| Animal food intake determines the exogenous amount of nutrients directly, which is the basis for evaluation of animal nutrient requirements and energy metabolism. Physiologically affecting animal feed intake is the most fundamental factor. Studies have been shown that appetite regulatory network (ARN) on hypothalamus has many regulatory factors improving or suppressing appetite, e.g. PYY is one of them. PYY plays an important role in regulation of appetite and body obesity. When animals reduce feed intake, the release of PYY will be increased, appetite will be improved, and there is a negative correlation between PYY and obesity. Regulation of PYY may be an important way to control appetite. In the present study, we commenced on the epigenetic regulation of PYY gene, cloned porcine PYY gene, and searched and validate SNPs leading to potential changes in miRNA binding sites in3’UTR of PYY gene. Simultaneously, we explored PYY gene methylation of5’flanking sequences, preliminary analyzed relationship between miRNA-652and PYY. Furthermore, we preliminarily revealed a theoretical base to PYY regulating feed intake in pigs, and provided materials for the use of PYY controlling food intake. Results are as follows.1. Q-PCR results showed that the expression of PYY gene in small intestine and stomach of both Meishan and Large White was significantly higher compared with that in brain and kidney. Whereas, PYY in various tissues in Meishan was expressed higher than that of Large White pigs. Both in small intestine and stomach, there is a trend gradually increasing expression of PYY gene from30day-old,75day-old to275day-old Meishan pig, and expression in intestinal was higher than that in stomach. While PYY gene both in small intestine and stomach was expressed highly at first and then declined from3day-old,30day-old and120day-old Large White pig, and expression in intestinal was higher than that in stomach.2.892bp flanking sequence including3’UTR of PYY gene both in Large White and Meishan pigs were cloned. By sequencing and blasting of57individuals from Meishan or Large White pigs, we found8mutant loci. Out of them, there are three relatively stable SNPs, C137T, C146T and C169T. In addition, genotype and allele frequency of these three SNPs were calculated. The genotype frequency of CC, CT and TT were29.41%,11.77%and58.82%, respectively; the allele frequency of C and T are35.29%and64.71%, respectively.3. Bioinformatic prediction showed that both miR-652and miR-4339target3’UTR of PYY gene corresponding to C146T and C169T, respectively. In order to further validate the prediction, we construct luciferase expression vector and transfect it to cells. The results showed that there is not a target relationship between miR-4339and PYY gene, whereas miR-652significantly downregulate the luciferase activity and the expression of PYY gene. Furthermore, the mutation of T146C resulted in miR-652absent in regulation of PYY.4. Prediction of5’flanking sequence of PYY gene found a CpG island. Using bisulphite sequencing for Meishan and Large White pigs in the small intestine and stomach. The results showed that there is different methylation status at different tissues or different stage pig PYY gene. In addition, combined with the result of expression profile, there is a negative correlation between them.To sum up, PYY plays role in appetite suppression via participating in ARN. Different expression level in various tissues or at different developmental stages revealed the change of PYY when functioned. The difference of DNA methylation rates at developing stages may be one of the reasons reducing PYY differential expression. We also found miRNA involved in the post-transcriptional regulation of PYY, indicating the complex of PYY functioned in appetite control. |
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