Studies On Regulation Of Porcine Immature Sertoli Cell Growth By MiR-638 Via SPAG1 Gene

MicroRNAs(miRNAs)can bind specific mRNA,and then directly degrade mRNA or inhibit the translation process and play important roles in the plant and animal.Many studies have suggested that miRNAs play vital roles in testicular development,maturation and spermatogenesis in animal testes.We have demonstrated that miR-638 was differentially expressed between the sexually mature and immature testes from Large White pigs,and miR-638 targets SPAG1(sperm-associated antigen 1)gene,and then plays vital roles on porcine immature Sertoli cell apoptosis.Here we study the effect of miR-638 on swine testicular(ST)cell growth and pig spermatogenesis.The main results in our study are as follows: 1.The effects of miR-638 and its target SPAG1 on the function of ST cellsThe effect of miR-638 on cell cycle to was detected using flow cytometer,and the results showed that the overexpression of miR-638 could inhibit the percentage of cells in S phase and thus inhibit the transition from G0/G1 phase to S phase.SPAG1 siRNA-transfected cells also inhibited the percentage of cells in S phase.The results showed that miR-638 and SPAG1 had the similar effects on ST cells,which indicated that miR-638 inhibited cell cycle through SPAG1.The effects of miR-638 on ST cells proliferation was detected using MTT(Methylthiazolyldiphenyl-tetrazolium bromide)assay.The results showed that overexpression of miR-638 could significantly inhibit proliferation and inhibition of miR-638 promoted proliferation.2.Studies on molecular mechanism of ST cell growth regulated by miR-638The expression of cell cycle-related factors was detected using Q-PCR and Western blot,and the results showed that overexpression of miR-638 could suppress the expression of c-MYC,CCND1 and CCNE1 both at mRNA and protein level.SPAG1 siRNA-transfected ST cells had the same effect on ST cells.The effect of miR-638 and its target gene SPAG1 on PI3 K / AKT pathway was detected using Western blot,the results suggested that overexpression of miR-638 could reduce the expression of P-PI3 K and P-AKT protein level,inhibition of miR-638 could promote P-PI3 K and P-AKT protein level;similarly,SPAG1 siRNA-transfected ST cells had the similar effect as overexpression of mi R-638 on ST cells.3.The effect of SPAG1 on ST cell mitosisImmunofluorescence experiment has confirmed that SPAG1 co-located with α-tubulin and localized to the spindle of the mitosis metaphase and telophase.The results suggested that SPAG1 might regulate mitosis by regulating spindle assembly,which thereby controlled ST cell growth.