Differentially Expressed MiR-638 Between Sexually Immature And Mature Pig Testes Influences Proliferation And Apoptosis Of Swine Testicular Cells By Targeting SPAG1 Gene

Micro RNAs(miRNAs)are 18-22 nt non-coding small RNA(ncs RNA)molecules,which can be combined with specific m RNAs during the translation of protein to regulate gene expression.And they are widely present in the eukaryotic genome.With in-depth study of miRNAs,more and more evidences show that miRNAs play an important role in testicular development in animals,functional differentiation of various cells and the process of spermatogenesis.However,the genetic mechanism of miRNAs targeting key m RNAs in spermatogenesis has not been fully understood.Therefore,the isolation and function research of miRNAs in pig testis is a new topic which is worth being investigated.We identified differentially expressed miR-638 from pig testes by miRNAs array and Real-time PCR in our previous study,and we got the predicted target gene SPAG1 for miR-638 by RNAhybrid online software.In this research,we transfected a dual luciferase reporter gene vector into ST cells to get the information about the relationship between miR-638 and its potential target gene SPAG1,and then we further studied their functions in ST cells proliferation and apoptosis.The results obtained were as follows:1.Preliminary validation of the miR-638 target SPAG1We constructed a SPAG1 3’UTR dual luciferase reporter vector Pmir GLO which contained predicted binding site of miR-638,and co-transfected with miR-638 mimic to ST cells.Dual luciferase detection system results showed that the interaction of SPAG1 and miR-638 was a negative regulation(P<0.05).miR-638 mimic or inhibitor was transfected into ST cells in order to increase or inhibit the expression of miR-638.The result showed that m RNA and protein expression of the target gene SPAG1 decreased significantly in the over-expression of miR-638 group(P<0.01).Inhibiting the expression of miR-638 showed a significant increase in the expression level of SPAG1 at both m RNA and protein level(P<0.01).2.Localization of the SPAG1We conducted an immunofluorescence experiment to located SPAG1 by using the scanning confocal microscopy.We can see that SPAG1 was located in cytoplasm.3.The effects of miR-638 and its target SPAG1 on the function of ST cellsWe conducted siRNA to inhibit the expression of SPAG1.From the x CELLigence system-based on real-time monitoring of ST cells proliferation,we knew that SPAG1-siRNA-transfected ST cells got a decreasing growth rate compared with their controls.Using flow cytometer to detect the apoptosis of miR-638 mimic-transfected ST cells,the result showed that the apoptosis of miR-638 mimic-transfected ST cells was higher than NC-transfected ST cells(P<0.01),so as SPAG1-siRNA-transfected ST cells.But the apoptosis of miR-638 inhibitor-transfected ST cells was significantly decreasing(P<0.05).Overexpression of miR-638 and SPAG1-siRNA got lower amount of ATP in ST cells by ELISA analysis(P<0.01).And conversely,inhibition of miR-638 had an opposite trend(P<0.05).To reveal the influence of miR-638 and target SPAG1 on ERK signal we detected ERK phosphorylation,and found the reduced expression of SPAG1 inhibited ERK phosphorylation;however,after inhibiting the expression of miR-638 promoted ERK phosphorylation.To detectiDNA damage marker H2 AX phosphorylation(γ-H2AX)by Western Blot,we got that reduced the expression SPAG1 promoted H2 AX phosphorylation.So perhaps SPAG1 inhibited DNA damage.