Studies On The Molecular Mechanism Of RNF4 Interacting Protein-AR In Regulating Immature Porcine Sertoli Cell Proliferation

Spermatogenesis is a process that multiple cells are involved in.In this process,Sertoli cells play vital roles due to their specific physiological functions.Numerous studies have shown that the growth of sertoli cells is regulated by a variety of miRNAs and genes.Our previous study found that RNF4(ring finger protein 4)co-localized with AR(androgen receptor)in ST(Swine Testicular)cells and may affect ST cell growth through AR.Based on this research,we studied the effect of AR on ST cells proliferation and pig spermatogenesis,and explored related molecular regulation mechanism.The main results are as follows:1.Study of the interaction of RNF4 with ARCo-IP proved that AR interacted with RNF4 in ST cells,and the interaction between AR and RNF4 disappeared when the DBD domain of AR was deleted.2.The expression of AR and its effects on the function of ST cellsThe expression of AR in testis of 60-day-old and 180-day-old Large White boars was examined by immunofluorescence technique.The experimental results showed that AR was expressed in both testis of 60-day-old and 180-day-old boars.The overexpression vector and siRNA of AR were constructed and their effects were verified by qRT-PCR and Western blot.The effect of AR on ST cells cycle was detected by flow cytometry.The effect of AR on cell viability was detected by CCK8.The results showed that AR could promote cell cycle progression and cell proliferation.When AR DBD was absent,the impact of AR on cell cycle and cell proliferation disappeared,indicating that AR can influence the function of ST cells through binding to RNF4.3.The effects of miR-124 a on the function of ST cellsmiR-124 a directly bound the 90bp-96 bp site of 3' un-translated region(3'UTR)of AR was demonstrated by dual luciferase reporter system.Moreover,miR-124 a mimic was used to demonstrate that AR expression was negatively regulated by miR-124 a,indicating that AR is a potential target of miR-124 a.The effects of miR-124 a on ST cell cycle progression and cell proliferation were detected by flow cytometry and CCK8,respectively.The results showed that miR-124 a mimic can inhibit cell cycle progression and cell proliferation.Taken together,the above results indicate that miR-124 a inhibits the proliferation of immature porcine sertoli cells via AR.