感染产肠毒素大肠杆菌的猪小肠上皮细胞microRNA表达谱分析

试验旨在探索产肠毒素大肠杆菌（enterotoxigenic Escherichia coli,ETEC）感染猪小肠上皮细胞（IPEC-J2）诱导的microRNA （miRNA）表达谱变化,为解析宿主miRNA在ETEC感染过程中的调控作用提供理论基础。利用Illumina 6000 Novoseq SE50测序平台分别对ETEC感染前后的IPEC-J2进行高通量测序,用Bowtie与参考基因组比对,用DESeq R Package进行miRNA差异性分析。通过miRanda和RNAhybid共同预测差异表达miRNA的靶基因,对差异表达miRNA靶基因进行GO功能和KEGG通路分析。随机选取5个miRNAs,对测序结果进行实时荧光定量PCR验证。结果显示,IPEC-J2在感染前后的sRNA文库经过滤分别得到12 889 260和11 203 056条clean reads。感染前后文库中,miRNA所占比例最高,分别为73.16%和54.10%;分别有97.98%和69.83%长度为18~40 nt的sRNA可比对到参考基因组,表明测序质控良好。长度在22~24 nt的序列大部分首位碱基偏向U,2~8位点出现频率最高的碱基分别为AGCUUAU。共发现311个已知miRNAs,128个新miRNAs。在2个文库中,长度为23 nt的miRNA序列占比最高,分别为41.42%和23.56%。感染后共筛选到140个差异表达miRNAs,其中74个表达上调,66个表达下调。GO分析表明,miRNA靶基因显著富集于代谢过程、正向调节代谢过程、细胞成分或生物合成、免疫系统、细胞内部分和细胞器等功能。KEGG分析表明,差异表达miRNA靶基因显著富集于赖氨酸降解、生产IgA的肠道免疫网络、NF-κB信号通路和T细胞受体信号通路等。实时荧光定量PCR验证结果表明,随机选取的5个miRNAs表达趋势与测序结果一致,表明测序准确可靠。综上所述,IPEC-J2的miRNAs参与了ETEC感染过程,为进一步揭示调控ETEC感染的关键miRNA及其作用机制提供科学依据。

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

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.