Circulating MicroRNA-486 and MicroRNA-146a serve as potential biomarkers of sarcopenia in the older adults

This study surveyed 597 participants who were recruited community-dwelling older adults from March 2016 to December 2019 at National Taiwan University Hospital, Bei-Hu Branch, Taipei, Taiwan. Exclusion criteria included the presence of inflammatory disease within the recent 3 months, acute or unstable cerebrovascular disease, chronic obstructive pulmonary disease, uncontrolled diabetes mellitus, alcohol or drug abuse during the previous 12 months, significant renal or acute hepatic disease. The physician asks the subject’s past medical history and the subject fills out the questionnaire to exclude inappropriate subjects. Afterward, eligible 77 subjects were enrolled in this study and then divided into three groups: normal (N, n = 24), dynapenic (D, n = 35), and sarcopenic groups (S, n = 18) (Fig. 1). Dynapenia and sarcopenia were classified using the Asian Working Group of Sarcopenia (AWGS) criteria: (i) dynapenia is normal muscle mass and the loss of muscle strength (handgrip strength) or declined physical performance (gait speed) and (ii) sarcopenia is a condition characterized by insufficient muscle mass, poor muscle strength (handgrip strength), or/and declined physical performance (gait speed) [15]. Additionally, the N group was carefully selected to recruit the older adult 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.

This study evaluated four c-miRs into two categories: myo-miRs (c-miR-486-5p and c-miR-133a-3p) and inflammation-related miRs (c-miR-146a-5p and c-miR-21-5p). RNA template was used a fixed amount (1 μL) in Reverse Transcription (RT). Sample miRNAs were transcribed into cDNA via miRNA specific reverse transcription reaction using miRNA specific 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 short Locked Nucleic Acid (LNA) probe for microRNAs specific forward primer, besides, using a universal reverse primer be the microRNAs reverse primer. Amplifications were conducted following the manufacturer’s instructions using a LightCycler® 96 Real-Time PCR System (Roche, Mannheim, Germany) [9]. For analysis quality, the acceptable Ct numbers are less than 35 and each qPCR reaction was performed in triplicate. To 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 use. In this formula, the ratio of the c-miRs is the relative expression. In addition, quantification of c-miRNAs in plasma samples were evaluated by measuring endogenous myo-miRs (c-miR-486-5p and c-miR-133a-3p) and inflammation-related miRs (c-miR-146a-5p and c-miR-21-5p) levels, as well as, exogenous control cel-miR-39 levels that was spiked in all the samples at the same concentration, method as described by Sourvinou and colleagues [25].

Obtain 5-mL blood samples from all subjects for inflammation-related cytokines and lipid peroxide assay and placed in a centrifuge EDTA tube (final concentration, 4 mM), and immediately centrifuged at 1500 g for 10 min at 4 °C. The plasma samples were then stored in 500 μl aliquots at − 80 °C until ELISA 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 quantified by commercially available ELISA kits.

This study enrolled eligible 77 participants (66–92 (78.7 ± 6.2) years old, 41 males and 36 females) from 597 older adults (Fig. 1). Both D and S group had higher PBF (P < 0.05) while only the S group exhibited lower PBL (P < 0.05), compared to those of the N group. Moreover, the S group exhibited a 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). The effects of sarcopenic parameters and c-miRNAs, in total participants the S group exhibited a smaller SMI (P < 0.001), grip strength (P < 0.001), gait speed (P < 0.001), c-miR-486 (P < 0.05), and c-miR-146a (P < 0.05) than those of the N groups. In addition, the S group exhibited a smaller SMI (P < 0.001), grip strength (P < 0.001), gait speed (P < 0.001), and c-miR-486 (P < 0.05) than those of the D groups. Moreover, only the Grip strength (P < 0.001) a smaller exhibited in the D group than in the N group. The characteristics of males and females have similar results in sarcopenic parameters and c-miRNAs, indicating that sex is not the main influence factor in sarcopenia (Table 2).

Compared to the N group, both D and S groups had lesser grip strength (P<0.01) while the S group alone exhibited slower gait speed (P<0.01) (Table 2). Moreover, the S group had lower c-miR-486 (P < 0.05) (Fig. 2a) and c-miR-146a (P < 0.05) (Fig. 2c), as well as, the D group had lesser c-miR-146a (P < 0.05) (Fig. 2c) than the N group did. Additionally, the SMI (P<0.01), grip strength (P < 0.01), gait speed (P<0.01) (Table 2), and c-miR-486 (P = 0.023) (Fig. 2a) in the S group were inferior than those in the D group. In addition, the c-miR-486 and c-miR-146a normalizing with respect to c-miR-133a and c-miR-21, used as an endogenous control (Fig. 3a); exogenous control cel-miR-39(Fig. 3b); and a combination of the two, and then ΔCt was estimated but were no significant changes (Fig. 3c).

There were no significant differences in plasma MPO, IL-1β, and IL-6 levels among the N, D, and S groups (Table 3).

Pearson’s correlation coefficient 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 4). Moreover, SMI and handgrip strength were directly related to c-miR-146a level (P < 0.05, Table 4).

Both c-miR-486 and c-miR-146a were placed into a forward and stepwise, multivariate regression model that includes BMI, LMI, SMI, and handgrip strength. After adjustment, c-miR-486 level was meaningfully associated with SMI (r = 0.334, P = 0.003) whereas c-miR-146a level was significantly correlated to handgrip strength (r = 0.253, P = 0.027) (Fig. 4).

The ROC curve analysis evaluated the diagnostic accuracy of c-miRNAs and sarcopenic parameters. The AUC in c-miR-486 was 0.708 (95% confidence interval, CI: 0.561 ~ 0.855, P = 0.008) with cut-off point was 0.391 (78% sensitivity, 61.1% specificity) (Fig. 5a). Moreover, the AUC in c-miR-146a was 0.676 (95% CI:0.551 ~ 0.801, P = 0.024) with cut-off point was 0.371 (59.3% sensitivity, 77.8% specificity) (Fig. 5b).

A limitation of this study is the lack of a control cohort of younger health subjects chosen base on the same criteria of exclusion. Because the ages of these three groups are similar, the main purpose of this study focuses on the effect of inflammation on sarcopenia in the elderly. Additionally, the cross-sectional study design is a major limitation of this study. The loss of muscle mass and poor muscle strength or physical performance in these older adult participants may be only partially attributable to physiological aging, and the influences 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 have not provided direct evidence to clarify how c-miRNAs regulate the sarcopenic processes in the older adults. Further longitudinal and interventional studies are needed in the future.