Study On The Molecular Mechanisms Of RFRP Signaling Pathway Regulated By MiRNA In The Central Nervous System

The reproductive regulation of mammals is the results of the combined action of central nervous system and endocrine system,and the key to reproduction is mainly determined by the synthesis and release of several major hormones from the hypothalamic-pituitary-gonadal(HPG)axis.RFRP-3 is a unique hypothalamic neuropeptide yet found that has an inhibitory effect on the HPG axis.As a key negative regulator,RFRP-3 plays an important role in the regulation of the HPG axis among different species in spite of few specialized study.However,it is not clear that the modulation of RFRP-3 mainly occurs in the hypothalamus level or the pituitary level,the discovery of miRNA provides a new view for exploring the mechanism of RFRP in reproductive process.In recent years,many studies have shown that miRNA plays an important role in the regulation of mammalian reproduction,which is widely involved in the regulation of many physiological functions in the organism.It is worth noting that the study of the physiological and pathological regulation of miRNA in different neuroendocrine cells is still insufficient,especially the regulation of HPG axis.Therefore,taking the RFRP signal pathway as the breakthrough point,we explored the important role of miRNA in reproductive regulation in order to provide a new theoretical basis for the reproductive research in mammals.In this topic,mouse hypothalamic cell line GT1-7 and hypophyseal cell line LβT2 cells as the research object.1)Treatment of GT1-7 cells with RFRP-3 analogs RFRP3-8,and then the main miRNAs(miR155,429)related to RFRP signaling pathway werescreened through high-throughput sequencing and bioinformatics analysis;2)On this basis,combined with dual luciferase reporter assay to investigate the relationship between the two miRNA and its target genes and find the specific site of action by UTR sequence truncation test in 293 T cells;3)Overexpression and interference miRNA and related genes expression in two kinds of cells to explore the effect of miRNA and target gene(Npffr1)on the expression of downstream genes GnRH,FSH and LH through RT-PCR and WB at mRNA and protein levels.The results showed that: 1)Compared with the control group,a total of 328 miRNA in GT1-7 cells treated by RFRP3-8 were differentially expressed through high throughput sequencing analysis,160 were up-regulated and 168 were down-regulated,among them,miR155,429 were both significantly down-regulated,and bioinformatics analysis showed that these two miRNAs could influence animal reproduction by targeting to Npffr1.Therefore,it is speculated that it may play an important regulatory role in RFRP signaling pathway;2)In 293 T cells,miR155,429 can act on the 3 ’UTR sequence of Npffr1,where the interaction sites are between 1430-1436bp(the seed sequence is CGUAAU)and 1614-1619bp(the seed sequence is UCAUA),respectively;3)In GT1-7 and LβT2 cells,the expression of Npffr1 was significantly reduced both at transcriptional and translational level,and the downstream genes expression,like GnRH,FSH and LH,were promoted by overexpression of these two miRNAs.More importantly,the enhancement can be partially offset by transfer to the overexpression vector of Npffr1 and even back to the base level.Likewise,the expression of Npffr1 was promoted when treated with miRNA inhibitors.Collectively,miR155,429 can promote the expression of reproductive related genes,such as GnRH,FSH and LH through targeted downregulation of Npffr1 gene expression and play a significant role in the reproductive regulation of the RFRP signaling pathway in GT1-7 and LβT2 cells,which provide a further theoretical foundation for understanding the regulatory mechanism of miRNA in mammalian reproduction.