Target Gene Prediction Analysis and Partial Target Gene Verification of Pig (Sus scrofa) miR-339-3p
WANG Wei1, HUANG Xiao-Yu1, YAN Zun-Qiang1, MA Xiao-Wen3, WANG Peng-Fei1, XIE Kai-Hui1, LUO Rui-Rui1, GAO Xiao-Li1, MA Yan-Ping4, GUN Shuang-Bao1, 2, *
1 College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China;
2 Gansu Research Center for Swine Production Engineering and Technology, Lanzhou 730070, China;
3 College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China;
4 Gansu Agricultural University Library, Lanzhou 730070, China
Abstract：miRNAs (microRNAs) play a role in a variety of physiological and pathological processes.miRNAs play biological functions through the corresponding target genes.By combining bioinformatics prediction and experimental verification, miRNA target genes can be rapidly and efficiently screened.In our early study, high-throughput sequencing revealed that ssc-miR-339-3p was differentially expressed between control group and treatment group on piglets (Sus scrofa) infected with Clostridium perfringens type C, suggesting that it might have played an important role in the infection process.This study aimed to bioinformatics prediction and analysis of the target genes of ssc-miR-339-3p, and to verify the ssc-miR-339-3p and partial target genes, to explore its possibility influence and regulating mechanism of piglets diarrhea.In this study, bioinformatics databases such as miRBase, Ensemble, NCBI, miRTarBase, and bioinformatics software such as PITA, RNAhybrid, miRanda, Promoter Scan, Alibaba2.1, DAVID, Cytoscape, were used to perform the prediction of transcription factor binding site and conservative analysis among different species of its target genes, involved Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.qRT-PCR was used to verify the expressions of ssc-miR-339-3p and some target genes.The results showed that miR-339-3p was highly conserved among species, and multiple transcription factor binding sites such as Sp1, AP-1, C/EBPα, NF- κB, SRF and USF, were found in the ssc-miR-339-3p promoter region.The obtained 160 target genes are significantly enriched in positive regulation of MAP kinase activity, positive regulation of apoptosis, negative regulation of RNA polymerase Ⅱ promoter transcription, apoptosis signaling pathway and other biological processes and participated in NF-κB, PI3K-Akt, FoxO and Toll-like receptors signal pathways.qRT-PCR results showed that the expression level of ssc-miR-339-3p was significantly lower in the resistance groups (IR) and sensitive groups (IS) than in the control groups (IC) (P<0.05), this result was consistent with the high-throughput sequencing results; the expression levels of the target genes nuclear factor kappa-B 1 (NFKB1) and B-cell surface antigen CD40 (CD40) were significantly higher between IR and IS groups than the IC group (P<0.01); the expression of TNF receptor-associated factor 3 (TRAF3) and interleukin 1 receptor associated kinase 1 (IRAK1) were not significantly different among the 3 groups (P>0.05).Therefore, we assumed that ssc-miR-339-3p is regulated by various transcription factors such as Sp1, NF-κB and USF, which may activate NF-κB, PI3K-Akt, FoxO and Toll-like receptors signal pathways to regulate the piglets diarrhea through the target genes NFKB1, TRAF3, IRAK1, CD40.This study predicts and preliminarily validates the obtained target genes, which may provide experimental and theoretical basis for the function and regulating mechanism of miR-339-3p in piglet's resistance to Clostridium perfringens infection, thereby it can provide a theoretical basis for searching for the effective molecular genetic markers of resistance to Clostridium perfringens type C in piglets.
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