金路恒, 杨培琰, 赵阿会, 田志鹏, 姚武, 翟若南, 郝长付. 转化生长因子-β1致肺成纤维细胞转分化过程中差异表达基因的筛选和验证[J]. 环境与职业医学, 2020, 37(3): 211-217. DOI: 10.13213/j.cnki.jeom.2020.19589
引用本文: 金路恒, 杨培琰, 赵阿会, 田志鹏, 姚武, 翟若南, 郝长付. 转化生长因子-β1致肺成纤维细胞转分化过程中差异表达基因的筛选和验证[J]. 环境与职业医学, 2020, 37(3): 211-217. DOI: 10.13213/j.cnki.jeom.2020.19589
JIN Lu-heng, YANG Pei-yan, ZHAO A-hui, TIAN Zhi-peng, YAO Wu, ZHAI Ruo-nan, HAO Chang-fu. Screening and identification of differentially expressed genes in transforming growth factor-β1-induced trans-differentiation of pulmonary fibroblasts[J]. Journal of Environmental and Occupational Medicine, 2020, 37(3): 211-217. DOI: 10.13213/j.cnki.jeom.2020.19589
Citation: JIN Lu-heng, YANG Pei-yan, ZHAO A-hui, TIAN Zhi-peng, YAO Wu, ZHAI Ruo-nan, HAO Chang-fu. Screening and identification of differentially expressed genes in transforming growth factor-β1-induced trans-differentiation of pulmonary fibroblasts[J]. Journal of Environmental and Occupational Medicine, 2020, 37(3): 211-217. DOI: 10.13213/j.cnki.jeom.2020.19589

转化生长因子-β1致肺成纤维细胞转分化过程中差异表达基因的筛选和验证

Screening and identification of differentially expressed genes in transforming growth factor-β1-induced trans-differentiation of pulmonary fibroblasts

  • 摘要: 背景

    肺成纤维细胞向肌成纤维细胞转分化是矽肺由炎症期进入纤维化期的标志性细胞事件,转化生长因子-β1(TGF-β1)是此环节中最重要的细胞因子。近年来,随着高通量测序和芯片技术的发展,越来越多TGF-β1刺激成纤维细胞转分化的数据被人们所关注。

    目的

    利用生物信息学相关方法探索TGF-β1刺激的人肺成纤维细胞转分化过程中差异共表达基因,筛选与肺成纤维细胞转分化密切相关的转录因子。

    方法

    从基因表达综合数据库GEO检索并下载TGF-β1与肺成纤维细胞相关的高通量数据集GSE17518、GSE97829和GSE119007,对数据集进行差异分析,筛选三组数据集共同上调和共同下调的差异表达基因;对筛选出的差异基因进行基因本体论(GO)和京都基因与基因组百科全书(KEGG)信号通路富集分析;通过HumanTFDB数据库,下载所有人转录因子基因名称和序列,并与上述获得的共表达差异基因进行比对,得到共表达差异转录因子;随后用TGF-β1刺激人胚肺成纤维细胞MRC-5构建转分化模型,对筛选出的差异转录因子进行初步验证。

    结果

    基于这三个数据集,共得到145个上调差异表达基因,88个下调差异表达基因。GO分析结果显示:上述共表达差异基因主要参与细胞外基质、肌动蛋白细胞骨架、内质网腔等组分的构成;并且与肌动蛋白结合、生长因子活性、细胞外基质结构性成分、胶原结合、TGF-β1受体结合等生物学过程相关;此外还与细胞外基质组织、细胞迁移和凋亡,以及丝裂原活化蛋白激酶(MAPK)通路活化、MAPK激活、肽基酪氨酸磷酸化的正调节等有关。通过KEGG信号通路富集分析,筛选出的主要相关信号通路包括TGF-β1信号通路、细胞外基质-受体相互作用、黏着斑和磷脂酰肌醇3激酶-蛋白激酶B(PI3K-Akt)信号通路等。将上述得到的差异基因与人转录因子序列进行比对,得到了7个上调的转录因子和11个下调的转录因子,从中挑选的上调转录因子早期生长应答因子2(EGR2)、SNAIL家族转录抑制因子1(SNAIL1)和下调转录因子胸腺选择相关高迁移转录因子(TOX)、过氧化物酶体增殖物激活受体γ(PPARG)在TGF-β1刺激MRC-5细胞中的qRT-PCR验证结果显示,EGR2和SNAIL1均高表达,而TOX和PPARG均低表达。

    结论

    筛选出18个与肺成纤维细胞转分化相关的转录因子基因,其中在TGF-β1刺激MRC-5细胞中EGR2和SNAIL1表达升高,TOX和PPARG表达降低。

     

    Abstract: Background

    Pulmonary fibroblast to myofibroblast trans-differentiation is a critical symbol of silicosis progression from inflammation to fibrosis, among which transforming growth factorbeta 1 (TGF-β1) is the most important cytokine. In recent years, with the development of highthroughput sequencing and microarray, more and more TGF-β1-induced fibroblast trans-differentiation data have been concerned.

    Objective

    The current study is designed to explore and screen differentially expressed genes (DEGs) and hub transcription factors (TFs) in trans-differentiation of pulmonary fibroblasts induced by TGF-β1 through bioinformatics strategies.

    Methods

    GSE17518, GSE97829, and GSE119007 datasets linking TGF-β1 and pulmonary fibroblasts were downloaded from Gene Expression Omnibus (GEO) database to screen upregulated and downregulated DEGs. Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed to the selected DEGs. All obtained DEGs sequences were blast with the TFs sequences downloaded from HumanTFDB database. The selected genes of TFs were validated in TGF-β1 stimulated MRC-5 cells.

    Results

    Altogether 145 upregulated DEGs and 88 downregulated DEGs were gained based on the three datasets. The results of GO functional enrichment analysis were mostly enriched in extracellular matrix organization, actin cytoskeleton, endoplasmic reticulum cavity, and other components; they were related to biological processes such as actin binding, growth factor activity, extracellular matrix structural components, collagen binding, and TGF-β1 receptor binding; in addition, they were also related to extracellular matrix tissue, cell migration and apoptosis, mitogen-activated protein kinase (MAPK) pathway activation, MAPK activation, and positive regulation of peptide tyrosine phosphorylation. The results of KEGG pathway enrichment analysis were significantly enriched in TGF-β1 signaling pathway, extracellular matrix-receptor interaction, focal adhesion, and phosphoinositide 3-kinase-protein kinase B (PI3K-Akt) signaling pathway, etc. After comparing with the sequences downloaded from HumanTFDB, 7 upregulated TFs and 11 downregulated TFs were obtained. Early growth response 2 (EGR2), SNAIL family transcriptional repressor 1 (SNAIL1), thymocyte selection-associated high mobility group box protein (TOX), and peroxisome proliferators-activated receptor gamma (PPARG) were validated by qRT-PCR in TGF-β1 stimulated MRC-5 cells, and the results showed that EGR2 and SNAIL1 were upregulated, and TOX and PPARG were downregulated.

    Conclusion

    Among the 18 screened genes of TFs related to pulmonary fibrosis trans-differentiation, EGR2 and SNAIL1 are upregulated, and TOX and PPARG are downregulated in TGF-β1 stimulated MRC-5 cells.

     

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