Abstract：Abstract Post-weaning diarrhea (PWD) is a very common infectious disease in pig (Sus scrofa) production, which has caused huge losses to the pig industry. MicroRNAs(miRNAs) play an important role in physiological and pathological processes by acting on the corresponding target genes. MiRNAs mainly perform complete or incomplete pairing with 3' UTR of target mRNA to degrade target mRNA or inhibit mRNA translation. Afterwards, the transcriptional expression of the target gene is regulated to affect the biological activity of the individual. Our previous studies have screened out miR-192 which may be important miRNAs for regulating Escherichia coli F18 infection in weaned piglets by high-throughput sequencing and verification test, and these studies have preliminarily screened out two key target genes discs large homolog 5 (DLG5) and activated leukocyte cell adhesion molecule (ALCAM) which regulate E. coli F18 infection. In this study, the dual-luciferase reporter system and Western blotting analysis were performed to explore whether DLG5 and ALCAM genes were under the specific regulation of miR-192. Recombinant plasmids which included miRNA target sites were constructed and were co-transfected with PRL-TK and miRNA-192 mimics, inhibitor or NC into 293 cells (miRNA-192 mimics and negative control). Cells were collected for detection of luciferase activity and protein levels after 24 h transfection. Then mRNA expression of two target genes in IPEC-J2 cells infected by E. coli was detected. The 3'UTR regions of 2 target genes were constructed into the dual-luciferase reporter vector. Then, this study successfully obtained luciferase reporter gene recombinant plasmids. The results of luciferase activity showed that miR-192 mimics could bind to binding sites of target genes and significantly inhibit luciferase activity of ALCAM and DLG5 3'UTR dual-luciferase reporter recombinant vector (P＜0.05), and miR-192 inhibitor could significantly enhance luciferase activity of ALCAM and DLG5 3'UTR dual-luciferase reporter recombinant vector (P＜0.05). Meanwhile, Western blotting analysis further indicated the target protein levels of miRNA-192 mimics group were significantly lower than the negative control group, which verified the preceding luciferase activity results. After being infected with three kinds of E. coli, the expression of 2 target genes significantly or extremely significantly increased. The results indicated that porcine mir-192 could inhibit the expression of DLG5 and ALCAM gene via targeting those 2 genes. MiR-192 and its targets played important regulatory function in replying the invasion of E. coli, maintaining stability of intestine and regulating immunization in weaned piglets. This study provides a theoretical and experimental evidence for participating in resistance to E. coli infection via porcine mir-192 targeting DLG5 and ALCAM gene, and lays the foundation for the further studies on the molecular mechanism of weaned piglets E. coli F18 diarrhea disease, thereby it can provide a theoretical basis for searching for the effective genetic markers of resistance to colibacillosis in pigs.
孙丽 吴森 吴嘉韵 赵呈祥 吴圣龙 包文斌. 猪miR-192对DLG5和ALCAM基因的靶向作用关系验证[J]. , 2017, 25(9): 1451-1459.
Sun Li Sen Wu Jiayun Wu Chenxiang Zhao. Validation of the Targeting Effects of Swine (Sus scrofa) miR-192 on the DLG5 and ALCAM Genes. , 2017, 25(9): 1451-1459.
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