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

摘要/Abstract

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27 818个区域在HLTF基因缺失时染色质可及性发生显著改变,其中14 225个区域染色质可及性增强,13 593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 factor β,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。

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

Abstract: 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 may be a potential downstream gene of HLTF, and our study provided directions and ideas to elucidate the mechanism of HLTF in HCC.

Key words: Assay for transposase-accessible chromatin using sequencing, RNA sequencing, Hepatocellular carcinoma, Helicase like transcription factor

张艺旋, 马亚锐, 王小兵, 焦宇辰. 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[J]. 肝癌电子杂志, 2023, 10(2): 7-15.

Zhang Yixuan, Ma Yarui, Wang Xiaobing, Jiao Yuchen. Assay for transposase-accessible chromatin using sequencing combined with RNA sequencing to explore the effect of helicase like transcription factor deletion on hepatocellular carcinoma[J]. Electronic Journal of Liver Tumor, 2023, 10(2): 7-15.

参考文献

[1] SUNG H, FERLAY J, SIEGEL R L, et al.Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries[J]. CA Cancer J Clin, 2021, 71(3): 209-249.
[2] SIEGEL R L, MILLER K D, FUCHS H E, et al.Cancer statistics, 2022[J]. CA Cancer J Clin, 2022, 72(1): 7-33.
[3] VITALE A, PECK-RADOSAVLJEVIC M, GIANNINI E G, et al.Personalized treatment of patients with very early hepatocellular carcinoma[J]. J Hepatol, 2017, 66(2): 412-423.
[4] UNK I, HAJDU I, BLASTYAK A, et al.Role of yeast Rad5 and its human orthologs, HLTF and SHPRH in DNA damage tolerance[J]. DNA Repair (Amst), 2010, 9(3): 257-267.
[5] ACHAR Y J, BALOGH D, NECULAI D, et al.Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling[J]. Nucleic Acids Res, 2015, 43(21): 10277-10291.
[6] CHAVEZ D A, GREER B H, EICHMAN B F.The HIRAN domain of helicase-like transcription factor positions the DNA translocase motor to drive efficient DNA fork regression[J]. J Biol Chem, 2018, 293(22): 8484-8494.
[7] SANDHU S, WU X, NABI Z, et al.Loss of HLTF function promotes intestinal carcinogenesis[J]. Mol Cancer, 2012, 11: 18.
[8] LI S P, WANG H Y, LI J Q, et al.Genome-wide analyses on loss of heterozygosity in hepatocellular carcinoma in Southern China[J]. J Hepatol, 2001, 34(6): 840-849.
[9] KANG S, KIM J W, KANG G H, et al.Comparison of DNA hypermethylation patterns in different types of uterine cancer: cervical squamous cell carcinoma, cervical adenocarcinoma and endometrial adenocarcinoma[J]. Int J Cancer, 2006, 118(9): 2168-2171.
[10] KIM J J, CHUNG S W, KIM J H, et al.Promoter methylation of helicase-like transcription factor is associated with the early stages of gastric cancer with family history[J]. Ann Oncol, 2006, 17(4): 657-662.
[11] ZHANG X, LI H M, LIU Z, et al.Loss of heterozygosity and methylation of multiple tumor suppressor genes on chromosome 3 in hepatocellular carcinoma[J]. J Gastroenterol, 2013, 48(1): 132-143.
[12] LAM T W, TONG J H, TO K F, et al.Correlative Analysis of DNA Methyltransferase Expression and Promoter Hypermethylation of Tumor Suppressor Genes in Hepatocellular Carcinoma[J]. Cancer Genomics Proteomics, 2006, 3(3-4): 271-277.
[13] THURMAN R E, RYNES E, HUMBERT R, et al.The accessible chromatin landscape of the human genome[J]. Nature, 2012, 489(7414): 75-82.
[14] CHEN S, ZHOU Y, CHEN Y, et al.fastp: an ultra-fast all-in-one FASTQ preprocessor[J]. Bioinformatics, 2018, 34(17): i884-i890.
[15] DOBIN A, DAVIS C A, SCHLESINGER F, et al.STAR: ultrafast universal RNA-seq aligner[J]. Bioinformatics, 2013, 29(1): 15-21.
[16] ROBINSON M D, MCCARTHY D J, SMYTH G K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data[J]. Bioinformatics, 2010, 26(1): 139-140.
[17] YANG J, BOGERD H P, WANG P J, et al.Two closely related human nuclear export factors utilize entirely distinct export pathways[J]. Mol Cell, 2001, 8(2): 397-406.
[18] VAN TOORN M, TURKYILMAZ Y, HAN S, et al. Active DNA damage eviction by HLTF stimulates nucleotide excision repair[J]. Mol Cell, 2022, 82(7): 1343-1358.e8.
[19] ACHAR Y J, BALOGH D, HARACSKA L.Coordinated protein and DNA remodeling by human HLTF on stalled replication fork[J]. Proc Natl Acad Sci U S A, 2011, 108(34): 14073-14078.
[20] DEBAUVE G, NONCLERCQ D, RIBAUCOUR F, et al.Early expression of the Helicase-Like Transcription Factor (HLTF/SMARCA3) in an experimental model of estrogen-induced renal carcinogenesis[J]. Mol Cancer, 2006, 5: 23.
[21] CHO S, CINGHU S, YU J R, et al.Helicase-like transcription factor confers radiation resistance in cervical cancer through enhancing the DNA damage repair capacity[J]. J Cancer Res Clin Oncol, 2011, 137(4): 629-637.
[22] HE S, TANG S.WNT/beta-catenin signaling in the development of liver cancers[J]. Biomed Pharmacother, 2020, 132: 110851.
[23] PERUGORRIA M J, OLAIZOLA P, LABIANO I, et al.Wnt-beta-catenin signalling in liver development, health and disease[J]. Nat Rev Gastroenterol Hepatol, 2019, 16(2): 121-136.
[24] LIANG J Q, TEOH N, XU L, et al.Dietary cholesterol promotes steatohepatitis related hepatocellular carcinoma through dysregulated metabolism and calcium signaling[J]. Nat Commun, 2018, 9(1): 4490.
[25] LI M O, FLAVELL R A.TGF-beta: a master of all T cell trades[J]. Cell, 2008, 134(3): 392-404.
[26] CHEN W, TEN DIJKE P.Immunoregulation by members of the TGFbeta superfamily[J]. Nat Rev Immunol, 2016, 16(12): 723-740.
[27] YUAN J H, YANG F, WANG F, et al.A long noncoding RNA activated by TGF-beta promotes the invasion-metastasis cascade in hepatocellular carcinoma[J]. Cancer Cell, 2014, 25(5): 666-681.
[28] SIA D, JIAO Y, MARTINEZ-QUETGLAS I, et al.Identification of an Immune-specific Class of Hepatocellular Carcinoma, Based on Molecular Features[J]. Gastroenterology, 2017, 153(3): 812-826.
[29] LI M W, SLETTEN A C, LEE J, et al.Nuclear export factor 3 regulates localization of small nucleolar RNAs[J]. J Biol Chem, 2017, 292(49): 20228-20239.
[30] KATCHMAN B A, CHOWELL D, WALLSTROM G, et al.Autoantibody biomarkers for the detection of serous ovarian cancer[J]. Gynecol Oncol, 2017, 146(1): 129-136.
[31] ZENG T, WANG D, CHEN J, et al.AF119895 regulates NXF3 expression to promote migration and invasion of hepatocellular carcinoma through an interaction with miR-6508-3p[J]. Exp Cell Res, 2018, 363(1): 129-139.