LOC102176306 Regulates Testosterone Synthesis In Goat Leydig Cells By Competitively Binding To MiR-1197-3p And Targeting PPARGC1A Gene

The growth,development and functional regulation mechanism of mammalian testes are important research contents of male reproduction.In the study of testicular development and testosterone synthesis in model animals,it was found that a large number of non coding RNA participated in these processes directly or indirectly involved in male reproductive process.In addition to providing energy for cells,mitochondria are also involved in processes such as cell differentiation,proliferation and apoptosis,which play an important role in testicular growth and development,steroid hormone synthesis and spermatogenesis.The production and function of mitochondria are regulated by nuclear genome,and mainly by peroxisome proliferator activated receptor gamma coactivator 1-alpha,PPARGC1 A/nuclear respiratory factors,NRFs/mitochondrial transcription factor a,TFAM pathway regulation.However,there are few studies on PPARGC1A/NRF1/TFAM pathway involved in the regulation of male testicular development and testosterone synthesis,there have been no reports of non-coding RNA targeting the key genes of PPARGC1A/NRF1/TFAM pathway involved in testicular development and testosterone synthesis in domestic animals.Therefore,explore the role of PPARGC1A/NRP1/TFAM pathway core genes in the process of goat sexual maturity,look for non-coding RNA targeting PPARGC1A/NRF1/TFAM pathway core genes,and clarify their molecular mechanisms involved in the synthesis of testosterone in goat leydig cells,not only can enrich the research of reproductive physiology of domestic animals,but also provide a theoretical basis for improving the reproductive capacity of male goats.The experiment divided into three main parts as follows:Experiment 1:Study on the expressions of PPARGC1A/NRF1/TFAM pathway genes in the development of goat testisThis study mainly used IHC,qRT-PCR and western blot to detect the localization and differential expression of core genes of PPARGC1A/NRF1/TFAM pathway in in testis of prepubertal(3-month-old)and postpubertal goats(9-month-old).ELISA results showed that the levels of testosterone,leptin,insulin and insulin-like growth factor Ⅰ in peripheral blood of 9-month-old goats were significantly higher than those in 3-month-old goats(P<0.05).The results of immunohistochemistry showed that PPARGC1A/NRF1/TFAM pathway core proteins were expressed in various cells of testis of 3-month-old and 9-month-old goats,qRT-PCR and western blot analysis revealed that mRNAs and proteins expressions of the PPARGC1A/NRF1/TFAM pathway core genes in testicular tissues of 9-month-old goats were significantly higher than those of 3-month-old goats(P<0.05).These results suggest that the core genes of PPARGC1A/NRF1/TFAM pathway may be involved in the regulation of testicular development and sexual maturity in goats.Experiment 2:Study on the mechanism of miR-1197-3p regulates testosterone synthesis in goat leydig cells via targeting PPARGC1A geneThe aim of this study was to investigate the molecular mechanism of miRNA regulating hormone secretion of Leydig cells(LCs)via the core genes of the PPARGC1A/NRF1/TFAM pathway.By predicting candidate miRNAs targeting the core genes of the PPARGC1A/NRF1/TFAM pathway,double luciferase reporter vector,overexpression and interference co-transfection assay were used to explore the mechanism of candidate miRNA regulatory target genes involved in the synthesis of testosterone in goat testis LCs.The results showed that miR-1197-3p targets PPARGC1A gene.Transfection of miR-1197-3p mimics or siRNA-PPARGC1A significantly reduced the synthesis of testosterone and the expression of steroidogenesis related genes(StAR,CYP11A1 and 3BHSD)(P<0.05),while transfection of miR-1197-3p inhibitor or pEX-4-PPARGC1A had the opposite effect.In addition,inhibition of PPARGC1A can significantly reduce the testosterone synthesis in goat LCs induced by miR-1197-3p inhibitor(P<0.05),while overexpression of PPARGC1A gene significantly attenuates the testosterone synthesis of goat LCs testosterone in goats induced by miR-1197-3p mimics.In conclusion,miR-1197-3p regulates the synthesis of testosterone in goat testis by targeting PPARGC1A gene.These results provide new insights into the regulatory mechanism of male sexual maturity and help to understand the role of PPARGC1A/NRF1/TFAM pathway in testosterone synthesis.Experiment 3:Study on the mechanism of LOC102176306 and miR-1197-3p regulates testosterone secretion in goat leydig cellsThe purpose of this study was to screen lncRNA combine with miR-1197-3p and target PPARGC1A gene to participate in the synthesis of testosterone in goats LCs,and to provide a reference for exploring the mechanism of testicular development and testosterone synthesis in male reproduction of goats.The co-expression network of candidate lncRNA-miR-1197-3p and PPARGC1A genes was screened and constructed by using the lncRNA database sequenced in this experiment.Moreover,the double luciferase report vector and the overexpression and interference experiments were used to determine the lncRNA that have ceRNA effect with miR-1197-3p.The results showed that LOC102176306 combined with miR-1197-3p and targeted PPARGC1A gene.Interference with LOC102176306 significantly reduced the synthesis of testosterone in LCs(P<0.05)and the expressions of key steroidogenesis related genes(StAR,CYP11A1 and 3BHSD)(P<0.05).LOC102176306 and miR-1197-3p may target the PPARGC1A gene through the ceRNA mechanism to regulate testosterone synthesis in goat LCs.These results enrich the related research of lncRNA in the testosterone secretion of LCs in goat testis,and lay a foundation for better exploring the mechanism of lncRNA in testicular development and testosterone synthesis.