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mir186 and mir145 In vivo Evaluation and Enrichment in Rats Submitted to Treadmill Strenuous Exercise

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Aims: The present study aimed to identify miRNAs differentially expressed in rats submitted to strenuous exercise and in silico investigation of the biological implication of the findings. Place and Duration of Study: The in vivo experiments and analyses were performed in the Laboratory of Biochemistry and Gene Expression – LABIEX of the Superior Institute of Biomedical Science – ISCB from the State University of Ceará. Between 2017-2020. Methodology: The study was performed using as subjects 2-month-old male wistar rats, which initially were submitted to 2-week adaptation training. Later the animals were separated in two distinct groups, control (C) and trained (T), where only T performed a single session of strenuous exercise, while C were not submitted to this treatment. The applied exercise protocol consisted in a running training in treadmill with speed constant increasing until the animal exhaustion which was measured by the animal refusal to keep running. After 24h, soleus muscle was desiccated and submitted to RNAseq sequencing protocols. Obtained data were statistically evaluated in R environment with EBSeq package, to characterize and predict the miRNAs and their targets were used bioinformatics tools Gene Cards, mi RBase enrichR and KEGG. Results: Two differentially expressed miRNAs were found, mir145 and mir 186, both with downregulated expression pattern in strenuous exercise. These miRNAs have a total of 1201 predicted target genes, 67 were repeated and mostly correlate to cardiovascular disease pathways, between those 5 were differentially expressed as down-regulated. Conclusion: In conclusion, the findings suggest that mir186 and mir145 down-regulation profile mediated by strenuous exercise implicates in the non-alteration of the target genes expression profile, and consequently did not mediate alterations in the pathways they are evolved, which are mainly related to signaling and disorders.
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*Corresponding author: E-mail: raquel.martins.rf@gmail.com;
International Journal of Biochemistry Research & Review
30(6): 31-44, 2021; Article no.IJBCRR.74616
ISSN: 2231-086X, NLM ID: 101654445
mir186 and mir145 In vivo Evaluation and
Enrichment in Rats Submitted to Treadmill
Strenuous Exercise
Raquel Martins De Freitas1*, Christina Pacheco2, Stela Mirla Da Silva Felipe1,
Jannison Karlly Ribeiro Cavalcante3, Paulo Elesson Oliveira1,
Denner Silvino Da Silva1 and Vânia Marilande Ceccatto1
1Instituto Superior de Ciências Biomédicas, ISCB Universidade Estadual do Ceará, Fortaleza,
Brazil.
2Núcleo de Medicina Tropical, Universidade de Brasília, Brasília, Distrito Federal, Brazi.
3HEMOCE -Centro de Hematologia e Hemoterapia do Ceará - Fortaleza, Ceará, Brazil.
Authors’ contributions
This work was carried out in collaboration among all authors. Authors RMF, CPSM and SMSF
designed the study methodology, conducted the investigation processes, data curation and validation
of the results. Authors DSS and PEO performed the text preparation for publication and review.
Authors JKRC and CPSM performed the formal analyses. Author VMC administrated, managed, and
supervised the study. All authors read and approved the final manuscript.
Article Information
DOI: 10.9734/IJBCRR/2021/v30i630274
Editor(s):
(1) Prof. Halit Demir, Yil University, Turkey.
Reviewers:
(1) Jean-François Picimbon, Qilu University of Technology, China.
(2) Sonu Mishra, Mewar University, India.
(3) Hani Nasser Abdelhamid, Assuit University, Egypt.
Complete Peer review History: https://www.sdiarticle4.com/review-history/74616
Received 09 August 2021
Accepted 19 October 2021
Published 25 October 2021
ABSTRACT
Aims: The present study aimed to identify miRNAs differentially expressed in rats submitted to
strenuous exercise and in silico investigation of the biological implication of the findings.
Place and Duration of Study: The in vivo experiments and analyses were performed in the
Laboratory of Biochemistry and Gene Expression LABIEX of the Superior Institute of Biomedical
Science ISCB from the State University of Ceará. Between 2017-2020.
Methodology: The study was performed using as subjects 2-month-old male wistar rats, which
initially were submitted to 2-week adaptation training. Later the animals were separated in two
distinct groups, control (C) and trained (T), where only T performed a single session of strenuous
exercise, while C were not submitted to this treatment. The applied exercise protocol consisted in a
Original Research Article
Freitas et al.; IJBCRR, 30(6): 31-44, 2021; Article no.IJBCRR.74616
32
running training in treadmill with speed constant increasing until the animal exhaustion which was
measured by the animal refusal to keep running. After 24h, soleus muscle was desiccated and
submitted to RNAseq sequencing protocols. Obtained data were statistically evaluated in R
environment with EBSeq package, to characterize and predict the miRNAs and their targets were
used bioinformatics tools Gene Cards, mi RBase enrichR and KEGG.
Results: Two differentially expressed miRNAs were found, mir145 and mir 186, both with
downregulated expression pattern in strenuous exercise. These miRNAs have a total of 1201
predicted target genes, 67 were repeated and mostly correlate to cardiovascular disease pathways,
between those 5 were differentially expressed as down-regulated.
Conclusion: In conclusion, the findings suggest that mir186 and mir145 down-regulation profile
mediated by strenuous exercise implicates in the non-alteration of the target genes expression
profile, and consequently did not mediate alterations in the pathways they are evolved, which are
mainly related to signaling and disorders.
Keywords: mir145 and mir186; RNAseq; strenuous exercise; data mining.
1. INTRODUCTION
MicroRNAs (miRNAs) are a non-coding RNA
class present in a variety of regulation
mechanisms, by performing gene expression
modulation in multicellular and unicellular
organisms [1,2]. miRNAs are recognized for their
role in a post-transcriptional silencing
mechanism, which consists of a molecular
recognition of a mRNA target by the miRNA,
based on nucleotide complementarity, this
interaction is mediated by a multiprotein complex
named RISC (RNA induced silencing complex)
that leads to the target inhibition. The inhibition
mechanisms consist of mRNA cleavage causing
its degradation or translation repression[3].
miRNA editing has become studied to elucidate
the mechanisms of RNA modulation[4].
miRNAs are expressed in a variety of tissues
having different patterns and regulatory roles
according to each organ. Some specific miRNAs
families are usually found in particular tissues, an
example of this is the expression of the mir-506
family members in abundance in testis tissue [5].
Besides this, the physiologic conditions also can
modify miRNA molecule expression profiles, in
cancer, there is being reported cases of aberrant
changes in miRNA patterns [6]. miRNA presence
is commonly described to have a role in the
regulation of diseases such as heart disorders,
diabetes, and Alzheimer’s [7]. Due to this, the
miRNA class is useful as a biomarker for studies
aiming to provide diagnosis and physiologic
process comprehension[8].
Physical activity is well-known as a positive
factor in the human biological regulation
processes; therefore, exercise can act as a factor
that can modify epigenetic factors. The
mechanisms related to this alteration are vast,
including, methylation, histone modifications, and
miRNAs expression. Exercise induces metabolic
alterations through changes in the signaling
pathways, mainly in skeletal muscle tissue,
leading to adaptations that alter the
transcriptome profile [9]. One bout exercise
activity is related to cellular energy balance
mechanisms through AMP-activated protein
kinase (AMPK) and Calcium/calmodulin-
dependent protein kinase II (CaMKII) signaling.
Besides this, there are related increases in
oxidative activity and myokines secretion.
Consequently, it leads to positive effects in the
major systems, such as neurological, immune,
and cardiovascular [10].
The effects of exercise in the regulation
pathways from humans and rats are similar,
having also a correspondence between the
transcriptional profile of these organism [11].
From rodents class, the rat model is widely used
in studies with for exercise in view of reproducing
similar behavior and physiologic patterns present
in humans [12]. Besides this, the cost-benefit of
this model is very pleasant when compared to
other organism models [13].
miRNAs are present in muscle regeneration and
are related to it aging retardation mediated by
exercise practice. MyomiRs are an example of
miRNA class expressed specifically in skeletal
and cardiac muscle and are known as an
important key on skeletal muscle exercise
adaptation, having each kind of exercise a
different role in circulant miRNAs expression
[14,15].
Endurance exercise is characterized by aerobic
activities such as running, swimming, and cycling
Freitas et al.; IJBCRR, 30(6): 31-44, 2021; Article no.IJBCRR.74616
33
[16]. Different doses of this exercise modality
lead to different pathways signatures. Strenuous
exercise, which consists in the exercise practice
until arrive exhaustion, is linked to the expression
of miRNAs in inflammatory alterations, while
moderate activity is related to the opposite [17].
Strenuous acute exercise is a modality
recognized for promoting changes in the redox
balance and is related to up-regulate antioxidant
pathways [18].
The miRNAs differentially expressed in aerobic
acute strenuous exercise are underexplored,
most of the published data have approaches
based on circulating blood analyses and there is
little information correlating miRNAs in this
exercise modality in muscle. Nevertheless, in the
present study, we evaluated miRNAs
differentially expressed in Rat submitted to
strenuous exercise by RNAseq, aiming to identify
miRNAs pattern and predict by in silico
evaluation its possible metabolic implications on
the biologic process, and possible interactions
with the physical exercise modality practiced.
2. MATERIALS AND METHODS
All exercise protocol, RNAseq experiment, and
bioinformatic differential expression evaluation
steps were performed as detailed described in
the “Transcriptional profile in rat muscle: down-
regulation networks in acute strenuous exercise”
[19], which is the main study of the project that
the present article composes.
2.1 Exercise Protocol
The experiment consisted of animal physical
training, through submission to strenuous
physical exercise in an adapted treadmill as
previously described [19]. The animals choose
for the study were healthy wistar male rats, 2-
months-old, with average weight of 240-280 g
provided by the Superior Institute of Biomedical
Sciences bioterium at the State University of
Ceará, where were kept in cycles of light/dark
(12h/12h), in a controlled temperature (2225◦C)
environment, with water and feed ad libitum.
The animals passed for a two-week adaptation
training in rat adapted treadmill in view to
promote animal familiarization with the applied
environment. Later the rats were randomly
separated into two groups, control (C) (n=4) and
trained (T) (n=4). The T group were submitted to
an only session of strenuous exercise training at
0.5 km/h initial speed, with an addition of 0.2
km/h every three minutes, until animal
exhaustion, parameter that was measured by the
animal refusal of keep running on the treadmill
and loss of the limb coordination.
2.2 Experimental Analysis
All RNAseq steps were performed as described
in the “Transcriptional profile in rat muscle: down-
regulation networks in acute strenuous exercise”
[19], main study of the project that the present
article composed.
After 24h of the training exercise all animals were
euthanized with Thiopental sodium (150 mg/kg,
the soleus muscle was dissected and submitted
to biochemistry protocols and total RNA
sequencing (RNA-seq). The extraction of total
RNA was performed with TRIzol® (Thermo
Fisher Scientific/Massachusetts, EUA) and
RNeasy Plus Mini Kit® (QIAGEN) following the
recommendations and specifications of
manufactures. Samples satisfied RNA quality
and integrity requirements for sequencing.
Quality of the extracted RNA was verified by the
RNA integrity number (RIN) as previously
described. All samples demonstrated integral
18S and 28S bands. RNA concentrations
obtained in the samples were of 466.6 ± 21.7
ng/mL and the 28S/18S ratio was of 1.4 ± 0.02.
The RIN values obtained were 8.1 ± 0.14.
Samples satisfied RNA quality and integrity
requirements for sequencing. The cDNA libraries
were built with Ilumina TruSeq Kit (kits/truseq-
rna-v2.html). Illumina HiSEQ 2500 Platform was
used to perform the sequencing (Illumina, San
Diego, CA, EUA). Sequencing coverage was 30
million reads per sample, with 100bp paired-end
sequence reads.
2.3 Bioinformatic Analysis
Gene differential expression evaluation was
using EBSeq v 1.22.1 package in R environment
(https://www.bioconductor.org/packages/release/
bioc/html/EBSeq .html) [20], considering RNO
4.0 reference genome with transcriptome version
dated from 27/01/2011 obtained in UCSC
Genome Browser (https://genome.ucsc.edu/)
with the Fold change ≥ 1.4, p ≤ 0:05 and FDR
0.05 parameters. Differentially expressed
miRNAs genes were characterized with
GeneCards (https://www.genecards .org/) [21] e
miRBase (mirbase.org)[22], in terms of standard
expression in muscle (GTEx-RNAseq),
nucleotide size, chromosomic location, orthologs
Freitas et al.; IJBCRR, 30(6): 31-44, 2021; Article no.IJBCRR.74616
34
correlation, and related physiological processes
reported in the literature.
Genes candidates for miRNAs target were
searched using miRBase through its prediction
tool. Each miRNA transcript (5p and 3p) can
have innumerous gene targets due to its
transcription sense. The gene targets from both
miRNAs were mapped according to their
chromosomic location with Idiographica
(http://rtools.cbrc.jp/idiographica/) [23]. The
genes that were recurrent in the results from
different miRNAs and transcripts lists were
identified over a Venn diagram with InteractiVenn
(http://www.interactivenn.net/) [24].
The Enrichr pathway analysis tool
(http://amp.pharm.mssm.edu/Enrichr/) [25] and
the Kyoto Encyclopedia of Genes and Genomes
database (KEGG; http://www.genome.jp/kegg/)
[26] was applied to predict pathways involved in
the interactions between miRNAs targets. The
genome used was human (KEGG: hsa
T01001) due to the absence of the rat genome in
the tool. Only pathways with a statistical
significance of p < 0.05 were considered of
interest.
3. RESULTS AND DISCUSSION
3.1 Differentially Expressed miRNAs
All the sequence data showed in the present
study are submitted to the National Center for
Biotechnology Information's Sequence Read
Archive (SRA) [27] through BioProject
PRJNA557195. Two miRNAs were found
differentially expressed in the RNAseq results
evaluations through EBSeq. These miRNAs were
mir145 e mi186, both demonstrated a down-
regulation pattern (Table 1), indicating that
strenuous exercise may have a potential role in
decreasing these miRNAs expression when
comparing to sedentary individuals.
mir186 is an RNA gene located in the long arm of
chromosome 2 in position 45 (2q45) constituted
by 21 nucleotides. The mir186 gene from rats is
ortholog in humans and mice[22]. Ortholog
genes share an evolutionary connection and
usually present similar roles in different
organisms, so some features of an ortholog gene
from a particular species can support the
understanding of this feature in another
specie[28]. Studies demonstrated a correlation
between mir186 expression in regulatory
processes involved in the development of human
disorders, such as hodgkin lymphoma [29],
multiple sclerosis [30], hepatocarcinogen [31],
and Head and neck squamous cell carcinoma
[32].
mir145 is an RNA gene located in the rat
genome at chromosome 18, in the short arm in
12.1 (18p12.1) position and constituted by 23
nucleotides[22]. This miRNA also is an
orthologous gene in humans. Mir145 expression
is usually related in the literature to play a part in
the regulation process from a variety of metabolic
pathways, such as miRNAs in cancer [33], heart
development, miRNAs in DNA damage
response, and smooth muscle differentiation and
proliferation[34,35]. This miRNA is also
described as a biomarker for cardiovascular
diseases [35], B cell lymphoma, Burkitt
lymphoma [36], coronary heart disease [34], and
diabetes mellitus[37].
3.2 mir145 and mir186 Predicted Targets
Prediction analysis revealed 1201 target genes,
distributed throughout the rat genome, being
present in all 21 chromosomes, including the X
chromosome. Chromosome 20 had the lowest
number of targets with 18 genes, while
chromosome 1, had the largest, with 112 genes.
Gene target distribution is represented below
(Fig. 1).
Evaluating the number of genes obtained in
prediction analyses from each of the four
miRNAs transcripts separately (Table 2) is
demonstrated that mir186-5p transcript stood out
when compared to the others, in the number of
target genes, having the largest number. While
mir145-3p gene predicted genes presented the
smallest number of targets. Indicating that
mir186-5p may be present in a wider variety of
processes by regulating these targets in a wider
variety of processes by regulating these targets.
Comparative analyses between the target lists
are demonstrated below in a Venn diagram in
Fig. 2. The results presented 67 repeated genes,
mostly present in two different transcripts list.
The only gene found three times in the
predictions analyses were Prcki being present in
mir186-5p, 186-3p, and 145-3p transcripts.
These findings suggest that Prcki has a higher
probability of being regulated when mir145 and
mir186 are expressed at the same time.
Freitas et al.; IJBCRR, 30(6): 31-44, 2021; Article no.IJBCRR.74616
35
Table 1. EBSeq expression parameters from miRNA
Gene
Log2 FC
P
GTEx (RNA-seq muscle skeletal
human / (100x FPKM)1/2)
Regulation
Profile
Mir186
-0.6950
0.0303
17
Down
Mir145
-1.2099
0.0295
2
Down
Table 2. MiRNAs transcripts gene targets number
mir145
mir186
5p
3p
5p
3p
321 genes
104 genes
469 genes
307 genes
Total: 427 genes
Total: 778 genes
mir186 and 145 target genes: 1201
Fig. 1. mir-145 and mir-186 predicted targets gene targets distribution in the rat genome about
strenuous exercise
Prcki is a kinase protein-coding gene involved in
several biologic processes, playing important
roles in metabolic pathways regulation. This
gene has a part in cascade regulation events.
Some examples of these are the Rap1 signaling
pathway (rno04015), Endocytosis (rno04144),
Hippo signaling pathway (rno04390), and insulin
signaling (rno04910) [21].
mir145 predicted targets presented a total of 425
genes, of which only the gene Psd3 were
repeated both in 5p and 3p transcript. This gene
is involved in endocytosis, being part of
cellular transport, and catabolism of cellular
process. Besides, Psd3 plays a role in the
regulation of membrane trafficking (rno04131) in
endocytosis and ARF protein signal in guanyl-
Freitas et al.; IJBCRR, 30(6): 31-44, 2021; Article no.IJBCRR.74616
36
nucleotide exchange in GTPase ARF mechanism
[21].
mir186 presented 776 target genes in total, 20 of
them present in both 5p and 3p transcripts.
Between the duplicated genes were found that all
of them are protein-coding genes, mainly related
to cellular process regulation. Five of them are
related to signal transduction[21]. Suggesting
that mir186 can perform important roles in the
response of the cellular mechanism.
3.3 Target Genes Expressed in the Study
A total of 12498 genes were found in RNAseq
study results wherefrom the predicted target
genes 1060 were present, while 142 were not
found expressed in any of the conditions[19].
This finding demonstrates that 88.18% of the
predicted targets genes are expressed in the
study, which agrees with the down-regulation
pattern of the miRNAs, since their decreased
expression disables its potential action in the
post-transcriptional silencing mechanism
releasing the targets to express without its
regulation interference.
The differentially expressed genes identified in
the RNAseq results with EBseq package were
compared to the 1201 target predicted gene from
mir145 and mir186, which resulted in the finding
of six corresponding genes (Table 3) between
them. All of these genes presented a down-
regulation profile in strenuous exercise
experiments considering the values of logFc
through EBseq statistics evaluation[19].
These findings suggest that other variants, not
these miRNAs, are promoting the decrease of
these genes regulation patterns mediated by
strenuous exercise, since the sedentary group
did not present differential expression. Although
the targets differentially expressed in the study
were found to represent only 0.5% of the
predicted targets. This implicates a low
probability of these genes being regulated
directly by the miRNAs studied.
Fig. 2. Venn diagram describing gene shared number between different transcripts
Freitas et al.; IJBCRR, 30(6): 31-44, 2021; Article no.IJBCRR.74616
37
Table 3. Genes in miRNAs target lists and differentially expressed in strenuous exercise.
Gene Symbol
Gene Name
LogFc
Regulation
Cxadr
CXADR, Ig-like cell adhesion molecule
-0.68
Down
Kbtbd8
Kelch repeat and BTB domain containing 8
-0.61
Down
Gria2
Glutamate ionotropic receptor AMPA type subunit 2
-0.70
Down
Pappa
Pappalysin 1
-1.28
Down
Slc7a11
Solute Carrier Family 7 Member 11
-1.08
Down
Unc5c
Unc-5 netrin receptor C
-1.09
Down
3.4 mir186 and mir145 Target Genes
Function Evaluation
3.4.1 Gene targets enrichment analyses
Gene enrichment analyses were performed with
the miRNAs and the 1201 predicted target
genes, based on the human genome in KEGG
database. Function characterization revealed
that the target genes cluster has a significant
statistical probability (p < 0.05) to be related to
26 pathways (Table 4) cataloged in KEGG
database[16] considering human genome. These
data were organized following each pathway
hierarchy to demonstrate the whole scenario in
which they are involved correlating the possible
biologic interactions that can exist in this
perspective.
Enrichment results include 10 pathways related
to human diseases, 7 to the nervous and
endocrine system, and 5 correlated to
environmental information processing. The main
finds at pathway level according to p value <
0.05 were nervous and endocrine system and
human disease, such as cancer, cardiopathies,
bacterial and viral diseases, metabolic and
endocrine disorders (Table 4).
Despite numerous metabolic pathways founded
in our statistical evaluation for miRNAs target
genes in enrichment, there is little information
exploring miRNAs role in target direct regulation
mechanisms and possible changes provoked by
them in physiology. However, there is a
significant number of studies about these
miRNAs correlating them to cardiopathy and
cancer development.
Studies demonstrated that mir186 plays a role in
the endocrine system being up-regulated
prolactin treatment, which promotes a decrease
in p21 protein expression. P21 protein plays a
role in the cellular regulation process and its
inhibition is related to tumorigenesis and drug
resistance in cancer treatment[38].
Erfg gene was present in the enrichment of the
oxytocin, Hypoxia-inducible factor 1-alpha (HIF-
1), forkhead box O (FoxO), proteoglycans in
cancer, and pancreatic cancer pathways. In
literature, there is data that relate Egfr increased
expression in several cancers. Mir145 in several
cancers is usually found as down-regulated. In
this scenario, mir145 has Ergf as a target, so
when mir145 is up-regulated there is a large
probability of Ergf gene suppression mediated by
mir145, reducing this gene’s role in its respective
pathological pathway [39].
mTOR (Mammalian/mechanistic target of
rapamycin) another pathway found relevant in
enrichment analyses, is present in signal
transduction, closely related to maintenance of
biological processes, such as protein synthesis
and cell growth. However, mTOR dysregulation
is correlated to pathological disorders, such as
diabetes and cancer[40] mTOR participates in
the insulin signaling pathway through lipid and
glucose metabolism, acting in homeostasis
combined with protein kinase B (AKT) and
FOXO1. Physical exercise acts in the mTOR
pathway in plenty of tissues leading to beneficial
results in the heart, muscle, liver, and brain.
Studies relate mTOR to endurance exercise[41].
The mir186-5p transcript has expression
correlated to cardiac disorders as important
elements in cytotoxicity and apoptosis process in
diabetic cardiopathy pathological (DCM), which
makes its inhibition a great therapeutic proposal
for DCM [42]. Up-regulation profile in patients
with coronary acute syndrome, is related to
glucose metabolism and HIF-1 signaling, making
it a promisor biomarker in disease diagnostic[43].
Dawson and collaborator [44] showed that in
humans acute strenuous exercise promotes
negative effects in cardiac function, provoking
cardiac depression, and a decrease in leg
vascular function.
miRNAs play essential roles in regulation
processes related to nervous system
development and functioning in mammals.
Bioinformatics analyses also predicted that
mir145 and mir186 present target genes in
Freitas et al.; IJBCRR, 30(6): 31-44, 2021; Article no.IJBCRR.74616
38
GABAergic synapse pathway[45]. Although, no
further experimental information was found about
the mechanisms used by mir145 in this pathway.
mir145 plays an important role in the regulation
of plasticity process and smooth muscle
cells[35]. These aspects implicate in mir145
presence in vascular system processes, being
part of atherosclerosis mechanisms, arterial
pulmonary hypertension, vasoconstriction and
vasodilatation. Besides that, mir145 also
presents an important role in heart protection
against cardiomyocyte hypertrophy. mir145 is
described with up-regulated profile in induced
hypertrophy rats and dilated cardiopathy in the
terminal stage, which lead it to be considered a
potential biomarker in cardiac disorders[46].
3.4.2 Duplicated targets enrichment analyses
Enrichment analysis was also performed with the
67 repeated genes in the target lists, considering
KEGG pathway database with p value < 0.05
(Table 5). Results demonstrate convergency
between Cacnb2, Dag1 e Actg1 genes to heart
pathologies pathways, such as Dilated
Cardiopathy, Cardiac Hypertrophy, and Right
Ventricle Arrhythmogenic Cardiomyopathy.
The correlation between the targets and
cardiovascular pathways evidence the likely role
of strenuous exercise performed in our study in
the expression modulation of mir145 and mir186.
The enriched genes Cacnb2, Actg1, Impad1,
Sacm1l, and Gria3 were found expressed in the
strenuous exercise RNAseq results, however,
they did not present differential expression when
related to control. In this case, it is supposed that
acute strenuous exercise provoked miRNA
down-regulation decreasing the possibilities of
these genes to be regulated by them, but other
mechanisms were responsible for modulating
them.
3.4.3 Target gene correlation to exercise and
enrichment analyses
Our study exhibits differentially expressed
miRNAs in rats’ muscle after one session of
strenuous exercise in an adapted treadmill. To
demonstrate the possible biologic implications of
these miRNA regulations through exercise was
performed a search for mir145 and mir186 and
their target genes in the compendium of physical
exercise-related human genes, previously
published by our research group also called The
FITNOME Catalogue. This compendium is a
catalog that gathered approximately five
thousand genes described in the literature to be
related to exercise in studies that used humans
as subjects, with an explanation on gene
expression context inside exercise study
scenario[47].
In The FITNOME catalog listed 66 miRNAs, but
only one was differentially expressed in our
study, mir186, characterized in the catalog to be
related to acute endurance exercise in male
subjects, in this condition mir186 presented an
up-regulation profile [47,48,39,38].
Comparisons between miRNAs targets and the
FITNOME genes set showed 351 genes in
similarity, in which only 4 were both present in
mir145 and mir186 target list. In Fig. 3, is
describing the interactions between the exercise,
miRNAs, miRNAs targets, and results pathway
enrichment analyses. Most of these genes had
down-regulation profile in the FITNOME reports,
and its expression was observed in studies
involving mainly to chronic endurance exercise
[47].
However, 17 genes, present in both FITNOME
genes set and miRNAs targets were related to
acute resistance exercise. Our study also was
with acute exercise, but on the other hand,
contemplated another exercise modality,
strenuous resistance exercise. However, our
prediction findings suggest that both kinds of
exercise affect these genes expression
modulations.
The presence of some of the target genes in the
FITNOME gene set confirms the relation
between them and exercise, even in different
modalities, which implicates selectivity in
expression regulation mediated by the exercise
type. Data found in our study demonstrated
strenuous exercise acts as a negative regulator
in mir145 and mir186 expression in rat skeletal
muscle, which implies the decrease of miRNAs
modulation in the target genes regulation, which
allows target genes to have regular expression
without miRNAs intervention.
Corresponding genes between miRNAs targets
and FITNOME catalog, in enrichment analysis,
presented a similar metabolic profile. Revealing
the correlation between predicted genes and
exercise expression patterns, being possible that
the specific exercise factor has a direct effect on
miRNAs expression, as demonstrated in our
study with strenuous exercise, and consequently
in its targets.
Freitas et al.; IJBCRR, 30(6): 31-44, 2021; Article no.IJBCRR.74616
39
Fig. 3. The relation between the profile of miRNAs expression in exercise and possible main pathways related to the targets enrichment analyses
considering the human KEGG database
Freitas et al.; IJBCRR, 30(6): 31-44, 2021; Article no.IJBCRR.74616
40
Table 4. Enrichment analyses from target genes with KEGG database [16]
Super pathway
Sub pathway
KEGG ID
Pathway
P-value
Organismal
System
Endocrine System
hsa04917
Prolactin signaling pathway
0.0071
hsa04921
Oxytocin signaling pathway
0.0175
Nervous System
hsa04727
GABAergic synapse
0.0004
hsa04723
Retrograde endocannabinoid
signaling
0.0045
hsa04725
Cholinergic synapse
0.0100
hsa04724
Glutamatergic synapse
0.0124
Environmental
information
Signal
transduction
hsa04066
HIF-1 signaling pathway
0.0006
hsa04150
mTOR signaling pathway
0.0018
hsa04630
Jak-STAT signaling pathway
0.0175
hsa04390
Hippo signaling pathway
0.0132
hsa04070
Phosphatidylinositol signaling
system
0.0227
hsa04068
FoxO signaling pathway
0.0386
Human
Diseases
Cancer
hsa05205
Proteoglycans in cancer
0.0012
hsa05202
Transcriptional misregulation
in cancer
0.0269
hsa05212
Pancreatic cancer
0.0323
Infectious
Disease: Bacteria
hsa05100
Bacterial invasion of epithelial
cells
0.0043
Infectious
Diseases: Virus
hsa05132
Salmonella Infection
0.0233
Cardiovascular
Disease
hsa05410
Hypertrophic cardiomyopathy
0.0186
hsa05414
Dilated cardiomyopathy
0.0308
Metabolic and
endocrine
diseases
hsa04931
Insulin resistance
0.0447
hsa04933
AGE-RAGE signaling
pathway in diabetic
complications
0.0277
Genetic
Information
Processing
Folding, sorting
and degradation
hsa04120
Ubiquitin mediated proteolysis
0.0186
hsa03018
RNA degradation
0.0296
Table 5. Top 5 metabolic pathways related to repeated gene targets
KEGG ID
Metabolic pathways enrichment based on
Human KEGG database
Genes
p value
hsa05412
Arrhythmogenic right ventricular cardiomyopathy
(ARVC)
CACNB2; DAG1;
ACTG1
0.0015
hsa05410
Hypertrophic cardiomyopathy (HCM)
CACNB2; DAG1;
ACTG1
0.002
hsa05414
Dilated cardiomyopathy (DCM)
CACNB2; DAG1;
ACTG1
0.003
hsa04070
Phosphatidylinositol signaling system
IMPAD1; SACM1L;
ITPR2
0.003
hsa04724
Glutamatergic synapse
SLC1A2; ITPR2;
GRIA3
0.007
Cardiovascular pathways were also the main
results in the enrichment analysis with the target
genes present in the FITNOME gene set, also
considering with p<0.05 value. The evaluation
presented enrichment based in Dag1 and Actg1
genes, which also were found duplicated in the
miRNAs target lists. These genes are described
in the FITNOME gene set as related to chronic
endurance exercise with up-regulated profile
[47]. Proteins coded by these genes when
associated with each other have a large
probability to be involved in cardiopathies
development [49].
Freitas et al.; IJBCRR, 30(6): 31-44, 2021; Article no.IJBCRR.74616
41
4. CONCLUSION
Our findings in the present study demonstrate
that mir186 and mir145 are down-regulated by
strenuous exercise. These miRNAs present a
relevant number of gene targets when
simultaneously expressed. The down-regulated
profile of the miRNAs found in strenuous
exercise exhibit a low probability of the target
genes being potentially regulated by them. Data
about the expression in both studied conditions
confirm the presence of those gene targets,
without differential expression.
Changes in the expression of these miRNAs
presents a high probability to alter several
essential regulation processes, mainly involved
in signaling transduction and human disorders. In
this scenario, metabolic pathways related to
signaling transduction are responsible for helping
biologic process occurrence, as a cascade
reaction, mTOR pathway is an example of this.
Another relevant finding is presence of the
miRNA predicted targets in FITNOME catalog,
which correlates the targets with exercise
annotation and its altered expression is related to
cardiac pathways modulation.
Nevertheless, the findings suggest that the
down-regulation of mir145 and mir186 prevent
the mediation of alteration of the gene
expression and its pathways.
DISCLAIMER
The products used for this research are
commonly and predominantly use products in our
area of research and country. There is no conflict
of interest between the authors and producers of
the products because we do not intend to use
these products as an avenue for any litigation but
the advancement of knowledge. Also, the
research was not funded by the producing
company rather it was funded by personal efforts
of the authors.
ETHICAL APPROVAL
All procedures of this study described
were reviewed and approved by the Ethics
for Animal Use Committee of the State University
of Ceará UECE, with the protocol number:
1592060/2014.
ACKNOWLEDGEMENTS
We cordially thank the Coordination for Personal
Improvement of Higher Education Personal
(CAPES) for the provided fellowship.
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
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