Functional Identification of MicroRNAs Counteracting Cellular Senescence

Ring, Nadja Anneliese Ruth (2017). Functional Identification of MicroRNAs Counteracting Cellular Senescence. PhD thesis The Open University.



Cellular senescence is a process closely linked to organismal ageing, and clearance of senescent cells has been shown to revert aspects of the ageing phenotype. We sought to identify miRNAs able to counteract replicative senescence at the cellular level, and investigate their effect in vivo. A library of 879 human miRNAs mimics was used for a high throughput screen using high content microscopy to search for miRNAs able to restore a high replicative phenotype in WI-38 human diploid fibroblasts exhibiting replicative senescence. Screening endpoints were the analysis of the incorporation of the nucleotide analogue EdU, as a measure of cell proliferation, and the level of the cell cycle kinase inhibitor p21 as an indicator of G1 cell cycle arrest.

Twenty miRNAs were identified that exerted a strong replicative effect on senescent cells (up to 40% EdU-positive cells compared to a basal incorporation of <10%) while markedly reducing p21 levels. Cells treated with these miRNAs had decreased expression of p16 and senescence-associated beta galactosidase. Importantly, all of the identified miRNAs were capable of exerting their pro-proliferative action even in the absence of serum stimulation.

Three miRNAs - hsa-miR-523-3p, hsa-miR-639, and hsa-let-7i-3p - were selected for deep-transcriptomic sequencing, to identify putative miRNA targets. CDKN1A and GRIN3B were identified as possible targets, while the mTOR pathway was observed to be necessary for miRNA induced proliferation. Finally, miR-523-3p and let-7i-3p were administered in vivo in aged mice to assess their effect on sarcopenia. The identified miRNAs provide important information on the molecular mechanisms underlying cellular senescence and with further research may constitute a new class of nucleic acid therapeutics to combat tissue degeneration in ageing.

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