MicroRNAs in Sarcopenia: A Systematic Review

Sarcopenia, which is characterized by the loss of skeletal muscle, has been reported to contribute to development of physical disabilities, various illnesses, and increasing mortality. MicroRNAs (miRNAs) are small non-coding RNAs that inhibit translation of target messenger RNAs. Previous studies have shown that miRNAs play pivotal roles in the development of sarcopenia. Therefore, this systematic review focuses on miRNAs that regulate sarcopenia.


INTRODUCTION
Sarcopenia, defined by the loss of skeletal muscle loss, contributes to developing physical disabilities, various illnesses, and increasing mortality (1,2). MicroRNAs (miRNAs) have attracted attention as potential biomarkers and targets for specific therapies. MiRNAs are small non-coding RNAs (21-25 bases) that are not translated into proteins but inhibit the function of their target messenger RNAs (mRNAs) by destabilizing them and inhibiting their translation (3,4). Previous studies have shown that miRNAs play pivotal roles in the development of sarcopenia . Therefore, this systematic review focuses on miRNAs that regulate sarcopenia.

MECHANISM OF THE DEVELOPMENT OF SARCOPENIA
Several factors, including chronic inflammation, increased reactive oxidative species, increased fibrosis of muscle, and increased loss of motor neurons, have been reported to contribute to development of sarcopenia by progressing muscle atrophy that results in lower muscle mass (26,46). These factors have been reported to be tightly controlled by many signaling pathways and effector proteins, including some crosstalk with the protein synthesis pathway (32). Among these signaling pathways, transforming growth factor-β 1 (TGF-β 1 ) is considered as the main signaling molecule in the development of sarcopenia (47). TGF-β 1 activates many downstream profibrotic signaling molecules, including mothers against decapentaplegic (Smad), extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and p38, which contribute to increasing transdifferentiation of myoblasts into myofibroblasts, resulting in development of muscle atrophy and fibrosis (47). Chronic inflammation has also been considered to contribute to the development of sarcopenia through the production of numerous proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β, which promote muscle catabolism (64). The words "frailty" and "frail" were used for this review because they are involved in the condition of sarcopenia. Studies whose titles and abstracts did not meet selection criteria were excluded from this review. The remaining studies were carefully checked for eligibility for inclusion in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [ Figure 1; (83)]. The studies were included if (1) they reported the utility of miRNAs as potential biomarkers or targets for specific therapies of sarcopenia; and (2) they were published as full-text journal articles in English. Exclusion criteria were as follows: (1) they did not discuss specific miRNAs in sarcopenia; and (2) they included no description of sample settings. We could not perform a meta-analysis because the number of studies reporting miRNAs for sarcopenia was small, so statistical power would have been low.

Search Results
A flow diagram of this study is shown in Figure 1 and Supplemental Table 1. Computer and manual searches identified 254 potentially suitable publications. After the removal of duplicates, the titles and abstracts of 98 remaining papers were screened. Of these, 15 publications were excluded because they did not describe specific miRNAs in sarcopenia, and one publication was excluded because it lacked a description of the sample setting; 82 studies were included in the final systematic review , and 12 studies were included for extracting data (1,5,6,16,28,38,40,42,60,64,71,82).

Effects of microRNA Modulation on Sarcopenia
Several studies have reported that modulation of miRNAs has significant effects on sarcopenia in cultured myocytes in vitro ( Table 3, Figures 2A,B). The specific miRNAs that have been reported to affect sarcopenia are described below.

MiRNA-181a
MiRNA-181a binds to the 3 ′ -untranslated region of Sirt1 that is implicated in influencing aging, apoptosis, and inflammation (71). Overexpression of miRNA-181a using an miRNA-181a mimic by lipofection was shown to significantly decrease the myotube diameter, which was mediated by inhibiting its target Sirt1 in cultured myotubes of differentiated C2C12 cells, a subclone of mouse myoblasts. However, miRNA-181a knockdown using a miRNA-181a inhibitor led to an increase in the myotube diameter of cultured myotubes of differentiated C2C12 cells (71).

MiRNA-203a-3p
MiRNA-203a-3p binds to the 3 ′ -untranslated region of baculoviral inhibitors of apoptosis repeat containing 5 (BIRC5), a member of the apoptosis inhibitor protein family that suppresses apoptosis via inhibition of the initiator caspase 9 and executers caspase 3 and 7 (84). MiRNA-203a-3p was upregulated in serum of colorectal cancer patients with sarcopenia as evaluated by a lower psoas muscle mass index compared with than in colorectal cancer patients without sarcopenia (1). Knockdown of miRNA-203a-3p using an miRNA-203-3p mimic by lipofection inhibited cell proliferation and induced apoptosis via increasing the expression level of the target BIRC5 in cultured human skeletal muscle cells (1).

MiRNA-455-3p
Overexpression of miRNA-455-3p using an miRNA-455-3p mimic by lipofection inhibited the expression levels of PITX1 and RXRB, which are involved in muscle dystrophy and aging, resulting in a significant increase of the diameter of cultured myotubes differentiated from cultured mouse C2C12 myoblasts (42).

DISCUSSION
Many miRNAs increase or decrease in muscles and blood of patients with sarcopenia and rodent models of sarcopenia. These expression changes are associated with the phenotypes of sarcopenia, such as lower physical functions and expression levels of many signaling molecules that mediate progression of sarcopenia. These lines of evidence suggest that miRNA levels in muscles and/or blood are potential biomarkers for sarcopenia. However, no study has reported the relationship between the expression changes of plasma/serum and muscle miRNAs in sarcopenia. It is necessary to investigate this relationship to clarify the mechanisms of miRNAs in each organ including muscles and their circulation form in blood for the development of sarcopenia as well as the utility of miRNAs in blood as biomarkers of sarcopenia. Additionally, several miRNAs have been demonstrated to affect sarcopenia in myocytes in vitro or rodent sarcopenia models in vivo.
All studies reported the effects of miRNAs on sarcopenia in the setting of overexpression of these miRNAs as shown in Table 3. These results suggest that miRNAs are potential targets of gene therapy for sarcopenia. However, further studies are needed to investigate the mechanisms, target cells, and adverse effects of modulating these miRNAs. Additionally, so far, there is no clinical study that has directly investigated the functions of miRNAs in sarcopenia by modulation of their expression using a mimic and/or inhibitor. Future clinical studies will be necessary to confirm the effects of miRNAs on sarcopenia and their potential targets for gene therapy of sarcopenia. Our review has a number of limitations. First, we only searched for studies published in English. Second, we only used the PubMed database to identify publications. Third, meta-analysis could not be performed because the number of studies reporting miRNAs for sarcopenia was small, so statistical power would have been low. Therefore, further research is warranted to verify our conclusions.

CONCLUSION
Many miRNAs increase or decrease in muscles and blood of patients with sarcopenia and rodent models Frontiers in Medicine | www.frontiersin.org of sarcopenia. Additionally, several miRNAs have been demonstrated to affect sarcopenia in myocytes in vitro or rodent sarcopenia models in vivo. These results suggest that miRNAs are potential biomarkers and targets of gene therapy for sarcopenia. Further studies including clinical studies will be necessary to confirm the utility of miRNAs as biomarkers and targets for gene therapy of sarcopenia.

AUTHOR CONTRIBUTIONS
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

ACKNOWLEDGMENTS
We thank Mitchell Arico from Edanz Group (www.edanzediting. com/ac) for editing a draft of this manuscript.