![Effect of miR-34a administration on wound healing in DLD1 cells under control and RF-EMF conditions. Cells were transfected with (A) miR-34a-5p (m-m), (B) pre-miR-34a (pre-m), mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding negative control of mature and pre-miRNA (mNC and preNC, respectively). Data obtained 24 h after wound generation were relativised to the corresponding cell-free area of the wound at time 0 h. Data are presented as the mean ± SEM (n = 4). Two-way ANOVA, Tukey's post hoc test, * p< 0.05, ** p< 0.01. MTS assay: The results indicate a significant effect of RF-EMF exposure (48 h) on proliferation (pre-m: F [1, 83] = 7.215, p< 0.01, Figure 10B) and the interaction between the RF-EMF and transfected oligos (pre-m: F [2, 83] = 6.330, p< 0.01). Post hoc analysis confirmed that miR-34a (48 h) reduced the proliferation of DLD1 cells compared to the corresponding control under Wi-Fi-free conditions (Tukey's post hoc test; p< 0.01; Figure](https://www.researchgate.net/publication/364971882/figure/fig2/AS:11431281094716042@1667555589424/Effect-of-miR-34a-administration-on-wound-healing-in-DLD1-cells-under-control-and-RF-EMF_Q320.jpg)
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Citation: Olejárová, S.; Moravˇcík, R.;
Herichová, I. 2.4 GHz Electromagnetic
Field Influences the Response of the
Circadian Oscillator in the Colorectal
Cancer Cell Line DLD1 to
miR-34a-Mediated Regulation. Int. J.
Mol. Sci. 2022,23, 13210. https://
doi.org/10.3390/ijms232113210
Academic Editors: Giuseppe
Damante and Rebecca Chin
Received: 1 October 2022
Accepted: 27 October 2022
Published: 30 October 2022
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4.0/).
International Journal of
Molecular Sciences
Article
2.4 GHz Electromagnetic Field Influences the Response of the
Circadian Oscillator in the Colorectal Cancer Cell Line DLD1 to
miR-34a-Mediated Regulation
So ˇna Olejárová, Roman Moravˇcík and Iveta Herichová*
Department of Animal Physiology and Ethology, Faculty of Natural Sciences, Comenius University Bratislava,
842 15 Bratislava, Slovakia
*Correspondence: iveta.herichova@uniba.sk; Tel.: +421-2-9014-9572
Abstract:
Radiofrequency electromagnetic fields (RF-EMF) exert pleiotropic effects on biological
processes including circadian rhythms. miR-34a is a small non-coding RNA whose expression is
modulated by RF-EMF and has the capacity to regulate clock gene expression. However, interference
between RF-EMF and miR-34a-mediated regulation of the circadian oscillator has not yet been
elucidated. Therefore, the present study was designed to reveal if 24 h exposure to 2.4 GHz RF-
EMF influences miR-34a-induced changes in clock gene expression, migration and proliferation in
colorectal cancer cell line DLD1. The effect of up- or downregulation of miR-34a on DLD1 cells was
evaluated using real-time PCR, the scratch assay test and the MTS test. Administration of miR-34a
decreased the expression of per2,bmal1,sirtuin1 and survivin and inhibited proliferation and migration
of DLD1 cells. When miR-34a-transfected DLD1 cells were exposed to 2.4 GHz RF-EMF, an increase
in cry1 mRNA expression was observed. The inhibitory effect of miR-34a on per2 and survivin was
weakened and abolished, respectively. The effect of miR-34a on proliferation and migration was
eliminated by RF-EMF exposure. In conclusion, RF-EMF strongly influenced regulation mediated by
the tumour suppressor miR-34a on the peripheral circadian oscillator in DLD1 cells.
Keywords: cry;per;bmal1;clock; survivin; sirtuin1; birc5
1. Introduction
The circadian system is a network of endogenous oscillators localised in the brain and
peripheral tissues that facilitate proper synchronisation of the organism to environmental
factors exerting a 24 h cycle [
1
]. The organisation of the circadian system is hierarchical,
as it is composed of the central oscillator situated in the suprachiasmatic nuclei of the
hypothalamus (SCN) and peripheral oscillators localised in most other tissues. Peripheral
oscillators are usually under the regulatory influence of the SCN. However, there are
circumstances in which peripheral oscillators can partially or completely uncouple from the
master oscillator. This phenomenon arises from the different responsiveness of central and
peripheral oscillators to synchronising cues. While the SCN is predominantly synchronised
by the light (L) and dark (D) cycle, due to its direct connection to the retina, peripheral
oscillators can be more responsive to other synchronising factors [2].
As the anticipation of cyclic changes in the environment was useful, the circadian
system was evolutionarily conserved [
1
]. Circadian oscillator functioning relies on the
expression of clock genes (period and cryptochrome), which negatively influences their own
transcription. In humans, there are three homologues of the period gene (pre1,per2 and per3)
and two homologues of the cryptochrome gene (cry1 and cry2). Transcription of clock genes
per and cry is typically induced by heterodimers composed of transcription factors BMAL1
and CLOCK, which exert their function via the regulatory region E-box. BMAL1/CLOCK-
induced transcription of clock genes promotes the accumulation of per and cry protein
products in the cytoplasm. When the concentrations of PER and CRY achieve critical levels,
Int. J. Mol. Sci. 2022,23, 13210. https://doi.org/10.3390/ijms232113210 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2022,23, 13210 2 of 21
they create heterodimers that are translocated back into the nucleus, where they inhibit the
transcription of their own mRNA [3,4].
In addition to E-box-dependent regulation, there are several additional loops that influ-
ence the transcription of clock genes via RORE and DBP regulatory regions [
2
]. Moreover,
the transcription of clock genes is also subjected to epigenetic regulation based on clock
gene mRNA 3
0
UTR interaction with small non-coding RNAs (miRNAs). All genes of the
basic feedback loop are subjected to miRNA-mediated control, although particular miRNAs
usually differ for specific clock genes [
5
–
9
]. The administration of miR-219 or miR-132
facilitates BMAL1/CLOCK-induced expression of per1 [
10
]. Similarly, per1 expression is
inhibited by miR-34a-5p [
11
], miR-24 and miR-29a [
12
]. clock expression is under the control
of miR-182 [
13
], miR-17-5p [
14
], miR-124 [
15
] and miR-455-5p [
16
]. Expression of bmal1 is
responsive to miR-27b-3p [
17
], miR-142-3p, miR-494 [
18
,
19
], miR-155 [
20
], miR-135b [
21
]
and miR-211 [
22
], and per2 expression is inhibited by miR-24-3p and 25-3p [
12
,
23
,
24
], miR-
30a [
12
,
23
] and miR-96 [
25
] and induced by miR-107 [
26
]. The expression of miR-34a-5p
is negatively associated with per2 mRNA levels in colorectal cancer tissue in humans [
27
].
The expression of the whole period family is inhibited by the miR-192/194 cluster [
28
]. cry1
expression is enhanced in response to the overexpression or sponging of miR-17-5p [
14
],
and miR-185 administration inhibits the expression of cry1 mRNA [
29
]. cry2 expression is
inhibited by miR-107 [26] and miR-181d [30].
Among the cues synchronizing the circadian system are the light–dark regimen, food
availability, reward, exercise and temperature [
1
,
2
,
4
]. In addition, the electromagnetic
field has also been implicated as a possible factor with the capacity to influence circadian
rhythms in the human body [
31
]. During evolution, living organisms were exposed only to
the naturally occurring electromagnetic field of Earth [
32
]. However, in response to progress
in science and technologies, artificially generated electromagnetic fields are growing ex-
ponentially (www.itu.int/en/ITU-D/Statistics/Documents/facts/FactsFigures2021.pdf
(accessed on 26 October 2022)), and concerns about their effects on human physiology have
increased, especially after the International Agency for Research on Cancer at the World
Health Organization classified non-ionising radio-frequency electromagnetic fields (RF-
EMF) as a Group 2B agent that is, possibly carcinogenic to humans [
33
]. RF-EMF include
a frequency range from 30 kHz to 300 GHz and is generated mainly by cellular antennas,
Wi-Fi access points and Bluetooth devices [
34
]. The intensity of radiofrequencies (RF)
increased 2.3 times from 2017 to 2020, with Wi-Fi as the most important contributor [35].
Except for indirect heat effects of electromagnetic fields on living organisms, numerous
direct non-thermal interactions have been reported recently. RF-EMF can induce electron
and/or ion vibrations and consequently interactions among molecules that would not
occur otherwise. It was shown that RF-EMF influence polarisation of charged particles
and generation of electric dipoles. Moreover, effects of RF-EMF on potentials of cellular
membranes and reactive oxygen species levels have been reported [
36
]. Considering
constantly growing intensities of electromagnetic fields used in urban areas, non-thermal
effects of RF-EMF definitely deserve attention.
There is little data on the effect of Wi-Fi on clock gene expression. The effect of RF-EMF
on the circadian system has been investigated with respect to reproductive functions, where
the expression of clock,bmal1 and ror
α
mRNA is strongly suppressed in Leydig cells of
male mice exposed to1.8 GHz RF for several hours [
35
]. Similarly, the expression of clock
genes per,clc and cyc is downregulated, while cry expression is strongly induced by3.5 GHz
RF-EMF in Drosophila [37].
Clock genes can influence the cell cycle [
38
,
39
], and deregulation of the circadian
system has been associated with cancer progression [
40
,
41
]. A regulatory relationship
between clock gene expression and cell cycle has been demonstrated in many types of
cancer [42].
Int. J. Mol. Sci. 2022,23, 13210 3 of 21
Downregulation of per2 was associated with tumour progression in non-small cell
lung cancer [
43
]. Similarly, over-expression of per2 inhibited growth of lung carcinoma and
murine mammary carcinoma cell lines LLC and EMT6, respectively [
44
]. Over-expression
of per2 inhibited growth and promoted apoptosis of K562 leukemia cells [
45
]. On the other
hand, bmal1 has been shown to induce metastasis, migration and invasion in ZR-75-30
and MDA-MB-231 lines of breast cancer [
46
]. Similarly, the clock gene has been reported
to induce proliferation of breast cancer cell lines MCF-7, T47D and MDA-MB-231 [
47
]. In
the HL-60 promyeloblast cell line, bmal1 downregulation induced apoptosis and inhibited
proliferation [
48
]. The opposite results were observed in tongue squamous carcinoma cell
lines SCC9, SCC25, and CAL27 [
49
] and nasopharyngeal carcinoma cell line [
50
], where
over-expression of bmal1 inhibited cell proliferation. Upregulation of bmal1 has been shown
to inhibit proliferation U87MG glioblastoma cells [
51
] and per2 inhibited proliferation and
invasion ability in glioma stem cells U87 and U251 [
52
]. The effect of bmal1 downregulation
on growth of colorectal cancer (CRC) cell lines (HCT116 and SW481) and the metastatic
CRC line SW620 was dependent on the functional p53 pathway and AKT/mTOR activity.
Proliferation was increased in HCT116 and SW620 cells while inhibition of cell growth was
observed in SW480 cells after bmal1 knockdown [
53
].A functional relationship between
β
-catenin and per2 stability in CRC cell lines HCT116 and SW480 implicates a role of the
circadian oscillator in β-catenin- mediated effects [54].
Among cancer-related diseases, colorectal cancer has been identified as the third
most commonly diagnosed malignancy, with the second highest mortality rate after lung
cancer [
55
]. Moreover, during the Covid-19 pandemic, cancer screening and routine
diagnostics dropped. The estimated increase in deaths because of colorectal cancer (
15–16%
)
was highest among the four types of cancer (colorectal, breast, lung, and oesophageal) that
were included in the analysis [56].
Studies focused on clock genes expression in colorectal cancer tissue have revealed
that their expression is frequently down- or deregulated in cancer tissue [
57
]. Previously,
we observed downregulation of cry2 and per2 expression in tumour tissue in comparison
to adjacent tissue [
27
], and higher expression of cry1 in right-sided tumours but not in
left-sided tumours compared to surrounding tissue [
58
]. A negative association between
per2 expression and tumour staging has also been described [
59
]. Similarly, the amplitude
and mesor of bmal1,per1,per2 and rev-erba decreased in cancer tissue compared to healthy
colon in a mice model with induced colorectal tumours [60].
Considering that clock genes are involved in the regulation of cancer progression
and that their expression is modulated by miRNAs, there is quite a complex regulatory
network influencing processes in cancer tissue during the 24 h cycle. Our previous research
implicated the role of miR-34a in per2 regulation in patients with higher TNM stages [
61
].
Accordingly, miR-34a has been shown to have strong potential in colorectal cancer treat-
ment [
62
,
63
] and its prognosis [
64
–
66
]. Recently, concern about the effect of RF-EMF on
clock gene expression has risen, as the use of RF-EMF have significantly increased during
recent years and colorectal cancer treatment has been weakened by overwhelmed medical
capacities due to the Covid-19 pandemic. Therefore, this study was designed to determine
whether RF-EMF influence miR-34a-mediated regulation of clock gene expression in the
colorectal cell line DLD1 and whether it can be associated with cancer cell line growth.
2. Results
qPCR: Two-way ANOVA confirmed that the administration of miR-34a mimics (
48 h
)
resulted in a significant increase in the miR-34a-5p level in transfected cells (m-m:
F[2, 15] = 31.42
,p< 0.0001, Figure 1A; pre-m: F
[2, 17]
= 83.92, p< 0.0001, Figure 1B). A
post hoc test revealed a significant increase in miR-34a levels in cells influenced by the
mimic or the mimic together with the inhibitor compared to the corresponding control
(Tukey’s post hoc test; Figure 1A,B). We did not observe a significant effect of RF-EMF
administration (24 h) on the intracellular levels of miR-34a (m-m: F
[1, 15]
= 0.61, p= 0.4474;
pre-m: F
[1, 15]
= 1.83, p= 0.1963), indicating that RF-EMF did not influence transfection
Int. J. Mol. Sci. 2022,23, 13210 4 of 21
and/or levels of miR-34a in transfected cells. Accordingly, the interaction between the
investigated factors was not confirmed (two-way ANOVA).
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 22
mimic together with the inhibitor compared to the corresponding control (Tukey’s post
hoc test; Figure 1A,B). We did not observe a significant effect of RF-EMF administration
(24 h) on the intracellular levels of miR-34a (m-m: F [1, 15] = 0.61, p = 0.4474; pre-m: F [1, 15] =
1.83, p = 0.1963), indicating that RF-EMF did not influence transfection and/or levels of
miR-34a in transfected cells. Accordingly, the interaction between the investigated factors
was not confirmed (two-way ANOVA).
m-m m-m + i mNC m-m m-m + i mNC
0.0
mRNA miR-34a (ln scale,a.u.)
Control
RF-EMF
✱
✱✱✱
✱✱
✱✱✱
A
1.0
2.0
3.0
4.0
pre-m m-m + i preNC pre-m m-m + i preNC
0.0
mRNA miR-34a (ln scale,a.u.)
Control
RF-EMF
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
B
1.0
2.0
3.0
4.0
Figure 1. Levels of miR-34a-5p in the DLD1 cells. Cells were transfected with (A) miR-34a-5p
(m-m), (B) pre-miR-34a (pre-m), mimic of mature miR-34a together with the miR-34a inhibitor
(m-m + i) or corresponding negative control of mature and pre-miRNA (mNC a preNC, respec-
tively). Data are presented as the mean ± SEM (n = 4) in logarithmic scale. a.u.—arbitrary units.
Two-way ANOVA, followed by Tukey’s post hoc test, * p< 0.05; ** p< 0.01; *** p< 0.001; **** p< 0.001.
A two-way ANOVA confirmed the interaction between the influences of transfected
oligos and RF-EMF (m-m: F [2, 17] = 3.759,p = 0.067; pre-m: F [2, 17] = 8.143, p< 0.01) and the
effect of miR-34a administration (m-m: F [2, 17] = 3.759,P = 0.045, Figure 2A; pre-m: F [2, 17] =
15.96, p< 0.0001, Figure 2B) on cry1 mRNA levels. Accordingly, cry1 mRNA expression
was significantly induced by miR-34a administration only in cells exposed to RF-EMF,
while no effect of mi-34a on cry1 mRNA expression was observed under control condi-
tions (Tukey’s post hoc test; Figure 2A,B). We also revealed an effect of RF-EMF on cry1
mRNA (m-m: F [1, 17] = 3.981, p = 0.062; pre-m: F [1, 17] = 12.03, p< 0.01; followed by Šídák’s
multiple comparisons test, p< 0.05 and p< 0.001, respectively, Figure 2A,B). cry1 mRNA
levels were suppressed by RF-EMF when miR-34a was not administered in cells trans-
fected with mature oligos (Šídák’s multiple comparisons test; Figure 2A).
Figure 2. Effect of miR-34a administration on cry1 mRNA expression in DLD1 cells under control
and RF-EMF conditions. Cells were transfected with (A) miR-34a-5p (m-m), (B) pre-miR-34a
(pre-m), mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding
negative control of mature and pre-miRNA (mNC and preNC, respectively). The data were rela-
tivised to the corresponding negative control measured under control conditions and presented as
the mean ± SEM (n = 4). a.u.—arbitrary units. Two-way ANOVA, Tukey’s post hoc test, * p< 0.05,
**** p< 0.0001—comparison between groups transfected with different oligos within the control or
RF-EMF treatment. Two-way ANOVA, Šídák’s multiple comparisons test, # p < 0.05, ### p<
0.001—comparison between the control and RF-EMF groups.
Figure 1.
Levels of miR-34a-5p in the DLD1 cells. Cells were transfected with (
A
) miR-34a-5p (m-m),
(
B
) pre-miR-34a (pre-m), mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or
corresponding negative control of mature and pre-miRNA (mNC a preNC, respectively). Data are
presented as the mean
±
SEM (n = 4) in logarithmic scale. a.u.—arbitrary units. Two-way ANOVA,
followed by Tukey’s post hoc test, * p< 0.05; ** p< 0.01; *** p< 0.001; **** p< 0.001.
A two-way ANOVA confirmed the interaction between the influences of transfected
oligos and RF-EMF (m-m: F
[2, 17]
= 3.759, p= 0.067; pre-m: F
[2, 17]
= 8.143, p< 0.01) and
the effect of miR-34a administration (m-m: F
[2, 17]
= 3.759, p= 0.045, Figure 2A; pre-m:
F
[2, 17]
= 15.96, p< 0.0001, Figure 2B) on cry1 mRNA levels. Accordingly, cry1 mRNA
expression was significantly induced by miR-34a administration only in cells exposed to
RF-EMF, while no effect of mi-34a on cry1 mRNA expression was observed under control
conditions (Tukey’s post hoc test; Figure 2A,B). We also revealed an effect of RF-EMF on
cry1 mRNA (m-m: F
[1, 17]
= 3.981, p= 0.062; pre-m: F
[1, 17]
= 12.03, p< 0.01; followed by
Šídák’s multiple comparisons test, p< 0.05 and p< 0.001, respectively, Figure 2A,B). cry1
mRNA levels were suppressed by RF-EMF when miR-34a was not administered in cells
transfected with mature oligos (Šídák’s multiple comparisons test; Figure 2A).
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 22
mimic together with the inhibitor compared to the corresponding control (Tukey’s post
hoc test; Figure 1A,B). We did not observe a significant effect of RF-EMF administration
(24 h) on the intracellular levels of miR-34a (m-m: F [1, 15] = 0.61, p = 0.4474; pre-m: F [1, 15] =
1.83, p = 0.1963), indicating that RF-EMF did not influence transfection and/or levels of
miR-34a in transfected cells. Accordingly, the interaction between the investigated factors
was not confirmed (two-way ANOVA).
m-m m-m + i mNC m-m m-m + i mNC
0.0
mRNA miR-34a (ln scale,a.u.)
Control
RF-EMF
✱
✱✱✱
✱✱
✱✱✱
A
1.0
2.0
3.0
4.0
pre-m m-m + i preNC pre-m m-m + i preNC
0.0
mRNA miR-34a (ln scale,a.u.)
Control
RF-EMF
✱✱✱✱
✱✱✱✱
✱✱✱✱
✱✱✱✱
B
1.0
2.0
3.0
4.0
Figure 1. Levels of miR-34a-5p in the DLD1 cells. Cells were transfected with (A) miR-34a-5p
(m-m), (B) pre-miR-34a (pre-m), mimic of mature miR-34a together with the miR-34a inhibitor
(m-m + i) or corresponding negative control of mature and pre-miRNA (mNC a preNC, respec-
tively). Data are presented as the mean ± SEM (n = 4) in logarithmic scale. a.u.—arbitrary units.
Two-way ANOVA, followed by Tukey’s post hoc test, * p< 0.05; ** p< 0.01; *** p< 0.001; **** p< 0.001.
A two-way ANOVA confirmed the interaction between the influences of transfected
oligos and RF-EMF (m-m: F [2, 17] = 3.759,p = 0.067; pre-m: F [2, 17] = 8.143, p< 0.01) and the
effect of miR-34a administration (m-m: F [2, 17] = 3.759,P = 0.045, Figure 2A; pre-m: F [2, 17] =
15.96, p< 0.0001, Figure 2B) on cry1 mRNA levels. Accordingly, cry1 mRNA expression
was significantly induced by miR-34a administration only in cells exposed to RF-EMF,
while no effect of mi-34a on cry1 mRNA expression was observed under control condi-
tions (Tukey’s post hoc test; Figure 2A,B). We also revealed an effect of RF-EMF on cry1
mRNA (m-m: F [1, 17] = 3.981, p = 0.062; pre-m: F [1, 17] = 12.03, p< 0.01; followed by Šídák’s
multiple comparisons test, p< 0.05 and p< 0.001, respectively, Figure 2A,B). cry1 mRNA
levels were suppressed by RF-EMF when miR-34a was not administered in cells trans-
fected with mature oligos (Šídák’s multiple comparisons test; Figure 2A).
Figure 2. Effect of miR-34a administration on cry1 mRNA expression in DLD1 cells under control
and RF-EMF conditions. Cells were transfected with (A) miR-34a-5p (m-m), (B) pre-miR-34a
(pre-m), mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding
negative control of mature and pre-miRNA (mNC and preNC, respectively). The data were rela-
tivised to the corresponding negative control measured under control conditions and presented as
the mean ± SEM (n = 4). a.u.—arbitrary units. Two-way ANOVA, Tukey’s post hoc test, * p< 0.05,
**** p< 0.0001—comparison between groups transfected with different oligos within the control or
RF-EMF treatment. Two-way ANOVA, Šídák’s multiple comparisons test, # p < 0.05, ### p<
0.001—comparison between the control and RF-EMF groups.
Figure 2.
Effect of miR-34a administration on cry1 mRNA expression in DLD1 cells under control
and RF-EMF conditions. Cells were transfected with (
A
) miR-34a-5p (m-m), (
B
) pre-miR-34a (pre-m),
mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding negative
control of mature and pre-miRNA (mNC and preNC, respectively). The data were relativised to the
corresponding negative control measured under control conditions and presented as the
mean ±SEM
(n = 4). a.u.—arbitrary units. Two-way ANOVA, Tukey’s post hoc test, * p< 0.05, **** p< 0.0001—
comparison between groups transfected with different oligos within the control or RF-EMF treatment.
Two-way ANOVA, Šídák’s multiple comparisons test, # p< 0.05, ### p< 0.001—comparison between
the control and RF-EMF groups.
Int. J. Mol. Sci. 2022,23, 13210 5 of 21
miR-34a administration significantly influenced cry2 mRNA expression (pre-m:
F[2, 17]= 5.116
,p< 0.05), which was reflected by a trend and an increase in cry2 mRNA levels
in cells transfected with miR-34a or miR-34a + inhibitor relative to the negative control
(Tukey’s post hoc test, p= 0.087 and p< 0.05, respectively, Figure 3B) under Wi-Fi-free
conditions. Two-way ANOVA also proved the effect of RF-EMF on cry2 mRNA expression
(m-m: F
[1, 17]
= 8.519, p< 0.01, Figure 3A; pre-m: F
[1, 17]
= 13.99, p< 0.01, Figure 3B), and the
post hoc test revealed a significant increase in cry2 mRNA expression in groups influenced
by the mimic together with the inhibitor of miR-34a with a significant decrease under
RF-EMF conditions compared to the Wi-Fi-free environment (Šídák’s multiple comparisons
test, Figure 3A,B). Two-way ANOVA did not show interference between the investigated
factors (miR-34a and RF-EMF).
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 5 of 22
miR-34a administration significantly influenced cry2 mRNA expression (pre-m: F [2,
17]= 5.116, p< 0.05), which was reflected by a trend and an increase in cry2 mRNA levels in
cells transfected with miR-34a or miR-34a + inhibitor relative to the negative control
(Tukey’s post hoc test, p = 0.087 and p< 0.05, respectively, Figure 3B) under Wi-Fi-free
conditions. Two-way ANOVA also proved the effect of RF-EMF on cry2 mRNA expres-
sion (m-m: F [1, 17] = 8.519, p< 0.01, Figure 3A; pre-m: F [1, 17] = 13.99, p< 0.01, Figure 3B), and
the post hoc test revealed a significant increase in cry2 mRNA expression in groups in-
fluenced by the mimic together with the inhibitor of miR-34a with a significant decrease
under RF-EMF conditions compared to the Wi-Fi-free environment (Šídák’s multiple
comparisons test, Figure 3A,B). Two-way ANOVA did not show interference between
the investigated factors (miR-34a and RF-EMF).
Figure 3. Effect of miR-34a administration on cry2 mRNA expression in DLD1 cells under control
and RF-EMF conditions. Cells were transfected with (A) miR-34a-5p (m-m), (B) pre-miR-34a
(pre-m), (A, B) mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or corre-
sponding negative control of mature and pre-miRNA (mNC and preNC, respectively). The data
were relativised to the corresponding negative control measured under control conditions and
presented as the mean ± SEM (n = 4). a.u.—arbitrary units. Two-way ANOVA, Tukey’s posthoc
test, * p< 0.05, comparison between groups transfected with different oligos within the control or
RF-EMF treatment; One-way ANOVA, Tukey’s post hoc test, $ p< 0.05, comparison within the
control or RF-EMF treatment. Two-way ANOVA, Šídák’s multiple comparisons test, # p< 0.05
comparison between the control and RF-EMF groups. For a more detailed statistical analysis,
please see the results.
As interference between factors was not proven, the results were also analysed using
simple ANOVA for oligo type and treatment. ANOVA was used when three groups were
compared (effect of oligos), and an unpaired t-test was employed to compare the treat-
ments. In this way, we revealed an RF-EMF-associated decrease in cry2 mRNA expression
in groups mNC, preNC and pre-m compared to Wi-Fi-free conditions (t-test, p< 0.05). In-
terestingly, despite this, significantly higher expression of cry2 mRNA was detected in cells
transfected with mir-34a compared to the corresponding control under RF-EMF conditions
(pre-m: One-way ANOVA: F [2, 9] = 5.598, p = 0.026, p< 0.05, Figure 3B).
Two-way ANOVA clearly indicated a significant influence of miR-34a administra-
tion (m-m: F [2, 17] = 28.14, p< 0.0001, Figure 4A; pre-m: F [2, 17] = 20.77; p< 0.0001, Figure 4B)
and the effect of RF-EMF (m-m: F [1, 17] = 5.356, p< 0.05, Figure 4A; pre-m: F [1, 17] = 6.039, p<
0.05, Figure 4B) on per2 mRNA expression. The interaction of the investigated factors,
however, was not indicated.
m-m m-m + i mNC m-m m-m + i mNC
0.0
mRNA cry2/s17 (a.u.)
Control
RF-EMF
A#
1.0
2.0
3.0
4.0
pre-m m-m + i preNC pre-m m-m + i preNC
0.0
mRNA cry2/s17 (a.u.)
Control
RF-EMF
B#
✱
p = 0.087
1.0
2.0
3.0
4.0
$
Figure 3.
Effect of miR-34a administration on cry2 mRNA expression in DLD1 cells under control
and RF-EMF conditions. Cells were transfected with (
A
) miR-34a-5p (m-m), (
B
)
pre-miR-34a (pre-m)
,
(A,B) mimic
of mature miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding
negative control of mature and pre-miRNA (mNC and preNC, respectively). The data were relativised
to the corresponding negative control measured under control conditions and presented as the
mean ±SEM
(n = 4). a.u.—arbitrary units. Two-way ANOVA, Tukey’s posthoc test, * p< 0.05,
comparison between groups transfected with different oligos within the control or RF-EMF treatment;
One-way ANOVA, Tukey’s post hoc test, $ p< 0.05, comparison within the control or RF-EMF
treatment. Two-way ANOVA, Šídák’s multiple comparisons test, # p< 0.05 comparison between the
control and RF-EMF groups. For a more detailed statistical analysis, please see the results.
As interference between factors was not proven, the results were also analysed using
simple ANOVA for oligo type and treatment. ANOVA was used when three groups were
compared (effect of oligos), and an unpaired t-test was employed to compare the treatments.
In this way, we revealed an RF-EMF-associated decrease in cry2 mRNA expression in groups
mNC, preNC and pre-m compared to Wi-Fi-free conditions (t-test, p< 0.05). Interestingly,
despite this, significantly higher expression of cry2 mRNA was detected in cells transfected
with mir-34a compared to the corresponding control under RF-EMF conditions (pre-m:
One-way ANOVA: F [2, 9] = 5.598, p= 0.026, p< 0.05, Figure 3B).
Two-way ANOVA clearly indicated a significant influence of miR-34a administration
(m-m: F
[2, 17]
= 28.14, p< 0.0001, Figure 4A; pre-m: F
[2, 17]
= 20.77; p< 0.0001, Figure 4B)
and the effect of RF-EMF (m-m: F
[1, 17]
= 5.356,
p< 0.05
, Figure 4A; pre-m: F
[1, 17]
= 6.039,
p< 0.05, Figure 4B) on per2 mRNA expression. The interaction of the investigated factors,
however, was not indicated.
Int. J. Mol. Sci. 2022,23, 13210 6 of 21
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 of 22
Figure 4. Effect of miR-34a administration on per2 mRNA expression in DLD1 cells cultured in
Wi-Fi-free conditions (white columns) or exposed to 24h lasting RF-EMF exposure (grey columns).
Cells were transfected with (A) miR-34a-5p (m-m), (B) pre-miR-34a (pre-m), mimic of mature
miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding negative control of mature
and pre-miRNA (mNC and preNC, respectively). The data were relativised to the corresponding
negative control measured under control conditions and presented as the mean ± SEM (n = 4).
a.u.—arbitrary units. Two-way ANOVA, Tukey’s post hoc test, * p< 0.05, ** p < 0.01, *** p < 0.001,
**** p< 0.0001—comparison between groups transfected with different oligos within the control or
RF-EMF treatment. Two-way ANOVA, Šídák’s multiple comparisons test, # p< 0.05 comparison
between the control and RF-EMF groups.
The post hoc test demonstrated that miR-34a strongly inhibits per2 expression
compared to the negative control under Wi-Fi-free conditions. Moreover, the miR-34a
inhibitor reversed this effect and caused an increase in per2 expression compared to the
control (Tukey’s post hoc test; p< 0.05; Figure 4A, B). Exposure of DLD1 cells to RF-EMF
inhibited miR-34a-mediated effects on per2 mRNA expression (Tukey’s post hoc test;
Figure 4A,B). We also observed a significant effect of RF-EMF on per2 expression in
groups that were treated with the mimic and inhibitor (m-m + i) together, where a sig-
nificant decrease in per2 mRNA expression was detected as a result of RF-EMF exposure
(Šídák’s multiple comparisons test; Figure 4A).
The statistical analysis implicated a significant effect of transfected oligos on clock
mRNA expression (m-m: F [2, 18] = 4.58, p< 0.05, Figure 5A; pre-m: F [2, 18] = 8.04, p< 0.01,
Figure 5B), and the post hoc analysis confirmed a difference between cells transfected
with the mimic + inhibitor compared to the control (Tukey’s post hoc test, p< 0.05, Figure
5B). Two-way ANOVA did not reveal an effect of RF-EMF exposure on clock mRNA ex-
pression or an interaction between the tested factors.
Figure 5. Effect of miR-34a administration on clock mRNA expression in DLD1 cells under control
and RF-EMF conditions. Cells were transfected with (A) miR-34a-5p (m-m), (B) pre-miR-34a
(pre-m), mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding
negative control of mature and pre-miRNA (mNC and preNC, respectively). The data were rela-
tivised to the corresponding negative control measured under control conditionspresented as the
mean ± SEM (n = 4). a.u.—arbitrary units. Two-way ANOVA, Tukey’s post hoc test, * p < 0.05.
Two-way ANOVA revealed a significant effect of miR-34a on bmal1 mRNA expres-
sion (m-m: F [2, 17] = 7.879, p< 0.01, Figure 6A; pre-m: F [2, 17] = 10.31, p< 0.01, Figure 6B), and a
m-m m-m + i mNC m-m m-m + i mNC
mRNA clock/s17 (a.u.)
Control
RF-EMF
0.09
1.0
2.0
3.0
4.0
0.0
A
pre-m m-m + i preNC pre-m m-m + i preNC
mRNA clock/s17 (a.u.)
Control
RF-EMF
1.0
2.0
3.0
4.0
0.0
B✱
✱
m-m m-m + i mNC m-m m-m + i mNC
mRNA per2/s17 (a.u.)
Control
RF-EMF
A
✱✱
#
✱
✱✱
✱✱✱✱
1.0
2.0
3.0
4.0
0.0
pre-m m-m + i preNC pre-m m-m + i preNC
mRNA per2/s17 (a.u.)
Control
RF-EMF
1.0
2.0
3.0
4.0
0.0
✱✱
✱✱✱
#
✱
✱✱
B
Figure 4.
Effect of miR-34a administration on per2 mRNA expression in DLD1 cells cultured in
Wi-Fi-free conditions (white columns) or exposed to 24 h lasting RF-EMF exposure (grey columns).
Cells were transfected with (
A
) miR-34a-5p (m-m), (
B
) pre-miR-34a (pre-m), mimic of mature miR-
34a together with the miR-34a inhibitor (m-m + i) or corresponding negative control of mature and
pre-miRNA (mNC and preNC, respectively). The data were relativised to the corresponding negative
control measured under control conditions and presented as the mean
±
SEM (n = 4). a.u.—arbitrary
units. Two-way ANOVA, Tukey’s post hoc test, * p< 0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001—
comparison between groups transfected with different oligos within the control or RF-EMF treatment.
Two-way ANOVA, Šídák’s multiple comparisons test, # p< 0.05 comparison between the control and
RF-EMF groups.
The post hoc test demonstrated that miR-34a strongly inhibits per2 expression com-
pared to the negative control under Wi-Fi-free conditions. Moreover, the miR-34a inhibitor
reversed this effect and caused an increase in per2 expression compared to the control
(Tukey’s post hoc test; p< 0.05; Figure 4A,B). Exposure of DLD1 cells to RF-EMF inhibited
miR-34a-mediated effects on per2 mRNA expression (Tukey’s post hoc test; Figure 4A,B).
We also observed a significant effect of RF-EMF on per2 expression in groups that were
treated with the mimic and inhibitor (m-m + i) together, where a significant decrease in
per2 mRNA expression was detected as a result of RF-EMF exposure (Šídák’s multiple
comparisons test; Figure 4A).
The statistical analysis implicated a significant effect of transfected oligos on clock
mRNA expression (m-m: F
[2, 18]
= 4.58, p< 0.05, Figure 5A; pre-m: F
[2, 18]
= 8.04, p< 0.01,
Figure 5B), and the post hoc analysis confirmed a difference between cells transfected with
the mimic + inhibitor compared to the control (Tukey’s post hoc test, p< 0.05, Figure 5B).
Two-way ANOVA did not reveal an effect of RF-EMF exposure on clock mRNA expression
or an interaction between the tested factors.
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 of 22
Figure 4. Effect of miR-34a administration on per2 mRNA expression in DLD1 cells cultured in
Wi-Fi-free conditions (white columns) or exposed to 24h lasting RF-EMF exposure (grey columns).
Cells were transfected with (A) miR-34a-5p (m-m), (B) pre-miR-34a (pre-m), mimic of mature
miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding negative control of mature
and pre-miRNA (mNC and preNC, respectively). The data were relativised to the corresponding
negative control measured under control conditions and presented as the mean ± SEM (n = 4).
a.u.—arbitrary units. Two-way ANOVA, Tukey’s post hoc test, * p< 0.05, ** p < 0.01, *** p < 0.001,
**** p< 0.0001—comparison between groups transfected with different oligos within the control or
RF-EMF treatment. Two-way ANOVA, Šídák’s multiple comparisons test, # p< 0.05 comparison
between the control and RF-EMF groups.
The post hoc test demonstrated that miR-34a strongly inhibits per2 expression
compared to the negative control under Wi-Fi-free conditions. Moreover, the miR-34a
inhibitor reversed this effect and caused an increase in per2 expression compared to the
control (Tukey’s post hoc test; p< 0.05; Figure 4A, B). Exposure of DLD1 cells to RF-EMF
inhibited miR-34a-mediated effects on per2 mRNA expression (Tukey’s post hoc test;
Figure 4A,B). We also observed a significant effect of RF-EMF on per2 expression in
groups that were treated with the mimic and inhibitor (m-m + i) together, where a sig-
nificant decrease in per2 mRNA expression was detected as a result of RF-EMF exposure
(Šídák’s multiple comparisons test; Figure 4A).
The statistical analysis implicated a significant effect of transfected oligos on clock
mRNA expression (m-m: F [2, 18] = 4.58, p< 0.05, Figure 5A; pre-m: F [2, 18] = 8.04, p< 0.01,
Figure 5B), and the post hoc analysis confirmed a difference between cells transfected
with the mimic + inhibitor compared to the control (Tukey’s post hoc test, p< 0.05, Figure
5B). Two-way ANOVA did not reveal an effect of RF-EMF exposure on clock mRNA ex-
pression or an interaction between the tested factors.
Figure 5. Effect of miR-34a administration on clock mRNA expression in DLD1 cells under control
and RF-EMF conditions. Cells were transfected with (A) miR-34a-5p (m-m), (B) pre-miR-34a
(pre-m), mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding
negative control of mature and pre-miRNA (mNC and preNC, respectively). The data were rela-
tivised to the corresponding negative control measured under control conditionspresented as the
mean ± SEM (n = 4). a.u.—arbitrary units. Two-way ANOVA, Tukey’s post hoc test, * p < 0.05.
Two-way ANOVA revealed a significant effect of miR-34a on bmal1 mRNA expres-
sion (m-m: F [2, 17] = 7.879, p< 0.01, Figure 6A; pre-m: F [2, 17] = 10.31, p< 0.01, Figure 6B), and a
m-m m-m + i mNC m-m m-m + i mNC
mRNA clock/s17 (a.u.)
Control
RF-EMF
0.09
1.0
2.0
3.0
4.0
0.0
A
pre-m m-m + i preNC pre-m m-m + i preNC
mRNA clock/s17 (a.u.)
Control
RF-EMF
1.0
2.0
3.0
4.0
0.0
B✱
✱
m-m m-m + i mNC m-m m-m + i mNC
mRNA per2/s17 (a.u.)
Control
RF-EMF
A
✱✱
#
✱
✱✱
✱✱✱✱
1.0
2.0
3.0
4.0
0.0
pre-m m-m + i preNC pre-m m-m + i preNC
mRNA per2/s17 (a.u.)
Control
RF-EMF
1.0
2.0
3.0
4.0
0.0
✱✱
✱✱✱
#
✱
✱✱
B
Figure 5.
Effect of miR-34a administration on clock mRNA expression in DLD1 cells under control
and RF-EMF conditions. Cells were transfected with (
A
) miR-34a-5p (m-m), (
B
) pre-miR-34a (pre-m),
mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding negative
control of mature and pre-miRNA (mNC and preNC, respectively). The data were relativised to the
corresponding negative control measured under control conditionspresented as the mean
±
SEM
(n = 4). a.u.—arbitrary units. Two-way ANOVA, Tukey’s post hoc test, * p< 0.05.
Two-way ANOVA revealed a significant effect of miR-34a on bmal1 mRNA expression
(m-m: F
[2, 17]
= 7.879, p< 0.01, Figure 6A; pre-m: F
[2, 17]
= 10.31, p< 0.01, Figure 6B), and
a post hoc test confirmed a significant decrease in bmal1 levels in cells transfected with
miR-34a compared to control and/or cells transfected with the mimic + inhibitor under
Int. J. Mol. Sci. 2022,23, 13210 7 of 21
control conditions (Tukey’s post hoc test, Figure 6A,B). RF-EMF exposure also influenced
bmal1 levels in DLD1 cells (m-m: F
[1, 17]
= 6.023; p< 0.05), and a post hoc test confirmed
a significantly higher level of bmal1 mRNA expression in cells transfected with the miR-
34a mimic alone cultured in the RF-EMF environment than expression measured in the
corresponding group cultured under Wi-Fi-free conditions (Šídák’s multiple comparisons
test, Figure 6A). This increase may be related to the diminishing of inhibitory influence of
miR-34a observed under the control conditions. Interference between tested factors was
not significant, although it was close (m-m: F [2, 17] = 3.389; p= 0.06).
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 7 of 22
post hoc test confirmed a significant decrease in bmal1 levels in cells transfected with
miR-34a compared to control and/or cells transfected with the mimic + inhibitor under
control conditions (Tukey’s post hoc test, Figure 6A,B). RF-EMF exposure also influenced
bmal1 levels in DLD1 cells (m-m: F [1, 17] = 6.023; p< 0.05), and a post hoc test confirmed a
significantly higher level of bmal1 mRNA expression in cells transfected with the miR-34a
mimic alone cultured in the RF-EMF environment than expression measured in the cor-
responding group cultured under Wi-Fi-free conditions (Šídák’s multiple comparisons
test, Figure 6A). This increase may be related to the diminishing of inhibitory influence of
miR-34a observed under the control conditions. Interference between tested factors was
not significant, although it was close (m-m: F [2, 17] = 3.389; p = 0.06).
Figure 6. Effect of miR-34a administration on bmal1 mRNA expression in DLD1 cells under control
and RF-EMF conditions. Cells were transfected with (A) miR-34a-5p (m-m), (B) pre-miR-34a
(pre-m), mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding
negative control of mature and pre-miRNA (mNC and preNC, respectively). The data were rela-
tivised to the corresponding negative control measured under control conditions and presented as
the mean ± SEM (n = 4). a.u.—arbitrary units. Two-way ANOVA, Tukey’s post hoc test, * p< 0.05, **
p < 0.01, comparison between groups transfected with different oligos within the control or RF-EMF
treatment. Two-way ANOVA, Šídák’s multiple comparisons test, # p< 0.05 comparison between
control and RF-EMF groups.
Two-way ANOVA confirmed the effect of transfected oligos (m-m: F [2, 17] = 9.988, p<
0.01, Figure 1A; pre-m: F [2, 17] = 11.17, p< 0.001) on sirt1 mRNA expression regardless of the
RF-EMF factor. Post hoc tests revealed a significant decrease in sirt1 mRNA expression in
miR-34a transfected cells compared to those also transfected with the inhibitor under
control and RF-EMF conditions (Tukey’s and Šídák’s multiple comparisons test, respec-
tively; Figure 7A,B). Statistical analysis did not indicate an interaction of the tested fac-
tors.
Figure 7. Effect of miR-34a administration on sirt1 (sirtuin 1) mRNA expression in DLD1 cells under
control and RF-EMF conditions. Cells were transfected with (A) miR-34a-5p (m-m), (B)
pre-miR-34a (pre-m), mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or
corresponding negative control of mature and pre-miRNA (mNC and preNC, respectively). The
data were relativised to the corresponding negative control measured under control conditions and
presented as the mean ± SEM (n = 4). a.u.—arbitrary units. Two-way ANOVA, Tukey’s post hoc
test, * p< 0.05, ** p < 0.01.
m-m m-m + i mNC m-m m-m + i mNC
mRNA bmal1/s17 (a.u.)
Control
RF-EMF
A
✱✱
✱
#
1.0
2.0
3.0
4.0
0.0
pre-m m-m + i preNC pre-m m-m + i preNC
mRNA bmal1/s17 (a.u.)
Control
RF-EMF
B
1.0
2.0
3.0
4.0
0.0
✱✱ 0.08 0.09 ✱✱
m-m m-m + i mNC m-m m-m + i mNC
mRNA sirt1/s17 (a.u.)
Control
RF-EMF
1.0
2.0
3.0
4.0 A
0.0
✱✱
✱
pre-m m-m + i preNC pre-m m-m + i preNC
0.0
1.0
2.0
3.0
4.0
mRNA sirt1/s17 (a.u.)
Control
RF-EMF
B
✱✱✱✱ ✱
Figure 6.
Effect of miR-34a administration on bmal1 mRNA expression in DLD1 cells under control
and RF-EMF conditions. Cells were transfected with (
A
) miR-34a-5p (m-m), (
B
) pre-miR-34a (pre-m),
mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding negative
control of mature and pre-miRNA (mNC and preNC, respectively). The data were relativised
to the corresponding negative control measured under control conditions and presented as the
mean ±SEM
(n = 4). a.u.—arbitrary units. Two-way ANOVA, Tukey’s post hoc test, * p< 0.05,
** p< 0.01
, comparison between groups transfected with different oligos within the control or RF-
EMF treatment. Two-way ANOVA, Šídák’s multiple comparisons test, # p< 0.05 comparison between
control and RF-EMF groups.
Two-way ANOVA confirmed the effect of transfected oligos (m-m: F
[2, 17]
= 9.988,
p< 0.01
, Figure 1A; pre-m: F
[2, 17]
= 11.17, p< 0.001) on sirt1 mRNA expression regardless of
the RF-EMF factor. Post hoc tests revealed a significant decrease in sirt1 mRNA expression in
miR-34a transfected cells compared to those also transfected with the inhibitor under control
and RF-EMF conditions (Tukey’s and Šídák’s multiple comparisons test, respectively;
Figure 7A,B). Statistical analysis did not indicate an interaction of the tested factors.
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 7 of 22
post hoc test confirmed a significant decrease in bmal1 levels in cells transfected with
miR-34a compared to control and/or cells transfected with the mimic + inhibitor under
control conditions (Tukey’s post hoc test, Figure 6A,B). RF-EMF exposure also influenced
bmal1 levels in DLD1 cells (m-m: F [1, 17] = 6.023; p< 0.05), and a post hoc test confirmed a
significantly higher level of bmal1 mRNA expression in cells transfected with the miR-34a
mimic alone cultured in the RF-EMF environment than expression measured in the cor-
responding group cultured under Wi-Fi-free conditions (Šídák’s multiple comparisons
test, Figure 6A). This increase may be related to the diminishing of inhibitory influence of
miR-34a observed under the control conditions. Interference between tested factors was
not significant, although it was close (m-m: F [2, 17] = 3.389; p = 0.06).
Figure 6. Effect of miR-34a administration on bmal1 mRNA expression in DLD1 cells under control
and RF-EMF conditions. Cells were transfected with (A) miR-34a-5p (m-m), (B) pre-miR-34a
(pre-m), mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding
negative control of mature and pre-miRNA (mNC and preNC, respectively). The data were rela-
tivised to the corresponding negative control measured under control conditions and presented as
the mean ± SEM (n = 4). a.u.—arbitrary units. Two-way ANOVA, Tukey’s post hoc test, * p< 0.05, **
p < 0.01, comparison between groups transfected with different oligos within the control or RF-EMF
treatment. Two-way ANOVA, Šídák’s multiple comparisons test, # p< 0.05 comparison between
control and RF-EMF groups.
Two-way ANOVA confirmed the effect of transfected oligos (m-m: F [2, 17] = 9.988, p<
0.01, Figure 1A; pre-m: F [2, 17] = 11.17, p< 0.001) on sirt1 mRNA expression regardless of the
RF-EMF factor. Post hoc tests revealed a significant decrease in sirt1 mRNA expression in
miR-34a transfected cells compared to those also transfected with the inhibitor under
control and RF-EMF conditions (Tukey’s and Šídák’s multiple comparisons test, respec-
tively; Figure 7A,B). Statistical analysis did not indicate an interaction of the tested fac-
tors.
Figure 7. Effect of miR-34a administration on sirt1 (sirtuin 1) mRNA expression in DLD1 cells under
control and RF-EMF conditions. Cells were transfected with (A) miR-34a-5p (m-m), (B)
pre-miR-34a (pre-m), mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or
corresponding negative control of mature and pre-miRNA (mNC and preNC, respectively). The
data were relativised to the corresponding negative control measured under control conditions and
presented as the mean ± SEM (n = 4). a.u.—arbitrary units. Two-way ANOVA, Tukey’s post hoc
test, * p< 0.05, ** p < 0.01.
m-m m-m + i mNC m-m m-m + i mNC
mRNA bmal1/s17 (a.u.)
Control
RF-EMF
A
✱✱
✱
#
1.0
2.0
3.0
4.0
0.0
pre-m m-m + i preNC pre-m m-m + i preNC
mRNA bmal1/s17 (a.u.)
Control
RF-EMF
B
1.0
2.0
3.0
4.0
0.0
✱✱ 0.08 0.09 ✱✱
m-m m-m + i mNC m-m m-m + i mNC
mRNA sirt1/s17 (a.u.)
Control
RF-EMF
1.0
2.0
3.0
4.0 A
0.0
✱✱
✱
pre-m m-m + i preNC pre-m m-m + i preNC
0.0
1.0
2.0
3.0
4.0
mRNA sirt1/s17 (a.u.)
Control
RF-EMF
B
✱✱✱✱ ✱
Figure 7.
Effect of miR-34a administration on sirt1 (sirtuin 1) mRNA expression in DLD1 cells
under control and RF-EMF conditions. Cells were transfected with (
A
) miR-34a-5p (m-m),
(B) pre-miR-34a (pre-m)
, mimic of mature miR-34a together with the miR-34a inhibitor (
m-m + i
) or
corresponding negative control of mature and pre-miRNA (mNC and preNC, respectively). The
data were relativised to the corresponding negative control measured under control conditions and
presented as the
mean ±SEM
(n = 4). a.u.—arbitrary units. Two-way ANOVA, Tukey’s post hoc test,
*p< 0.05, ** p< 0.01.
Expression of survivin was inhibited by miR-34a transfection (m-m: F
[2, 17]
= 8.753,
p< 0.01, Figure 8A; pre-m: F
[2, 17]
= 5.412, p< 0.05, Figure 8B) and Tukey’s post hoc test
confirmed a significant decrease in survivin expression in cells transfected with miR-34a
compared to the control or compared to the mimic + inhibitor (Šídák’s multiple comparisons
Int. J. Mol. Sci. 2022,23, 13210 8 of 21
test, Figure 8A,B) in Wi-Fi-free conditions. Two-way ANOVA was not significant for RF-
EMF (m-m: F
[1, 17]
= 3.259; p= 0.08); however, the decrease in survivin expression after
miR-34a administration observed in the Wi-Fi-free environment, according to Tukey’s post
hoc, diminished in cells exposed to RF-EMF (Figure 8A,B).
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 8 of 22
Expression of survivin was inhibited by miR-34a transfection (m-m: F [2, 17] = 8.753, p<
0.01, Figure 8A; pre-m: F [2, 17] = 5.412, p< 0.05, Figure 8B) and Tukey’s post hoc test con-
firmed a significant decrease in survivin expression in cells transfected with miR-34a
compared to the control or compared to the mimic + inhibitor (Šídák’s multiple compar-
isons test, Figure 8A,B) in Wi-Fi-free conditions. Two-way ANOVA was not significant
for RF-EMF (m-m: F [1, 17] = 3.259; p = 0.08); however, the decrease in survivin expression
after miR-34a administration observed in the Wi-Fi-free environment, according to
Tukey’s post hoc, diminished in cells exposed to RF-EMF (Figure 8A,B).
Figure 8.Effect of miR-34a administration on survivin mRNA expression in DLD1 cells under con-
trol and RF-EMF conditions. Cells were transfected with (A) miR-34a-5p (m-m), (B) pre-miR-34a
(pre-m), mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding
negative control of mature and pre-miRNA (mNC and preNC, respectively). The data were rela-
tivised to the corresponding negative control measured under control conditions and presented as
the mean ± SEM (n = 4). a.u.—arbitrary units. Two-way ANOVA, Tukey’s post hoc test, * p< 0.05, **
p < 0.01.
Scratch assay: A two-way ANOVA indicated a significant inhibitory effect of
miR-34a (48 h) on wound closure (pre-m: F [2, 18] = 8.232; p< 0.01, Figure 9B) and Tukey’s
post hoc test confirmed significantly slower wound closure in cells transfected with
miR-34a compared to cells transfected with the mimic together with the inhibitor or to
the negative control under Wi-Fi-free conditions (Figure 9B). This effect was not observed
in cells transfected with miR-34a exposed to RF-EMF for 24 h (Tukey’s post hoc test,
Figure 9B).
Figure 9.Effect of miR-34a administration on wound healing in DLD1 cells under control and
RF-EMF conditions. Cells were transfected with (A) miR-34a-5p (m-m), (B) pre-miR-34a (pre-m),
mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding negative
control of mature and pre-miRNA (mNC and preNC, respectively). Data obtained 24 h after
wound generation were relativised to the corresponding cell-free area of the wound at time 0 h.
Data are presented as the mean ± SEM (n = 4). Two-way ANOVA, Tukey’s post hoc test, * p< 0.05, **
p< 0.01.
MTS assay: The results indicate a significant effect of RF-EMF exposure (48 h) on
proliferation (pre-m: F [1, 83] = 7.215, p< 0.01, Figure 10B) and the interaction between the
RF-EMF and transfected oligos (pre-m: F [2, 83] = 6.330, p< 0.01). Post hoc analysis con-
firmed that miR-34a (48 h) reduced the proliferation of DLD1 cells compared to the cor-
responding control under Wi-Fi-free conditions (Tukey’s post hoc test; p< 0.01; Figure
m-m m-m + i mNC m-m m-m + i mNC
mRNA survivin/s17 (a.u.)
Control
RF-EMF
✱✱
A
p = 0.08
1.0
2.0
3.0
4.0
0.0
pre-m m-m + i preNC pre-m m-m + i preNC
mRNA survivin/s17 (a.u.)
Control
RF-EMF
✱
B
1.0
2.0
3.0
4.0
0.0
Figure 8.
Effect of miR-34a administration on survivin mRNA expression in DLD1 cells under control
and RF-EMF conditions. Cells were transfected with (
A
) miR-34a-5p (m-m), (
B
) pre-miR-34a (pre-m),
mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding negative
control of mature and pre-miRNA (mNC and preNC, respectively). The data were relativised to the
corresponding negative control measured under control conditions and presented as the
mean ±SEM
(n = 4). a.u.—arbitrary units. Two-way ANOVA, Tukey’s post hoc test, * p< 0.05, ** p< 0.01.
Scratch assay: A two-way ANOVA indicated a significant inhibitory effect of miR-34a
(48 h) on wound closure (pre-m: F
[2, 18]
= 8.232; p< 0.01, Figure 9B) and Tukey’s post hoc test
confirmed significantly slower wound closure in cells transfected with miR-34a compared
to cells transfected with the mimic together with the inhibitor or to the negative control
under Wi-Fi-free conditions (Figure 9B). This effect was not observed in cells transfected
with miR-34a exposed to RF-EMF for 24 h (Tukey’s post hoc test, Figure 9B).
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 8 of 22
Expression of survivin was inhibited by miR-34a transfection (m-m: F [2, 17] = 8.753, p<
0.01, Figure 8A; pre-m: F [2, 17] = 5.412, p< 0.05, Figure 8B) and Tukey’s post hoc test con-
firmed a significant decrease in survivin expression in cells transfected with miR-34a
compared to the control or compared to the mimic + inhibitor (Šídák’s multiple compar-
isons test, Figure 8A,B) in Wi-Fi-free conditions. Two-way ANOVA was not significant
for RF-EMF (m-m: F [1, 17] = 3.259; p = 0.08); however, the decrease in survivin expression
after miR-34a administration observed in the Wi-Fi-free environment, according to
Tukey’s post hoc, diminished in cells exposed to RF-EMF (Figure 8A,B).
Figure 8.Effect of miR-34a administration on survivin mRNA expression in DLD1 cells under con-
trol and RF-EMF conditions. Cells were transfected with (A) miR-34a-5p (m-m), (B) pre-miR-34a
(pre-m), mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding
negative control of mature and pre-miRNA (mNC and preNC, respectively). The data were rela-
tivised to the corresponding negative control measured under control conditions and presented as
the mean ± SEM (n = 4). a.u.—arbitrary units. Two-way ANOVA, Tukey’s post hoc test, * p< 0.05, **
p < 0.01.
Scratch assay: A two-way ANOVA indicated a significant inhibitory effect of
miR-34a (48 h) on wound closure (pre-m: F [2, 18] = 8.232; p< 0.01, Figure 9B) and Tukey’s
post hoc test confirmed significantly slower wound closure in cells transfected with
miR-34a compared to cells transfected with the mimic together with the inhibitor or to
the negative control under Wi-Fi-free conditions (Figure 9B). This effect was not observed
in cells transfected with miR-34a exposed to RF-EMF for 24 h (Tukey’s post hoc test,
Figure 9B).
Figure 9.Effect of miR-34a administration on wound healing in DLD1 cells under control and
RF-EMF conditions. Cells were transfected with (A) miR-34a-5p (m-m), (B) pre-miR-34a (pre-m),
mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding negative
control of mature and pre-miRNA (mNC and preNC, respectively). Data obtained 24 h after
wound generation were relativised to the corresponding cell-free area of the wound at time 0 h.
Data are presented as the mean ± SEM (n = 4). Two-way ANOVA, Tukey’s post hoc test, * p< 0.05, **
p< 0.01.
MTS assay: The results indicate a significant effect of RF-EMF exposure (48 h) on
proliferation (pre-m: F [1, 83] = 7.215, p< 0.01, Figure 10B) and the interaction between the
RF-EMF and transfected oligos (pre-m: F [2, 83] = 6.330, p< 0.01). Post hoc analysis con-
firmed that miR-34a (48 h) reduced the proliferation of DLD1 cells compared to the cor-
responding control under Wi-Fi-free conditions (Tukey’s post hoc test; p< 0.01; Figure
m-m m-m + i mNC m-m m-m + i mNC
mRNA survivin/s17 (a.u.)
Control
RF-EMF
✱✱
A
p = 0.08
1.0
2.0
3.0
4.0
0.0
pre-m m-m + i preNC pre-m m-m + i preNC
mRNA survivin/s17 (a.u.)
Control
RF-EMF
✱
B
1.0
2.0
3.0
4.0
0.0
Figure 9.
Effect of miR-34a administration on wound healing in DLD1 cells under control and
RF-EMF conditions. Cells were transfected with (
A
) miR-34a-5p (m-m), (
B
) pre-miR-34a (pre-m),
mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding negative
control of mature and pre-miRNA (mNC and preNC, respectively). Data obtained 24 h after wound
generation were relativised to the corresponding cell-free area of the wound at time 0 h. Data are
presented as the mean
±
SEM (n = 4). Two-way ANOVA, Tukey’s post hoc test, * p< 0.05, ** p< 0.01.
MTS assay: The results indicate a significant effect of RF-EMF exposure (48 h) on
proliferation (pre-m: F
[1, 83]
= 7.215, p< 0.01, Figure 10B) and the interaction between the
RF-EMF and transfected oligos (pre-m: F
[2, 83]
= 6.330, p< 0.01). Post hoc analysis confirmed
that miR-34a (48 h) reduced the proliferation of DLD1 cells compared to the corresponding
control under Wi-Fi-free conditions (Tukey’s post hoc test; p< 0.01; Figure 10B). Proliferation
showed a decreasing trend (p= 0.08) in miR-34a-treated cells compared to cells transfected
with the mimic together with the inhibitor under control conditions (Tukey’s post hoc test;
p< 0.01
; Figure 10B). Exposure of cells to RF-EMF caused a diminishing of miR-34a-induced
inhibition in cell proliferation under Wi-Fi-free conditions (Šídák’s multiple comparisons
test, Figure 10B). Instead, we observed an increase in proliferation in cells exposed to
miR-34a together with the inhibitor compared to the corresponding control under RF-EMF
conditions (Šídák’s multiple comparisons test, Figure 10A,B).
Int. J. Mol. Sci. 2022,23, 13210 9 of 21
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 9 of 22
10B). Proliferation showed a decreasing trend (p = 0.08) in miR-34a-treated cells com-
pared to cells transfected with the mimic together with the inhibitor under control con-
ditions (Tukey’s post hoc test; p< 0.01; Figure 10B). Exposure of cells to RF-EMF caused a
diminishing of miR-34a-induced inhibition in cell proliferation under Wi-Fi-free condi-
tions (Šídák’s multiple comparisons test, Figure 10B). Instead, we observed an increase
in proliferation in cells exposed to miR-34a together with the inhibitor compared to the
corresponding control under RF-EMF conditions (Šídák’s multiple comparisons test,
Figure 10A, B).
Figure 10. Effect of miR-34a administration on the proliferation of DLD1 cells under control and
RF-EMF conditions. Cells were transfected with (A) miR-34a-5p (m-m), (B) pre-miR-34a (pre-m),
mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding negative
control of mature and pre-miRNA (mNC and preNC, respectively) and incubated for 48 h; then,
cell proliferation was assessed by a colorimetric MTS assay. Data are presented as the mean ± SEM
(n = 4). Two-way ANOVA, Tukey’s post hoc test, * p< 0.05, ** p< 0.01, comparison between groups
transfected with different oligos within control or RF-EMF treatment. Two-way ANOVA, Šídák’s
multiple comparisons test, ## p< 0.01 comparison between control and RF-EMF groups.
In most cases, the administration of the mature dominant strand and precursor form
of miR-34a-5p caused a similar effect. In some cases, however, more pronounced results
were observed when pre-miR-34a was administered instead of miR-34a-5p. This finding
is addressed in the discussion in more detail. However, as the administration of precur-
sor and mature forms of miR-34a increased the intracellular level of miR-34a-5p identi-
cally (Figure 1A,B), we consider both treatments to be equally informative.
m-m m-m + i mNC m-m m-m + i mNC
cell proliferation (a.u.)
Control
RF-EMF
A
1.0
2.0
3.0
4.0
0.0
0.09
pre-m m-m + i preNC pre-m m-m + i preNC
cell proliferation (a.u.)
Control
RF-EMF
B
1.0
2.0
3.0
4.0
0.0
✱
0.08
✱✱
##
Figure 10.
Effect of miR-34a administration on the proliferation of DLD1 cells under control and
RF-EMF conditions. Cells were transfected with (
A
) miR-34a-5p (m-m), (
B
) pre-miR-34a (pre-m),
mimic of mature miR-34a together with the miR-34a inhibitor (m-m + i) or corresponding negative
control of mature and pre-miRNA (mNC and preNC, respectively) and incubated for 48 h; then,
cell proliferation was assessed by a colorimetric MTS assay. Data are presented as the mean
±
SEM
(
n=4
). Two-way ANOVA, Tukey’s post hoc test, * p< 0.05, ** p< 0.01, comparison between groups
transfected with different oligos within control or RF-EMF treatment. Two-way ANOVA, Šídák’s
multiple comparisons test, ## p< 0.01 comparison between control and RF-EMF groups.
In most cases, the administration of the mature dominant strand and precursor form
of miR-34a-5p caused a similar effect. In some cases, however, more pronounced results
were observed when pre-miR-34a was administered instead of miR-34a-5p. This finding is
addressed in the discussion in more detail. However, as the administration of precursor
and mature forms of miR-34a increased the intracellular level of miR-34a-5p identically
(Figure 1A,B), we consider both treatments to be equally informative.
3. Discussion
Recent data indicate that 2.4 GHz RF-EMF influence the effect of miR-34a on the
transcription of clock genes and survivin (birc5), an inhibitor of apoptosis. Under control
conditions, miR-34a administration caused a significant decrease in per2,bmal1,sirt1 and
survivin mRNA expression. However, the exposure of DLD1 cells to RF-EMF resulted in
weakening or diminishing of the miR-34a effect on per2 and survivin, respectively. Moreover,
RF-EMF administration was accompanied by a significant increase in cry1 expression in
cells treated with miR-34a. The effect of RF-EMF was also reflected at the level of cell
migration and viability, as miR-34a significantly decreased wound closure and metabolism
intensity in cell culture under control conditions, while this effect was not observed when
cells were exposed to RF-EMF.
A decrease in per2 expression after miR-34a has been implicated previously by in silico
analysis [
67
], cross-linking immunoprecipitation sequencing (GSE161238, GSE161239), a
negative correlation between per2 expression and miR-34a was observed in colorectal cancer
tissue [
27
] and recent results clearly confirmed that miR-34a inhibits the expression of per2
under in vitro conditions.
A decrease in bmal1 expression observed after miR-34a administration is in agreement
with the in silico analysis performed by miRWalk [
68
]. However, we did not observe a
decrease in clock gene expression in miR-34a-treated cells, which was implicated by in silico
analysis [
67
] and observed previously in human oesophageal epithelium [
69
], which can
be attributed to different cell types and/or methodological differences in the experimental
setups. cry1 is a predicted target of miR-34a according to miRWalk [
68
], and cry2 has been
predicted to be a target of miR-34a by TargetScan [
67
]. However, in a recent study, we
observed an increase in cry genes expression induced by miR-34a administration, which
implicates other than 30UTR-mediated regulation.
It was shown that miRNAs (typically inhibitors) can also induce gene expression. In
particular, one of the pioneer studies showed that miR-373 induces the gene expression of E-
cadherin via its complementary region in the promoter sequence [
70
]. A similar mechanism
of gene expression induction was also shown for other genes, and it was revealed that
miRNAs localised in the nucleus can, in cooperation with Argonaute proteins (AGO),
Int. J. Mol. Sci. 2022,23, 13210 10 of 21
interact with gene promoters and either activate or inhibit transcription [
71
]. In silico
analysis indicated the presence of a miRNA-responsive element with 75% homology with a
seed sequence of miR-34a-5p in the promoter region of cry1 and no responsive element in the
UTR region [
67
,
72
]. Thus, upregulation of cry1 expression via AGO-mediated interaction
is a possible mechanism for how miR-34a could induce cry1 expression under constant
conditions. Interestingly, this upregulation was potentiated under RF-EMF conditions.
In accordance with an in silico analysis [
67
], we observed an inhibition of sirt1 ex-
pression after mir-34a administration. Inhibition of sirt1 expression had been previously
shown in colorectal cancer cell lines HCT-116 [
73
,
74
], DLD1 [
75
], SW480 [
76
] and a kidney
epithelial cell line [77], and our data are in accordance with these observations.
The inhibition of survivin expression by miR-34a was also predicted by in silico anal-
ysis [
67
]. The level of survivin mRNA expression was significantly decreased after 72 h
of incubation with miR-34a in the triple-negative breast cancer cell lines MDA-MB-231
and SUM-159 [
78
]. A similar regulatory relationship between survivin and miR-34a was
demonstrated in several gastric cancer cell lines [
79
–
81
]. The p53-dependent inhibition
of survivin expression induced by mir-34a administration was demonstrated in colorectal
cancer cell line HCT116 [
82
]. We observed pronounced downregulation of survivin expres-
sion after miR-34a administration in DLD1 cells, which is in accordance with previously
reported results.
Under recent experimental conditions, RF-EMF treatment was associated with signifi-
cant changes in the miR-34a-mediated regulation of five out of seven investigated target
genes. In cells exposed to RF-EMF, the downregulation of per2 and survivin expression
caused by miR-34a was weakened or eliminated, respectively, and expression of cry1 was
stimulated more by miR-34a in RF-EMF-treated cells compared to the control. In addition
to these major effects, two-way ANOVA also indicated a significant influence of RF-EMF
on the cry2 and bmal1 mRNA levels.
Recently, it is not known how 2.4 GHz RF-EMF cause these effects. As mentioned
above, clock genes are functionally interconnected via feedback loops and influence the
expression and/or functioning of each other [
83
–
87
]. If one of them is a functional sensor
of RF-EMF, this sensor can mediate the influence of RF-EMF on the expression of other
clock genes.
In this respect, the most likely candidates are cryptochromes. Cryptochromes are
evolutionary descendants of light-activated DNA repair enzymes that in mammals lost
the capacity to bind DNA and repair UV-induced DNA photoproducts. Instead, the role
of mammalian CRY1 and CRY2 proteins in the circadian loop influences gene expression
via the E-box, which emerged during evolution. The functioning of cry genes in the
generation of the circadian pattern of locomotor activity [
88
] and their capacity to influence
synchronisation by light has been proven in double mutant mice cry1-/-and cry2-/- [89,90].
In several insect and vertebrate species, the role of magnetoreception has been at-
tributed to CRY proteins [
91
,
92
], and the role of the CRY protein as an effector molecule
transmitting magnetosensing to the circadian system has been proven, at least in Drosophila
[
93
,
94
]. However, this function is believed to be based on photo-excitation of flavin ade-
nine dinucleotide (FAD) facilitated by the Trp-triad located in the CRY molecule, and the
structure of the FAD-binding domain in mammalian CRY proteins does not allow sufficient
FAD binding. Therefore, the role of CRY proteins in the mediation of electromagnetic fields
to the circadian system is not expected to be executed in this way in humans [91].
Despite the weak flavin binding affinity for mammalian CRYs, the role of human
CRY2 (hCRY2) in light-dependent magnetoreception was demonstrated, as in cry-deficient
Drosophila, responsiveness to the magnetic field was rescued by the insertion of human cry2
into the fly genome [
95
]. In a similar experiment with the use of humanised Drosophila,
the human cry1 (hCRY1) insert [
96
] restored the behavioural response of cry-deficient flies
to a weak pulsed electromagnetic field (PEMF) with a frequency of 10 Hz. Moreover,
the PEMF strongly induced reactive oxygen species (ROS) generation and influenced the
transcriptome [
97
]. ROS generation in response to RF-EMF was also reported in human
Int. J. Mol. Sci. 2022,23, 13210 11 of 21
HEK293 cells where the 1.8 GHz field strongly modified the expression of antioxidative
and oxidative enzymes [
98
]. The response of CRY1 to a magnetic field up to 1.32 mT under
in vitro conditions has also been demonstrated [99].
Therefore, there is still uncertainty about the role of FAD activation in the CRY-
mediated response to the magnetic and/or electromagnetic field, and several alternative
hypotheses have been proposed. One of them, based on a mutational study of CRY pro-
tein, showed that extremely low frequencies (3–50 Hz) modify the free running period in
Drosophila, even in the case where only the C-terminus of the CRY protein, which does not
contain the Trp-triad and FAD-binding pocket, is preserved. This observation suggests a
mechanism of electromagnetic field-induced effects on the circadian oscillator other than
that based on radical pairs theory and FAD activation [94].
Another open question is whether sensing of experimentally tested magnetic and
electromagnetic fields generally share a common mechanism and, if so, how broad a
range of intensities and frequencies are covered by this pathway. The argument for a
unique mechanism relies on the fact that a moving charge produces a magnetic field and
therefore electric and magnetic fields usually go together. Functional similarity between
magnetosensing and sensing of RF-EMF is implicated by studies evidencing that RF-EMF
can influence pathway(s) involved in magnetoreception, as RF-EMF of anthropogenic
origin, even with very low magnitude, disturb magnetoreceptor-based orientation [
100
].
The present results implicate the role of the CRY1 protein in RF-EMF-mediated effects
under the specific conditions used in our study.
Although the deregulation of clock genes under RF-EMF conditions could be ex-
plained by the sensory role of cry genes, the mechanism by which survivin expression can
respond to RF-EMF is completely unknown. A functional E-box was not revealed in the
survivin sequence, and survivin mRNA did not show rhythmic expression [
101
]. However,
previously, survivin expression was shown to have a negative correlation with the clock
gene per2 in patients with colorectal cancer [
102
]; thus, it is possible that survivin expres-
sion is functionally related to the circadian oscillator indirectly, at least in some specific
biological context.
Interestingly, RF-EMF effects of miR-34a are gene-specific, which implies the possible
involvement of modified interactions of miRNA and mRNA. This hypothesis is supported
by the observation that the electromagnetic field can influence hybridisation [
103
]; however,
in the context of RF-EMF frequency and the type of nucleic acid used, this assumption
needs to be evaluated.
Taken together, it is likely that more than one factor contributes to the gene-specific
response of RF-EMF-modulated effects of mR-34a.
Previously, the expression of miRNA was shown to be influenced by a wide spectrum
of electromagnetic field wavelengths. A 50 Hz electromagnetic field exposure lasting for
60 days
caused a decrease in miRNA levels in the brain and circulation of rats in a sex- and
age-dependent manner [
104
]. Long-time exposure to a 900 MHz RF-EMF caused a decrease
in miR-107 expression in the brain [
105
], and long-time exposure to a 2.4 GHz RF-EMF
caused changes in miR-106b, miR-107 and miR-181 expression in rat brain [106,107].
Under recent experimental conditions, we did not observe the effect of RF-EMF
directly on the miR-34a level in cells, but the effects exerted by this miRNA changed. As
many target genes of miR-34a, including sirt1 [
108
] and survivin [
109
], have been shown to
promote cancer progression and/or were associated with poor patient prognosis, miR-34a
is frequently referred to as a tumour suppressor [
62
]. However, diverse roles of newly
recognised target genes of miR-34a with respect to tumorigenesis were reported.
Overexpression of cry1 stimulated proliferation, colony formation and cell migration
in the HCT116 colorectal cell line and silencing of cry1 caused the inhibition of SW480 cell
proliferation. Under
in vivo
conditions, cry1 overexpression promoted colorectal cancer
growth [
110
]. High levels of cry1 were associated with the development of metachronous
metastasis [
111
] and worse survival in patients with colorectal cancer [
27
,
110
–
112
]. Up-
regulation of cry1 inhibited apoptosis in colorectal cell lines HT29 and SW480, increased
Int. J. Mol. Sci. 2022,23, 13210 12 of 21
proliferation in HCT116, HT29 and SW480, and decreased responsiveness to 5-fluorouracil
(5-FU) in HCT116 and SW480 cells [
112
]. However, downregulation of cry1 caused an
increase in osteosarcoma cell proliferation and migration and stimulated tumour growth
in nude mice under
in vivo
conditions [
113
]. Despite some contradictory reports in CRC
models, oncogenic features are usually attributed to cry1 in respect to CRC and gastric
cancer progression [114].
Similarly, as in the case of cry1, oncogenic effects were predominantly referred with
respect to the cry2 gene, as worse survival was associated with high cry2 expression in
colorectal cancer tissue [
27
,
112
,
115
]. Downregulation of cry2 expression in DLD1 and
SW480 cells showed pro-apoptotic effects of oxaliplatin (OXA) [
115
]. cry2 overexpression
in colorectal cell lines CaCo2 HT29 and SW480 inhibited apoptosis, increased proliferation
in HT29 and SW480 cells and decreased the response to 5-fluorouracil (5-FU) in HCT116
and SW480 CRC cells [112].
On the other hand, bmal1 exerts tumour-suppressive effects. bmal1 silencing increased
C26 cell proliferation in mice bearing C26 cell-derived tumours. Similarly, bmal1 downreg-
ulation caused a decrease in Etoposid-induced apoptosis and DNA damage induced by
cisplatin administration [
116
]. bmal1 silencing also caused a decreased rate of proliferation
in the SW480 CRC cell line, an increase in glycolytic activity, and a modified response to the
glucose transport inhibitor (VZB117) and OXA in SW480 and SW620 CRC cell lines [
117
].
Moreover, a high level of bmal1 was associated with better survival compared to low bmal1
expression in CRC patients [111].
Previously, the tumour-suppressive effects of per2 were referred to [
118
], as per2-
deficient mice were more susceptible to tumour induction by
γ
radiation compared to
wild-type mice. per2 mutant mice also showed a decrease in p53 and an increase in c-Myc
and cytochrome cresponse to
γ
radiation compared to the control. Therefore, per2 can
influence the DNA damage response [
119
]. In accordance with a previous observation,
per2 downregulation was associated with attenuated apoptosis and a delay in CHK2
response to double-stranded DNA breaks induced by doxorubicin in the HCT116 cell
line [
120
]. per2 inactivation increased cell proliferation,
β
-catenin and D cyclin levels in
HCT116 and SW480 cell lines and increased small intestinal and colon polyp numbers
in Apc mutant mice [
121
]. Overexpression of per2 sensitised cancer cell lines Panc1 and
Aspc1 to cisplatin, inhibited cell proliferation and induced apoptosis [
85
]. The tumour-
suppressive effect of per2 was demonstrated in oral squamous cell carcinoma (OSCC), as
per2 silencing inhibited autophagy, apoptosis and increased the proliferation rate [
122
,
123
].
Moreover, per2 silencing significantly decreased the expression of p53, p16 and p21 mRNA
and increased mRNA expression of cyclin A2, B1 and D1, and CDK4, CDK6 and E2F1 in
OSCC Tca8113 cells. These results imply that per2 plays an important role in cell cycle
progression [
123
]. Although better survival was found in CRC patients with high compared
to low per2 expression [111], in this aspect complete agreement has not yet been achieved,
as the association of per2 with survival has not been consistently observed [27,124].
In respect to the known capacity of miR-34a to attenuate tumour growth [
66
], its
influence on the circadian oscillator differs from that usually reported, as clock genes that
are inhibited by miR-34 administration (per2 and bmal1) are more often mentioned in the
context of tumour suppression than the opposite. However, clock genes are not major
effectors in miR-34a-mediated effects on cancer progression, and we suppose that effects
mediated by per2 and bmal1 are under control conditions overwhelmed by the effect of
other target genes of miR-34a, whose oncogenic capacity (e.g., sirt1 and survivin) is higher
than the tumour-suppressive capacity of the above-mentioned clock genes.
However, transfection of miR-34a increased the expression of cry1. This observation is
in accordance with a previous study referring to the positive correlation between miRNAs
and their target clock genes [
125
], although the mechanism of this regulation has not
been entirely elucidated. Cry1 is frequently referred to as a tumour promoter in colorectal
cancer [
114
]. Under the conditions of a recent study, miR-34a induced cry1 expression
only when cells were exposed to RF-EMF. Interestingly, the inhibitory effect of miR-34a on
Int. J. Mol. Sci. 2022,23, 13210 13 of 21
survivin expression diminished when the cells were exposed to RF-EMF, implicating the
gene-specific effect of RF-EMF on miR-34a-induced regulation. Although the mechanism
of this regulation is not known, it seems that RF-EMF have the capacity to switch the role
of miR-34a in cancer progression from typical tumour suppressor to neutral or slightly
oncogenic (at least in respect to the studied target genes).
This observation is in line with the RF-EMF-induced elimination of the decrease in cell
proliferation and migration observed after miR-34a administration under
control conditions
.
Generally, miR-34a is known to inhibit cell proliferation and migration [
82
,
126
,
127
],
which was confirmed in our study. In contrast, both cry1 and survivin have previously
been shown to possess the capacity to exert opposite effects [
110
,
112
,
128
,
129
]. Compre-
hensive meta-analysis confirmed that RF-EMF predominantly influence the growth of
human and faster-growing cells (45% and 47% of the included studies, respectively),
and the observed effects are cell type- and dose-dependent under
in vitro
[
130
] and
in vivo
[
131
–
133
] conditions. However, in HCT-116 and DLD1 cells, RF-EMF from
900–2100 MHz
applied up to 4 h did not induce changes in proliferation, as assessed
by the MTT test [
134
]. These results are in line with our study, as we also did not observe
a significant effect of RF-EMF on cell proliferation when miR-34a was not administered.
However, human colonic adenocarcinoma cells (Caco-2) were responsive to2.5GHz RF-
EMF, which caused a decrease in cell proliferation [
135
]. Therefore, even within clusters of
colorectal cell lines, the genetic background and/or transcriptome state of the cells can be
important in the manifestation of RF-EMF-mediated effects.
Although in most cases the effect of the mature form of the miR-34a dominant strand
and pre-miRNA showed very similar effects on gene expression, in some cases, a more
significant effect was observed (usually, administration of pre-miRNA was more potent).
We suspect that this effect can be caused by different rates of mature and premature miRNA
incorporation into the RISC complex (under normal conditions, pre-miRNA is bound by
DICER and incorporated into the RISC complex) and/or turnover half-time of pre-miRNA
and mature miRNA [
136
,
137
]. We previously observed a different pattern in miR-34a-5p
and its premature form, implicating a non-uniform rate of their processing [
138
]. An alterna-
tive explanation for differences in the output of miRNA vs. pre-miRNA administration can
be related to the involvement of the loop structure in the interference reaction. This feature
of miRNA-mediated regulation was observed previously when it was revealed [
139
] that
pre-miRNA can be more effective in the treatment of human gastric cancer cells compared
to the miR-34a mimic, as in some cases loop sequences can contribute to the efficiency
of gene targeting [
140
]. Knowledge about the possible role of loop sequences in target
recognition is also being incorporated into new miRNA-based therapeutics [141].
As studies aimed at the effects of RF-EMF often bring inconclusive results, observed
effects frequently do not show dose and/or time dependence or the response curve has
several peaks [
31
,
130
], it is difficult to make firm conclusions about the impact of RF-EMF
on the state of a particular biological system. Because of this broadly observed obstacle, the
attributive hypothesis has been postulated. Attributive theory is based on the existence of
pre-existing conditions whose presence allows to reveal the RF-EMF-mediated effect [
142
].
Our results are in complete agreement with this hypothesis, as under the conditions of a
recent study, RF-EMF exerted most of their effects only in cases when another treatment
was administered concurrently.
4. Materials and Methods
The human colon adenocarcinoma cell line DLD1 (CCL-221) was obtained from the
American Type Culture Collection (ATCC, Manassas, VA, USA). DLD1 cells were cultured
in RPMI 1640 medium (Thermo Fisher Scientific,Waltham, MA, USA) supplemented with
10% foetal bovine serum (FBS, Biosera, Nuaille, France), penicillin (50 U/mL) (Gibco,
St. Louis, MO, USA), streptomycin (50
µ
L/mL) (Gibco, USA), and ampicillin (50
µ
g/mL)
(Oasis-lab, San Francisco, CA, USA) in biological Celculture
®
Incubator CCL-050B-8 (Esco
Int. J. Mol. Sci. 2022,23, 13210 14 of 21
medical, Egå, Denmark) with a humidified atmosphere containing 5% CO
2
at 37
◦
C. The
24-well plates were covered with 1% sterile gelatin.
To test the effect of RF-EMF on gene expression, DLD1 cells were exposed to a pulsed
electromagnetic field, generated by a D-Link GO-RT-N150 Wi-Fi router (D-Link, Taipei,
Taiwan), during 24 h. The radiofrequency range was 2426 to 2448 MHz (Wi-Fi channel 6),
pulse length was 2.76 ms, pulse frequency was 9.7 Hz, pulse risetime was 0.06–0.08
µ
s, and
pulse falltime was 0.067–0.107 µs.
Radiofrequency field power flux density at the level of the cell layer was 1 W/m
2
(19 V/m) peak, 0.12 W/m
2
(6.6 V/m) RMS. Plates with sham-exposed cells were during
this time covered by radiofrequency protective foil YSHIELD HNV100 (YSHIELD GmbH
& Co. KG, Ruhstorf an der Rott, Germany). Other conditions were identical for both
groups: powerline frequency electric field (50 Hz) at the level of cell layer was below
1 V/m
, powerline frequency magnetic field at the level of cell layer was 0.3
µ
T in both
control and experimental groups.
To manipulate intracellular miR-34a levels in the DLD1 cells, Lipofectamine
®
RNAiMAX
Reagent (Thermo Fisher Scientific, USA) and Opti-MEM medium (Thermofisher, USA) were
used according to the manufacturer’s instructions. Cells were transfected with a mimic
of miR-34a mature dominant strand miR-34a-5p (m-m), either with a precursor of miR-
34a mimic (pre-m) or with a miR-34a-inhibitor and m-m (m-m + i, Catalogue #4464066,
#AM17100, #4464066, respectively, Life Technologies, Carlsbad, CA, USA) at a concentration
of 100 nM. Control cells were transfected with the corresponding negative controls (mNC
#4464058, preNC #AM17110, respectively, Life Technologies, USA). The effect of RF-EMF
was tested in the following groups: mimic of pre-miR-34a, NC pre-miR-34a, mimic miR-34a-
5p, NC miR-34a-5p and mimic + inhibitor miR-34a-5p. Oligos were added to cell culture
immediately after cell trypsinisation to facilitate the entrance of nucleotides into cells, and
cells were seeded into 24- or 96-well plates in concentrations of 1 mil. or 0.25 mil. cells per
well, respectively (Supplementary Figure S1).
Scratch assay: The effect of 24 h exposure to RF-EMF on cell migration was tested when
the cell culture reached a confluence of 80–90% (24 h after transfection). The monolayers
were scratched using a 10
µ
Lsterile tip. Pictures were taken immediately after wound
generation and 24 h after the scratch assay with an inverted fluorescence microscope
NIB-100F (Nanjing Jiangnan Novel Optics Co.,Ltd., Nanjing, China) and BEL Capture
3.2 software
(BEL Engineering s.r.l., Monza, Italy). Wound closing was analysed using
TScratch 1.0 software (ETH, Zurich, Switzerland, [
130
]. Consequently, the cells were
harvested for gene expression analysis, as described below.
MTS assay: The effect of 24 h RF-EMF exposure on the metabolism of viable prolifer-
ating cells was determined by an MTS test (CellTiter 96 AQ
ueous
Cell Proliferation Assay,
Promega, Madison, WI, USA) employing the modified tetrazolium compound (MTS),
whose conversion reflects the activity of mitochondrial dehydrogenase producing NADPH
or NADH in metabolically active cells. The MTS test was performed according to the
manufacturer’s instructions 3 h after MTS administration, during which the cells were
exposed to RF-EMF or incubated under control conditions at 37
◦
C and 5% CO
2
. The
reaction was terminated by adding 10% sodium dodecyl sulphate, and absorbance was
measured at 490 nm using a UV spectrophotometer (Epoch, Agilent Technologies, Inc.,
Santa Clara, CA, USA).
qPCR: The extraction of mRNA and miRNA from DLD1 cells was performed after
24 h
of RF-EMF treatment using RNAzol according to the manufacturer’s instructions (MRC,
Washington, DC, USA, Protocol for Isolation of Large RNA and Small RNA Fractions).
To synthesise cDNA from mRNA, 0.34
µ
g of mRNA was used. cDNA synthesis was
performed using the ImProm-II Reverse Transcription System (Promega, USA) and random
hexamers, according to the manufacturer’s instructions.
Before cDNA synthesis from miRNA, a small RNA fraction obtained by extraction
was polyadenylated using the Poly(A)tailing kit (Life Technologies, USA), and cDNA was
subsequently synthesised from 0.15
µ
g of the polyadenylated template using the ImProm-
Int. J. Mol. Sci. 2022,23, 13210 15 of 21
II
™
Reverse Transcription kit (Promega, USA) and employing a primer with a universal
tag [48] to extend the miRNA sequence.
To analyse mRNA and miRNA expression, a QuantiTect SYBR Green PCR Kit and a
miScript SYBR green PCR kit (Qiagen, Hilden, Germany) were used, respectively. Quantifi-
cation was performed using the StepOnePlus
™
Real-Time PCR System (Applied Biosys-
tems, Waltham, MA, USA). We used subsequent real-time PCR conditions: activation of
hot start polymerase at 95
◦
C for 15 min, followed by 40 cycles at 94
◦
C for 15 s, 49–62
◦
C
for 30 s (depending on a particular gene, see Supplementary Table S1) and extension at
72 ◦C
for 30 s. The specificity of the PCR products was validated by melting curve analysis.
Ribosomal protein s17 was used for gene expression normalisation. The primers used in
PCR reactions are provided in Supplementary Table S1.
5. Conclusions
In conclusion, the present study revealed new target genes of miR-34a-5p with key
roles in the circadian oscillator functioning– per2,bmal1 and cry1. In the case of per2
and bmal1, miR-34a-5p downregulated gene expression, and this effect was most likely
mediated via the 3
0
UTR region. cry1 mRNA expression was upregulated after miR-34a
administration, and this influence is more likely to be mediated by an alternative way of
miRNA functioning.
Interestingly, exposure to RF-EMF potentiates the capacity of miR-34a to induce
the expression of clock genes with oncogenic capacity—cry1 and cry2. Moreover, the
functioning of the miR-34a inhibitor was weakened when cells were exposed to RF-EMF,
and consequently, the increase in tumour-suppressive per2 mRNA expression induced by
the miR-34a inhibitor was less pronounced under RF-EMF conditions compared to the
control. Similarly, miR-34a inhibited the expression of anti-apoptotic protein survivin was
diminished when cells were exposed to RF-EMF. RF-EMF have been classified by the WHO
as possibly carcinogenic for humans. Our data are in line with this conclusion, as the results
indicate that RF-EMF can shift the influence of miR-34a in cancer progression manifested
by analysed target genes from typical tumour-suppressive to neutral or slightly oncogenic.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/ijms232113210/s1.
Author Contributions:
Conceptualization, S.O. and I.H.; methodology, S.O., R.M. and I.H.; validation,
S.O. and R.M.; formal analysis, S.O.; investigation, S.O. and I.H.; resources, S.O., R.M. and I.H.;
writing—original draft preparation, S.O. and I.H.; writing—review and editing, I.H.; visualization,
S.O. and I.H.; supervision, I.H.; project administration, I.H.; funding acquisition, I.H. All authors
have read and agreed to the published version of the manuscript.
Funding:
This research was funded by “Slovak Research and Development Agency”, grant numbers
APVV-16-0209 and APVV-20-0241; and “Scientific Grant Agency of the Ministry of Education, science,
research and sport of the Slovak Republic and the Slovak Academy of Sciences”, grant number VEGA
1/0679/19.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
All relevant data within the document will be made available
at Figshare
.
Conflicts of Interest: The authors declare no conflict of interest.
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