罗伊氏乳杆菌对仔猪空肠miRNAs表达谱的影响

Effect of Lactobacillus reuteri on the Expression Profile of miRNAs in the Jejunum of Piglets

试验旨在研究罗伊氏乳杆菌对仔猪空肠miRNAs表达的影响。选用1日龄健康约克×荣昌仔猪6窝,随机分为两组,每组3窝(共27头)。即对照组(每天每头灌喂0.1%的无菌蛋白胨溶液),LR组(每天每头灌喂活菌总数为1.0×10^10CFU的罗伊氏乳杆菌),分别在10和20日龄,每组选取6头进行屠宰,采集空肠样品。通过Solexa高通量测序技术检测空肠miRNA的表达谱,利用生物信息学技术进行靶基因预测和功能分析。结果显示:1)试验构建了8个文库,共获得187103840条纯净reads序列,其中22 nt长度序列占的比例最大;2)10和20日龄分别鉴定出354和352个已知的miRNAs,其中有329个miRNAs共表达;3)灌喂罗伊氏乳杆菌组仔猪空肠miRNA差异表达,10和20日龄分别获得7和9个差异显著的miRNAs,ssc-miR-218在两个日龄中都差异表达;4)对差异表达的miRNAs进行靶基因预测和KEGG通路分析,发现有10221个靶基因富集到293条通路上,有66条通路显著富集(P<0.05),包括与肠道发育及免疫调控相关的MAPK和PI3K-Akt信号通路;5)随机挑选6个差异表达miRNAs进行荧光定量PCR验证,验证结果与测序结果基本一致。本研究表明,罗伊氏乳杆菌可能通过影响空肠miRNAs表达来调控有关肠道健康的信号通路。

This study was conducted to investigate the effect of Lactobacillus reuteri on the expression of miRNAs in the jejunum of piglets.Six litters of healthy 1-day-old York×Rongchang piglets were selected and randomly divided into the two group,3 litters per group(n=27).The two groups were control group(daily orally adminitrated with 0.1%sterile peptone solution per piglet),and the LR group(daily orally administrated with 1.0×10^10 CFU of L.reuteri per piglet),respectively.At 10 and 20 days of age,six piglets per group were selected for slaughter,respectively,and the jejunum samples were collected.After RNA extraction,the library was constructed and the miRNA expression profile of the jejunum was detected by Solexa high-throughput sequencing technology.Bioinformatics technology was used to conduct target genes prediction and functional analysis.The results showed that:1)A total of 8 libraries were constructed and a total of 187103840 clean reads were obtained,of which the 22 nt length sequence accounted for the largest proportion;2)Three hundred and fifty-four and 352 known miRNAs were respectively identified at 10 and 20 days of age,of which 329 miRNAs were co-expressed;3)Differentially expressed(DE)miRNAs in the jejunum were found after L.reuteri administration,7 and 9 DE miRNAs were obtained at 10 and 20 days of age,respectively.Notably,ssc-miR-218 was expressed differentially at two ages.4)KEGG pathway analysis and target gene prediction on the differentially expressed miRNAs revealed that 10221 target genes were enriched in 293 biological pathways,of which 66 were significantly enriched(P<0.05),including the MAPK signaling pathway and PI3K-Akt signaling pathway,which were known to regulate intestinal development and immune.5)Six miRNAs were selected to validate the sequencing results by qRT-PCR,the qRT-PCR expression results corresponded well with those from the sequencing.The results indicates that L.reuteri may regulate intestinal health related pathways by influencing the expression of jejunal miRNAs.