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

The effects of exposure of mouse spermatocyte-derived cells to a 50 Hz magnetic field on the general microRNA expression and influence of the microRNA miR-26b-5p on cellular reactions should be investigated.

MicroRNAs are small non-coding RNAs pieces, which regulate gene expression at the post-transcriptional level. Mouse spermatocyte-derived cells were investigated to explore the mechanisms of action of possible adverse effects of magnetic fields on the male reproductive system.
Cells were divided into the following groups: exposure to a magnetic field of 1) 1 mT, 2) 2 mT and 3) 3 mT. For each of these groups a respective sham exposure was conducted. Moreover, cells were 4) transfected with miR-26b-5p (100 nmol/l) or 5) a miR-26b-5p inhibitor (100 nM) to induce an experimental overexpression or blocked expression, respectively. For both groups, an individual negative control was conducted. Finally, cells were transfected with 6) miR-26b-5p and after 6 hours exposed to a magnetic field of 3 mT and 7) with a miR-26b-5p negative control and after 6 hours exposed to a magnetic field of 3 mT. For groups 6 and 7, separate sham exposures were conducted.
All data were derived from at least 3 independent experiments that were performed in duplicate.

• intact cell/cell culture
• GC-2 cells (murine spermatocyte-derived cell line)

MiR-26b-5p was identified as the microRNA with the most differential expression in response to the exposure to a magnetic field (other microRNAs were found but not further investigated). There was no difference in the miR-26b-5p expression between group 1 (1 mT) and the sham exposure, but in group 2 (2 mT) it was significantly increased and in group 3 (3 mT) significantly reduced in comparison to the sham exposure group.
The methylation status of the miR-26b-5p host gene did not show any significant differences between the exposure and the sham exposure groups.
An experimentally induced overexpression of miR-26b-5p (group 4) or blocked expression (group 5) did not change cell viability, apoptosis or cell cycle in cells compared to the respective negative controls.
However, an overexpression of miR-26b-5p in combination with exposure to a 3 mT magnetic field (group 6) altered the cell cycle significantly compared to a exposure without this overexpression (group 7).
Software analysis identified cyclin D2 as a direct target of miR-26b-5p and real-time RT-PCR and Western Blot confirmed that miR-26b-5p overexpression (group 4) and exposure to a magnetic field of 2 mT (group 2) and 3 mT (group 3) as well as a combination of overexpression and exposure (group 6) altered the mRNA and protein expression of cyclin D2 significantly.
The authors conclude that exposure of mouse spermatocyte-derived cells to a 50 Hz magnetic field could change the expression rate of some microRNAs and miR-26b-5p could serve as a potential marker. Furthermore, miR-26b-5p-cyclin D2-mediated cell cycle regulation might play a role in the biological effects of exposure to extremely low frequency magnetic fields.