The Regulatory Mechanism Of MiR-322 In Germ Cells And Its Diagnostic Value For Male Infertility With High DFI Rate

Objective:1.To investigate the changes of cell proliferation,apoptosis and the expression of apoptosis-related genes in GC-2 cells of miR-322 inhibition.2.To find out the target gene of miR-322 and confirm the regulation function for GC-2 cell proliferation and apoptosis.3.To evaluate the etiologically diagnostic and therapeutic value of male infertility with high DFI rate by co-measuring the expression of miR-322 and its target gene in seminal plasma.Method:1.For cell proliferation test:According to the difference of miRNA mimics,inhibitors and NCs(Negative Controls)transfected into GC-2 cells,cells were divided into 7 groups(A～G group).The cell proliferation rate of each group was detected by MTT and CCK8 assay,and the results between groups were compared.2.For apoptosis test:GC-2 cells transfected with miR-322 inhibitors and inhibitor NCs as well as blank control cells were divided in H～J group.The apoptosis rate of each group was detected by flow cytometry,and the results between groups were compared.3.For tests of expression of apoptosis-related genes(Caspase-3,Caspase-9,Caspase-8,Bax,Bcl-2):The RNA and protein of H-J group cells were extracted.The expression levels of apoptosis-related genes in each group were detected by real-time PCR and Western blot.Results between groups were compared.4.For prediction and verification test of target gene:Bioinformatics method was used to predict the target gene of miR-322 by comparing online databases.Compared with the predicted gene in H group,the gene in I group were identified as candidate gene.The wild type and mutant plasmids containing within the 3’UTR of candidate gene were successfully established.GC-2 cells were divided in 4 groups for the differences of co-transfection with the wild type/mutant vector and miR-322 mimics/miRNA mimic NCs(K～N group).The luciferase activity of each group was detected by double luciferase assay to confirm the possible gene we predicted.5.For contrast detection in human specimen:DFI greater than 30%and DFI less than 10%were defined as the high DFI male sterile group(O group,experimental group)and the normal male group(P group,control group).Detection of the expression of miR-322 and its possible target gene Ddx3x in the seminal plasma of each group was performed by real-time PCR and Western blot.Results were compared between two groupsResult:1.Compared with the control group,the proliferation rate of GC-2 cells was significantly decreased only in the group transfected with miR-322 inhibitor(*P<0.05).Other groups didn’t demonstrate significance(P>0.05).2.Compared with the control group,the expression of apoptosis-promoted genes Caspase-3,Caspase-9,Caspase-8,Bax were significantly increased(*P<0.05)in the group transfected with miR-322 inhibitors,while the expression of anti-apoptosis Bcl-2 was significantly decreased(*P<0.05).3.Compared with the control group,the luciferase activity was significantly decreased in the experimental group(*P<0.05).4.The expression of miR-322 in seminal plasma of male sterile with high rate of DFI was significantly lower than that in the control group(*P<0.05),and the expression of possible target gene Ddx3x was significantly higher than control group(*P<0.05).Conclusion:1.Proliferation rate was significantly decreased in GC-2 cells with miR-322 inhibition,and the apoptosis was obviously promoted.2.Ddx3x was direct binding site of miR-322 leading the decline of proliferation and the promotion of apoptosis of GC-2 cells.3.In the seminal plasma of male infertility patients with high DFI rate,the expression of miR-322 and its possible target gene Ddx3x were of value in the etiological diagnosis and treatment.