Identification of functional single nucleotide polymorphisms in the porcine SLC6A4 gene associated with aggressive behavior in weaned pigs after mixing.

Variation in genes of the serotonergic system influences aggressive behavior by affecting serotonin levels in the central and cortical nervous system. SLC6A4 (serotonin transporter) is a master regulator of 5-HT signaling and involved in the regulation of aggressive behavior in humans and rodents. To identify potential functional single nucleotide polymorphisms (SNPs) for the porcine SLC6A4 gene associated with aggressive behavior, a total of 500 pigs (268 barrows and 232 gilts) were selected and mixed in 51 pens. Their behavior was recorded and observed for 72 h after mixing. Based on a composite aggressive score (CAS), the most aggressive and the least aggressive pigs within each pen were selected separately (a total of 204 pigs). Ear tissue was sampled to extract genomic DNA. Eight SNPs in the 5'-flanking region, coding region, and 3'-untranslated region (3'-UTR) of SLC6A4 were genotyped, of which 6 SNPs had significant differences (P < 0.05) in allele frequency between the most aggressive and least aggressive pigs. Luciferase activity was greater in plasmids of genotype GG than plasmids of genotype CC of rs345058216 (P < 0.01). Computational analysis nominated MAZ as putative transcription factor (TF) with higher probability to bind the SLC6A4 promoter at the SNP (rs345058216) site. Also, we demonstrated that MAZ overexpression modulates SLC6A4 promoter activity in allele-specific manner with an in vitro assay. In addition, we demonstrated that SLC6A4 was a direct target of miR-671-5p. The dual luciferase reporter gene assay and cell transfection were performed to examine the role of miR-671-5p in regulating SLC6A4 expression. The luciferase assays revealed that the SNP rs332335871 affects regulation of miR-671-5p in SLC6A4 expression. After overexpression of miR-671-5p in porcine primary neural cells, the SLC6A4 mRNA levels can be significantly reduced. In conclusion, we here found that miR-671-5p and MAZ mediated porcine SLC6A4 expression level, which provides the possible molecular mechanism of aggressive behavior.