Introduction
Successful ageing is a multi-domain concept which comprises different biopsychosocial factors that can counteract the progressive decline of biological functions. Although the biological ageing process is unstoppable, it has now been established that regular exercise can counter some of the adverse physiological and cognitive consequences of ageing. In recent years, it has been reported that different genes, proteins and microRNAs (miRNAs) are involved in gene expression regulation during skeletal muscle ageing (Schmidt et al.
2019; Fochi et al.
2020).
MiRNAs are a family of single-stranded, non-coding, short RNA molecules which play a role in cellular metabolism and processes and are emerging as key regulators in gene expression at the post-transcriptional level. We previously performed a GeneChip array analysis to identify differentially expressed genes and miRNAs in skeletal muscles from veteran football players (VPG) compared to active untrained elderly (CG) subjects and we demonstrate that the expression of key messengers involved in the proteasome promotion and autophagy processes are up-regulated in VPG muscle (Mancini et al.
2019). Here, we further analysed Gene chip data, focussing our attention on the miR-1303 MiR-1303 is involved in the tumorigenesis and progression of several cancers as prostate cancer (Bo et al.
2019), neuroblastoma (Li et al.
2016), gastric (Zhang et al.
2014) and colorectal cancer (El-Murr et al.
2012). Moreover, miR-1303 results up-regulated in patients with type 2 diabetes mellitus (T2DM), and is indicated as a novel biomarker for this pathology (Wang et al.
2016). To date, there are no evidence on miR-1303 and on the effects mediated by exercise training on the expression in skeletal muscle.
Furthermore, some miRNAs, together with heat-shock proteins (HSPs), play an important role in autophagy-associated pathways (Talebian et al.
2020). HSPs are molecular chaperones and have been implicated in longevity and ageing in many species. The autophagy pathway, being a highly regulated process for recycling intracellular protein and organelles, results in up-regulation in response to danger and differentiation signals (Au et al.
2016; Levine
2005; Shintani and Klionsky
2004). Exercise induces the activation of HSPs in skeletal muscle cells (Koh and Escobedo
2004; Paulsen et al.
2009). It has also been demonstrated that not only acute exercise but also long-term recreational training can lead to an increase in HSPs in veteran skeletal muscle (Mancini et al.
2019), together with systemic improvement: anti-oxidative potential, metabolic adaptations and cardiovascular capacity (Alfieri et al.
2015; Andersen et al.
2016; Bangsbo et al.
2015; Krustrup et al.
2010a,
b; Krustrup and Krustrup
2018; Mancini et al.
2017; Schmidt et al.
2015).
Within this frame, the aims of the present study were: (a) to analyse the effects of football training on the expression of miR-1303 (b) to identify its putative targets and the possible involvement in the longevity pathways in skeletal muscle from veteran football players (VPG) compared to active untrained elderly subjects (CG).
Discussion
The main objectives of this study were to assess the effects of football training on skeletal muscle miR-1303 expression level and to find its putative interactors. We demonstrate that miR-1303 expression was down-regulated in muscle from VPG compared to active untrained elderly subjects and we identify BAG-2, a chaperon protein involved in preventing unregulated ubiquitination of misfolded protein by CHIP, as a down-regulated molecule by miR-1303 in human myoblast LHCN-M2 cells. To the best of authors’ knowledge, this is the first report describing miR-1303 regulation by football training and the identification of the putative target protein.
MiRNAs regulate different cellular processes including proliferation, motility and apoptosis (Bartel
2004). MiRNAs with preferred expression in skeletal muscle are termed “MyomiRs” and regulate muscle development, plasticity and functionality (Güller and Russell
2010; Moresi et al.
2015). Regular exercise promotes positive adaptations in skeletal muscle. In particular, it improves muscle mass, resistance to fatigue and cardiovascular fitness, increasing general quality of life (Petriz et al.
2017; Naseeb et al.
2017; Gries et al.
2018). Several studies have investigated the effects of different types of exercise on miRNA expression (Da Silva et al.
2020; Falzone et al.
2020; Fochi et al.
2020). Only a few studies have investigated the correlation between exercise and miRNA expression in the elderly, particularly in acute or short-term training. A recent meta-analysis evidenced only nine circulating miRNAs differently expressed in young compared to elderly subjects after acute exercise (Margolis et al.
2017; Gopinath et al.
2018).
Regular exercise training mediates the reduction in the risk of chronic non-communicable diseases (NMCT), including cancer (Cartee et al.
2016; Gebel et al.
2015; Kyu et al.
2016) and promotes wellbeing and longevity (Garatachea et al.
2015), also through the regulation of miRNAs and protein expression. In the last few years, adapted sport training, particularly football, has been reported as holistic positive paradigm, linking training to improved cardiovascular, metabolic and musculoskeletal fitness, also in the elderly (Krustrup et al
2010a,
2018; Bangsbo et al.
2015; Andersen et al.
2016; Krustrup and Krustrup
2018; Imperlini et al.
2020). Regular training promotes successful ageing, activating the autophagy process in muscle tissue (Fan et al.
2016). Despite growing evidence linking regular exercise to longevity, the underlying molecular mechanisms are not completely understood. In particular, the effects mediated by football training on miRNA muscle expression associated with longevity, to our knowledge, have not been reported until now. In this context, we provide evidence that miR-1303 expression was down-regulated in skeletal muscle from VPG compared to active untrained elderly subjects. MiR-1303 also plays an important role in cancer development by acting as an oncogene in different types of tumours, such as in neuroblastoma where it promotes proliferation (Li et al.
2016) and in gastric cancer to modulate proliferation and invasion (Zhang et al.
2014), in colorectal cancer (El-Murr et al.
2012) and in prostate cancer progression and development (Liu et al.
2019). In addition, miR-1303 overexpression was associated with microvascular complications in T2DM patients (Wang et al.
2016).
We recently demonstrated that in veteran muscle, the autophagy pathways were enhanced: we found up-expression of Beclin (Bcl-2), ATG (ATG5-ATG12 complex), heat-shock (HSC70/90) and PSMD13 (proteasome complex) proteins, suggesting a more efficient protein quality control process in veteran trained muscle compared to untrained active elderly subjects, which correlates with longevity (Quan and Lee
2013; Walczak et al.
2013; Wedel et al.
2018; Mancini et al.
2019). The maintenance of proteostasis is fundamental for the function and viability of cells. On the other hand, the deterioration of these pathways is associated with several diseases such as Alzheimer’s, Parkinson’s and T2DM; moreover, proteostasis pathway impairment is a hallmark of ageing (Harlt et al.
2011; He and Klionsky
2009; Ross and Poirier
2004). Molecular chaperon proteins, like HSPs, are involved in correct protein-folding and in the autophagic lysosomal pathway (Douglas and Dillin
2010; Lopez-Otin et al.
2013). BAG-2 belongs to a family of 6 BCL2-associated athanogene members (BAGs) (Behl
2016) which are conserved in different non-human, mammal and plant species, suggesting a key biological role in cell physiology (Doukhanina et al.
2006; Takayama and Reed
2001). BAG-2 improves correct protein folding by interacting with HSP70/CHIP complex and prevents aggregations of misfolded proteins (Arndt et al.
2005; Dai et al.
2005; Schönbühler et al.
2016; Wang et al.
2008); furthermore, BAG proteins can bind to various transcriptional factors, to regulate various processes such as cell apoptosis and differentiation (Qin et al.
2016).
Our results indicate that BAG-2 protein is up-regulated in muscle from veterans compared to active untrained elderly subjects, and it is in turn associated with a healthier profile although, up to now, no direct evidence correlating miR-1303 expression and longevity pathways is known. It is worth noting that BAG-2 up-expression in muscle from VPG subjects is in line with the increase of Bcl-2, ATG5-ATG12 complex, HSC70/90 and PSMD13 protein expression involved in the lysosomal and proteasome pathways as we previously reported (Mancini et al.
2019). Therefore, we can speculate that football training positively is related to a healthier profile and longevity promotion in veterans.
In conclusion, we provide evidence, for the first time, that miR-1303 expression and BAG-2 protein, its putative target, are regulated by football training in veterans muscle tissue. We also contribute to enhance the knowledge of molecular mechanisms linking l football training to longevity. The limited number of subjects investigated represents the main limitation of this study, as well as the absence of females. Further elucidation of the effects mediated by different types of sports training on muscle miR-1303 and BAG-2 protein expression will contribute to greater understanding of this process in females too. In the same way, it will be useful to investigate these processes on subjects who have been playing football and/or other types of sports for a shorter time and to discriminate whether these effects are due to the life-long football training or only to football training.
Finally, research into circulating miR-1303 represents a future goal to define healthy ageing biomarkers.
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