CeRNA Network In Bovine Hypothalamic And Research Of Bta-miR-377 On The Regulation Of CART Expression

A variety of hormones secreted by the hypothalamic-pituitary-ovary(HPO)axis regulate gonadal development and reproductive activity in bovine.Cocaine and amphetamine regulated transcript peptide(CART)is an endogenous neuropeptide secreted by the hypothalamus,which negatively regulates the proliferation of granulesa cells(GCs)and the secretion of estrogen(E2)in bovine follicles.microRNA(miRNA)is a kind of non-coding shortRNA with a length of 18~24 nt,which realizes post-transcriptional regulation of mRNA by binding target genes,inhibiting their translation process or degradation of mRNA.Studies have shown that miRNA plays an important role in regulating the expression of HPO axis related genes,hormone synthesis and secretion.Based on bioinformatics targeted prediction,this study explored the effect of bta-miR-377 on CART gene expression in bovine hypothalamus through dual luciferase reporter gene method,cell culture in vitro and gene overexpression technology.The main content includes the following aspects:(1)The circRNA/lncRNA-miRNA-CART regulatory network was successfully constructed using bioinformatics techniques,and 7 miRNAs targeting CART 3′UTR were obtained(bta-miR-377,bta-miR-331-3p,bta-miR-491,bta-miR-877,bta-miR-758,bta-miR-381 and bta-miR-493).Based on the binding site and secondary structure,the interaction between CART 3′UTR and miRNA was further verified,and the complete complementary pairing and strong specificity of seed region were confirmed.RT-PCR detection showed that the expression of CART,bta-miR-377,bta-miR-331-3p,bta-miR-491,bta-miR-877,bta-miR-758,bta-miR-381 and bta-miR-493 was verified in bovine hypothalamus(2)Seven pairs of wild-type and mutant-type double luciferase reporter gene vectors of having miRNA-CART 3′UTR binding sites were successfully constructed,and the resultsd of double luciferase activity detection showed that bta-miR-377,bta-miR-331-3p,bta-miR-491,bta-miR-877,bta-miR-758,bta-miR-381 and bta-miR-493 only had significant inhibitory effects on the double luciferase activity expressed by the CART 3′UTR wild-type recombinant vector,indicating that these 7 miRNAs interacted with CART,and bta-miR-377 had a strong targeted inhibitory effect on CART.(3)Base on the fact that core region of amino acid sequence of bovine was consistent with rat,and the binding site of bta-miR-377-CART 3′UTR was the same,so rat adrenal pheochromotoma cells(PC-12)were overexpressed with bta-miR-377 mimics.q RT-PCR showed that the relative expression level of CART gene in the overexpressed bta-miR-377 mimics group was significantly lower than that in the control group(P<0.01),indicating that bta-miR-377 had an inhibitory effect on the expression of CART gene.In conclusion,bta-miR-377,bta-miR-331-3p,bta-miR-491,bta-miR-877,bta-miR-758,bta-miR-381、bta-miR-493 and CART 3′UTR have targeted binding ability and all expressed in bovine hypothalamus.bta-miR-377 has a strong targeted inhibitory effect on CART,significantly reduce the expression of CART mRNA,affect the secretion of CART peptide in bovine hypothalamic neurons,and then participate in the regulation of bovine follicle development.