Abstract 1231: The identification of novel senescence-associated miRNAs
Authors: Klinger Rut W. 
Published: June 2012

Cellular senescence was first described by Len Hayflick as the finite replicative lifespan of human fibroblasts in culture. Since then, it has emerged that senescence can be induced via multiple mechanisms, including radiochemotherapy and oncogenic stimuli, and that senescence rather than apoptosis occurs when damage to the cellular genome threatens to overwhelm the DNA repair machinery. Moreover, stress-induced premature senescence may have serious implications for the success of conventional chemotherapeutic regimes, as senescent cells adopt an anti-apoptotic and mitogenic secretory phenotype that can influence the growth of both normal and malignant neighbouring cells. Induction of p21 expression (via a stably integrated, IPTG-inducible transgene) in HT1080 fibrosarcoma cells causes a senescent-like phenotype characterised by an inability to divide or form colonies, activation of senescence-associated β-galactosidase expression, increased granularity and a large, flattened morphology. Indeed, along with p53, p21 has been shown to act as a potent regulator of accelerated senescence in tumour cells and a key mediator of the senescence-associated secretory phenotype. Here we describe a microRNA (miRNA) profiling study to identify novel miRNAs that are differentially expressed upon p21-mediated senescence.

Total RNA from p21-induced (senescent) and p21-uninduced (cycling) HT1080 cells was isolated in duplicate using the mirVana miRNA isolation kit (Ambion) and miRNAs were reverse transcribed using the Megaplex RT primer human pool set (Applied Biosystems). miRNA profiling was performed on TaqMan Human MicroRNA Array Sets (Applied Biosystems) which enable the simultaneous relative quantification of the expression 667 human miRNAs. Differentially expressed miRNAs were identified using the ααCT method with RealTime Statminer data analysis software (Integromics) using 4 endogenous controls for data normalisation. Twenty statistically significant (p&lt;0.05) differentially expressed miRNAs were identified following the induction of senescence. Four miRNAs were expressed only in cycling cells (hsa-miR-100*, hsa-let-7b, hsa-miR-30d, hsa-miR-422a) while expression was lost in senescent cells, and 3 miRNAs expressed only after the induction of p21 expression (hsa-miR-188-5p, hsa-miR-345, hsa-miR-425). Expression levels of miRNAs hsa-miR-100*, hsa-let-7b were examined in individual Taqman assays (Applied Biosystems) and higher expression of hsa-miR-100* and hsa-let-7b in cycling cells was confirmed. All of the differentially expressed miRNAs have previously been related to cancer in the literature, but there are no findings yet about their role in cellular senescence. Although their potential to act as regulators of senescence requires further validation, we believe these miRNAs represent novel biomarkers of p21-mediated senescence.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1231. doi:10.1158/1538-7445.AM2011-1231