Overexpression of miR-26b-5p regulates the cell cycle by targeting CCND2 in GC-2 cells under exposure to extremely low frequency electromagnetic fields

The increasing prevalence of extremely low frequency electromagnetic fields (ELF-EMFs) exposure has raised considerable public concern regarding the potential hazardous effects of ELF-EMFs on male reproductive function. Increasing evidence indicates that miRNAs are necessary for spermatogenesis and male fertility. However, the regulation of miRNA expression and the roles of miRNAs in response to ELF-EMFs remain unclear. In our study, mouse spermatocyte-derived GC-2 cells were intermittently exposed to a 50 Hz ELF-EMF for 72 h (5 min on/10 min off) at magnetic field intensities of 1 mT, 2 mT and 3 mT. MiR-26b-5p was differentially expressed in response to different magnetic field intensities of ELF-EMFs. The host gene CTDSP1 showed an unmethylation status in GC-2 cells at different magnetic field intensities of ELF-EMF exposure. MiR-26b-5p had no significant, obvious influence on the cell viability, apoptosis or cell cycle of GC-2 cells. However, the overexpression of miR-26b-5p significantly decreased the percentage of G0/G1 phase cells and slightly increased the percentage of S phase cells compared to the sham group that was exposed to a 50 Hz ELF-EMF. Computational algorithms identified Cyclin D2 (CCND2) as a direct target of miR-26b-5p. MiR-26b-5p and a 50 Hz ELF-EMF altered the expression of CCND2 at both the mRNA and protein levels. Overexpressed miR-26b-5p in GC-2 cells can change the mRNA expression of CCND2 following 50 Hz ELF-EMF at 3 mT. These findings demonstrate that miR-26b-5p could serve as a potential biomarker following 50 Hz ELF-EMF exposure, and miR-26b-5p-CCND2-mediated cell cycle regulation might play a pivotal role in the biological effects of ELF-EMFs.

极低频电磁场（extremely low frequency electromagnetic fields，ELF-EMFs）暴露的发生率日益升高，其对男性生殖功能的潜在危害已引发公众广泛担忧。越来越多的证据表明，微RNA（microRNAs，miRNAs）在精子发生与男性生育能力维持中发挥必要作用。然而，关于微RNA的表达调控及其在极低频电磁场暴露应答中的功能，目前仍未明确。本研究中，我们将小鼠精母细胞来源的GC-2细胞以1 mT、2 mT及3 mT的磁场强度，间歇性暴露于50 Hz极低频电磁场中72小时（开启5分钟/关闭10分钟）。MiR-26b-5p的表达水平随极低频电磁场的不同磁场强度呈现显著差异性变化。在不同磁场强度的极低频电磁场暴露下，GC-2细胞中的宿主基因CTDSP1均呈未甲基化状态。MiR-26b-5p对GC-2细胞的细胞活力、凋亡或细胞周期无明显影响。然而，与接受50 Hz极低频电磁场暴露的假照射组相比，过表达MiR-26b-5p可显著降低G0/G1期细胞占比，并小幅升高S期细胞占比。通过计算算法预测发现，细胞周期蛋白D2（Cyclin D2，CCND2）是MiR-26b-5p的直接靶基因。MiR-26b-5p与50 Hz极低频电磁场均可在mRNA及蛋白水平上调控CCND2的表达。在3 mT的50 Hz极低频电磁场暴露条件下，GC-2细胞中过表达MiR-26b-5p可改变CCND2的mRNA表达水平。本研究结果表明，MiR-26b-5p可作为50 Hz极低频电磁场暴露后的潜在生物标志物，且MiR-26b-5p-CCND2介导的细胞周期调控可能在极低频电磁场的生物学效应中发挥关键作用。