非洲猪瘟病毒对猪肺泡巨噬细胞mRNAs和microRNAs表达模式的影响

doi:10.7668/hbnxb.20193131

摘要/Abstract

摘要：

旨在探究非洲猪瘟(ASFV)病毒感染猪肺泡巨噬细胞后在体内的作用方式。以感染ASFV的猪肺泡巨噬细胞(PAMs)的mRNAs和microRNAs测序数据为基础,利用生物信息学软件对数据进行处理,得到了一批差异表达基因;利用基因功能富集和对应蛋白互作解析生物学通路;再利用mRNAs和microRNAs的互作方式确认重要的靶标关系。结果表明:ASFV改变了PAMs中1 181个基因的表达量;主要调控宿主在能量代谢过程(如脂质分解、有机羟基化合物代谢、碳水化合物分解代谢、一元羧酸代谢),以及细胞周期过程(如有丝分裂细胞周期过程、细胞周期相变、细胞周期的正向调控);ASFV还引起了PAMs中25个microRNAs的差异表达,包括病毒相关的miR-27b-3p、miR-193a-3p、miR-132等;而miR-27b-3p作为表达丰度最高的差异microRNA,它与一些重要的差异基因(如PLK2、HSP90AA1等)存在着靶标关系,进而调控机体免疫和细胞周期相关通路。综上所述,ASFV感染PAMs后的作用方式包括破坏宿主在能量代谢和细胞周期方面的稳态,以及打破了机体microRNAs调节平衡;其中miR-27b-3p等小RNAs可以作为ASFV研究的靶向分子。

关键词: 猪, ASFV, 猪肺泡巨噬细胞, mRNAs, microRNAs

Abstract:

The aim of this study was to investigate the effect of African swine fever virus(ASFV) after infecting swine alveolar macrophages in vivo. Based on the mRNAs and microRNAs sequencing data of pig alveolar macrophages(PAMs)infected with ASFV,using bioinformatics software to process the data to obtain a number of differentially expressed genes;using gene function enrichment and corresponding protein interaction to analyze biological pathways;and using mRNAs and microRNAs interaction to confirm the important target relationships. The results showed that ASFV changed the expression level of 1 181 genes in PAMs;mainly regulate the host in the energy metabolism processes(eg.,lipid decomposition,organic hydroxy compound metabolism,carbohydrate catabolism,monocarboxylic acid metabolism),and also in the cell-cycle process(eg.,mitotic cell cycle process,cell cycle phase transition,cell cycle positive regulation);ASFV also elicited the differential expression of 25 microRNAs in PAMs,including the virus-related miR-27b-3p,miR-193a-3p,and miR-132,etc.;however,miR-27b-3p,as the differential microRNA with the highest expression abundance,had a target relationship with some important differential genes(eg.,PLK2,HSP90AA1,etc.),and regulated the body's immunity and cell cycle-related pathways. In conclusion,this study showed that the mode of action after ASFV infection with PAMs included destroying the host homeostasis through energy metabolism and cell cycle,and breaking the microRNAs regulation balance of the body;and small RNAs such as miR-27b-3p could be used as target molecules for ASFV research.

Key words: Swine, ASFV, Porcine alveolar macrophages, mRNAs, microRNAs

张廷焕, 柴捷, 陈力, 龙熙, 郭宗义. 非洲猪瘟病毒对猪肺泡巨噬细胞mRNAs和microRNAs表达模式的影响[J]. 华北农学报, 2022, 37(S1): 348-357. doi: 10.7668/hbnxb.20193131.

ZHANG Tinghuan, CHAI Jie, CHEN Li, LONG Xi, GUO Zongyi. Effects of African swine fever virus on mRNAs and microRNAs Expression Patterns of Porcine Alveolar Macrophages[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(S1): 348-357. doi: 10.7668/hbnxb.20193131.

http://www.hbnxb.net/CN/Y2022/V37/IS1/348

图/表 9

图1 基因表达差异分析 A.PCA分析;B.试验组ASFV_24 h和对照组Mock_24 h的差异分析;C.试验组ASFV_24 h和对照组ASFV_0 h的差异分析;D.2组差异基因集的交集。

Fig.1 Difference analysis of genes expression A.PCA analysis;B.Difference analysis of ASFV_24 h group and Mock_24 h group;C.Difference analysis of ASFV_24 h group and ASFV_0 h group;D.The overlaps of two sets of differential genes.

图2 基因表达量的变化 A-C.LPL、FABP4、FABP5的表达量;D-F.KLF4、FOSB、CCL4的表达量。不同分组之间的显著性用大写字母表示,相同字母表示表达量不显著(P>0.05),不同字母表示表达量极显著(P<0.01)。图9同。

Fig.2 Changes of genes expression A-C. The expression levels of LPL, FABP4 and FABP5, respectively; D-F. The expression levels of KLF4, FosB and CCl4, respectively. The significance between different groups is indicated by capital letters. The same letter indicates that the expression amount is not significant (P>0.05), and the different letters indicate that the expression amount is very significant (P< 0.01).The same as Fig.9.

图3 差异表达基因功能富集分析 A.ASFV_上调基因富集的通路;B. ASFV_下调基因富集的通路。

Fig.3 Functional enrichment analysis of differential genes A.ASFV_ Up genes enrichment pathways;B. ASFV_ Down gene enrichment pathways.

图4 ASFV_上调基因相关蛋白互作分析 A-B. 所有蛋白互作网络进行聚类和功能富集分析;C-D. MCODE1的蛋白互作和功能富集分析;E~F. MCODE2的蛋白互作和功能富集分析。图5同。

Fig. 4 The protein-protein interaction analysis of ASFV_Up genes A-B.The interaction networks and functional enrichment analysis of ASFV_ up genes related proteins;C-D.The interaction networks and functional enrichment analysis of MCODE1;E-F.The interaction networks and functional enrichment analysis of MCODE2.The same as Fig.5.

图5 ASFV_下调基因相关蛋白互作分析

Fig.5 The protein-protein interaction analysis of ASFV_Down genes

图6 microRNAs表达差异分析 A.PCA分析;B. 试验组ASFV_24 h和对照组ASFV_0 h的差异分析;C. 试验组ASFV_24 h和对照组Mock_24 h的差异分析;D. 2组差异micoRNAs集的交集;E. 差异micoRNAs表达量热图。

Fig.6 Difference analysis of microRNAs expression A.PCA analysis;B. Difference analysis of ASFV_24 h group and ASFV_0 h group;C. Difference analysis of ASFV_24 h group and Mock_24 h group;D. The overlaps of two sets of differential micoRNAs;E. The heat map of differential microRNAs expression.

图7 差异microRNAs的表达丰度和靶基因预测 A.ASFV_上调的microRNAs;B.ASFV_下调的microRNAs。圆点的颜色代表microRNAs的平均表达丰度,其中红色代表RPM>10⁴,橙色代表RPM>100,灰色代表RPM<10,黑色代表RPM<1;圆点的大小代表靶基因的个数,圆点越大靶基因越多;不同microRNAs之间连接线的粗细代表共有靶基因的数量,线条越粗共有靶基因越多。

Fig.7 Expression abundance and targets prediction of differential microRNAs A. ASFV_ Up microRNAs;B. ASFV_Down microRNAs. The color of dots represent the average expression abundance of microRNAs,in which red represents RPM>10⁴,orange represents RPM>100,gray represents RPM <10 and black represents RPM<1;The size of the dots represent the number of targets. The larger the dot,the more targets;The thickness of the line between different microRNAs represents the number of shared targets. The thicker the line,the more shared targets.

图8 miR-27b-3p成熟体的保守性以及靶基因功能富集 A.miR-27b-3p成熟体在不同物种间序列保守性分析;B-C. miRanda和Targetscan预测miR-27b-3p靶基因的交集;D. miR-27b-3p的靶基因的功能富集分析。

Fig.8 Conservation of miR-27b-3p and Enrichment function of its targets A.Sequence conservation analysis of miR-27b-3p among different species;B-C. The overlaps of miR-27b-3p targets by miRanda and Targetscan;D. Functional enrichment analysis of miR-27b-3p targets.

图9 miR-27b-3p靶位点的保守性以及靶基因的表达量变化 A.miR-27b-3p与PLK2基因3'UTR区结合位点在不同物种间保守性;B.PLK2基因的表达量变化;C.miR-27b-3p与HSP90AA1基因3'UTR区结合位点在不同物种间保守性;D.HSP90AA1基因的表达量变化。

Fig.9 Conservation of miR-27b-3p target sites and the expression change of targets A.The binding sites of miR-27b-3p and 3'UTR region of PLK2 gene were conserved among different species;B.Changes in the expression of PLK2 gene;C.The binding sites of miR-27b-3p and 3'UTR region of HSP90AA1 gene were conserved among different species;D.Changes in the expression of HSP90AA1 gene.

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