Abstract
Senescence is a highly regulated process that limits cellular replication by enforcing a G1 arrest in response to various stimuli. Replicative senescence occurs in response to telomeric DNA erosion, and telomerase expression can offset replicative senescence leading to immortalization of many human cells. Limited data exists regarding changes of microRNA (miRNA) expression during senescence in human cells and no reports correlate telomerase expression with regulation of senescence-related miRNAs. We used miRNA microarrays to provide a detailed account of miRNA profiles for early passage and senescent human foreskin (BJ) fibroblasts as well as early and late passage immortalized fibroblasts (BJ-hTERT) that stably express the human telomerase reverse transcriptase subunit hTERT. Selected miRNAs that were differentially expressed in senescence were assayed for expression in quiescent cells to identify miRNAs that are specifically associated with senescence-associated growth arrest. From this group of senescence-associated miRNAs, we confirmed the ability of miR-143 to induce growth arrest after ectopic expression in young fibroblasts. Remarkably, miR-143 failed to induce growth arrest in BJ-hTERT cells. Importantly, the comparison of late passage immortalized fibroblasts to senescent wild type fibroblasts reveals that miR-146a, a miRNA with a validated role in regulating the senescence associated secretory pathway, is also regulated during extended cell culture independently of senescence. The discovery that miRNA expression is impacted by expression of ectopic hTERT as well as extended passaging in immortalized fibroblasts contributes to a comprehensive understanding of the connections between telomerase expression, senescence and processes of cellular aging.
Highlights
Senescence is a cellular state characterized by loss of replicative potential and continued metabolic activity that appears to function as a tumor suppressor mechanism and contributes to aging
In this study we present the miRNA profile of replicative senescence in human BJ fibroblasts, which in contrast to WI-38 fibroblasts express negligible amounts of p16 [6], and compare this to the miRNA expression profile of BJ fibroblasts immortalized by the stable transfection of the catalytic subunit of human telomerase
BJ fibroblasts were passaged to approximately 50 population doublings before population doubling time and morphologic changes indicated senescence in the wild type (WT) cell line, and senescence was confirmed by beta-galactosidase staining (Fig. 1a, b)
Summary
Senescence is a cellular state characterized by loss of replicative potential and continued metabolic activity that appears to function as a tumor suppressor mechanism and contributes to aging. Several diverse stimuli including DNA damage, oncogene expression, and telomere attrition can lead to senescence. Even though diverse stresses are capable of inducing senescence, p53, Rb, and more recently Skp have been identified as critical pathways common to initiation, execution and maintenance of senescence-associated growth arrest [1,2,3]. The critical pathways of senescence are controlled by a complex network that regulates chromatin remodeling, proliferation arrest, cell remodeling, activation of the senescence associated secretory pathway, and inhibition of apoptosis [2]. While major effectors of these critical pathways have been identified, a complete understanding of this molecular network is still limited
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