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主管单位 工业和信息化部 主办单位 哈尔滨工业大学 主编 任南琪 国际刊号ISSN 1672-5565 国内刊号CN 23-1513/Q

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引用本文:伊梦杰,张锦锦,郭强,唐慧.联合应用RNA-seq和ATAC-seq寻找FOXQ1转录因子的下游靶基因[J].生物信息学,2019,17(4):227-236.
YI Mengjie,ZHANG Jinjin,GUO Qiang,TANG Hui.Combining ATAC-seq and RNA-seq to find downstream target genes of FOXQ1 transcription factor[J].Chinese Journal of Bioinformatics,2019,17(4):227-236.
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联合应用RNA-seq和ATAC-seq寻找FOXQ1转录因子的下游靶基因
伊梦杰1,2,张锦锦3,郭强2,唐慧2
(1.昆明理工大学 医学院, 昆明 650504;2.云南省第一人民医院,昆明理工大学附属医院,云南省消化内镜临床医学中心,云南省临床病毒学重点实验室,昆明市肿瘤分子与免疫防治重点实验室,昆明 650032;3.中国科学院昆明动物研究所,昆明 650223)
摘要:
结直肠癌(Colorectal cancer, CRC)是一种全球高发的恶性肿瘤,发病原因复杂且预后较差。近年来发现叉头框Q1(Forkhead box Q1, FOXQ1)基因作为一类核转录因子在结直肠癌中高表达,可控制下游基因转录活性。本实验拟探究CRC细胞中FOXQ1的转录调控功能并寻找其下游基因。方法:(1)构建低表达FOXQ1基因的稳定转染CRC细胞株;(2)应用RNA-seq检测FOXQ1敲低前后表达量显著差异的基因;(3)应用转座酶可接近性核染色质区域测序分析(Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq)检测FOXQ1敲低前后细胞染色质易接近性的变化;(4)进一步对FOXQ1敲低前后的RNA-seq和ATAC-seq数据进行一系列生物信息学分析,寻找CRC中FOXQ1转录调控的潜在下游基因。结果:应用RNA-seq筛选出了敲低FOXQ1后表达显著差异的基因EI24、TLR2、SMAD3,通过联合分析两细胞系的测序结果,发现FOXQ1基因敲低后,在DLD1和SW480两个细胞系中染色质易接近性均增强且表达量均上调的基因有61个,染色质易接近性均减弱且表达量均下调的基因有70个,且EI24、TLR2、SMAD3基因均位于重叠分析结果中,其中TLR2、SMAD3基因的染色质区域有明显变化,而EI24基因的染色质区域变化不明显。通过代谢通路分析找到了EI24、TLR2、SMAD3基因所富集的代谢通路。其中SMAD3、TLR2基因在炎症性肠病(Inflammatory bowel disease , IBD)通路中显著富集。EI24基因在p53信号通路(p53 signaling pathway)通路中显著富集。结论:基于染色质易接近性的变化和转录水平的研究发现:敲低FOXQ1基因对CRC细胞系中染色质的开放情况有较大的影响,且影响FOXQ1转录调控的下游基因的表达。找到了FOXQ1敲低后在SW480、DLD1中均发生变化的基因,为丰富FOXQ1转录因子的下游调控网络提供了研究基础。
关键词:  ATAC-seq  RNA-seq  结直肠癌  靶基因
DOI:10.12113/j.issn.1672-5565.201906003
分类号:R735.3
文献标识码:A
基金项目:国家自然科学基金项目(No.6,2);云南省卫生和计划生育委员会医学学科带头人培养基金(No.D-201642);云南省临床病毒学重点实验室基金(No.2018DG010),昆明市肿瘤分子与免疫防治重点实验室基金(No.2018-1-A-17334).
Combining ATAC-seq and RNA-seq to find downstream target genes of FOXQ1 transcription factor
YI Mengjie1,2, ZHANG Jinjin3, GUO Qiang2, TANG Hui2
(1. Medical School, Kunming University of Science and Technology, Kunming 650504, China; 2. Kunming Key Laboratory of CancerMolecular and Immunological Control, Yunnan Provincial Key Laboratory of Virology, Institute of Basic Medical Sciences, Affiliated Hospital of Kunming University of Science and Technology , First Peoples Hospital of Yunnan Province, Kunming 650032, China; 3. Kunming Institute of Zoology. CAS, Kunming 650223, China)
Abstract:
Colorectal cancer (CRC) is a kind of malignant tumor with high incidence in the world, which has complicated causes and poor prognosis. In recent years, Forkhead box Q1 (FOXQ1) gene has been found to be highly expressed in CRC as a nuclear transcription factor, which can control the transcriptional activity of downstream genes. This study aims aims to explore the transcriptional regulation function of FOXQ1in CRC cells and search for its downstream genes. Methods of the study include 1)constructing constructing stable transfected CRC cell lines with knockdown FOXQ1gene, 2) applying RNA-seq to detect genes with significant differences in expression level before and after FOXQ1, 3) assaying for the Transposase-Accessible Chromatin using sequencing (ATAC-seq) for the determination of Chromatin accessibility in cells before and after FOXQ1knockdown; and 4) conducting a series of bioinformatics analysis on the RNA-seq and ATAC-seq data before and after FOXQ1knockdown to search for potential downstream genes of FOXQ1in CRC. EI24, TLR2,and SMAD3genes were picked out using RNA-seq, which expressed significant difference. Through joint analysis of two cell lines sequencing results, we found that after knocking down FOXQ1,there there were 61 genes with enhanced chromatin accessibility and up-regulation in both cell lines of DLD1 and SW480, and 70 genes with reduced chromatin accessibility and down-regulated expression.EI24, TLR2,and SMAD3genes were located in the overlapping analysis. The chromatin region of TLR2and SMAD3genes showed significant changes, while that of EI24gene did not change significantly. Metabolic pathways enriched by EI24, TLR2,and SMAD3 geneswere found by metabolic pathway analysis. SMAD3and TLR2genes were significantly enriched in the Inflammatory Bowel Disease (IBD) pathway, and EI24gene was significantly enriched in the p53 signaling pathway. Based on the changes in chromatin accessibility and transcriptional levels, it was found that knocking down the FOXQ1gene had a great effect on the chromatin opening in CRC cell lines and it influenced the expression of downstream genes for FOXQ1transcriptional regulation. Genes that changed in SW480 and DLD1 after FOXQ1knockdown were found, which provides a research basis for enriching the downstream regulatory network of FOXQ1 transcription factors.
Key words:  ATAC-seq  RNA-seq  Colorectal cancer  Target genes

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