The Role And Mechanism Of The MiR-17-92 Cluster MiRNAs In Abnormal Spermatogenesis Induced By Genistein

Genistein (GEN) is one of the isoflavones exhibiting weak estrogenic effect and has the adverse effect on male reproduction. However, the underlying mechanism remains unknown. miRNAs are a type of small non-coding RNAs that silence their target genes by comnination of 3’-UTR region of target genes and often play important roles in spermatogenesis. It has been reported that miR-17-92 cluster is involved in spermatogenesis, and estrogen can induce changes in expression of miR-17-92 cluster. In order to investigate whether miR-17-92 plays roles in GEN induced abnormal spermatogenesis, we measured the GEN levels and miR-17-92 cluster expression in infertile subjects and found that expressions of miR-19b-1, miR-20a and miR-92a-1 were higher in relative high GEN exposure group, while the sperm motility was lower in corresponding group. These results suggested that miR-17-92 cluster miRNAs might be involved in GEN induced abnormal spermatogenesis. To clarify, we fed adult ICR mice with different doses of GEN (0, 0.5,5,50 and 250 mg/kg/day) for 35 days to study the underlying mechanism. We found that average path velocity (VAP), straight-line velocity (VSL) and eurvilinear velocity (VCL) of the mice orally with GEN at 5mg/kg/day were significantly decreased. We also measured the expression levels of miR-17-92 in mice testis and found the expression levels of miR-17 and miR-20a was higher in corresponding group. By comparing the results in human and mice, we found that miR-20a was the only miRNA that differentially expressed both in human and mice. By applying bioinformatics methods, Limkl was predicted to be the target gene of miR-20a that is involved in spermatogenesis. Furthermore, we determined that mRNA and protein levels of Limkl were down-regulated in corresponding group. Dual-luciferase report assay also proved that miR-20a could directly target Limkl. These results implied that Limkl might be the target gene of miR-20a that is involved in slightly abnormal spermatogenesis induced by GEN.Part I:The association between the levels of seminal plasma miR-17-92 cluster and GEN exposureObjectiveIn order to study the association of miR-17-92 cluster and the GEN exposure induced abnormal spermatogenesis in Han population.Methods1.We examined the urine GEN exposure levels of total 130 infertile subjects by UPLC-MS/MS method;2.The expression levels of seminal plasma miR-17-92 of these subjects were analyzed by qRT-PCR method;3. We divided the subjects into four groups according to the exposure levels of GEN, and then study the correlation of seminal plasma miR-17-92 in the GEN exposure induced abnormal spermatogenesis.Results1. We compared the subjects’ sperm characteristics in different groups and found that the sperm motility was lower in group 3 (the range of the GEN concentration were 76.32-281.96 ng/ml);2. We found that expressions of miR-19b-1, miR-20a and miR-92a-1 were higher in group3, and the expressions of other 3 miRNAs were no significant differences.ConclusionRelative high GEN exposure is associated with abnormal spermatogenesis, mainy with low sperm motility, and miR-17-92 might be involved in this progress.Part Ⅱ:The role and mechanism of miRNAs of miR-17-92 cluster in GEN induced abnormal spermatogenesis in miceObjectiveIn order to study the role and mechanism of miRNAs of miR-17-92 cluster in GEN exposure induced abnormal spermatogenesis in mice model.Methods1. To study the effects of different doses of GEN on mouse spermatogenesis, 7-weeks-old male ICR mice were fed at different doses of GEN (including 0,0.5,5, 50 and 250 mg/kg/day) for 35 days;2.The expression levels of miR-17-92 in mouse testis were detected by qRT-PCR method;3. Combining the results in human, we selected miRNAs which were significantly changes both in human and mice, then we screened target genes by target gene prediction software and dual luciferase reporter assay to define the target genes;4. The expression levels of target genes were detected by qRT-PCR and western blotting methods.Results1. We found that average path velocity (VAP), straight-line velocity (VSL) and eurvilinear velocity (VCL) of the mice orally with GEN at 5mg/kg/day were significantly decreased;2. The expression levels of miR-17 and miR-20a in mouse testis was significantly higher in groups at dose of 5mg/kg/day. After combining the results in human and mice, we defined miR-20a to be the key miRNA;3. By the target gene prediction software and comparison of pathway enrichment results in human and mouse, we find that actin cytoskeleton pathway was reported to involve in the microtubule-based processes of spermatogenesis, and LIMK1 plays an important role in this pathway;4. Dual luciferase reporter assay suggested that Limkl is the target gene of miR-20a;5. Limkl was significantly lower in the testis of mice orally with GEN at 5 mg/kg/day, and the protein expression was consistent with the results of gene expression.Conclusion1. Orally with GEN at 5 mg/kg/day for 35 days could lead to abnormal spermatogenesis in male adult ICR mice;2. Limkl as a target gene of miR-20a, may play a role in the dysfunction of spermatogenesis caused by GEN exposure.