潜在下游基因。Objective:To investigate the potential regulatory mechanism of helicase like transcription factor(HLTF)in hepatocellular carcinoma(HCC).Method:HLTF was knockout through clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)in HCC cell line.RNA sequencing(RNA-seq)was applied to detect and analyze the differentially expressed genes of the wild type and HLTF knockout cells. Assay for transposase-accessible chromatin using sequencing (ATAC- seq) was applied to detect the changes in chromatin accessibility of the wild type and HLTF knockout cells. The multi-omics analysis of RNA-seq and ATAC-seq was used to find the key signaling pathways and downstream genes of HLTF. Result: The cancer genome atlas (TCGA) database analysis showed that HLTF was highly expressed in HCC and was associated with poor prognosis;RNA-seq sequencing showed that 563 genes were up-regulated and 656 genes were down- regulated in HLTF knockout cells compared to wild-type cells;ATAC-seq analysis showed a total of 27 818 regions had significantly altered in chromatin accessibility with HLTF deletion, including 14 225 regions with enhanced chromatin accessibility and 13 593 regions with weakened chromatin accessibility;motif enrichment analysis showed that Atf3, Fra1 and BATF were enriched in regions with enhanced chromatin accessibility, Fra1, Fra2 and JunB were enriched in regions with weakened chromatin accessibility;the combination of RNA-seq and ATAC-seq analysis showed that the overlapping genes were mainly enriched in the arachidonic acid metabolism pathway, Wnt signaling pathway, calcium signaling pathway and the transforming growth factor β (TGF-β) signaling pathway;the deletion of HLTF increased the expression of nuclear RNA export factor 3 (NXF3) and highly-expressed NXF3 was associated with better prognosis in HCC patients. Conclusion: Our study pointed out that HLTF may be involved in regulating arachidonic acid metabolism pathway, Wnt signaling pathway, TGF-β signaling pathway, etc. NXF3

关 键 词：转座酶可及性染色质测序法 RNA测序法 肝细胞癌 解旋酶样转录因子 

分 类 号：R735.7[医药卫生—肿瘤]