| Intersex pig caused by intensive breeding,single provenance and high inbreeding restricts the cultivation of high-quality breeding pigs in China and even in the world.However,the molecular mechanism of the pathogenesis of intersexual pigs has not been fully explained.Hypothalamic-pituitary-gonadal(HPG)axis regulates the reproductive physiological process of mammals from fetal development to puberty and then to sexual maturity,and is an important neuroendocrine mechanism in the development of mammalian reproductive system.Studies have shown that the structure and function of the HPG axis in intersex pigs are abnormal and the secretion of reproductive hormones is disordered.Micro RNA(mi RNA)is a kind of endogenous non coding RNA,which can regulate the endocrine function of hypothalamus and pituitary,and then achieve its intervention in the process of reproductive physiological activities.The mi RNA molecular regulation mechanism on intersex pigs through central organs(hypothalamus and pituitary)has not been elucidated.In order to explore the molecular regulatory mechanism of mi RNA on intersex pig reproductive hormone disorder,intersex pigs(disease group)and normal sows(control group)were used as experimental materials.High-throughput sequencing and bioinformatics methods were used to identify the differentially expressed mi RNAs in hypothalamus and pituitary tissues of the two groups.The target gene of differentially expressed mi RNAs were predicted(mi Randa and RNAhybrid).Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were performed on the target genes to screen the regulatory pathways related to the synthesis and secretion of reproductive hormones.Protein-protein interaction(PPI)network analysis was performed on the candidate target genes,and the key modules of them were examined by Molecular complex detection(MCODE)of Cytoscape software.Study of the expression relationships between mi RNA and its target gene were analysis using realtime fluorescence quantitative PCR.Target relationship between the two candidate mi RNAs and target genes was verified by dual luciferase reporter system.The main results are as follows:(1)Analysis of mi RNA expression characteristics in the hypothalamus and pituitary of intersex pigs.The length distributions of small RNA in both hypothalamus and pituitary were mainly concentrated in the range of 21 ～ 23 nt,of which 22 nt was the most.A total of 361 known mi RNAs and 234 novel mi RNAs were identified in the hypothalamus.Compared with the control group,100 differentially expressed mi RNA were screened in the disease group,of which 47 were up-regulated and 53 were downregulated.A total of 360 known mi RNAs and 174 novel mi RNAs were identified in the pituitary,and 17 differentially expressed mi RNAs were screened,of which 4 were upregulated and 13 were down-regulated.(2)Analysis of target genes of differentially expressed mi RNAs in the hypothalamus.The differential expression of mi RNAs in hypothalamus had 7572 target gene pairs.Synthesis and secretion of hypothalamic reproductive hormones pathways were enriched by GO and KEGG analysis,incuding regulation of hormone levels,positive regulation of peptide hormone secretion,endocrine and other factors regulating calcium reabsorption,c AMP signaling pathway and Gn RH secretion.Ssc-mi R-4334-3p and Arginine vasopressin(AVP)was consider as the candidate target gene pair by MCODE and RT-q PCR.(3)Analysis of target genes of differentially expressed mi RNAs in the pituitary.The differential expression of mi RNAs in the pituitary had 1442 target gene pairs.Synthesis and secretion of pituitary reproductive hormones pathways were enriched by GO and KEGG analysis,incuding protein secretion,protein phosphorylation,cellular response to calcium ion,Gn RH signaling pathway,c AMP signaling pathway,phosphatidylinositol signaling system.Ssc-mi R-143-3p and AKT Serine/Threonine Kinase 1(AKT1)was consider as the candidate target gene pair by MCODE and RTq PCR.(4)Identification of the target relationship between ssc-mi R-4334-3p and AVP and between ssc-mi R-143-3p and AKT1.The results of the dual luciferase reporter gene detection system showed that ssc-mi R-4334-3p combined with the 3’-UTR of the AVP gene and ssc-mi R-143-3p did not have a direct targeting relationship with the AKT1 gene.In summary,this study revealed the expression characteristics of mi RNAs in the hypothalamus and pituitary gland of intersex pigs.It also lays a foundation for elucidating the molecular regulatory mechanisms of reproductive hormone disorders in intersex pigs.Ssc-mi R-4334-3p may affect the disorder of reproductive hormone secretion in hypothalamic Gn RH neurons of intersex pigs through the action of AVP gene.However,ssc-mi R-143-3p has no direct affected on AKT1 gene,and the function of ssc-mi R-143-3p needs to be further investigated.This study was conducted to elucidate the molecular mechanism of intersex pig pathogenesis from the perspective of non-coding RNA regulation.It provides a theoretical foundation for the development of genetic diagnosis and the elucidation of pathogenesis from multiple perspectives. |
