Circulating MicroRNA-486 and MicroRNA-146a Serve as Potential Biomarkers of Sarcopenia in the Elderly

Background: Age-related sarcopenia meaningfully increases the risks of functional limitations and mortality in the elderly. Although circulating microRNAs (c-miRNAs) are associated with aging-related cellular senescence and inammation, the relationships between c-miRNAs and sarcopenia in the elderly remain unclear. This study investigates whether circulating myo-miRNAs and inammation-related miRNAs are associated with sarcopenia in the elderly. Methods: This investigation recruited 77 eligible subjects (41 males and 36 females) from 597 community-dwelling older adults, and then divided into normal (n=24), dynapenic (loss of muscular function without mass, n=35), and sarcopenic groups (loss of muscular function with mass, n=18). Moreover, myo- (c-miRNA-133a and c-miRNA-486) and inammation- (c-miRNA-21 and c-miRNA-146a) related miRNAs, as well as, inammatory-related cytokine and peroxide levels in plasma were determined using quantitative polymerase chain reaction and ELISA, respectively. Results: Sarcopenic group exhibited lesser skeletal muscle mass index (SMI), handgrip strength, and gait speed, as well as, lower c-miR-486 and c-miR-146a levels, compared to those of normal and dynapenic groups. Moreover, c-miR-486 level was positively related to SMI (r=0.334, P=0.003), whereas c-miR-146a level was positively associated with SMI (r=0.240, P=0.035) and handgrip strength (r=0.253, P=0.027). In the receiver operating characteristic analysis for predicting sarcopenia, the area under the curve in c-miR-486 was 0.708 (95% condence interval: 0.561-0.855, P=0.008) and c-miR-146a was 0.676 (95% CI: 0.551-0.801, P=0.024). However, no signicant relationships were observed between SMI/handgrip strength/gait speed and plasma myeloperoxidase/interleukin-1 𝛽 /interleukin-6 levels. Conclusions: Myo-miRNA (c-miR-486) and inammation-related miRNA (c-miR-146a) are superior to inammatory peroxide/cytokines in plasma for serving as critical biomarkers of age-related sarcopenia. and Each qPCR reaction was performed in triplicate. For the estimated ratio of circulating hsa-miRs repeat cycle number to a cel-miR- 39-3p cycle number (spike-in control), formula [Ct (c-miRs assay) /Ct (cel-miR- 39 assay)] were used, in this formula, indicates the relative expression ratio of c-miRs.


Background
Sarcopenia is de ned as a loss of muscle mass and either a loss of muscle strength or physical performance [1]. Age-related sarcopenia meaningfully decreases the quality of life, leading to a loss of independence, ultimately increasing morbidity and mortality in elderly people [1,2]. Handgrip strength has been reported to be a useful predictor of whole-body muscler strength, further applications to predict health conditions in older adults [3,4]. Multi-factorial mechanisms contribute to age-declined handgrip strength, including muscular senescence, sedentary lifestyle, poor nutritional status, hormonal dysregulation, and pro-in ammation status [5,6].
MicroRNAs (miRs) play important roles in age-related changes in muscle mass and strength, including cellular proliferation, differentiation, metabolism, and in ammation responses [7]. Recent investigations have demonstrated that muscle-related microRNAs (myo-miRs) and in ammation-related miRs might be useful for estimating physical performance [8,9] and health conditions [10,11]. Moreover, circulating microRNAs (c-miRs) have been discovered in the bloodstream and bodily uids, mature miRs can be packaged in micro-particles (exosomes, micro-vesicles, and apoptotic bodies) or complexed with miRNA-binding proteins including Argonaute 2 or high-density lipoproteins [12]. Therefore, c-miRs have important functions as intercellular communication and the potential to function as the biomarkers of a physiopathological state [12,13]. Although c-miRs are associated with aging-related processes such as cellular in ammation and senescence [7,10], the relationship between c-miRs and muscle function in the aging process remains unclear. Accordingly, we hypothesized that myo-or in ammation-related c-miRs are associated with muscular mass and strength or physical performance in the elderly.
To answer the abovementioned questions, this study evaluated the sarcopenic parameters that included body composition, handgrip strengths, and gait speed in the elderly. Furthermore, myo-miRs (c-miR-133a and c-miR-486) and in ammation-related miRs (c-miR-21 and c-miR-146a), as well as, plasma in ammatory-related peroxide and cytokine levels were determined, respectively. The present study aims to establish a critical biomarker for age-related sarcopenia.

Methods
Participants poor muscle strength (handgrip strength), and declined physical performance (gait speed) [14]. Additionally, the N group was carefully selected to recruit the elderly subjects who were normal values in handgrip strength, gait speed, and muscular mass. The study was conducted according to the guidelines of the Declaration of Helsinki. The study was approved by the ethics committees of the National Taiwan University Hospital and all subjects provided written informed consent before participation.

Sarcopenic parameters
Grip strength. Subjects' handgrip strengths were evaluated using the analogue isometric dynamometer (BASELINE® Hydraulic Hand Dynamometer; Fabrication Enterprises Inc., White Plains, NY, USA). Appropriate position as recommended by the American Society of Hand Therapists protocols was used [15]. Each handgrip strength test consisted of three maximal repeated trials, and the maximal value was calculated. According to the recommendation of AWGS operational de nitions, cut-off values for handgrip strength (<26 kgs for men and <18 kgs for women) to clinical de ne poor muscle strength [14].
Gait speed. Gait speed time was assessed with a 5-meter walk test, walking time was measured for all subjects over a 5-meter distance as quickly as possible. Subjects were allowed to use their own walk aids during the test. Times were measured three times and fastest to calculate a gait speed in distance (meters) divided by walking time (sec) [16]. According to the recommendation of AWGS, a low physical performance cut point is de ned as a gait speed ≤ 0.8 m/sec. Body composition. Total body mass and composition were determined using dual-energy x-ray absorptiometry (DXA; Stratos dR; DMS Inc., Maugio, France). Scan acquisition and analysis were performed according to manufacturer guidelines. Total body measurements of fat and lean mass were reported. Moreover, percentages of body lean (PBL) and fat (PBF), lean mass index (LMI, total lean mass/ height 2 ), skeletal muscle mass index (SMI, appendicular muscle mass/ height 2 ), and bone mass density (T score) were determined using DXA, respectively.

Plasma sampling and RNA extraction
All subjects arrived at the testing center at 09:00 h to eliminate any possible diurnal effects. Participants were instructed to fast for at least 8 h and to refrain from strenuous physical exercise for at least 48 h before blood sampling. Ten mL of venous blood samples were drawn from venipuncture into a polypropylene tube that contained 4 mM ethylenediaminetetraacetic acid (EDTA, Sigma). Cell-free plasma was prepared through centrifugation at 1500 g for 20 min at 4℃. The plasma samples were then stored in 500 μl aliquots at 80℃, prior to RNA extraction. Afterward, 200 μl of plasma was used as a constant input amount in the RNA extraction. Synthetic C. Elegans cel-miR-39-3p (Concentration 0.5 fM, determined by dilution series) was added to all samples prior to the extraction of RNA as a spike-in control in order to monitor the e ciency and reproducibility of the RNA extraction and qPCR procedure. The total RNA was extracted from plasma samples using a Direct-zol™ RNA MiniPrep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's protocol.

Reverse transcription and c-miRNA quanti cation
This study evaluated four c-miRs into two categories: myo-miRs (c-miR-486-5p and c-miR-133a-3p) and in ammation-related miRs (c-miR-146a-5p and c-miR-21-5p). Reverse transcription (RT) was used as a xed amount (1 μL) of RNA. Sample miRNAs were transcribed into cDNA via miRNA speci c reverse transcription reaction using miRNA speci c stem loop-RT primer and SuperScript™ III Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA, USA). To quantify levels of c-miRNA, real-time quantitative polymerase chain reaction (RT-qPCR) using microRNAs speci c forward primer, short Locked Nucleic Acid (LNA) probe and the universal reverse primer were utilized. Ampli cations were conducted following the manufacturer's instructions using a LightCycler® 96 Real-Time PCR System (Roche, Mannheim, Germany) [19]. Ct values less than 35 were accepted for the analysis and Each qPCR reaction was performed in triplicate. For the estimated ratio of circulating hsa-miRs repeat cycle number to a cel-miR-39-3p cycle number (spike-in control), formula [Ct (c-miRs assay) /Ct (cel-miR-39 assay)] were used, in this formula, indicates the relative expression ratio of c-miRs.
In ammation-related cytokines and peroxide in plasma An additional 5-mL blood sample was obtained from all subjects, placed in a cold centrifuge tube containing EDTA ( nal concentration, 4 mM), and immediately centrifuged at 1500 g for 10 min at 4 °C. The plasma samples were then stored at −80 °C until assay. Plasma myeloperoxidase (MPO) (Immunology Consultants Laboratory, Newberg, OR), interleukin-1β (IL-1β) (eBioscience, San Diego, CA), and interleukin-6 (IL-6) (eBioscience, San Diego, CA) concentrations were quanti ed by commercially available ELISA kits.

Statistical analysis
Subjects characteristics are presented in the text as means± standard deviation (SD). Data were normally distributed; data was assessed using Shapiro-Wilk tests for dependent variables. The differences in plasma c-miRs, cytokine, and sarcopenic parameters among the normal, dynapenic, and sarcopenic groups were compared by one-way ANOVA followed by Bonferroni's post hoc test. Pearson's correlation coe cient was used to measure the strength of the association between variables. Logistic linear regression analysis was conducted to determine the relationship of c-miRs to handgrip strength, gait speed, and body composition. The receiver operating characteristic (ROC) curve analysis was constructed using the expression values with c-miRs and sarcopenia to distinguish between normal and sarcopenic subjects. The area under the curve (AUC) was estimated to assess the diagnostic performance of c-miRs and sarcopenia. The α level for statistical signi cance was set at p < 0.05. Data were analyzed using IBM SPSS Statistics for Windows Version 21 (IBM Corp., Armonk, NY).

Participant characteristics
This study enrolled eligible 77 participants (66-92 (78.7 ± 6.2) years old, 41 males and 36 females) from 597 elderly people (Fig. 1). Both D and S group had higher PBF (P < 0.05) while only S group exhibited lower PBL (P < 0.05), compared to those of the N group. Moreover, the S group exhibited smaller BMI (P < 0.01), LMI (P < 0.001) and T score (P < 0.01) than those of the N groups. On the other hand, the BMI (P< 0.001), waistline (P < 0.001) and LMI (P< 0.001) in the S group were inferior than those in the D group (Table 1).

Plasma Lipid Peroxide And In ammatory Cytokines
There were no signi cant differences in plasma MPO, IL-1b, and IL-6 levels among the N, D, and S groups ( Table 2).

Associations Between C-mirs And Sarcopenic Parameters
Pearson's correlation coe cient was used to analyze the association between the c-miRNAs and sarcopenic variables. This present study observed that BMI, LMI and SMI were positively associated with c-miR-486 level (P < 0.05, Table 3). Moreover, SMI and handgrip strength were directly related to c-miR-146a level (P < 0.05, Table 3).

Discussion
This study is the rst to report that c-miR-486 and c-miR-146a serve as potential biomarkers of sarcopenia-related declines of muscle mass and strength, respectively. However, plasma peroxide and in ammatory cytokines are not associated with sarcopenia-declined muscle mass and functions in the elderly.
Sarcopenia is likely by multifactorial contributors in sub-health conditions, includes muscular senescence, sedentary lifestyle, poor nutritional status, hormonal dysregulation, and pro-in ammatory status [17,18]. Previous studies have focused on muscle size as the major cause of age-declined muscle dysfunction. However, loss of muscle mass plays a relatively minor role in age-declined muscle function [17,18].
According to the ROC curve analysis for predicting sarcopenia, the c-miR-486 and c-miR-146a with sarcopenia are AUCs of 0.708 and 0.676 with signi cant differences, respectively. These ndings imply that either c-miR-486 or c-miRNA-146a acts as diagnostic and potential biomarkers for the prediction of sarcopenia in the elderly.
The c-miR-486 is highly expressed in skeletal muscle, that directly targets Pax7 to promote myoblast differentiation [19]. It also reduces the expressions of PTEN and FoxO1a, in turn phosphorylating AKT and activating PI3K/AKT pathway [20,21]. Therefore, lowered c-miR-486 level observed from the sarcopenic elderly may represent as a result of progressive loss in muscle mass. The c-miR-146a serves as an antiin ammatory miRNA, that negatively regulates the in ammatory response by targeting TNF receptor-associated factor 6 (TRAF-6) and IL-1Rassociated kinase (IRAK-1) to inactivate NF-κB in cytoplasm [22][23][24]. The miR146a also modulates cellular senescence, mitochondrial metabolism, and in ammation responses [25]. Conversely, downregulation of miR146a could accelerate the aging process and lead to immunosenescence [25]. In this study, sarcopenic elderly had lower c-miR-146a along with lesser handgrip strength, suggesting that poor muscle strength is associated with pro-in ammatory state in sarcopenic process.
Increasing evidence has demonstrated that aging process deteriorates mechanisms to maintain protein homeostasis and proteostasis.
Proteostasis is the maintenance of protein homeostasis through mechanisms that involve the location, concentration, conformation, and turnover of individual proteins [26]. Decrease of c-miR-486 in the aging process could impair protein turnover of skeletal muscle, leading to the loss of contractile protein and accumulation of protein damage [19].
Age-related declines in mitochondrial function also contribute to the disturbance of proteostasis processes in skeletal muscle [27]. When mitochondrial dysfunction, the electron transport system leads to an imbalance, which results in the formation of reactive oxygen species (ROS). Furthermore, miR-146a regulates mitochondria of NOX4 protein expression, thus modulating cellular senescence and redox status [28].
Downregulated miR-146a is associated with exacerbated ROS production from mitochondria and oxidative damage in aging process [29]. In contrast, maintain mitochondrial function can facilitate mechanisms of proteostasis. Thus, mitochondrial dysfunction caused by downregulated miR-146a may depress energy production and impair skeletal muscle function under progression of sarcopenia [30].

Limitation Of The Study
The cross-sectional study design is a major limitation in this study. The loss of muscle mass and poor muscle strength or physical performance in these elderly participants may be only partially attributable to physiological aging, and the in uences of genetic selection, lifestyle, and nutritional status or the differences in other characteristics among the three groups cannot be excluded. Moreover, the current experimental results may not provide direct evidence to clarify how c-miRNAs regulate the sarcopenic processes in the elderly people.

Declarations
The study was conducted according to the guidelines of the Helsinki Declaration of 1975, as revised in 2008. The study was approved by the ethics committees of the National Taiwan University Hospital and all subjects provided written informed consent before participation.

Consent for publication
Not applicable, no individual person's data in any form is visible in the paper.
Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Finding
The study is funded by the Ministry of Science and Technology, Taiwan  This funding source had no role in the design of this study and will not have any role during its execution, analyses, interpretation of the data, or decision to submit results. Although the study has been awarded a grant from this governmental funding institution, it was not peer reviewed by the funding agency.

Figure 1
Flowchart of enrolled community-dwelling elderly included normal, dynapenic, and sarcopenic subjects during following-up. This study surveyed 597 participants who were recruited community-dwelling elderly. Exclusion criteria listed in the gure were used to recruit eligible candidates. Afterwards, eligible 77 subjects were enrolled into this study, and then divided into three groups: normal (n=24), dynapenic (n=35), and sarcopenic groups (n=18).    The receiver operating characteristic curve analysis was constructed using the expression values with c-miRs and sarcopenia to distinguish between normal and sarcopenic subjects. The area under the curve (AUC) was estimated to assess the diagnostic performance of c-miRs and sarcopenia. The AUC in c-miR-486 was 0.708 (95% con dence interval, CI: 0.561~0.855, P=0.008) with cut-off point was 0.391 (78% sensitivity,