Preliminary Study On Molecular Mechanisms Of Pig Litter Size Regulated By MiR-429

Litter size,one of reproductive traits,play an important role in the modern pig-raising industry.The hypothalamic-pituitary-gonadal axis exert significant function on regulating reproduction,which is affected by many genes and environment interaction.Studies on pig miRNA increased in recent years.However,researches of miRNA on reproductive regulation are scarce report.Through the results reported in the literature and our preview studies,we found that mi R-429,mi R-143 and miR-145 may have a regulatory role in mammals reproductive traits.And in mice pituitary miR-429 can down-regulate the expression of ZEB1 further to up-regulate the expression of LH?,therefore to regulate the process of ovulation.Since miRNA is highly conserved in mammals,so these findings may also established in pigs.Therefore,fluorescence quantitative PCR,dual luciferase reporter gene,electrophoretic mobility and other experimental techniques were utilized in this experiment,mainly to study the expression of miR-429,gene(ZEB1 and LH?)associated with mi R-429,miR-143-3p and miR-145-3p in Yorkshire and Wannan Black sows reproductive axis(pituitary,hypothalamus,ovaries,uterus,fallopian tubes)with high and low litter size,and to study possible molecular mechanisms of miR-429 regulating pig litter size.The results are as follows:1.The expression of miR-429,gene(ZEB1 and LH?)associated with miR-429,miR-143-3p and miR-145-3p in sows reproductive axis: miR-429 was higher expressed in the oviduct and pituitary;LH? was only higher expressed in the pig pituitary;ZEB1was widely expressed in porcine reproductive axis tissues;miR-143-3p and miR-145-3p showed high expression in the uterus,ovaries,fallopian tubes.2.The different expression of miR-429,gene(ZEB1 and LH?)associated with miR-429,miR-143-3p and mi R-145-3p in Yorkshire and Wannan Black sows reproductive axis with high and low litter size: In the pituitary,the expression of miR-429 and LH? were significantly higher in high litter group than low litter group,while the results of ZEB1 just opposite;in the uterus,the expression of miR-143-3p and miR-145-3p were significantly lower in high litter group than in low litter group.The result showed that: miR-429,miR-143-3p and miR-145-3p may affect the litter size insows,miR-429,ZEB1 and LH? may exist relations of regulation.3.Targeting relationship between miR-429 and ZEB1: dual-luciferase experi-ments carried out in 293 T cells,we found that the fluorescence value of transfection with pmr-mcherry vector significantly higher than transfection with pmr-mcherry-ssc-miR-429(P <0.01).Showing miR-429 have binding sites in the 3'UTR region of ZEB1,indicating that miR-429 may be targeted regulation ZEB1.4.Targeting relationship between ZEB1 and LH?: According to results of EMSA,indi-cating that nuclear protein samples stored in the transcription factor ZEB1 can occur with the promoter region of the gene LH? interaction.There may be targeting relationship betwe-en transcription factors ZEB1 and LH? in pigs.In summary,miR-429,miR-143-3p and miR-145-3p may affect the performance of litter size in sows;in the pig pituitary mi R-429 may down-regulating the expression of ZEB1 gene and thus indirectly up-regulating expression of LH?,to regulate the litter size performance of sow.