Project description:Human skeletal myoblast cell line LHCN-M2 (Zhu et al., Aging Cell (2007), vol. 6, pp 515-523) were plated on collagen-coated 6-well Falcon cell culture plates at 500 000 cells/well, differentiation was induced 24h later by switching to DMEM supplemented with 0,01 mg/ml insulin and 0,1 mg/ml transferrin. Cells were collected at 0, 3, and 7 days of differentiation time points. Total RNA was extracted and miRNA expression evaluated by quantitative real-time PCR. MiRNA expression profiling in proliferating and differentiating (3 and 7 days) human skeletal myoblasts LHCN-M2.
Project description: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.
