5-氟尿嘧啶对结直肠癌基因组三维结构及基因表达的影响

最新的全球癌症调查报告结果表明，结直肠癌是世界上第三大最常见的新发癌症，占所有癌症病例的10%，同时结直肠癌是导致癌症病例死亡的第二大原因，占癌症死亡总人数的9.4%。近年来随着三维基因组学的发展，研究发现染色质的远程交互以及基因组的三维空间构象在癌症中发生了改变，表明癌症的发生与基因组三维结构的变化相关。

抗肿瘤药物5-氟尿嘧啶（5- fluorouracil, 5-FU）是结直肠癌治疗中的重要化疗药物，但在临床治疗中的副作用较大。本课题尝试探寻5-FU治疗结直肠癌中潜在的基因组三维结构的变化和基因远程调控机制，以寻找结直肠癌的新型药靶。本研究使用三维基因组研究技术in situ ChIA-PET，捕获RNA转录聚合酶 （RNA Polymerase II, RNAPII）介导的染色质交互信息，结合转录组测序技术RNA-seq获取基因表达的情况，对5-FU处理不同时间段的结直肠癌细胞系HCT116进行研究，从而捕获HCT116中活跃转录的基因组互作位点信息。同时为了多角度了解5-FU的作用机制，本研究同时使用了p53敲除型的细胞，以辨别5-FU不依赖于p53的作用路径。

本研究展示了5-FU处理对结直肠癌细胞基因表达和染色质远程互作的影响，发现了一类高度互作的核小体组蛋白基因簇HIST，其基因表达直接受5-FU的影响，并且5-FU处理也改变了其染色质的互作程度。结合其他的研究分析，发现5-FU通过短时间内增强染色质的互作频率来影响基因表达。本研究在基因组的三维结构和基因表达的层面进一步揭示了5-FU治疗结直肠癌的作用机制，为结直肠癌的治疗提供了一个新的研究思路。

[1] BONEV B, CAVALLI G. Organization and function of the 3D genome[J/OL]. Nature Reviews Genetics, 2016, 17(11): 661-678.
[2] JERKOVIC´ I, CAVALLI G. Understanding 3D genome organization by multidisciplinary methods[J/OL]. Nature Reviews Molecular Cell Biology, 2021, 22(8): 511-528.
[3] PRADO F, JIMENO-GONZÁLEZ S, REYES J C. Histone availability as a strategy to control gene expression[J/OL]. RNA Biology, 2016, 14(3): 281-286.
[4] BOGENBERGER J M, LAYBOURN P J. Human T Lymphotropic Virus Type 1 protein Tax reduces histone levels[J/OL]. Retrovirology, 2008, 5(1): 9.
[5] CHEN R, KANG R, FAN X G, et al. Release and activity of histone in diseases[J/OL]. Cell Death & Disease, 2014, 5(8): e1370-e1370.
[6] BUENROSTRO J, WU B, CHANG H, et al. ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide[J/OL]. Current protocols in molecular biology, 2015, 109: 21.29.1-21.29.9.
[7] BAYANI J, SQUIRE J A. Fluorescence in situ Hybridization (FISH)[J/OL]. Current Protocols in Cell Biology, 2004, Chapter 22: Unit 22.4.
[8] DEKKER J, RIPPE K, DEKKER M, et al. Capturing chromosome conformation[J/OL]. Science, 2002, 295(5558): 1306-1311.
[9] ZHAO Z, TAVOOSIDANA G, SJÖLINDER M, et al. Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions[J/OL]. Nature Genetics, 2006, 38(11): 1341-1347.
[10] DOSTIE J, RICHMOND T A, ARNAOUT R A, et al. Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements[J/OL]. Genome Research, 2006, 16(10): 1299-1309.
[11] LIEBERMAN-AIDEN E, VAN BERKUM N L, WILLIAMS L, et al. Comprehensive mapping of long range interactions reveals folding principles of the human genome[J/OL]. Science, 2009, 326(5950): 289-293.
[12] FULLWOOD M J, LIU M H, PAN Y F, et al. An oestrogen-receptor-alpha-bound human chromatin interactome[J/OL]. Nature, 2009, 462(7269): 58-64.
[13] SATI S, CAVALLI G. Chromosome conformation capture technologies and their impact in understanding genome function[J/OL]. Chromosoma, 2017, 126(1): 33-44.
[14] LI G, CAI L, CHANG H, et al. Chromatin Interaction Analysis with Paired-End Tag (ChIA-PET) sequencing technology and application[J/OL]. BMC Genomics, 2014, 15(S12): S11.
[15] TANG Z, LUO O J, LI X, et al. CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription[J/OL]. Cell, 2015, 163(7): 1611-1627.
[16] LI X, LUO O J, WANG P, et al. Long-read ChIA-PET for base-pair-resolution mapping of haplotype-specific chromatin interactions[J/OL]. Nature Protocols, 2017, 12(5): 899-915.
[17] WANG P, FENG Y, ZHU K, et al. In situ Chromatin Interaction Analysis Using Paired-End Tag Sequencing[J/OL]. Current Protocols, 2021, 1(8): e174.
[18] FULLWOOD M J, LIU M H, PAN Y F, et al. An oestrogen-receptor-α-bound human chromatin interactome[J/OL]. Nature, 2009, 462(7269): 58-64.
[19] LI G, RUAN X, AUERBACH R K, et al. Extensive promoter-centered chromatin interactions provide a topological basis for transcription regulation[J/OL]. Cell, 2012, 148(1-2): 84-98.
[20] VOGELSTEIN B, KINZLER K W. Cancer genes and the pathways they control[J/OL]. Nature Medicine, 2004, 10(8): 789-799.
[21] GIBCUS J H, DEKKER J. The Hierarchy of the 3D Genome[J/OL]. Molecular Cell, 2013, 49(5): 773-782.
[22] ZEITZ M J, AY F, HEIDMANN J D, et al. Genomic Interaction Profiles in Breast Cancer Reveal Altered Chromatin Architecture[J/OL]. PLOS ONE, 2013, 8(9): e73974.
[23] CHAKRABORTY A, AY F. The role of 3D genome organization in disease: From compartments to single nucleotides[J/OL]. Seminars in Cell & Developmental Biology, 2019, 90: 104-113.
[24] KEIM C, KAZADI D, ROTHSCHILD G, et al. Regulation of AID, the B-cell genome mutator[J/OL]. Genes and Development, 2013, 27(1): 1-17.
[25] QIAN J, WANG Q, DOSE M, et al. B cell super-enhancers and regulatory clusters recruit AID tumorigenic activity[J/OL]. Cell, 2014, 159(7): 1524-1537.
[26] TRIMARCHI T, BILAL E, NTZIACHRISTOS P, et al. Genome-wide Mapping and Characterization of Notch-Regulated Long Noncoding RNAs in Acute Leukemia[J/OL]. Cell, 2014, 158(3): 593-606.
[27] JIA Q, CHEN S, TAN Y, et al. Oncogenic super-enhancer formation in tumorigenesis and its molecular mechanisms[J/OL]. Experimental & Molecular Medicine, 2020, 52(5): 713-723.
[28] AMJADI-MOHEB F, PANIRI A, AKHAVAN-NIAKI H. Insights into the Links between MYC and 3D Chromatin Structure and Epigenetics Regulation: Implications for Cancer Therapy[J/OL]. Cancer Research, 2021, 81(8): 1925-1936.
[29] MIFSUD B, TAVARES-CADETE F, YOUNG A N, et al. Mapping long-range promoter contacts in human cells with high-resolution capture Hi-C[J/OL]. Nature Genetics, 2015, 47(6): 598-606.
[30] KLOETGEN A, THANDAPANI P, NTZIACHRISTOS P, et al. Three-dimensional chromatin landscapes in T cell acute lymphoblastic leukemia[J/OL]. Nature Genetics, 2020, 52(4): 388-400.
[31] SCHUIJERS J, MANTEIGA J C, WEINTRAUB A S, et al. Transcriptional Dysregulation of MYC Reveals Common Enhancer-Docking Mechanism[J/OL]. Cell Reports, 2018, 23(2): 349-360.
[32] SIEGEL R L, MILLER K D, WAGLE N S, et al. Cancer statistics, 2023[J/OL]. CA: A Cancer Journal for Clinicians, 2023, 73(1): 17-48.
[33] 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/OL]. CA: A Cancer Journal for Clinicians, 2021, 71(3): 209-249.
[34] ENG C, JÁCOME A A, AGARWAL R, et al. A comprehensive framework for early-onset colorectal cancer research[J/OL]. The Lancet Oncology, 2022, 23(3): e116-e128.
[35] DEKKER E, TANIS P J, VLEUGELS J L A, et al. Colorectal cancer[J/OL]. The Lancet, 2019, 394(10207): 1467-1480.
[36] PINO M S, CHUNG D C. The Chromosomal Instability Pathway in Colon Cancer[J/OL]. Gastroenterology, 2010, 138(6): 2059-2072.
[37] BATTAGLIN F, NASEEM M, LENZ H J, et al. Microsatellite Instability in Colorectal Cancer: Overview of Its Clinical Significance and Novel Perspectives[J]. Clinical advances in hematology & oncology: H&O, 2018, 16(11): 735-745.
[38] MALKI A, ELRUZ R A, GUPTA I, et al. Molecular Mechanisms of Colon Cancer Progression and Metastasis: Recent Insights and Advancements[J/OL]. International Journal of Molecular Sciences, 2020, 22(1): 130.
[39] MAGZOUB M M, PRUNELLO M, BRENNAN K, et al. The impact of DNA methylation on the cancer proteome[J/OL]. PLoS Computational Biology, 2019, 15(7): e1007245.
[40] OGINO S, CANTOR M, KAWASAKI T, et al. CpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative DNA methylation analysis and prospective cohort studies[J/OL]. Gut, 2006, 55(7): 1000-1006.
[41] PUCCINI A, BERGER M D, NASEEM M, et al. Colorectal cancer: epigenetic alterations and their clinical implications[J/OL]. Biochimica et biophysica acta, 2017, 1868(2): 439-448.
[42] FEARON E R, VOGELSTEIN B. A genetic model for colorectal tumorigenesis[J/OL]. Cell, 1990, 61(5): 759-767.
[43] CHURCH J. Molecular genetics of colorectal cancer[J/OL]. Seminars in Colon and Rectal Surgery, 2016, 27(4): 172-175.
[44] JAVIERRE B M, BURREN O S, WILDER S P, et al. Lineage-Specific Genome Architecture Links Enhancers and Non-coding Disease Variants to Target Gene Promoters[J/OL]. Cell, 2016, 167(5): 1369-1384.e19.
[45] YOUNGER S T, RINN J L. ’Lnc’-ing enhancers to MYC regulation[J/OL]. Cell Research, 2014, 24(6): 643-644.
[46] ORLANDO G, LAW P J, CORNISH A J, et al. Promoter capture Hi-C-based identification of recurrent noncoding mutations in colorectal cancer[J/OL]. Nature Genetics, 2018, 50(10): 1375-1380.
[47] JOHNSTONE S E, REYES A, QI Y, et al. Large-Scale Topological Changes Restrain Malignant Progression in Colorectal Cancer[J/OL]. Cell, 2020, 182(6): 1474-1489.e23.
[48] DIASIO R B, HARRIS B E. Clinical pharmacology of 5-fluorouracil[J/OL]. Clinical Pharmacokinetics, 1989, 16(4): 215-237.
[49] LONGLEY D B, HARKIN D P, JOHNSTON P G. 5-Fluorouracil: mechanisms of action and clinical strategies[J/OL]. Nature Reviews Cancer, 2003, 3(5): 330-338.
[50] SANTI D V, MCHENRY C S, SOMMER H. Mechanism of interaction of thymidylate synthetase with 5-fluorodeoxyuridylate[J/OL]. Biochemistry, 1974, 13(3): 471-481.
[51] YOSHIOKA A, TANAKA S, HIRAOKA O, et al. Deoxyribonucleoside triphosphate imbalance. 5-Fluorodeoxyuridine-induced DNA double strand breaks in mouse FM3A cells and the mechanism of cell death[J]. The Journal of Biological Chemistry, 1987, 262(17): 8235-8241.
[52] AHERNE G W, HARDCASTLE A, RAYNAUD F, et al. Immunoreactive dUMP and TTP pools as an index of thymidylate synthase inhibition; effect of tomudex (ZD1694) and a nonpolyglutamated quinazoline antifolate (CB30900) in L1210 mouse leukaemia cells[J/OL]. Biochemical Pharmacology, 1996, 51(10): 1293-1301.
[53] GREM J L, FISCHER P H. Enhancement of 5-fluorouracil’s anticancer activity by dipyridamole[J/OL]. Pharmacology & Therapeutics, 1989, 40(3): 349-371.
[54] GLAZER R I, LLOYD L S. Association of cell lethality with incorporation of 5-fluorouracil and 5-fluorouridine into nuclear RNA in human colon carcinoma cells in culture[J]. Molecular Pharmacology, 1982, 21(2): 468-473.
[55] Efficacy of adjuvant fluorouracil and folinic acid in colon cancer. International Multicentre Pooled Analysis of Colon Cancer Trials (IMPACT) investigators[J]. Lancet, 1995, 345(8955): 939-944.
[56] DOUILLARD J Y, CUNNINGHAM D, ROTH A D, et al. Irinotecan combined with fluorouracil compared with fluorouracil alone as first-line treatment for metastatic colorectal cancer: a multicentre randomised trial[J/OL]. Lancet, 2000, 355(9209): 1041-1047.
[57] AHN D H, WU C, WEI L, et al. The Efficacy of Adjuvant Chemotherapy in Patients with Stage II/III Resected Rectal Cancer Treated with Neoadjuvant Chemoradiation Therapy[J/OL]. American Journal of Clinical Oncology: Cancer Clinical Trials, 2017, 40(6): 531-534.
[58] SARGENT D, SOBRERO A, GROTHEY A, et al. Evidence for cure by adjuvant therapy in colon cancer: Observations based on individual patient data from 20,898 patients on 18 randomized trials[J/OL]. Journal of Clinical Oncology, 2009, 27(6): 872-877.
[59] MICHEL M, KAPS L, MADERER A, et al. The Role of p53 Dysfunction in Colorectal Cancer and Its Implication for Therapy[J/OL]. Cancers, 2021, 13(10): 2296.
[60] VODENKOVA S, BUCHLER T, CERVENA K, et al. 5-fluorouracil and other fluoropyrimidines in colorectal cancer: Past, present and future[J/OL]. Pharmacology & Therapeutics, 2020, 206: 107447.
[61] LEE B, WANG J, CAI L, et al. ChIA-PIPE: A fully automated pipeline for comprehensive ChIA-PET data analysis and visualization[J/OL]. Science Advances, 2020, 6(28): eaay2078.
[62] KHO P S, WANG Z, ZHUANG L, et al. p53-regulated Transcriptional Program Associated with Genotoxic Stress-induced Apoptosis *[J/OL]. Journal of Biological Chemistry, 2004, 279(20): 21183-21192.
[63] YANG C M, KANG M K, JUNG W J, et al. p53 expression confers sensitivity to 5‑fluorouracil via distinct chromatin accessibility dynamics in human colorectal cancer[J/OL]. Oncology Letters, 2021, 21(3): 1-1.