The Function Of MiR-124 Targeted To Flotillin-2 In Mouse Testes During Acrosome Biogenesis

Objective:To explore whether miR-124 could play an important role in flotillin-2 expression in mouse testes during acrosome biogenesis, and to further explore the mechanism of miR-124 expression in mouse acrosome biogenesis.Methods:Firstly, bioinformatics analysis was used to predict that whether miR-124 could regulate the expression of flotillin-2 through targeting its 3’-UTR. Secondly, the effect of miR-124 on flotillin-2 expression was investigated in vitro using a dual luciferase reporter assay system and in vivo using intratesticular injection in three-week-old male mice. Third, quantitative real-time RT-PCR and Western blot analysis were employed to confirm the function of miR-124 in regulating flotillin-2 after 48 hours. At last, sperm morphology was analyzed by ordinary optical microscopy and transmission electron microscopy three weeks after intratesticular injection.Results:Bioinformatics analysis validated that miR-124 was a direct and functional target of flotillin-2 via binding to its 3-UTR. MiR-124 was a negative factor which could down-regulate the expression of flotillin-2. Compared with the control group, sperm with significant increased abnormal heads in the miR-124 mimic group. Approximately 19.9% of sperm were abnormal in the control group; by contrast, nearly 28.5% of sperm were abnormal in the miR-124 injected group (P=4.68*10-5 vs. control). The proportion of sperm head abnormalities increased markedly to 20.7% in the miR-124 mimic group, which was significantly higher than that of the control group (13.7%, P=0.025). Transmission electron microscopy showed abnormal acrosome formation with discrete unfused small acrosomal vesicles around the nucleus and accumulation of many vesicles in the cytoplasm. In the caudal epididymis that corresponded to the testes treated with miR-124 mimic, sperm with malformed nuclei with an abnormal acrosome, fragmented discrete acrosome vesicles, residual Golgi apparatus, the degradation of Golgi membrane structure, large acrosome vesicle, or no acrosome were observed.Conclusion:This study provides insight into a novel molecular control mechanism of miR-124 through the expression of flotillin-2 during mouse acrosome biogenesis.