Study type: Medical/biological study (experimental study)

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.

Published in: Cell Cycle 2016; 15 (3): 357-367

Aim of study (acc. to author)

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.

Background/further details

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.

Endpoint

Exposure

Exposure Parameters
Exposure 1: 50 Hz
Exposure duration: intermittent (5 min on/10 min off) for 72 h
Exposure 2: 50 Hz
Exposure duration: intermittent (5 min on/10 min off) for 72 h
Exposure 3: 50 Hz
Exposure duration: intermittent (5 min on/10 min off) for 72 h

Exposure 1

Main characteristics
Frequency 50 Hz
Type
Exposure duration intermittent (5 min on/10 min off) for 72 h
Exposure setup
Exposure source
Chamber metal chamber in incubator
Setup exposure setup was composed of two four-coil systems (two coils with 56 windings and two coils with 50 windings) that generated a vertical MF inside a metal chamber; exposed and sham-exposed cell dishes were simultaneously placed in a separate incubator in which the environmental conditions were constant (37°C, 5% CO2); the temperature difference between sham exposure and exposure chamber never exceeded 0.3°C
Sham exposure A sham exposure was conducted.
Additional info the system was composed of two identical chambers; one of the chambers was used for sham-exposure, the other chamber was used for exposure; exposed and sham-exposed cell dishes were simultaneously placed in an incubator
Parameters
Measurand Value Type Method Mass Remarks
magnetic flux density 1 mT - measured - -

Exposure 2

Main characteristics
Frequency 50 Hz
Type
Exposure duration intermittent (5 min on/10 min off) for 72 h
Exposure setup
Exposure source
Sham exposure A sham exposure was conducted.
Parameters
Measurand Value Type Method Mass Remarks
magnetic flux density 2 mT - measured - -

Exposure 3

Main characteristics
Frequency 50 Hz
Type
Exposure duration intermittent (5 min on/10 min off) for 72 h
Exposure setup
Exposure source
Sham exposure A sham exposure was conducted.
Parameters
Measurand Value Type Method Mass Remarks
magnetic flux density 3 mT - measured - -

Reference articles

  • Ma Q et al. (2014): Extremely low-frequency electromagnetic fields affect transcript levels of neuronal differentiation-related genes in embryonic neural stem cells.
  • Schuderer J et al. (2004): In vitro exposure apparatus for ELF magnetic fields.

Exposed system:

Methods Endpoint/measurement parameters/methodology

Investigated system:
Time of investigation:
  • after exposure

Main outcome of study (acc. to author)

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.

Study character:

Study funded by

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