感染产肠毒素大肠杆菌的猪小肠上皮细胞microRNA表达谱分析

doi:10.16431/j.cnki.1671-7236.2021.11.034

Abstract

Abstract: This study was aimed to explore the changes of microRNA (miRNA) expression profile in porcine small intestine epithelial cells (IPEC-J2) infected with enterotoxigenic Escherichia coli (ETEC), and provide a theoretical basis for the analysis of the regulatory role of host miRNA in the process of ETEC infection. IPEC-J2 pre- and post-infection of ETEC were sequenced by Illumina 6000 Novoseq SE50 sequencing platform, Bowtie was used to compare it with reference genome, and DESeq R Package was used to analysis the miRNA differential expression. The target genes of the differentially expressed miRNA were predicted jointly by miRanda and RNAhybid, and the GO and KEGG analysis of the target genes were performed. Five miRNAs were randomly selected to verify the sequencing results by Real-time quantitative PCR. The results showed that after filtration, 12 889 260 and 11 203 056 clean reads were obtained in the sRNA libraries of IPEC-J2 before and after ETEC infection, respectively. In the pre-infection and post-infection libraries, miRNA accounted for the highest proportion, which were 73.16% and 54.10%. A total of 97.98% and 69.83% of sRNA with length of 18-40 nt matched to the reference genome, indicating the sequencing quality was well controlled. Most of the first bases of the 22-24 nt sequences were inclined to U, and AGCUUAU was the most frequent base at 2-8 sites. A total of 311 known miRNAs and 128 novel miRNAs were discovered. The miRNA sequences of 23 nt length accounted for the highest proportion, taking up 41.42% and 23.56% in the two libraries, respectively. A total of 140 differentially expressed miRNAs were revealed in the comparison between the two libraries, among which 74 were up-regulated and 66 were down-regulated after ETEC infection. GO analysis showed that miRNA target genes were significantly enriched in metabolic processes, positive regulation of metabolic processes, cell composition or biosynthesis, immune system, intracellular parts and organelles. KEGG analysis showed that the differentially expressed miRNA target genes were significantly enriched in lysine degradation, IgA-producing intestinal immune network, NF-κB signaling pathway and T cell receptor signaling pathway. Verification by Real-time quantitative PCR showed that the expression trends of 5 randomly selected miRNAs were consistent with the sequencing results, indicating that the sequencing was accurate and reliable. In conclusion, the miRNAs in IPEC-J2 were involved in the process of ETEC infection, which provided a scientific basis for further revealing the key miRNA regulating ETEC infection and its mechanism of action.

Key words: porcine small intestine epithelial cells (IPEC-J2); enterotoxigenic Escherichia coli (ETEC); miRNA; expression profile

CLC Number:

S852.23

LIU Yingying, ZHANG Di, LIU Guomei, LI Pei, LI Yan, ZUO Yuzhu. Analysis of microRNA Expression Profile in Porcine Small Intestinal Epithelial Cells Infected with Enterotoxigenic Escherichia coli[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(11): 4242-4253.

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Analysis of microRNA Expression Profile in Porcine Small Intestinal Epithelial Cells Infected with Enterotoxigenic Escherichia coli

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