Melatonin Inhibits Testosterone Synthesis In Rooster Leydig Cells Through MiR-7481-3p/CXCL14

Testosterone is mainly produced by Leydig cells,which play an important role in the development of male reproductive organs,the regulation of spermatogenesis,and the maintenance of secondary sexual characteristics.As a hormone secreted by pineal gland,melatonin can regulate the synthesis of steroid hormones,circadian rhythm,immune system and other functions in animals.Micro RNA(miRNA)is a kind of endogenous small RNA molecule with the length of 19–24 nucleotides,which performs the function of regulating gene expression after transcription.A host of studies indicate that miRNA can participate in the regulation of testosterone synthesis in interstitial cells.Our previous study indicate that melatonin could regulate the cyclic adenosine monophosphate(c AMP)/ protein kinase A(PKA)/cyclic-AMP response binding protein(CREB)signaling pathway and inhibit testosterone synthesis in rooster Leydig cells.However,whether melatonin regulated testosterone synthesis by miRNA has not been reported yet.To explore whether melatonin could regulate testosterone synthesis in rooster Leydig cells through the miRNA,we conducted the following studies.The rooster Leydig cells were treated with 1 ng/m L melatonin for 36 h by isolating and culturing the primary Leydig cells,and the expression levels of testosterone synthesis key enzymes St AR,3β-HSD,and CYP11A1 as well as the changes of testosterone levels were detected by qRT-PCR and ELISA.In order to screen differentially expressed miRNA and m RNA after melatonin treatment,miRNA sequencing and transcriptome sequencing(RNA-Seq)were used to screen differentially expressed miR-7481-3p and CXCL14,and the targeting relationship between them was verified by double luciferase reporter gene test,qRT-PCR and Western Blot.To prove whether melatonin could inhibit testosterone synthesis in rooster Leydig cells by miR-7481-3p,we transfected miR-7481-3p simulator to pretreat rooster Leydig cells and added melatonin.The levels of testosterone and the expressions of the key testosterone synthesis enzymes St AR,3β-HSD and CYP11A1 were detected by qRT-PCR,Western blot and ELISA.In addition,in order to prove that melatonin inhibited testosterone synthesis in rooster Leydig cells by CXCL14 and interfered with CXCL14,melatonin was added.The levels of testosterone and the expressions of the key testosterone synthesis enzymes St AR and CYP11A1,3β-HSD were detected by using qRT-PCR,Western blot and ELISA.It was concluded that melatonin could inhibit testosterone synthesis in rooster Leydig cells through the miR-7481-3p/CXCL14 pathway.The main research results are summarized as follows:(1)Primary rooster Leydig cells were divided into control groups(CTR)and melatonin treatment groups(MT).Compared with the CTR group,MT group significantly reduced testosterone levels and m RNA expression levels of the key enzymes of testosterone synthesis,St AR,CYP11A1 and 3β-HSD(P<0.05).The results of miRNA sequencing and RNA-Seq showed that melatonin treatment could significantly down-regulate miR-7481-3p and upregulate CXCL14 in rooster Leydig cells(P<0.01).qRT-PCR,Western blot and double luciferase assays showed that miR-7481-3p could target and inhibit the expression of CXCL14(P<0.05).The above results suggested that melatonin might inhibit the synthesis of testosterone in rooster Leydig cells through the MIRI-7481-3P/CXCL14 pathway.(2)qRT-PCR and Western blot results showed that the 48 h transfection of miR-7481-3p mimetics significantly promoted testosterone levels and the expressions of the key testosterone synthesis enzymes St AR,3β-HSD and CYP11A1(P<0.05).In addition,the inhibition of melatonin on the testosterone level in Leydig cells and the expression of the key testosterone synthesis enzymes St AR,3β-HSD and CYP11A1 was reversed by the overexpression of miR-7481-3p(P<0.05).The results showed that melatonin inhibited the synthesis of testosterone in rooster Leydig cells through miR-7481-3p.(3)qRT-PCR and Western blot results showed that interference with CXCL14 48 h significantly promoted testosterone levels and the expressions of St AR,3β-HSD and CYP11A1,the key enzymes in testosterone synthesis(P<0.05).In addition,interference with CXCL14 reversed the inhibitory effects of melatonin on testosterone levels in Leydig cells and the expressions of St AR,3β-HSD and CYP11A1,key enzymes in testosterone synthesis(P<0.05).The results showed that melatonin inhibited the synthesis of testosterone in rooster Leydig cells through CXCL14.The results of the above experiments indicated that melatonin inhibited the synthesis of testosterone in rooster Leydig cells by targeting CXCL14 with miR-7481-3p.