Plasma microRNA signature associated with skeletal muscle wasting in post-menopausal osteoporotic women

Abstract

Background: Skeletal muscle mass wasting almost invariably accompanies bone loss in elderly, and the coexistence of these two conditions depends on the tight endocrine crosstalk existing between the two organs, other than the biomechanical coupling. Since the current diagnostics limitation in this field, and given the progressive population aging, more effective tools are needed. The aim of this study was to identify circulating microRNAs (miRNAs) as potential biomarkers for muscle mass wasting in post-menopausal osteoporotic women.

Methods: One hundred seventy-nine miRNAs were assayed by quantitative real-time polymerase chain reaction in plasma samples from 28 otherwise healthy post-menopausal osteoporotic women (73.4 ± 6.6 years old). The cohort was divided in tertiles based on appendicular skeletal muscle mass index (ASMMI) to better highlight the differences on skeletal muscle mass (first tertile: n = 9, ASMMI = 4.88 ± 0.40 kg·m^-2; second tertile: n = 10, ASMMI = 5.73 ± 0.23 kg·m^-2; third tertile: n = 9, ASMMI = 6.40 ± 0.22 kg·m^-2). Receiver operating characteristic (ROC) curves were calculated to estimate the diagnostic potential of miRNAs. miRNAs displaying a statistically significant fold change ≥ ±1.5 and area under the curve (AUC) > 0.800 (P < 0.05) between the first and third tertiles were considered. A linear regression model was applied to estimate the association between miRNA expression and ASMMI in the whole population, adjusting for body mass index, age, total fat (measured by total-body dual-energy X-ray absorptiometry [DXA]) and bone mineral density (measured by femur DXA). Circulating levels of adipo-myokines were evaluated by bead-based immunofluorescent assays and enzyme-linked immunosorbent assays.

Results: Five miRNAs (hsa-miR-221-3p, hsa-miR-374b-5p, hsa-miR-146a-5p, hsa-miR-126-5p and hsa-miR-425-5p) resulted down-regulated and two miRNAs (hsa-miR-145-5p and hsa-miR-25-3p) were up-regulated in the first tertile (relative-low ASMMI) compared with the third tertile (relative-high ASMMI) (fold change ≥ ±1.5; P-value < 0.05). All the corresponding ROC curves had AUC > 0.8 (P < 0.05). Two signatures hsa-miR-126-5p, hsa-miR-146a-5p and hsa-miR-425-5p; and hsa-miR-126-5p, hsa-miR-146a-5p, hsa-miR-145-5p and hsa-miR-25-3p showed the highest AUC, 0.914 (sensitivity = 77.78%; specificity = 100.00%) and 0.901 (sensitivity = 88.89%; specificity = 100.00%), respectively.

Conclusions: In this study, we identified, for the first time, two miRNA signatures, hsa-miR-126-5p, hsa-miR-146a-5p and hsa-miR-425-5p; and hsa-miR-126-5p, hsa-miR-146a-5p, hsa-miR-145-5p and hsa-miR-25-3p, specifically associated with muscle mass wasting in post-menopausal osteoporotic women.

Keywords: osteoporosis; plasma miRNAs; plasma myokines; sarcopenia.

Figure 1

Fold change and receiver operating characteristic (ROC) curve of plasma microRNAs (miRNAs) of post‐menopausal osteoporotic women with different skeletal muscle mass. Differences of circulating level and ROC curve analysis of up‐regulated miRNAs (A) and down‐regulated miRNAs (B) in osteoporotic women within the first tertile (relative‐low appendicular skeletal muscle mass index [ASMMI]) compared with osteoporotic women within the third tertile (relative‐high ASMMI). Circulating levels of miRNAs are expressed as min to max and compared using a Mann–Whitney t‐test. The relative area under the curve (AUC) and P‐value are shown for each ROC curve analysis. The statistics were considered significant when P < 0.05 (*P < 0.05, **P < 0.01 and ***P < 0.001). Statistical analysis was performed with Prism® Version 6.01 (GraphPad Software).

Figure 2

Gene ontology (GO) of target genes predicted for up‐regulated and down‐regulated microRNAs differently expressed in post‐menopausal osteoporotic women with different skeletal muscle mass. GO has classified target genes in cellular component (CC), molecular function (MF) and biological process (BP). The most enriched categories (maximum 20) are shown. GO was performed with Panther Version 17.0 (http://www.pantherdb.org/), and the related statistic is shown in Table S4 .

Figure 3

Pathway enrichment analysis of predicted target genes predicted for up‐regulated and down‐regulated microRNAs (miRNAs) differently expressed in post‐menopausal osteoporotic women with different skeletal muscle mass. Analysis of the pathway enriched for target genes of the up‐regulated and down‐regulated miRNAs in osteoporotic women of first tertile (relative‐low appendicular skeletal muscle mass index [ASMMI]) compared with osteoporotic women of third tertile (relative‐high ASMMI). All the enriched pathways are shown. The analysis was performed using Panther Version 17.0 (http://www.pantherdb.org/), and the related statistic is shown in Table S4 .

Figure 4

Adipo‐myokines plasma profile in post‐menopausal osteoporotic women with different skeletal muscle mass. Differences in circulating levels of adipo‐myokines in osteoporotic women of first tertile (relative‐low appendicular skeletal muscle mass index [ASMMI]) compared with osteoporotic women of third tertile (relative‐high ASMMI). All data are expressed as min to max and compared through a parametric unpaired t‐test. Statistical analysis was performed with Prism® Version 6.01 (GraphPad Software). The differences were considered significant when P < 0.05 (*P < 0.05, **P < 0.01 and ***P < 0.001). IL‐8, interleukin‐8; PAI‐1, plasminogen activator inhibitor‐1.