Aging and MicroRNA Expression in Human Skeletal Muscle

A common characteristic of aging is the loss of skeletal muscle (sarcopenia) which can lead to falls and fractures. MicroRNAs (miRNA) are novel post-transcriptional modulators of gene expression with a potential role as a regulator of skeletal muscle mass and function. The purpose of this study was to profile miRNA expression patterns in aging human skeletal muscle using a miRNA array followed by in-depth functional and network analysis. Muscle biopsy samples from 36 men (young: 31±2; n=19; older: 73±3; n=17) were: 1) analyzed for the expression of miRNAs using a microRNA array 2) validated with Taqman quantitative real-time PCR assays, and 3) identified (and later validated) for potential gene targets using the bioinformatics knowledge base software, Ingenuity Pathways Analysis. We found that 18 miRNAs were differentially expressed in older humans (P<0.05 and >500 expression level). The Let-7 family members, Let-7b and Let-7e, were significantly elevated and further validated in older subjects (P<0.05). Functional and network analysis from Ingenuity determined that gene targets of the Let-7’s were associated with molecular networks involved in cell cycle control such as cellular proliferation and differentiation. We confirmed with real-time PCR that the mRNA expression of the cell cycle regulators, CDK6, CDC25A and CDC34 were downregulated in older subjects compared to the young (P<0.05). These data suggest that aging is characterized by an increased expression of Let-7 family members which may downregulate genes related to cellular proliferation. We propose that the increased Let-7 expression in older human muscle may be an indicator of impaired cell cycle function. We analyzed skeletal muscle biopsy samples from 19 young and 17 older male subjects that have participated in our previous and current research experiments. The subjects were not engaged in any regular exercise training at the time of the enrollment; however they were physically independent and overall healthy. Screening of subjects were performed with clinical history, physical exam, and laboratory tests including complete blood count with differential, liver and kidney function tests, coagulation profile, fasting blood glucose and oral glucose tolerance test, hepatitis B and C screening, HIV test, TSH, urinalysis, and drug screening. All subjects gave informed written consent before participating in the study, which was approved by the Institutional Review Board of the University of Texas Medical Branch (which is in compliance with the Declaration of Helsinki). Once recruited, a dual-energy X-ray absorptiometry (DEXA) scan (Hologic QDR 4500W, Bedford, MA) was performed to measure body composition and lean mass. Study design. All subjects were admitted to the Clinical Research Center the day prior to the experiment, were provided a standardized dinner and were studied following an overnight fast under basal conditions. Subjects were studied during the same time of day to avoid potential circadian changes and refrained from exercise 48h prior to study participation. A muscle biopsy was obtained from the vastus lateralis of the leg. The biopsy was performed using a 5 mm Bergström biopsy needle, under sterile procedure and local anesthesia (1% lidocaine). Muscle biopsy sample was immediately blotted and frozen in liquid nitrogen and stored at -80oC until analysis. The muscle biopsy sample was used for microRNA and gene analysis.

衰老的共同特征之一是骨骼肌量流失（肌少症，sarcopenia），可引发跌倒与骨折。微小核糖核酸（microRNAs, miRNA）是一类新型转录后基因表达调控因子，在骨骼肌质量与功能的调控中具有潜在作用。本研究旨在通过miRNA芯片分析衰老人类骨骼肌的miRNA表达谱，并开展深入的功能与网络分析。本研究纳入36名男性受试者（青年组：31±2岁，n=19；老年组：73±3岁，n=17），对其肌肉活检样本依次开展以下分析：1）采用miRNA芯片检测miRNA表达水平；2）通过Taqman定量实时PCR进行验证；3）借助生物信息学知识库软件Ingenuity通路分析（Ingenuity Pathways Analysis）筛选并验证潜在基因靶点。研究发现，老年受试者体内共有18种miRNA存在差异表达（P<0.05，且表达水平差异幅度超过500）。其中Let-7家族成员Let-7b与Let-7e在老年群体中显著上调，并经实验验证（P<0.05）。通过Ingenuity通路分析得到的功能与网络注释显示，Let-7的靶基因参与了细胞周期调控相关的分子网络，包括细胞增殖与分化过程。我们通过实时PCR进一步证实，与青年组相比，老年受试者体内细胞周期调控因子CDK6、CDC25A及CDC34的mRNA表达水平显著下调（P<0.05）。上述数据表明，衰老特征表现为Let-7家族成员表达上调，该现象可能下调与细胞增殖相关的基因。我们推测，老年人骨骼肌中Let-7表达升高可作为细胞周期功能受损的潜在标志物。本研究分析的骨骼肌活检样本来自19名青年与17名老年男性受试者，这些受试者均参与过我们此前及当前的研究实验。受试者入组时未进行规律运动训练，但身体独立且整体健康。受试者筛选通过临床病史问询、体格检查及实验室检测完成，检测项目包括全血细胞计数及分类、肝肾功能检测、凝血功能检测、空腹血糖与口服葡萄糖耐量试验、乙型/丙型肝炎筛查、HIV检测、促甲状腺激素（TSH）检测、尿液分析及药物筛查。所有受试者在参与研究前均签署了书面知情同意书，本研究经德克萨斯大学医学分校伦理审查委员会批准（符合《赫尔辛基宣言》要求）。受试者入组后，接受双能X线骨密度仪（dual-energy X-ray absorptiometry, DEXA，Hologic QDR 4500W，Bedford, MA）扫描，以评估身体成分与瘦体重。研究设计：所有受试者于实验前一日入住临床研究中心，食用标准化晚餐，并在过夜空腹的基础状态下开展实验。所有实验均在每日相同时段进行，以避免昼夜节律变化的影响，且受试者在实验前48小时内未进行运动。从受试者腿部股外侧肌获取肌肉活检样本，活检操作采用5 mm Bergström活检针，全程遵循无菌操作并使用局部麻醉（1%利多卡因）。获取的肌肉活检样本立即用吸水纸吸干，置于液氮中速冻，并保存于-80℃直至分析。该样本用于miRNA与基因表达分析。