基于ChIP-seq技术分析心肌成纤维细胞中KDM5A调控的下游靶基因网络

魏婷; 亓先杰; 张旭; 王亚涛; 姜亦瑶; 施超

doi:10.16766/j.cnki.issn.1674-4152.003149

基于ChIP-seq技术分析心肌成纤维细胞中KDM5A调控的下游靶基因网络

doi: 10.16766/j.cnki.issn.1674-4152.003149

蚌埠医学院第一附属医院心脏外科，安徽蚌埠 233004

基金项目:

国家自然科学基金青年基金项目 81800214

蚌埠医学院2021年度研究生科研创新项目 Byycx21077

详细信息

通讯作者:
施超，E-mail：wuweishichao@126.com

中图分类号: R542.2 R654.2
计量
- 文章访问数: 225
- HTML全文浏览量: 125
- PDF下载量: 13
- 被引次数: 0
出版历程
- 收稿日期: 2022-08-11
- 网络出版日期: 2023-10-19

Investigation of the downstream target gene network regulated by KDM5A using ChIP-seq in Cardiac fibroblasts

Department of Cardiovasular Surger, the First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui 233004, China

摘要

摘要: 目的探讨KDM5A调控心肌成纤维细胞生物学功能的潜在机制。方法获取2019年1月—2020年3月于蚌埠医学院第一附属医院心脏外科就诊的扩张型心肌病患者的心室肌组织。分离培养心肌纤维化患者的心肌成纤维细胞，采用染色质免疫共沉降测序(ChIP-seq)对细胞中KDM5A富集的DNA序列进行分析，对筛选出的差异基因进行基因本体论(GO)分析和京都基因和基因组百科全书(KEGG)通路富集分析。结果 KDM5A的结合峰在基因启动子区、内含子区和外显子区所占的比例分别为14.59%、44.14%和1.84%。其中，≤1 kb、＞1~2 kb和＞2~3 kb的启动子区比例分别为5.91%、4.56%和4.12%。KDM5A结合在第一内含子区和其他内含子区的比例分别为12.19%和31.95%。KDM5A结合峰相对于转录起始位点(TSS)的分布主要集中在-3~3 kb，在TSS坐标-1.0 kb和2.5 kb处的reads计数频率最大。利用Homer软件分别识别出KDM5A结合的motif保守序列包括FOXE1、PB、ZNF460等20个。GO功能注释分析提示，与KDM5A结合的基因涉及细胞形态发生的调控、轴突发育、神经元投射发育的调节、细胞质膜蛋白复合物、阳离子通道、肌动蛋白骨架、金属离子跨膜转运蛋白活性、阳离子跨膜转运蛋白活性、GTP酶结合等。KEGG信号通路富集结果显示，KDM5A相关基因显著富集在磷脂酶D信号通路、胆碱能突触、钙信号通路、Wnt信号通路、生长激素的合成、分泌和作用、胰岛素分泌、ErbB信号通路、Rap1信号通路等通路。结论 KDM5A通过调节基因的转录表达，调控信号通路表达，进而影响心肌成纤维细胞功能，参与心肌纤维化的发生与发展。
- KDM5A /
- 心肌成纤维细胞 /
- 染色质免疫共沉降测序 /
- 生物信息学分析
Abstract: Objective To investigate the potential mechanism of KDM5A in regulating the biological function of cardiac fibroblasts. Methods Cardiac fibroblasts from patients with myocardial fibrosis were isolated and cultured, chromatin immunoprecipitation sequence (ChIP-seq) was performed to analyze immunoaffinity separation of specific binding DNA fragments with KDM5A for sequence identification, differentially expressed genes (DEGs) were screened for gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. Results The binding peak of KDM5A in the gene promoter region, intron region, and exon region accounted for 14.59%, 44.14% and 1.84%, respectively. Among them, the proportion of ≤1 kb, >1-2 kb and >2-3 kb promoter regions were 5.91%, 4.56% and 4.12%, respectively. The binding ratio of KDM5A to the first intron region and other intron regions were 12.19% and 31.95%, respectively. The distribution of KDM5A binding peaks relative to the transcription start site (TSS) was mainly concentrated at -3 to 3 kb, and the reads count frequency was the highest at the TSS coordinates -1.0 kb and 2.5 kb. Homer software was applied to identify 20 motifs that bind to KDM5A, including FOXE1, PB, ZNF460, etc. GO functional annotation analysis suggested that the KDM5A binding genes were involved in the regulation of cell morphogenesis, axon development, regulation of neuron projection development, plasma membrane protein complex, cation channel, actin cytoskeleton, metal ion transmembrane transporter activity, cation transmembrane transporter activity and GTP enzyme binding, etc. The KEGG signaling pathway enrichment results showed that KDM5A-related genes were significantly enriched in the phospholipase D signaling pathway, cholinergic synapse, calcium signaling pathways, Wnt signaling pathway, growth hormone synthesis, secretion and action, insulin secretion, ErbB signaling pathway and Rap1 signaling pathways, etc. Conclusion KDM5A regulates the transcription expression of genes and the expression of signal pathways, thereby affecting the function of cardiac fibroblasts and participating in the onset and development of myocardial fibrosis.
- KDM5A /
- Cardiac fibroblasts /
- Chromatin immunoprecipitation sequence /
- Bioinformatics analysis

HTML全文

图 1 KDM5A蛋白结合基因交集GO功能注释

Figure 1. GO functional annotation for intersection of KDM5A target gene

下载: 全尺寸图片幻灯片

图 2 KDM5A蛋白结合基因交集KEGG Pathway分析

Figure 2. KEGG pathway analysis for intersection of KDM5A target genes

下载: 全尺寸图片幻灯片

表 1 KDM5A蛋白结合motif序列特征分析

Table 1. Characteristic analysis of KDM5A protein binding motif sequence

结合motif	最佳匹配转录因子	P值	背景序列占比(%)	模体序列数占KDM5A 结合序列数比例(%)
1	FOXE1	1× 10^-246	28.02	41.68
2	PB	1× 10^-208	12.38	22.05
3	ZNF460	1× 10^-191	12.90	22.28
4	NFIC	1× 10^-184	22.87	34.00
5	PITX2	1× 10^-166	19.47	29.49
6	TFAP2A	1× 10^-165	8.91	16.44
7	RXRG	1× 10^-125	15.04	22.95
8	YY1	1× 10^-122	20.48	29.14
9	VDR	1× 10^-120	7.73	13.70

下载: 导出CSV

参考文献(20)

[1]	巫辰, 王耀晟. 心外膜脂肪组织与心房颤动发生的关联机制及研究进展[J]. 上海医学, 2022, 45(6): 428-433. https://www.cnki.com.cn/Article/CJFDTOTAL-SHYX202206010.htm WU C, WANG Y S. Mechanism and research progress of the association between epicardial adipose tissue and atrial fibrillation[J]. Shanghai Med. J, 2022, 45(6): 428-433. https://www.cnki.com.cn/Article/CJFDTOTAL-SHYX202206010.htm
[2]	陈智慧, 田海萍, 刘艳阳. 心肌纤维化相关通路的研究进展[J]. 医学综述, 2022, 28(2): 259-264. https://www.cnki.com.cn/Article/CJFDTOTAL-YXZS202202009.htm CHEN Z H, TIAN H P, LIU Y Y. Research progress of myocardial fibrosis related pathways[J]. Med Recapitulate, 2022, 28(2): 259-264. https://www.cnki.com.cn/Article/CJFDTOTAL-YXZS202202009.htm
[3]	雷洋, 张彤瑜, 郭志华, 等. 益心泰醇提取物对Ang Ⅱ诱导的心肌成纤维细胞增殖与胶原合成的影响[J]. 中药药理与临床, 2021, 37(6): 124-128. https://www.cnki.com.cn/Article/CJFDTOTAL-ZYYL202106023.htm LEI Y, ZHANG T Y, GUO Z H, et al. Effects of alcohol extract of Yixintai on the proliferation and collagen synthesis of cardiac fibroblasts induced by Ang Ⅱ[J]. Pharmacol Clin Chin Mater Med, 2021, 37(6): 124-128. https://www.cnki.com.cn/Article/CJFDTOTAL-ZYYL202106023.htm
[4]	洪雪冬, 郑穗生, 许玲, 等. 心脏磁共振对原发扩张性心肌病左心整体功能和心肌纤维化的应用研究[J]. 中华全科医学, 2019, 17(4): 633-636, 665. doi: 10.16766/j.cnki.issn.1674-4152.000754 HONG X D, ZHENG S S, XU L, et al. Evaluation of left ventricular global function and myocardial fibrosis in patients with primary dilated cardiomyopathy by cardiac magnetic resonance imaging[J]. Chinese Journal of General Practice, 2019, 17(4): 633-636, 665. doi: 10.16766/j.cnki.issn.1674-4152.000754
[5]	KIRTANA R, MANNA S, PATRA S K. Molecular mechanisms of KDM5A in cellular functions: facets during development and disease[J]. Exp Cell Res, 2020, 396(2): 112314. DOI: 10.1016/j.yexcr.2020.112314.
[6]	汪黎明, 冯同富, 陈竹, 等. 沉默KDM5A基因对卵巢癌顺铂耐药SKOV3/DDP细胞迁移及侵袭的影响[J]. 中华肿瘤防治杂志, 2021, 28(12): 903-908. https://www.cnki.com.cn/Article/CJFDTOTAL-QLZL202112002.htm WANG L M, FENG T F, CHEN Z, et al. Silencing KDM5Agene inhibits the migration and invasion of ovarian cancer[J]. Chin J Cancer Prev Treat, 2021, 28(12): 903-908. https://www.cnki.com.cn/Article/CJFDTOTAL-QLZL202112002.htm
[7]	冯同富, 李力. 组蛋白赖氨酸去甲基化酶KDM5A在肿瘤中的研究进展[J]. 肿瘤, 2019, 39(11): 942-948. https://www.cnki.com.cn/Article/CJFDTOTAL-ZZLL201911009.htm FENG T F, LI L. Research progress of histone lysine demethylase KDM5A in tumors[J]. Tumor, 2019, 39(11): 942-948. https://www.cnki.com.cn/Article/CJFDTOTAL-ZZLL201911009.htm
[8]	任芳, 韩品, 李彩瑜, 等. MiR-421靶向调控KDM5A基因对卵巢癌细胞增殖和侵袭能力的影响[J]. 郑州大学学报(医学版), 2021, 56(5): 628-632. https://www.cnki.com.cn/Article/CJFDTOTAL-HNYK202105008.htm REN F, HAN P, LI C Y, et al. Effect of Mir-421 on proliferation and invasion of ovarian cancer cells by targeting KDM5A gene[J]. J Zhengzhou Univ Med Sci, 2021, 56(5): 628-632. https://www.cnki.com.cn/Article/CJFDTOTAL-HNYK202105008.htm
[9]	冯同富, 刘玉兰, 王波, 等. 沉默KDM5A基因对卵巢上皮癌顺铂耐药细胞SKOV3/DDP耐药性的影响[J]. 现代妇产科进展, 2020, 29(12): 886-891. https://www.cnki.com.cn/Article/CJFDTOTAL-XDFC202012002.htm FENG T F, LIU Y L, WANG B, et al. Effect of silencing KDM5A gene on cisplatin resistance of ovarian cancer cell line SKOV3/DDP[J]. Current Adv Obstetr Gynecol, 2020, 29(12): 886-891. https://www.cnki.com.cn/Article/CJFDTOTAL-XDFC202012002.htm
[10]	DAS S, FRISK C, ERIKSSON M J, et al. Transcriptomics of cardiac biopsies reveals differences in patients with or without diagnostic parameters for heart failure with preserved ejection fraction[J]. Sci Rep, 2019, 9(1): 3179. DOI: 10.1038/s41598-019-39445-2.
[11]	JIANG Y Y, KONG X R, XUE F L, et al. Incidence, risk factors and clinical outcomes of acute kidney injury after heart transplant ation: a retrospective single center study[J]. J Cardiothorac Surg, 2020, 15(1): 302.
[12]	DING Z, KE R, ZHANG Y, et al. FOXE1 inhibits cell proliferation, migration and invasion of papillary thyroid cancer by regulating PDGFA[J]. Mol Cell Endocrinol, 2019, 493: 110420. DOI: 10.1016/j.mce.2019.03.010.
[13]	HAO T, XU J, FANG S, et al. Overexpression of ZNF460 predicts worse survival and promotes metastasis through JAK2/STAT3 signaling pathway in patient with colon cancer[J]. J Cancer, 2021, 12(11): 3198-3208.
[14]	LONGBOTHAM J E, CHIO C M, DHARMARAJAN V, et al. Histone H3 binding to the PHD1 domain of histone demethylase KDM5A enables active site remodeling[J]. Nat Commun, 2019, 10(1): 94.
[15]	CUI J, QUAN M, XIE D, et al. A novel KDM5A/MPC-1 signaling pathway promotes pancreatic cancer progression via redirecting mitochondrial pyruvate metabolism[J]. Oncogene, 2020, 39(5): 1140-1151.
[16]	ZHANG Y, GUO Y, GOUGH S M, et al. Mechanistic insights into chromatin targeting by leukemic NUP98-PHF23 fusion[J]. Nat Commun, 2020, 11(1): 3339.
[17]	YANG G J, ZHU M H, LU X J, et al. The emerging role of KDM5A in human cancer[J]. J Hematol Oncol, 2021, 14(1): 30.
[18]	袁航, 褚婉飞, 徐强. 心脏交感神经对自发性高血压大鼠心脏的影响研究[J]. 中华全科医学, 2022, 20(5): 752-755. doi: 10.16766/j.cnki.issn.1674-4152.002446 YUAN H, CHU W F, XU Q. The effect of cardiac sympathetic nerve on the heart of spontaneously hypertensive rats[J]. Chinese Journal of General Practice, 2022, 20(5): 752-755. doi: 10.16766/j.cnki.issn.1674-4152.002446
[19]	刘俊雄, 王毅. 丹参素与小鼠心脏中的内质网Ca²⁺渗漏通道SEC61α相互调节心肌梗死后纤维化的作用[J]. 河北医药, 2021, 43(12): 1844-1847. https://www.cnki.com.cn/Article/CJFDTOTAL-HBYZ202112020.htm LIU J X, WANG Y. Interaction between Danshansu and endoplasmic reticulum Ca~(2+) leakage channel Sec61α in mouse heart to regulate fibrosis after myocardial infarction[J]. Hebei Med J, 2021, 43(12): 1844-1847. https://www.cnki.com.cn/Article/CJFDTOTAL-HBYZ202112020.htm
[20]	彭霞, 夏悦, 杨艺娇, 等. 成纤维细胞生长因子2在纤维化疾病中表达的研究进展[J]. 中国医药, 2022, 17(2): 305-307. https://www.cnki.com.cn/Article/CJFDTOTAL-ZGYG202202035.htm PENG X, XIA Y, YANG Y J, et al. Research progress on the expression of fibroblast growth factor 2 in fibrotic diseases[J]. Chinese Journal of Medicine, 2022, 17(2): 305-307. https://www.cnki.com.cn/Article/CJFDTOTAL-ZGYG202202035.htm