Downhill running increases markers of muscle damage and impairs the maximal voluntary force production as well as the late phase of the rate of voluntary force development.

PURPOSE: To examined the time-course of the early and late phase of the rate of voluntary force development (RVFD) and muscle damage markers after downhill running. METHODS: Ten recreational runners performed a 30-min downhill run at 10 km h-1 and -20% (-11.3°) on a motorized treadmill. At baseline and each day up to 4 days RVFD, knee extensors maximum voluntary isometric force (MVIC), serum creatine kinase (CK) concentration, quadriceps swelling, and soreness were assessed. The early (0-50 ms) and late (100-200 ms) phase of the RVFD, as well as the force developed at 50 and 200 ms, were also determined. RESULTS: MVIC showed moderate decrements (p < 0.05) and recovered after 4 days (p > 0.05). Force at 50 ms and the early phase were not impaired (p > 0.05). Conversely, force at 200 ms and the late phase showed moderate decrements (p < 0.05) and recovered after 3 and 4 days, respectively (p > 0.05). CK concentration, quadriceps swelling, and soreness increased (p < 0.05) were overall fully resolved after 4 days (p > 0.05). CONCLUSION: Downhill running affected the knee extensors RVFD late but not early phase. The RVFD late phase may be used as an additional marker of muscle damage in trail running.

Effect of collection matrix, platelet depletion, and storage conditions on plasma extracellular vesicles and extracellular vesicle-associated miRNAs measurements.

OBJECTIVES: The interest around circulating extracellular vesicles and their cargo in diagnostics has greatly increased; however, several pre-analytical variables affect their determination. In this study, we investigated the effects of sample matrix, processing, and plasma storage delay and temperature on extracellular vesicles and their miRNA content. METHODS: Blood was collected from 10 male volunteers in dipotassium ethylendiaminotetraacetate-coated tubes (K2EDTA), either with plasma-preparation tube (PPT) or without (K2E) gel separator. A stepwise centrifugation was applied to K2E aliquots to obtain platelet-poor plasma (PPP). K2E, PPP and PPT plasma, stored under different conditions, were assayed for extracellular vesicles concentration and size distribution, through dynamic laser light scattering, and microRNAs content, by qPCR. RESULTS: PPP samples were characterized by the lowest extracellular vesicles count and miRNA detectability. Although having no effects on extracellular vesicles total concentration, storage conditions influenced microRNAs detectability, mainly in PPP and PPT samples. Extracellular vesicles-associated miRNAs levels in K2E were, in general, higher than in PPP and to a very limited extent to PPT. Storage temperature and delay did not affect their profile in K2E samples. CONCLUSIONS: Extracellular vesicles count and extracellular vesicles miRNA profile changed under the analyzed pre-analytical variables, showing the greatest stability in K2E samples. Since pre-analytical variables differently affected extracellular vesicles and their miRNA content, they should be considered in each experimental setting and clinical routine.

Another Weapon against Cancer and Metastasis: Physical-Activity-Dependent Effects on Adiposity and Adipokines.

Physically active behavior has been associated with a reduced risk of developing certain types of cancer and improved psychological conditions for patients by reducing anxiety and depression, in turn improving the quality of life of cancer patients. On the other hand, the correlations between inactivity, sedentary behavior, and overweight and obesity with the risk of development and progression of various cancers are well studied, mainly in middle-aged and elderly subjects. In this article, we have revised the evidence on the effects of physical activity on the expression and release of the adipose-tissue-derived mediators of low-grade chronic inflammation, i.e., adipokines, as well as the adipokine-mediated impacts of physical activity on tumor development, growth, and metastasis. Importantly, exercise training may be effective in mitigating the side effects related to anti-cancer treatment, thereby underlining the importance of encouraging cancer patients to engage in moderate-intensity activities. However, the strong need to customize and adapt exercises to a patient's abilities is apparent. Besides the preventive effects of physically active behavior against the adipokine-stimulated cancer risk, it remains poorly understood how physical activity, through its actions as an adipokine, can actually influence the onset and development of metastases.

Correction to: Interplay between low plasma RANKL and VDR-FokI polymorphism in lumbar disc herniation independently from age, body mass, and environmental factors: a case-control study in the Italian population.

Under the headline "Correlation of RANKL concentrations and VDR-FokI polymorphism on disc herniation" in the description text for Table 2, the term "allelic frequency" was used erroneously for "genotypic frequency".

Differences in Osteoimmunological Biomarkers Predictive of Psoriatic Arthritis among a Large Italian Cohort of Psoriatic Patients.

(1) Background: In literature it is reported that 20-30% of psoriatic patients evolve to psoriatic arthritis over time. Currently, no specific biochemical markers can either predict progression to psoriatic arthritis or response to therapies. This study aimed to identify osteoimmunological markers applicable to clinical practice, giving a quantitative tool for evaluating pathological status and, eventually, to provide prognostic support in diagnosis. (2) Methods: Soluble (serum) bone and cartilage markers were quantified in 50 patients with only psoriasis, 50 psoriatic patients with psoriatic arthritis, and 20 healthy controls by means of multiplex and enzyme-linked immunoassays. (3) Results: Differences in the concentrations of matrix metalloproteases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), receptor activator of nuclear factor kappa-B- ligand (RANK-L), procollagen type I N propeptide (PINP), C-terminal telopeptide of type I collagen (CTx-I), dickkopf-related protein 1 (DKK1), and sclerostin (SOST) distinguished healthy controls from psoriasis and psoriatic arthritis patients. We found that MMP2, MMP12, MMP13, TIMP2, and TIMP4 distinguished psoriasis from psoriatic arthritis patients undergoing a systemic treatment, with a good diagnostic accuracy (Area under the ROC Curve (AUC) > 0.7). Then, chitinase-3-like protein 1 (CHI3L1) and MMP10 distinguished psoriasis from psoriatic arthritis not undergoing systemic therapy and, in the presence of onychopathy, MMP8 levels were higher in psoriasis than in psoriatic arthritis. However, in these latter cases, the diagnostic accuracy of the identified biomarkers was low (0.5 < AUC < 0.7). (4) Conclusions. By highlighting never exploited differences, the wide osteoimmunological biomarkers panel provides a novel clue to the development of diagnostic paths in psoriasis and psoriasis-associated arthropathic disease.

Changes in urinary amino acids excretion in relationship with muscle activity markers over a professional cycling stage race: in search of fatigue markers.

The aim of this study was to identify the relationship between metabolic effort, muscular damage/activity indices, and urinary amino acids profile over the course of a strenuous prolonged endurance activity, as a cycling stage race is, in order to identify possible fatigue markers. Nine professional cyclists belonging to a single team, competing in the Giro d'Italia cycling stage race, were anthropometrically characterized and sampled for blood and urine the day before the race started, and on days 12 and 23 of the race. Diet was kept the same over the race, and power output and energy expenditure were recorded. Sera were assayed for muscle markers (lactate dehydrogenase, aspartate aminotransferase, and creatine kinase activities, and blood urea nitrogen), and creatinine, all corrected for plasma volume changes. Urines were profiled for amino acid concentrations, normalized on creatinine excretion. Renal function, in terms of glomerular filtration rate, was monitored by MDRD equation corrected on body surface area. Creatine kinase activity and blood urea were increased during the race as did serum creatinine while kidney function remained stable. Among the amino acids, taurine, glycine, cysteine, leucine, carnosine, 1-methyl histidine, and 3-methyl histidine showed a net decreased, while homocysteine was increased. Taurine and the dipeptide carnosine (ß-alanyl-L-histidine) were significantly correlated with the muscle activity markers and the indices of effort. In conclusion, the metabolic profile is modified strikingly due to the effort. Urinary taurine and carnosine seem useful tools to evaluate the muscle damage and possibly the fatigue status on a long-term basis.

Systemic and Local Administration of Antimicrobial and Cell Therapies to Prevent Methicillin-Resistant Staphylococcus epidermidis-Induced Femoral Nonunions in a Rat Model.

S. epidermidis is responsible for biofilm-related nonunions. This study compares the response to S. epidermidis-infected fractures in rats systemically or locally injected with vancomycin or bone marrow mesenchymal stem cells (BMSCs) in preventing the nonunion establishment. The 50% of rats receiving BMSCs intravenously (s-rBMSCs) died after treatment. A higher cytokine trend was measured in BMSCs locally injected rats (l-rBMSCs) at day 3 and in vancomycin systemically injected rats (l-VANC) at day 7 compared to the other groups. At day 14, the highest cytokine values were measured in l-VANC and in l-rBMSCs for IL-10. µCT showed a good bony bridging in s-VANC and excellent both in l-VANC and in l-rBMSCs. The bacterial growth was lower in s-VANC and l-VANC than in l-rBMSCs. Histology demonstrated the presence of new woven bone in s-VANC and a more mature bony bridging was found in l-VANC. The l-rBMSCs showed a poor bony bridging of fibrovascular tissue. Our results could suggest the synergic use of systemic and local injection of vancomycin as an effective treatment to prevent septic nonunions. This study cannot sustain the systemic injection of BMSCs due to high risks, while a deeper insight into local BMSCs immunomodulatory effects is mandatory before developing cell therapies in clinics.

Interplay between low plasma RANKL and VDR-FokI polymorphism in lumbar disc herniation independently from age, body mass, and environmental factors: a case-control study in the Italian population.

PURPOSE: Aim of this study was to investigate RANKL and osteoprotegerin plasma concentrations in patients affected by disc herniation, the most common epiphenomenon of disc degenerative diseases, and in a matched cohort of healthy subjects and whether the expression of these markers was associated to a polymorphism of the vitamin D receptor gene. METHODS: For this case-control study, 110 consecutive cases affected by lumbar disc herniation (confirmed by MRI) and 110 healthy age- and sex-matched controls were enrolled. Subjects affected by any other pathology were excluded. RANKL and osteoprotegerin were measured in plasma by immunoassays. The difference in these markers between cases and controls was assessed by t test. The correlation between osteoimmunological markers concentrations, anthropometrical variables, and the expression of the pathology was statistically assessed (Pearson's test) along with the association (Fisher's exact test) with the vitamin D receptor gene genotype, determined elsewhere. RESULTS: Despite comparable osteoprotegerin concentrations, cases, altogether or grouped for gender, express lower RANKL and, consequently, RANKL-to-osteoprotegerin ratio. While in cases RANKL and osteoprotegerin concentrations were independent from age and BMI, in controls they increased with age. Disc herniation was strongly associated with RANKL and the presence of the F allele of the VDR gene. CONCLUSIONS: Whether vertebral bone changes precede or follow cartilage deterioration in intervertebral disc degeneration is not known. Our results suggest a reduced bone turnover rate, associated to a specific genetic background, in patients affected by lumbar disc herniation which could be one of the favoring factors for disc degeneration.

Osteocartilaginous metabolic markers change over a 3-week stage race in pro-cyclists.

Evidence suggests that endurance and even recreational cycling may stimulate bone resorption; however, little is known about cartilage response to endurance cycling exercise. We investigated effort-dependent changes in bone turnover and cartilage biomarkers in blood and urine samples from elite cyclists during a 3-week stage race. Whole blood and urine samples were collected the day before the start of the race, at mid and end-race for serum and urinary CTx-I, NTx-I, PINP, COMP (only in serum), and CTx-II analysis by enzyme-linked immunosorbent assay. The values were corrected for plasma volume or creatinine excretion, respectively, and correlated with power output (corrected for body weight) and net energy expenditure. Bone marker concentrations in both serum and urine were slightly but significantly decreased. Among the cartilage degradation markers, only CTx-II was decreased, while COMP remained unchanged. The changes in bone and cartilage turnover indexes were correlated with the indexes of physical effort and energy consumption. Strenuous physical effort, in the absence of mechanical loading, slows bone metabolism and only minimally affects cartilage turnover. Since changes in plasma and urine volume, which normally occur in exercising athletes, can mask these effects, biomarker concentrations need to be corrected for shifts in plasma volume and urinary creatinine for correct interpretation of the data.

Long non-coding and circular RNAs in osteoporosis: Translation to clinical practice.

Non-coding RNAs (ncRNAs) belong to a class of untranslated nucleic acids involved in regulation of gene expression. ncRNAs are categorized as small (<200 ribonucleotides in length), i.e., microRNAs (miRNAs), and long ncRNAs (lncRNAs) (200 to thousands of ribonucleotides in length) and circular RNAs (circRNAs). In contrast to miRNAs, the roles of lncRNAs in general and circRNAs in bone metabolism specifically are not well understood. As such, a comprehensive understanding of these RNA species in bone turnover could be of great value in the development of new diagnostic tools and therapeutic targets. Unfortunately, measurement of these unique RNAs lacks standardization, a component critical to clinical translation. This review examines the potential role of lncRNA and circRNA as bone biomarkers, the need for validated and standardized measurement and challenges thereof.

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

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.

Murine Myoblasts Exposed to SYUIQ-5 Acquire Senescence Phenotype and Differentiate into Sarcopenic-Like Myotubes, an In Vitro Study.

The musculoskeletal system is one of the most affected organs by aging that correlates well with an accumulation of senescent cells as for other multiple age-related pathologies. The molecular mechanisms underpinning muscle impairment because of senescent cells are still elusive. The availability of in vitro model of skeletal muscle senescence is limited and restricted to a small panel of phenotypic features of these senescent cells in vivo. Here, we developed a new in vitro model of senescent C2C12 mouse myoblasts that, when subjected to differentiation, the resulting myotubes showed sarcopenic features. To induce senescence, we used SYUIQ-5, a quindoline derivative molecule inhibitor of telomerase activity, leading to the expression of several senescent hallmarks in treated myoblasts. They had increased levels of p21 protein accordingly with the observed cell cycle arrest. Furthermore, they had enhanced SA-ßgalactosidase enzyme activity and phosphorylation of p53 and histone H2AX. SYUIQ-5 senescent myoblasts had impaired differentiation potential and the resulting myotubes showed increased levels of ATROGIN-1 and MURF1, ubiquitin ligases components responsible for protein degradation, and decreased mitochondria content, typical features of sarcopenic muscles. Myotubes differentiated from senescent myoblasts cultures release increased levels of MYOSTATIN that could affect skeletal muscle cell growth. Overall, our data suggest that a greater burden of senescent muscle cells could contribute to sarcopenia. This study presents a well-defined in vitro model of muscle cell senescence useful for deeper investigation in the aging research field to discover new putative therapeutic targets and senescence biomarkers associated with the aged musculoskeletal system.

Circulating Carboxylated Osteocalcin Correlates With Skeletal Muscle Mass and Risk of Fall in Postmenopausal Osteoporotic Women.

Background: Bone and skeletal muscle represent a single functional unit. We cross-sectionally investigated body composition, risk of fall and circulating osteocalcin (OC) isoforms in osteoporotic postmenopausal women to test the hypothesis of an involvement of OC in the bone-muscle crosstalk. Materials and Methods: Twenty-nine non-diabetic, non-obese, postmenopausal osteoporotic women (age 72.4 ± 6.8 years; BMI 23.0 ± 3.3 kg/m2) underwent to: 1) fasting blood sampling for biochemical and hormone assays, including carboxylated (cOC) and uncarboxylated (uOC) osteocalcin; 2) whole-body dual energy X-ray absorptiometry (DXA) to assess total and regional body composition; 3) magnetic resonance imaging to determine cross-sectional muscle area (CSA) and intermuscular adipose tissue (IMAT) of thigh muscles; 4) risk of fall assessment through the OAK system. Results: Appendicular skeletal muscle index (ASMMI) was low in 45% of patients. Forty percent got a low OAK score, consistent with moderate-severe risk of fall, which was predicted by low legs lean mass and increased total fat mass. Circulating cOC levels showed significantly correlated with ßCTx-I, lean mass parameters including IMAT, and OAK score. Fractured and unfractured women did not differ for any of the analyzed parameters, though cOC and uOC positively correlated with legs lean mass, OAK score and bone markers only in fractured women. Conclusions: Data supported the relationship between OC and skeletal muscle mass and function in postmenopausal osteoporotic women. Serum cOC, but not uOC, emerges as mediator in the bone-muscle crosstalk. Circulating cOC and uOC levels may be differentially regulated in fractured and unfractured osteoporotic women, suggesting underlying differences in bone metabolism.

Short and long-term effects of high-intensity interval training applied alone or with whole-body cryostimulation on glucose homeostasis and myokine levels in overweight to obese subjects.

Background: COVID-19 pandemic has exacerbated the problem of physical inactivity and weight gain. Consequently, new strategies to counteract weight gain are being sought. Because of their accessibility, interval training and cold therapy are the most popular such strategies. We here aimed to examine the effect of 6 units of high-intensity interval training (HIIT), applied alone or in combination with 10 sessions of whole-body cryotherapy (WBC; 3 min at -110 ∘C per session) on incretins, myokines, and adipokines levels. Materials and methods: The study involved 65 subjects (body mass index of approximately 30 kg•m-2). The subjects were randomly divided into training group (TR; n = 27) and training supported by WBC group (TR-WBC; n = 38). Blood samples were collected before, immediately following, and 4 weeks after the intervention. Results: Fibroblast growth factor 21 (FGF21) levels significantly increased (p = 0.03) and adiponectin levels increased in the TR group (p = 0.05) compared with those recorded in TR-WBC group 24 h after the end of experimental protocol. Beneficial changes in the lipid profile (p = 0.07), a significant drop in visfatin levels (p < 0.05), and the improvement in ß-cell function (HOMA-B; p = 0.02) were also observed in the TR group in the same time point of study. While TR-WBC did not induce similar changes, it ameliorated blood glucose levels (p = 0.03). Changes induced by both interventions were only sustained for 4 weeks after treatment. Conclusion: Collectively, HIIT, alone and in combination with WBC, positively affects metabolic indicators, albeit, most likely, different mechanisms drive the beneficial effects of different treatments.

A Physically Active Status Affects the Circulating Profile of Cancer-Associated miRNAs.

Circulating miRNAs are ideal diagnostics and prognostics biomarkers in cancer since altered levels of specific miRNAs have been associated to development/progression of several cancers. Physical activity is a recognized preventive strategy against several cancers, but it may also modify the baseline levels of cancer-associated miRNAs and, hence, may act as a confounding pre-analytical variable. This study aimed at understanding whether physical activity-dependent changes in cancer-associated circulating miRNAs profile could act as a confounding variable. A panel comprising 179 miRNAs was assayed in plasma from 20 highly trained and 10 sedentary men. RT-qPCR data were analyzed with the 2-2ΔΔCT methods and normalized on hsa-miR-320d, as determined by bioinformatics analysis. miRNAs associated with the diagnosis of the most prevalent cancers were considered. Only those miRNAs, relevantly associated with cancers, found ≥2-fold up- or downregulated in highly trained subjects compared to sedentary were disclosed. The results reveal that chronic physical activity determined modifications altering the baseline level of several cancer-associated miRNAs and, hence, their diagnostic and prognostic potential. In conclusion, based on our results, a physically active status emerges as an important pre-analytical variable able to alter the basal level of circulating miRNAs, and these alterations might be considered as potentially misleading the analytical output.

Peri-Surgical Inflammatory Profile Associated with Mini-Invasive or Standard Open Lumbar Interbody Fusion Approaches.

BACKGROUND: Different surgical approaches are available for lumbar interbody fusion (LIF) to treat disc degeneration. However, a quantification of their invasiveness is lacking, and the definition of minimally invasive surgery (MIS) has not been biochemically detailed. We aimed at characterizing the inflammatory, hematological, and clinical peri-surgical responses to different LIF techniques. METHODS: 68 healthy subjects affected by single-level discopathy (L3 to S1) were addressed to MIS, anterior (ALIF, n = 21) or lateral (LLIF, n = 23), and conventional approaches, transforaminal (TLIF, n = 24), based on the preoperative clinical assessment. Venous blood samples were taken 24 h before the surgery and 24 and 72 h after surgery to assess a wide panel of inflammatory and hematological markers. RESULTS: martial (serum iron and transferrin) and pro-angiogenic profiles (MMP-2, TWEAK) were improved in ALIF and LLIF compared to TLIF, while the acute phase response (C-reactive protein, sCD163) was enhanced in LLIF. CONCLUSIONS: MIS procedures (ALIF and LLIF) associated with a reduced incidence of post-operative anemic status, faster recovery, and enhanced pro-angiogenic stimuli compared with TLIF. LLIF associated with an earlier activation of innate immune mechanisms than ALIF and TLIF. The trend of the inflammation markers confirms that the theoretically defined mini-invasive procedures behave as such.

Engineering the early bone metastatic niche through human vascularized immuno bone minitissues.

Bone metastases occur in 65%-80% advanced breast cancer patients. Although significant progresses have been made in understanding the biological mechanisms driving the bone metastatic cascade, traditional 2Din vitromodels and animal studies are not effectively reproducing breast cancer cells (CCs) interactions with the bone microenvironment and suffer from species-specific differences, respectively. Moreover, simplifiedin vitromodels cannot realistically estimate drug anti-tumoral properties and side effects, hence leading to pre-clinical testing frequent failures. To solve this issue, a 3D metastatic bone minitissue (MBm) is designed with embedded human osteoblasts, osteoclasts, bone-resident macrophages, endothelial cells and breast CCs. This minitissue recapitulates key features of the bone metastatic niche, including the alteration of macrophage polarization and microvascular architecture, along with the induction of CC micrometastases and osteomimicry. The minitissue reflects breast CC organ-specific metastatization to bone compared to a muscle minitissue. Finally, two FDA approved drugs, doxorubicin and rapamycin, have been tested showing that the dose required to impair CC growth is significantly higher in the MBm compared to a simpler CC monoculture minitissue. The MBm allows the investigation of metastasis key biological features and represents a reliable tool to better predict drug effects on the metastatic bone microenvironment.

Study of the preanalytical variables affecting the measurement of clinically relevant free-circulating microRNAs: focus on sample matrix, platelet depletion, and storage conditions.

INTRODUCTION: Circulating microRNAs (miRNAs) are emerging as potential biomarkers. However, the lack of preanalytical and analytical standardization limits their use. The aim of this study was to determine the expression of different miRNAs in plasma according to different collection and storage conditions. MATERIALS AND METHODS: Venous blood from 10 volunteers was collected in tubes spray-coated with dipotassium salt of ethylendiaminetetraacetic acid, either with (plasma-preparation tube, PPT) or without (K2EDTA) gel separator. Platelet-poor plasma (PPP) was also obtained from K2EDTA plasma. After storage under different conditions, miRNA-enriched total RNA was isolated from plasma and reverse transcribed. A panel of 179 miRNAs was assayed by quantitative polymerase chain reaction and the results were analysed by GenEx software. Detectability and stability of miRNAs were determined. RESULTS: The number of undetected miRNAs was: 18, 24, and 22 in PPT; 83, 43, and 20 in K2EDTA; and 76, 106, and 104 in PPP samples, for plasma immediately frozen at - 80°C and plasma stored for 24h at room temperature or 4°C, respectively. Circulating miRNA expression in PPT samples was not affected by storage delay or temperature, while the percentage of up- and down-regulated miRNA in K2EDTA and PPP samples ranged from 2%, and 1% to 7%, and 5%, respectively. CONCLUSIONS: Sample matrix, temperature and delay in storage strongly influence the expression level of plasma miRNAs. Our results indicate PPT tubes as the most suitable matrix to improve total miRNA detectability and stability, independently of temperature.

Normalization strategies differently affect circulating miRNA profile associated with the training status.

MicroRNAs are fine regulators of the whole-body adaptive response but their use as biomarkers is limited by the lack of standardized pre- and post-analytical procedures. This work aimed to compare different normalization approaches for RT-qPCR data analyses, in order to identify the most reliable and reproducible method to analyze circulating miRNA expression profiles in sedentary and highly-trained subjects. As the physically active status is known to affect miRNA expression, they could be effective biomarkers of the homeostatic response. Following RNA extraction from plasma, a panel of 179 miRNAs was assayed by RT-qPCR and quantified by applying different normalization strategies based on endogenous miRNAs and exogenous oligonucleotides. hsa-miR-320d was found as the most appropriate reference miRNA in reducing the technical variability among the experimental replicates and, hence, in highlighting the inter-cohorts differences. Our data showed an association between the physically active status and specific skeletal muscle- and bone-associated circulating miRNAs profiles, revealing that established epigenetic modifications affect the baseline physiological status of these tissues. Since different normalization strategies led to different outputs, in order to avoid misleading interpretation of data, we remark the importance of the accurate choice of the most reliable normalization method in every experimental setting.