Study Of Proliferation Of Porcine Sertoli Cells Regulated By Circ01801/mir-30a-3p/ATP1A1

Sertoli cells are the first somatic cells to differentiate in embryonic gonads,which play an important role in boar spermatogenesis and testicular development.The ability of boar to produce sperm is limited by the number of mature Sertoli cells,which is determined by the proliferation activity of immature Sertoli cells.Some studies have shown that circRNAs play a key role in supporting cell proliferation or apoptosis,but the functions of most of the circRNAs identified so far are still unknown.In this study,we identified the circular structure of circ01801,detected its expression levels in the testis of Meishan,Duroc and Yorkshire boars at different times.Edu,CCK-8 and flow cytometry were used to explore the function of circ01801 in Sertoli cells.Then,qPCR,Western blot and dual-luciferase reporter system were used to explore the regulatory mechanism involved in circ01801.The main results are as follows:1.The whole transcriptome sequencing results of testis showed that circ01801 was differentially expressed in the testis of 210 day old Duroc boar and Yorkshire boars.The sequencing data were verified by qPCR and it was found that the expression of circ01801 in Sertoli cells was significantly higher than that in Leydig cells.Subsequently,circ01801 was mainly localized in the cytoplasm of Sertoli cells by karyoplasmic localization experiment.2.The back-splice site of circ01801 was identified by divergent primers,convergent primers amplification and Sanger sequencing,and then its circular structure was further confirmed by RNase R enzyme tolerance assay.3.The effects of overexpression and interference of circ01801 on Sertoli cells proliferation and cell phase were detected by qPCR,Western blot,Edu,CCK-8 and flow cytometry.The results showed that circ01801 could promote cell proliferation and accelerate the cycle process of Sertoli cells.4.Through dual-luciferase detection system,we found circ01801 could sponge miR-30a-3p in Sertoli cells.Using qPCR,Western blot,Edu,CCK-8 and flow cytometry,it was found that miR-30a-3p could inhibit the proliferation and cycle process of Sertoli cells.5.Through dual-luciferase detection system,it was found that miR-30a-3p could combine with ATP1A1 3＇UTR.Using qPCR and Western blot,it was found that miR-30a-3p negatively regulated the expression of ATP1A1,while circ01801 positively regulated the expression of target gene ATP1A1 by sponging miR-30a-3p.In conclusion,miR-30a-3p can target ATP1A1 gene and inhibit the proliferation of Sertoli cells.Meanwhile,circ01801 acted as a sponge for the miR-30a-3p and further facilitated the expression of target gene ATP1A1 in Sertoli cells,so as to promote the proliferation of Sertoli cells,to lay a foundation for the analysis of boar spermatogenesis and testicular development mechanism.