Abstract
Stem cells functions are regulated by different factors and non-conding RNAs, such as microRNA. MiRNAsplay an important role in modulating the expression of genes involved in the commitment and differentiation of progenitor cells. MiRNAs are post transcriptional regulators which may be modulated by physical exercise. MiRNAs, by regulating different signaling pathways, play an important role in myogenesis as well as in muscle activity. MiRNAs quantification may be considered for evaluating physical performance or muscle recovery. With the aim to identify specific miRNAs potentially involved in myogenesis and modulated by physical activity, we investigated miRNAs expression following physical performance in Peripheral Blood Mononuclear Cells (PBMCs) and in sera of half marathon (HM) runnners. The effect of runners sera on Myogenesis in in vitro cellular models was also explored. Therefore, we performed Microarray Analysis and Real Time PCR assays, as well as in vitro cell cultures analysis to investigate myogenic differentiation. Our data demonstrated gender-specific expression patterns of PBMC miRNAs before physical performance. In particular, miR223-3p, miR26b-5p, miR150-5p and miR15-5p expression was higher, while miR7a-5p and miR7i-5p expression was lower in females compared to males. After HM, miR152-3p, miR143-3p, miR27a-3p levels increased while miR30b-3p decreased in both females and males: circulating miRNAs mirrored these modulations. Furthermore, we also observed that the addition of post-HM participants sera to cell cultures exerted a positive effect in stimulating myogenesis. In conclusion, our data suggest that physical activity induces the modulation of myogenesis-associated miRNAs in bothfemales and males, despite the gender-associated different expression of certain miRNAs, Noteworthy, these findings might be useful for evaluating potential targets for microRNA based-therapies in diseases affecting the myogenic stem cells population.
Graphical Abstract
Similar content being viewed by others
Introduction
Specific miRNA signatures either allow stemness e maintenance or support stem cells differentiation [1]. However, miRNAs can also affect stem cells activity; their involvement in the dysregulation of stem cells has been demonstrated in lung injury, in Systemic Sclerosis, Ischemic Myocardium, in osteoporosis and in osteopenia as well as in Autism Spectrum Disorder [2]. In the complex scenario of gene expression regulation, micro RNAs (miRNAs) play an important role by targeting specific genes at the post-transcriptional level. In addition, microRNA based-therapies have been exploited as potential treatments for traumatic or degenerative diseases [3, 4]. MiRNAs regulation is also involved in myogenesis and muscle stem cell (MuSC) ageing as well as in the osteogenic commitment of MSCs in health and disease [5, 6]. MiRNAs have been identified as cell signaling mediators after cellular stress [7]. In mammals, miRNAs have been associated to the regulation of > 50% protein-coding genes expression [8].The molecular mechanisms involved in body adaptation following physical activity have not been clarified yet. However, it has been suggested that physical activity is able to modulate the expression of various genes by inducing or reducing the expression of different miRNAs [9, 10].
In the last years, running has been gaining increasing attractiveness due to its positive effects on overall health. A constantly growing number of recreational runners is taking part mainly in 10 km and half-marathon races, where the physical effort is lower than in longer distance races (such as marathon); yet the health benefits of endurance exercise are appreciable [11].
Body adaptation to exercise depends on the modulated expression of genes coding for proteins associated to physical activity [12, 13]. The exact molecular mechanisms responsible for body adaptation to exercise have not been defined.
During a half marathon race, recreational runners usually sustain an exercise intensity between 75% and 85% of the maximal aerobic capacity (VO2max) for about 80 to 120 min [11, 14]. Many acute cellular responses are detectable, e.g. an increase in mitochondrial oxidative phosphorylation, which in turn raises the production of reactive oxygen species (ROS) [15]. Furthermore, an increased anoxic regeneration of the oxidized form of nicotinamide adenine dinucleotide (NAD+) during glycolysis, produces lactates and H+ [16].
All these processes are responsible for muscle fatigue and exercise-induced muscle injury [7, 8]. They underlie chronic training adaptations and the positive effects of endurance exercise, which include cardiomyogenesis, optimization of peripheral tissue microcirculation, muscular satellite cells increase and modulation of mesenchymal cells differentiation [17]. In this context, miRNAs expression is enhanced under injury conditions or for excessive stress [18]. In fact, miRNAs can be considered modulators of inflammation and mitochondrial metabolism; they are involved in muscle recovery and hypertrophy [19]. Myogenesis and muscle activity are regulated by different signaling pathways which in turn are modulated by a large spectrum of miRNAs. In particular, both ubiquitous miRNAs and tissue-specific miRNAs are involved during muscle differentiation and activity [20].
Therefore, miRNAs expression modulation plays an important role during physical exercise; varying miRNA levels can be considered useful biomarkers for evaluating physical performance or stress, as well as muscle recovery.
Materials and Methods
Participants
38 age-matched runners (18 women and 20 men) carried out a 21.1 km half marathon. Study participants were enrolled during the sport event called “run for science,” held in Verona (Italy) in April 2019. The participants (median age: women 41.4 ± 7.2; men 41.7 ± 8.3) were all physically fit recreational runners. Bone Mineral Index (BMI) and CTX value were: median BMI (women 21.6 ± 1.3; men 23.1 ± 1.8); median CTX (women 0.34 ± 0.08 mg/ml; men 0.36 ± 0.03 mg/ml). All runners underwent a clinical evaluation as well as a medical history interview to exclude comorbidities or drugs intake. Written informed consent was obtained from all participants, and the study was approved by the ethical committee of Azienda Ospedaliera Universitaria Integrata of Verona, Italy (number 1538; Dec. 3, 2012; local ethical committee of Azienda Ospedaliera Integrata di Verona). The study design and methods comply with the Declaration of Helsinki.
Peripheral Blood Mononuclear Cells (PBMCs) and Sera Collection
Peripheral blood was collected by venipuncture before the run and immediately after, upon informed consent. Mononuclear cells (PBMCs) and sera were obtained with standard procedures as previously reported 12. Then, PBMCs and sera were stored at − 80 ◦C until use.
In Vitro Myogenic Differentiation
Human Skeletal muscle cells (SKMC) were purchased from PromoCells (C-12,580; PromoCell, GMBH Heidelberg, Germany) and cultured with Skeletal Muscle Cell Growth medium (Low serum) (C-23060PromoCell, GMBH Heidelberg, Germany) as recommended by manufacturer’s instructions., SKMCs were differentiated by using Skeletal Muscle Differentiation Medium Supplements (C-23,061; PromoCell, GMBH Heidelberg, Germany) with or without pooled sera of partecipants at 5% final concentration. The medium was changed every 3 days after initial plating.
RNAs Extraction and Reverse Transcription
miRNAs were extracted from PBMCs by using miRNeasy kit (Qiagen, Hilden, Germany) accordingly to manufacturer’s instruction. Circulating miRNAs were exctracted from serum samples with the miRNeasy Serum/Plasma Advanced Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. [21]. Total RNA from differentiating SKMCs treated or untreated with pooled sera was exctracted with a “RNeasy® protect mini kit” (Qiagen, Hilden, Germany), following the manufacturer’s protocol. miRNAs or RNA samples were quantified by a Qubit™ 3 fluorometer using a “Qubit™ RNA HS assay kit” (Invitrogen, Carlsbad, USA). Two micrograms of the extracted RNAs were reverse transcribed with a TaqMan microRNA Reverse Transcription kit (Thermofisher Corporation, Waltham, MA, USA) or First Strand cDNA Synthesis kit (GE Healthcare, Little Chalfont, UK), as previously reported. RNA and cDNA samples were stored at -80 °C.
PCR Array and Real Time RT-PCR
MiRNAs expression profiling was performed using TaqMan Advanced miRNA Human A Card (A34714; ThermoFisher Scientific) according to manufacturer’s instruction. The assay was carried out by using ViiA™7 Real-Time PCR System (Applied Biosystems). Four TaqMan Advanced miRNA Assay endogenous controls included in the card were used to data normalization and the ∆∆Ct method was used to evaluate the fold change as previously reported [22]. For mRNA or miRNAs expression analysis or array validation we used Real-time PCR using TaqMan Universal PCR Master Mix (Thermofisher Corporation, Waltham, MA, USA) and TaqMan pre-designed probes for each gene (miR 22-3p, 000398; miR 152-3p, 000475; miR 100 5p, 000437; miR 143-3p, 002249; miR 216-5p, 002220; miR 27a-3p 000408; miR 30b-5p, 000602; miR 200b-3p, 002251; U6 snRNA, 001973; MYOD, Hs00159528_m1; MYH2, Hs00430042_m1; ACTB, Hs99999903_m1).
Immunofluorescence
Immunofluorescence analyses were performed as previously reported [23]. Briefly, cells were fixed and processed according to the manufacturer’s protocols. The Primary antibody MYOD (cat. #MA1-41017; Thermofisher) was diluted (as reported in the datasheet) in antibody dilution buffer and incubated overnight at 4 °C. Slides were then incubated with secondary antibodies goat mouse fluorescein conjugated (cat. Ap124f, Millipore, Burlington, Massachusetts, USA). Nuclear staining was performed by ProLong™ Gold Antifade Mountant with DAPI. Images were recorded using a Leica (Wetzlar, Germany) inverted microscope at 10x. To express data in a semiquantitative way, four different fields were measured for each sample, in three independent experiments with about 80–100 total cells.
Western Blotting
Proteins were extracted by using Ripa buffer (Thermo Fisher Scientific, Waltham, MA,USA) and concentrations were calculated with BCA assay (Thermo Scientific, Waltham, MA, USA) as previously reported [22]. Protein samples were diluted in Laemmli’s sample buffer (Biorad, CA, US), heated for 5 min at 95 ◦C, and separated by sodium dodecyl sulfate − polyacrylamide gel electrophoresis (SDS PAGE), followed by transfer onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Waltham, MA, USA). PVDF membranes were probed with the primary (antiMyoD (MA1-41017; Thermo Scientific, Waltham, MA, USA), β ACTIN (BA3R; Thermo Scientific, Waltham, MA, USA); and secondary antibodies ( Anti-mouse (Cell Signaling, 7076). Signals were detected with a chemiluminescence reagent (ECL, Millipore, Burlington, MA, USA) and Images were recorded using a LAS4000 Digital Image Scanning System (GE Healthcare, Little Chalfont, UK). Densitometric analysis was performed as we previously reported [24].
Statistical Analysis
Results were expressed as mean ± SD. Statistical analysis was assessed by two-tailed Student’s paired-test. Differences were considered positive when p < 0.05. For in vitro data, analyses were applied to experiments carried out at least three times. We used SPSS for Windows, version 22.0 (SPSS Inc., Chicago, IL, USA) to analyse the data.
Results
In order to identify potential miRNAs modulation following HM, at first we evaluated microRNAs expression by array analysis in PBMCs collected before in male and female before HM. This initial evaluation was performed to explore potential difference gender associated. Then, we performed array analysis in PBMCs collected after HM and, by comparing miRNAs profiles of PBMCs collected before and after HM, we evaluated miRNAs modulation following physical activity. The modulated miRNAs identified in PBMCs were also tested in sera to evaluate the modulation of circulating miRNAs following HM. Therefore, we investigated the effects of runners sera participants on myogenesis by performing in vitro experiments.
Gender Associated microRNAs Profiles in Peripheral Blood
We analysed microRNAs expression in mononuclear cells collected from peripheral blood of age-matched females and males (18 women and 20 men). The array profiles analysed in females and males (Fig. 1A, Tables 1S and 2S) showed different expression profiles only for some miRNAs (Fig. 1B). Therefore, we observed some gender-associated PBMC miRNAs profiles. In detail:, miR223-3p. miR26b-5p, miR150-5p and miR15-5p expression was higher, while miR7a-5p and miR7i-5p expression was lower in females compared to males. In particular, miR223 expression was > 17x and miR26b > 14x in the female/male comparison.
Half Marathon Modulates Similarly the Expression of Other PBMCs miRNAs Regardless of Gender
After physical activity, we analysed miRNAs profiles in PBMCs of both females and males (Fig. 2; Tables 3S and 4S).
By comparing the expression profiles, we observed similar miRNAs modulation in female and male PBMCs for some miRNAs, which did not appear to be gender-associated. In particular, we observed an increased expression of miR152-3p, miR143-3p, miR27a-3p, and a reduced expression of miR30b-3p after the half marathon (Fig. 2). However, we also observed an increased expression of miR 22-3p, miR100-5p, and miR216-5p in females post half marathon (pHM)-PBMCs but not in males. Real Time PCR gene expression analysis for some miRNAs confirmed array results (Fig. 3).
Modulation of Circulating miRNAs After Half Marathon
For some of the above-mentioned miRNAs, we evaluated the corresponding expression levels in circle, by collecting sera before and after the performance. We observed an increased expression of circulating miRNAs 143-3p and 27a-3p and a reduced expression of miR30b-5p and miR200b-3p following HM, in agreement with the observations in PBMCs (Fig. 4).
Half Marathon Performance Enhances MYOD Expression in Myoblasts
As post HM myogenesis-related circulating miRNAs appeared to be modulated, we investigated the effects of runners’ sera on cultured cells during myogenic differentiation. Monitored markers were: MYOD, the earliest marker of myogenic commitment and MHY2, the gene coding for muscle myosin heavy chain 2.
Myoblast cells cultured for 3 days in the presence of sera collected before and after the half marathon (pHM, post Half Marathon) did not show any change in MHY2 expression in both genders, (Fig. 5B). However, we observed increased MYOD gene expression in pHM sera conditioned-cells (Fig. 5C). Expression of MHY2 gene increased (Fig. 5D) and the expression of MYOD decreased (Fig. 5E) after 7 days cultivation in the presence of pHM sera.
An increased number of MYOD positive cells (Fig. 6A) as well as increased MYOD protein levels (Fig. 6B) were observed in both female and male pHM sera conditioned-cells after 3 days of cultivation. However, we observed increased MYOD protein levels in both female and male pHM sera conditioned-cells also after 7 days (Fig. 6C).Furthermore, no differences in MYOD gene expression/protein levels were observed in both genders pHM sera conditioned-cells after 14 days of culture (Fig. 6D).
Discussion
MicroRNAs (miRNAs or miRs) are short (∼22 nucleotides) single strand molecules which act as key regulators of a wide spectrum of cellular and physiological processes such as proliferation, differentiation, repair mechanisms, signaling and apoptosis. They can modulate the expression of target genes by binding to the 3′ untranslated region (3′UTR) of their transcripts, causing their degradation, or preventing the translational process [25]. These regulators are found in all cell types and also in extracellular vesicles, or associated to proteins that allow their transportation through extracellular fluids [26, 27]. Therefore, miRNAs are considered useful biomarkers in human health and disease [6, 28]. In particular, circulating miRNAs levels can be associated to exercise intensity and activity especially in relation to maximum oxygen uptake [29]. It has been suggested that exercise increases the number of Bone-Marrow-Derived Mesenchymal Stem Cells along with the enhancement of their osteogenic potential at the expense of the adipogenic commitment [30]; we recently demonstrated that physical activity promotes osteogenic differentiation by modulating different miRNAs expression [31]. However, in a previous study we did not observe different numbers of total progenitor cells but we found that osteogenic progenitors increased while adipogenic progenitors decreased after HM [22]. We analysed the effects of HM on the modulation of progenitor cells numerosity and differentiation trend immediately after HM; it might be interesting to analyse these cells after a longer interval from the physical performance.
Previous studies of miRNAs response to exercise have focused almost exclusively on male athletes. However, it is well known that females show gender-specific responses in cardiovascular [32], musculoskeletal [33] and metabolic [34] patterns following physical exercise. Sex-specific miRNAs modulation has been observed as a pathophysiological response to environmental stress [35]. Sex-specific miRNA patterns are thought to occur in response to gonadal steroids, such as dihydrotestosterone and progesterone. Notably, as gonadal steroids are affected during exercise, sex specific miRNAs expression levels have been considered in response to dihydrotestosterone and progesterone stimulation [36]. Therefore, in this study we aimed to analyse the gender role in miRNAs modulation following a half-marathon. In order to avoid any bias due to miRNAs profiles before the competition, we firstly evaluated eventual miRNAs expression differences between men and women. Interestingly, we observed similar miRNAs expressions, with rare exceptions. Mir223-3p and mir 26b levels appeared 17 and 14fold higher, respectively, in women compared to men. On the basis of our results, we hypothesized that miRNAs expression profiles observed in PBMCs before the competition might be reliable. In particular, the expression of some gender-related miRNAs is consistent with female physiology. Increased levels of mir223-3p in female PBMCs are in agreement with the role of this miRNA in endometrium, as suggested by Dong et al. [37]. In mice it has been demonstrated that mir223-3p interacts with LIF 3’UTR. In the female reproductive system, LIF (Leukemia Inhibitory Factor) is expressed in endometrial epithelial cells; its expression is higher in the luteal phase compared to proliferative phase [38, 39]. Therefore, the expression of mir223-3p correlates negatively with LIF levels in females. MiR-26b is abundantly expressed in the adipose tissue [40]. During adipogenic differentiation the expression of miR-26b is upregulated [41]. In addition, it has been suggested that the expression of miR 26b is directly related to the composition of milk fatty acids [41]. MiR-26b expression is also involved in the regulation of mammary epithelial cells together with lipid-related transcription factors in goat [40]. Therefore, our data suggest that differences in gender associated miRNAs modulation might be associated to female physiology and therefore to different hormonal statuses.
When we compared miRNA modulation in PBMCs following HM, we observed increased expression of miR152-3p, miR143-3p, miR27a-3p and reduced expression of miR30b in both women and men. However, the increased expression of several miRNAs such as miR22-3p and miR 100-5p was observed only in feminine PBMCs. Reduced levels of miR-22-3p have been observed in patients with premature ovarian failure [42]. Estrogen receptor 1 (ESR1) and phosphatase and tensin homolog (PTEN) have been indicated as candidate genes for ovarian failure [43]. In addition, by bioinformatic analyses miR-22-3p has been predicted to be a negative regulator of ESR1 and PTEN. Accordingly, higher miR100 -5p expression has been observed in granulosa cells of women with normal ovarian reserve compared to subjects with lower ovarian reserve [44]. Therefore, our data suggest a protective role of physical activity for the prevention of ovary dysfunctions.
Several miRNAs that were positively modulated following the half marathon in PBMCs from both genders, are supposed to be involved in skeletal muscle activity. MiR 143-3p expression is elevated in skeletal muscle tissue whereas its downregulation has been observed during aging [45]. It has been demonstrated that miR-143 acts as regulator of the insulin growth factor‐binding protein 5 (IGFBP-5) and that the expression of miR-143 and its target gene are disrupted in primary myoblasts affecting muscle regeneration [46]. It has been observed that miR-152 inhibits proliferation and promotes myoblast differentiation in C2C12 cell line [47]; in porcine myotube, the upregulation of miR-152 has been associated to the promotion of slow-twitch myofibers formation and myogenesis [48]. Accordingly, it has been demonstrated that miR-27a favors myoblasts proliferation by targeting the inhibitor of skeletal myogenesis Myostatin [49]. Recently, it has been demonstrated that miR‐23a and miR‐27a, both located in clusters, regulate proteins involved in the atrophy process. Interestingly, the authors showed that the overexpression of miR‐23a/27a in muscle is able to counteract the diabetes‐induced muscle cachexia [50]. As PBMCs have been validated as surrogate models for skeletal muscle tissue [51], we believe that miRNAs modulation observed in PBMCs may also occur in cells involved in myogenesis. To strengthen this hypothesis, we also evaluated the expression of circulating miRNAs and the possible modulation of ci-miRNAs following exercise. Our data demonstrate that ci-miRNAs reflect what was observed in PBMCs. In addition, we observed downregulation of circulating miRNA 200b-3p following HM and this variation was statistically significant. MiR 200b has been demonstrated to target the GATA transcription factor, which is involved in cardiac myocytes proliferation and differentiation [52].
To reinforce the observation of modulated myogenesis-related circulating miRNAs upon HM, we cultured the cells in the presence of sera collected from runners before and after the half marathon. Our results regarding MYOD (transcription factor for the myogenic commitment) expression, confirmed an increased myogenic differentiation in cells treated with sera collected after physical activity.
Therefore, despite the gender-associated differential expression of some miRNAs observed in PBMCs in physiological conditions, we can state that physical activity induces the modulation of muscle differentiation-associated miRNAs in both women and men.
Data Availability
The datasets used and/or analysed during the current study are available from the corresponding author upon a reasonable request.
Change history
21 July 2022
Missing Open Access funding information has been added in the Funding Note.
References
Mathieu, J., & Ruohola-Baker, H. (2013). Regulation of stem cell populations by microRNAs (pp. 329–351). Transcriptional and Translational Regulation of Stem Cells
Ghafouri-Fard, S., et al. (2021). The impact of non-coding RNAs on normal stem cells. Biomedicine & Pharmacotherapy, 142, 112050
Gu, X. Y., et al. (2022). MicroRNA is a potential target for therapies to improve the physiological function of skeletal muscle after trauma. Neural Regeneration Research, 17(7), 1617
Saad, N. Y., et al. (2021). Human miRNA miR-675 inhibits DUX4 expression and may be exploited as a potential treatment for Facioscapulohumeral muscular dystrophy. Nature Communications, 12(1), 1–18
Yang, W., et al. (2022). Msi2-mediated MiR7a-1 processing repression promotes myogenesis. Journal of Cachexia, Sarcopenia and Muscle, 13(1), 728–742.
Valenti, M. T., et al. (2018). Role of microRNAs in progenitor cell commitment and osteogenic differentiation in health and disease. International Journal of Molecular Medicine, 41(5), 2441–2449
Mendell, J. T., & Olson, E. N. (2012). MicroRNAs in stress signaling and human disease. Cell, 148(6), 1172–1187
Krol, J., et al. (2010). The widespread regulation of microRNA biogenesis, function and decay. Nature Reviews Genetics, 11(9), 597–610
Rivas, D. A., et al. (2014). Diminished skeletal muscle microRNA expression with aging is associated with attenuated muscle plasticity and inhibition of IGF-1 signaling. The FASEB Journal, 28(9), 4133–4147
Fyfe, J. J., et al. (2016). Concurrent exercise incorporating high-intensity interval or continuous training modulates mTORC1 signaling and microRNA expression in human skeletal muscle. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 310(11), R1297–R1311
Lippi, G., et al. (2015). The baseline serum value of α-amylase is a significant predictor of distance running performance. Clinical Chemistry and Laboratory Medicine (CCLM), 53(3), 469–476
Flück, M., & Hoppeler, H. (2003). Molecular basis of skeletal muscle plasticity–from gene to form and function. Review of Physiology Biochemistry and Pharmacology, 146, 159–216.
Dalle Carbonare, L., et al. (2018). Can half-marathon affect overall health? The yin-yang of sport. Journal of Proteomics, 170, 80–87
Williams, C., & Nute, M. (1983). Some physiological demands of a half-marathon race on recreational runners. British Journal of Sports Medicine, 17(3), 152–161
Ji, L. L. (1999). Antioxidants and oxidative stress in exercise. Proceedings of the Society for Experimental Biology and Medicine, 222(3), 283–292
Fitts, R. H. (2008). The cross-bridge cycle and skeletal muscle fatigue. Journal of Applied Physiology, 104(2), 551–558
Valenti, M. T., et al. (2020). Effects of physical exercise on the prevention of stem cells senescence. Stem Cell Reviews and Reports, 16(1), 33–40
Leung, A. K., & Sharp, P. A. (2010). MicroRNA functions in stress responses. Molecular Cell, 40(2), 205–215
Polakovičová, M., et al. (2016). Circulating microRNAs as potential biomarkers of exercise response. International Journal of Molecular Sciences, 17(10), 1553
van Rooij, E., et al. (2008). MicroRNAs flex their muscles. Trends in Genetics, 24(4), 159–166
Valenti, M. T., et al. (2014). Ascorbic acid induces either differentiation or apoptosis in MG-63 osteosarcoma lineage. Anticancer Research, 34(4), 1617–1627
Dalle Carbonare, L., et al. (2019). Increased GENE EXPRESSION of RUNX2 and SOX9 in mesenchymal circulating progenitors is associated with autophagy during physical activity. Oxidative Medicine and Cellular Longevity, 15, 8426259
Dalle Carbonare, L., et al. (2021). Methylsulfonylmethane enhances MSC chondrogenic commitment and promotes pre-osteoblasts formation. Stem Cell Research & Therapy, 12(1), 1–12
Deiana, M., et al. (2018). New insights into the runt domain of RUNX2 in melanoma cell proliferation and migration. Cells, 7(11), 220
Tian, B., et al. (2015). let-7a and its target, insulin-like growth factor 1 receptor, are differentially expressed in recurrent prostate cancer. International Journal of Molecular Medicine, 36(5), 1409–1416
Dinger, M. E., et al. (2008). RNAs as extracellular signaling molecules. Journal of Molecular Endocrinology, 40(4), 151–159
Eisenberg, I., et al. (2009). miRNAS in normal and diseased skeletal muscle. Journal of Cellular and Molecular Medicine, 13(1), 2–11
Michael, A., et al. (2010). Exosomes from human saliva as a source of microRNA biomarkers. Oral Diseases, 16(1), 34–38
Mooren, F. C., et al. (2014). Circulating microRNAs as potential biomarkers of aerobic exercise capacity. American Journal of Physiology-Heart and Circulatory Physiology, 306(4), H557–H563
Marędziak, M., et al. (2015). Physical activity increases the total number of bone-marrow-derived mesenchymal stem cells, enhances their osteogenic potential, and Inhibits their adipogenic Properties. Stem Cells International, 2, 379093.
Valenti, M. T., et al. (2019). Physical exercise modulates miR-21-5p, miR-129-5p, miR-378-5p, and miR-188-5p expression in progenitor cells promoting osteogenesis. Cells, 8(7), 742
Blumenthal, J. A., et al. (1989). Cardiovascular and behavioral effects of aerobic exercise training in healthy older men and women. Journal of Gerontology, 44(5), M147–M157
Magkos, F., et al. (2007). The bone response to non-weight-bearing exercise is sport-, site-, and sex-specific. Clinical Journal of Sport Medicine, 17(2), 123–128
Tarnopolsky, M. A. (2000). Gender differences in substrate metabolism during endurance exercise. Canadian Journal of Applied Physiology, 25(4), 312–327
Morgan, C. P., & Bale, T. L. (2012). Sex differences in microRNA regulation of gene expression: no smoke, just miRs. Biology of Sex Differences, 3(1), 1–9
Hicks, S. D., et al. (2018). Distance running alters peripheral microRNAs implicated in metabolism, fluid balance, and myosin regulation in a sex-specific manner. Physiological Genomics, 50(8), 658–667
Dong, X., et al. (2016). MicroRNA-223-3p suppresses leukemia inhibitory factor expression and pinopodes formation during embryo implantation in mice. American Journal of Translational Research, 8(2), 1155
Yue, Z. P., et al. (2000). Leukemia inhibitory factor, leukemia inhibitory factor receptor, and glycoprotein 130 in rhesus monkey uterus during menstrual cycle and early pregnancy. Biology of Reproduction, 63(2), 508–512
Chen, D. B., et al. (1995). Implantation: Leukaemia inhibitory factor in human endometrium during the menstrual cycle: Cellular origin and action on production of glandular epithelial cell prostaglandin in vitro. Human Reproduction, 10(4), 911–918
Xu, G., et al. (2015). MiR-26b modulates insulin sensitivity in adipocytes by interrupting the PTEN/PI3K/AKT pathway. International Journal of Obesity, 39(10), 1523–1530
Wang, H., et al. (2018). Association between the expression of miR-26 and goat milk fatty acids. Reproduction in Domestic Animals, 53(6), 1478–1482
Dang, Y., et al. (2015). MicroRNA-22-3p is down-regulated in the plasma of Han Chinese patients with premature ovarian failure. Fertility and Sterility, 103(3), 802–807
Qin, Y., et al. (2015). Genetics of primary ovarian insufficiency: new developments and opportunities. Human Reproduction Update, 21(6), 787–808
Woo, I., et al. (2018). Micro-RNAs involved in cellular proliferation have altered expression profiles in granulosa of young women with diminished ovarian reserve. Journal of Assisted Reproduction and Genetics, 35(10), 1777–1786
Kim, J. Y., et al. (2014). Genome-wide profiling of the microRNA-mRNA regulatory network in skeletal muscle with aging. Aging (Albany NY), 6(7), 524
Soriano-Arroquia, A., et al. (2016). Age‐related changes in miR‐143‐3p: Igfbp5 interactions affect muscle regeneration. Aging Cell, 15(2), 361–369
Gan, M., et al. (2018). miR-152 regulates the proliferation and differentiation of C2C12 myoblasts by targeting E2F3. In Vitro Cellular & Developmental Biology-Animal, 54(4), 304–310
Zhang, Y., et al. (2019). Microrna-152 promotes slow-twitch myofiber formation via targeting uncoupling protein-3 gene. Animals, 9(9), 669
Huang, Z., et al. (2012). MicroRNA-27a promotes myoblast proliferation by targeting myostatin. Biochemical and Biophysical Research Communications, 423(2), 265–269
Zhang, A., et al. (2018). miRNA-23a/27a attenuates muscle atrophy and renal fibrosis through muscle‐kidney crosstalk. Journal of Cachexia, Sarcopenia and Muscle, 9(4), 755–770
Rudkowska, I., et al. (2011). Validation of the use of peripheral blood mononuclear cells as surrogate model for skeletal muscle tissue in nutrigenomic studies. Omics: a Journal of Integrative Biology, 15(1–2), 1–7
Yao, C. X., et al. (2013). miR-200b targets GATA-4 during cell growth and differentiation. RNA Biology, 10(4), 465–480
Acknowledgements
We thank the “Centro Piattaforme Tecnologiche (CPT-UNIVR)” of the University of Verona for technical support.
Funding
Open access funding provided by Università degli Studi di Verona within the CRUI-CARE Agreement. This study was supported by FUR-Dep of Medicine, University of Verona (LDC).
Author information
Authors and Affiliations
Contributions
LDC, interpretation of data and statistical analysis, drafting of the manuscript; MM and GI, interpretation of data, data curation; GD, VLV, JB, AM, MC, SC, MD, and CT, generation, collection, and assembly of data; interpretation of data; MTV and FS, conception and design, interpretation of data, critical revision of the manuscript, study supervision. The authors approved the final version of the manuscript.
Corresponding author
Ethics declarations
Ethical Approval
The procedures were approved by the ethical committee of Azienda Ospedaliera Universitaria Integrata of Verona, Italy (number 1538; 3 December 2012; local ethical committee of Azienda Ospedaliera Integrata di Verona).
Consent to Participate
The donors of peripheral blood provided informed consent. Informed consent was obtained from all subjects involved in the study.
Consent to Publish
Not Applicable.
Conflict of Interest
The authors have no disclosures to report and declare no competing financial interests.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Dalle Carbonare, L., Dorelli, G., Li Vigni, V. et al. Physical Activity Modulates miRNAs Levels and Enhances MYOD Expression in Myoblasts. Stem Cell Rev and Rep 18, 1865–1874 (2022). https://doi.org/10.1007/s12015-022-10361-9
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12015-022-10361-9