关键词: 广州管圆线虫, 长链非编码RNA, 转录组学, 竞争性内源RNA

Abstract:

Objective To understand the expression levels in brain tissue transcriptome of rat infected with Angiostrongylus cantonensis. Methods Thirty-two SD rats were randomly divided into control group (12 rats) and infected group (20 rats). The rats in the infected group were infected with 40 A. cantonensis stage Ⅲ larvae by gavage and in the control group with the same volume of saline. 8 rats (3 of the control group and 5 of the infected group) were randomly dissected to collect brain tissues, of which paraffin sections were prepared and stained with hematoxylin-eosin (HE) to observe the pathological changes 1, 7, 14 and 21 days after infection. Brain tissue RNA was extracted 14 days after infection for detection of differentially expressed mRNA and lncRNA by using RNA sequencing technique. Gene ontology (GO) enrichment analysis and Kyoto encyclopedia of genes and genomes (KEGG) metabolic pathway analysis of differentially expressed mRNA were performed. The STRING database was used to predict protein-protein interaction (PPI) between differentially expressed mRNA target proteins. Bioinformatics was utilized to construct the competitive endogenous RNA (ceRNA) regulatory network of differentially expressed genes. The differential expression of lncRNA was verified by qPCR. Results HE staining showed that pathological changes appeared in the rat brains of the infected group 14 days after infection with A. cantonensis, with cytoplasmic consolidation in the hippocampal neurons and parasite-like tissues could be seen at the meninges 21 days after infection. RNA sequencing result showed that the number of differentially expressed mRNAs in the rat brains was 955 (890 up-regulated and 65 down-regulated) 14 days after infection; the number of differentially expressed lncRNA was 193 (122 up-regulated and 71 down-regulated). GO enrichment analysis showed that differentially expressed mRNAs were mainly enriched in biological processes such as inflammatory response and immune response, the cellular components were mainly the extracellular space outside of the plasma membrane and the cell surface, and the molecular functions were mainly the chemokine activity and chemokine receptor binding. KEGG metabolic pathway analysis showed that the differentially expressed mRNAs were mainly involved in signaling pathways such as cytokine-cytokine receptor interactions and chemokines. PPI analysis showed that the main targets were chemokine ligand 11, RT1-Da, and serine family E member 1, all of which were associated with immune responses. ceRNA results showed that significantly enriched miRNAs such as mir-466b-3p, mir-1956, mir-207 and mir-328a-5p were associated with immune responses, apoptosis, angiogenesis and other processes. qPCR results showed that the H19 relative transcription level of rats in the infected group increased gradually, reaching a peak (15.074 ± 3.366) 21 days after infection, which was higher than rats in the control group (1.000 ± 0.113) (t = 13.190, P < 0.05). The relative transcript levels of RT1-CE6, LOC100910973 and lncR-ncf1 peaked 14 days after infection at 9.702 ± 1.408, 6.683 ± 1.299, and 7.733 ± 0.717, respectively, which were higher than rats in the control group (1.003 ± 0.039, 1.001 ± 0.156 and 0.999 ± 0.076) (t = 20.760, 13.830, 28.810, all P < 0.01). The AABR07030796.1 relative transcript level began to increase 14 days and peaked at 21 days after infection at 4.485 ± 0.236 and 5.068 ± 1.608, respectively, which were higher than rats in the control group (1.000 ± 0.159 and 1.001 ± 0.256) (t = 7.049, 8.229, both P < 0.01). The qPCR results and RNA sequencing results of H19, RT1-CE6, LOC100910973, lncR-ncf1 and AABR07030796.1 showed up-regulation in the rat brains of the infected group 14 days after infection with A. cantonensis with a consistent expression trend. Conclusion Total of 955 differentially expressed mRNA and 193 differentially expressed lncRNA were detected in the brain tissue of rats infected with A. cantonensis, which were mainly enriched in the inflammatory response, immune response and other biological processes.

Key words: Angiostrongylus cantonensis, Long non-coding RNA, Transcriptomics, Competitive endogenous RNAs