[Bioinformatics analysis of ventilator-induced lung injury genome microarray based on gene expression omnibus database and key gene verification].
[Bioinformatics analysis of ventilator-induced lung injury genome microarray based on gene expression omnibus database and key gene verification].
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DOI：
10.3760/cma.j.cn121430-20210823-01260
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发表时间：
2022-01-01
影响因子：
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通讯作者：
Zhan, Qingyuan
中科院分区：
文献类型：
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作者：
Chen, Shengsong;Zhang, Yi;Zhan, Qingyuan
目的：通过生物信息学分析研究呼吸机所致肺损伤（VILI）小鼠的差异表达基因（DEG），并通过复制VILI小鼠模型来验证关键基因。方法：（1）实验1（生物信息学分析）：从基因表达综合（GEO）数据库中下载VILI小鼠和自主呼吸对照组的GSE9368和GSE11662芯片数据集。通过R和维恩图获得的DEG进一步用于获得共同的DEG。使用DAVID在线数据库获得基因本体（GO）和京都基因和基因组百科全书（KEGG）通路分析。最后，利用互作基因数据库检索工具(STRING)对常见DEG进行蛋白质-蛋白质相互作用(PPI)分析，并利用CytoScape软件、分子复合物检测(MCODE)分析插件和CytoHubba插件以最大聚类中心性(MCC)、最大邻域连接性(MNC)和程度筛选出关键基因。 (2)实验2(相关蛋白验证)：采用高潮气量(20mL/kg)呼吸机复制VILI小鼠模型。设立自主呼吸对照组。采用苏木精-伊红（HE）染色评估肺损伤，并通过免疫组化染色验证实验1筛选的关键基因。 结果：（1）实验1结果：从GSE9368数据集中筛选出114个DEG，其中上调基因99个，下调基因15个。从GSE11662数据集中筛选出258个DEG，其中上调基因188个，下调基因70个。此外，还获得了66个常见DEG，其中上调基因61个，下调基因5个。 GO分析显示，常见的DEG主要参与炎症反应、免疫反应、白细胞和中性粒细胞趋化作用。 KEGG分析显示，常见的DEG涉及细胞粘附、细胞因子受体相互作用和肿瘤坏死因子（TNF）信号通路。利用STRING和CytoScape分析构建基因PPI网络图和重要子模块。使用带有MCC、MNC和度算法的CytoHubba插件进行拓扑分析，然后进行交集，得到细胞因子信号抑制因子3（SOCS3）、白介素-1β（IL-1β）、基质金属蛋白酶-9（MMP-9）、整合素Itgam、CXC趋化因子配体2（CXCL2）、CXC趋化因子受体2（CXCR2）、Sell和CC等8个基因。趋化因子受体 1 (CCR1)。 (2)实验2结果：复制高潮气量VILI小鼠模型。与自主呼吸对照组相比，通气结束后0小时肺组织损伤较轻，通气结束后6小时肺组织结构明显破坏，表现为肺泡腔出血、肺泡壁厚度明显增加、塌陷，炎症细胞浸润。通过免疫组织化学染色验证了交叉和拓扑分析的前三个基因，包括IL-1beta、SOCS3和MMP-9。结果显示，随着通气时间的延长，IL-1β、SOCS3、MMP-9的表达逐渐升高，6 h时与自主呼吸对照组相比差异明显[IL-1β（积分A值）：8.40±2.67 vs. 5.10±0.94，SOCS3（积分A值）：9.74±1.80 vs. 5.95±1.31， MMP-9（积分A值）：11.45±6.20 vs. 5.36±1.28，均P < 0.05]。结论：基于GSE9368和GSE11662数据集的生物信息学分析发现，VILI主要与炎症损伤、细胞因子和免疫细胞浸润有关； IL-1beta、SOCS3和MMP-9可能是VILI的生物标志物。
OBJECTIVE: To investigate differential expression gene (DEG) in mice with ventilator-induced lung injury (VILI) by bioinformatics analysis, and to verify the key genes by reproducing the VILI mouse model.METHODS: (1) Experiment 1 (bioinformatics analysis): the microarray dataset of GSE9368 and GSE11662 regarding VILI mice and those in the spontaneous breathing control group were downloaded from the gene expression omnibus (GEO) database. DEG obtained by R and Venn map was further used to obtain common DEG. DAVID online database was used to obtain gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Finally, the protein-protein interaction (PPI) analysis of common DEG was carried out by using Search Tool for the Retrieval of Interacting Genes Database (STRING) and the key genes were screened out by using CytoScape software, molecular complex detection (MCODE) analysis plug-in and CytoHubba plug-in with maximum cluster centrality (MCC), maximum neighbor connectivity (MNC) and degree. (2) Experiment 2 (related protein verification): VILI mouse model was reproduced by high tidal volume (20 mL/kg) ventilator. Spontaneous breathing control group was set up. Hematoxylin-eosin (HE) staining was performed to assess lung injury and the key genes screened in experiment 1 were verified by immunohistochemical staining.RESULTS: (1) Experiment 1 results: a total of 114 DEG were screened from GSE9368 dataset, including 99 up-regulated genes and 15 down-regulated genes. A total of 258 DEG were screened from GSE11662 dataset, including 188 up-regulated genes and 70 down-regulated genes. Furthermore, 66 common DEG were obtained, including 61 up-regulated genes and 5 down-regulated genes. GO analysis showed that the common DEG were mainly involved in inflammatory response, immune response, leukocyte and neutrophil chemotaxis. KEGG analysis showed that the common DEG were involved cell adhesion, cytokine receptor interaction and tumor necrosis factor (TNF) signaling pathway. STRING and CytoScape analysis were used to construct gene PPI network diagram and important sub modules. And the CytoHubba plug-in with MCC, MNC and degree algorithms was used to perform topology analysis and then taken an intersection to obtain eight genes including suppressor of cytokine signaling 3 (SOCS3), interleukin-1beta (IL-1beta), matrix metalloproteinase-9 (MMP-9), integrin Itgam, CXC chemokine ligand 2 (CXCL2), CXC chemokine receptor 2 (CXCR2), Sell and CC chemokine receptor 1 (CCR1). (2) Experiment 2 results: a mouse model of high tidal volume VILI was reproduced. Compared with the spontaneous breathing control group, the lung tissue was injured slightly at 0 hour after the end of ventilation, and the lung tissue structure was significantly damaged at 6 hours after the end of ventilation, showing bleeding in alveolar cavity, significant increase and collapse of alveolar wall thickness, and infiltration of inflammatory cells. The top three genes from intersection and topological analysis including IL-1beta, SOCS3 and MMP-9 were verified by immunohistochemical staining. The results showed that the expressions of IL-1beta, SOCS3 and MMP-9 were gradually increased with time of ventilation, the differences were found at 6 hours as compared with those in the spontaneous breathing control group [IL-1beta (integral A value): 8.40±2.67 vs. 5.10±0.94, SOCS3 (integral A value): 9.74±1.80 vs. 5.95±1.31, MMP-9 (integral A value): 11.45±6.20 vs. 5.36±1.28, all P < 0.05].CONCLUSIONS: Bioinformatics analysis based on GSE9368 and GSE11662 data sets found that VILI is mainly related to inflammatory injury, cytokines and immune cell infiltration; IL-1beta, SOCS3 and MMP-9 might be biomarkers of VILI.