Study On The Effect And Mechanism Of Exosomal MiR-145a-5p Derived From Sertoli Cells In Regulating Testosterone Synthesis In Leydig Cells

Objective:During the development of mammalian testes,Sertoli cells(SCs)play an important role in regulating development and function of Leydig cells(LCs).In this process,there are two types of Sertoli cells at different developmental stages---immature Sertoli cells(ISCs)and adult Sertoli cells(ASCs).However,it is not clear whether SCs at different developmental stages regulate the function of LCs in the same way.In this study,we clarify the different miRNAs of SCs at different stages,namely miR-145a-5p,and study the regulation of miR-145a-5p on the testosterone synthesis function of LCs.Method:In vitro experiments:(1)The two-step enzymatic hydrolysis method of collagenase and trypsin was used to separate SCs and LCs from the testis of 4-week-old or 8-week-old mice,and treat the ALCs with the supernatant of the conditioned medium of ISCs or ASCs,q PCR was to detect the expression of genes related to testosterone synthesis in ALCs;(2)q PCR and in situ hybridization were used to verify the expression of miRNA in ISCs and ASCs,and conduct the follow-up studies with miR-145a-5p as the research object;(3)ALCs was transfected with the miR-145a-5p mimic(mimic-miR-145)or miR-145a-5p inhibitor(inhibitor-miR-145),radioimmunoassay(RIA)was used to detect the change of testosterone level in the culture supernatant,q PCR was used to detect the change of gene expression related to testosterone synthesis,and the cells lipid droplet morphology was observed by oil red O staining;(4)Target Scan.org was used to predict the target genes of miR-145a-5p,and dual luciferase reporter system was used to examine the target genes of miR-145a-5p;mimic-miR-145 was used to transfected ALCs,q RT-PCR and Western blot for target verification;(5)Using the exosome extraction kit to separate the exosomes of ISCs and ASCs,identified morphology of the exosomes by transmission electron microscopy(TEM),analyze the particle sized of the exosomes by the nanoparticle tracking analysis system(NTA),and detected the expression of exosomal marker protein of exosomes by Western blot;(6)Exosomes of ISCs or ASCs were used to treat ALCs,and q PCR was used to detect expression of genes related to testosterone synthesis in ALCs;(7)ISCs were transfected with FAM-labelled miR-145a-5p and co-cultured with LCs,and the exosome inhibitor GW4869 was added to the ISCs medium.The pathway that miR-145a-5p transfered from SCs to LCs by exosome was verified by tracer experiment,Western blot for target verification.In vivo experiment:(1)In vivo agonist(Agomir-145)or in vivo antagonist(Antagomir-145)of miR-145a-5p were used to inject mouse testis,RIA was used to detect the testosterone levels in serum,Western blot for target verification;(2)TUNEL and HE staining were used to observe the state of testis tissue,and oil red O staining was used to observe the morphology of intracellular lipid droplets in LCs.Results:(1)The expression level of miR-145a-5p in ISCs is higher than that of ASCs.Transfection of LCs with mimic-miR-145 inhibit the expression of testosterone synthesis pathway genes such as Star、Cyp11a1、Hsd3b1 and Hsd17b3,and lead to lipid accumulation in LCs cells and reduce testosterone level in the supernatant of ALCs medium,while transfection of LCs with inhibitor-miR-145 have the opposite effect.(2)The target gene of miR-145a-5p is Sf-1,inhibiting the expression of Sf-1 lead to down-regulation of the expression levels of the downstream testosterone synthesis-related genes such as Star、Cyp11a1、Hsd3b1 and Hsd17b3,and lead to intracellular lipid accumulation in LCs.(3)Treating LCs with exosomes derived from ISCs inhibits the expression of testosterone synthesis pathway genes such as Sf-1、Star、Cyp11a1 and Hsd17b3,while exosomes derived from ASCs have the opposite effect.(4)The expression of miR-145a-5p in ISCs exosomes is positively correlated with the expression in ISCs.Inhibiting the secretion of ISCs exosomes significantly reduces the amount of miR-145a-5p entering LCs.Conclusion:miR-145a-5p is highly expressed in ISCs and transported to LCs through ISCs-derived exosomes,targeting 3’-UTR of Sf-1 and inhibiting the expression of the target gene Sf-1,and ultimately inhibiting the production of testosterone.