To further extend understanding of the role of miRNAs in cell senescence, we used deep sequencing to interrogate the miRNA transcriptomes of young and senescent IMR90 fibroblasts. Even though microarrays have been used to profile miRNA expression in senescence 
, the microarray technology suffers from limitations in sensitivity and specificity 
. Deep sequencing technology overcomes the disadvantages of microarrays and generates millions of small RNA sequence reads, to measure absolute abundance and to discover novel microRNAs that have evaded previous discovery efforts. MiRDeep2 analysis of the sequencing reads detected 141 known miRNAs that were induced by senescence and 131 that were repressed, and discovered 20 novel miRNAs. Some of the novel miRNAs are differentially expressed between young and senescent fibroblasts, while still showing the characteristic pattern of higher expression of the mature miRNA over the star and loop sequences. These novel sequences were missed by traditional analyses because they tend to be expressed at low levels and they are located within unannotated regions of the genome. The low expression levels of novel miRNAs are effectively detected because of the high sensitivity of deep sequencing; low expression of novel miRNAs has been observed in other studies, suggesting that the more abundant miRNAs have largely already been discovered 
. The targets and functions of the novel miRNAs remain to be investigated.
Among the known miRNAs detected in our study, members of the highly conserved let-7 family (let-7a, let-7f, let-7e, let-7i and let-7g) were the most abundant, with read counts up to 1.7 million and highly significant miRDeep2 detection scores. We found that senescence increased let-7 expression, as previously observed in microarray miRNA profiling studies 
. Let-7 is among several miRNAs currently considered as tumor suppressors 
, which is consistent with the view that senescence evolved as a tumor-suppressive mechanism to mitigate the hazard that cancer poses to longevity 
. Expression of let-7 is abnormal in 9 cancer types 
. Let-7 miRNA overexpression was shown to be associated with senescence in fibroids 
and in skeletal muscle of aged humans, and was proposed to contribute to decreased muscle cell renewal and regeneration 
. Taken together, these findings suggest that let-7 miRNAs play a key role in the control of cell senescence.
Members of the miR-449 family were among the most highly induced miRNAs in senescent fibroblasts. Consistent with a possible role of this miR-449 induction in cell cycle arrest during senescence, miR-449 has been shown to inhibit cell cycle progression at G1 phase by targeting CDK6 and CDC25A, which are pivotal to G1/S-phase transition 
. Also, miR-499 was found to be significantly upregulated in senescent human mesenchymal stem cells, with the potential to regulate all four of the senescence induction types namely, telomere attrition, oxidative stress, oncogene expression and DNA damage signaling 
. Among the miRNAs we found to be downregulated by senescence is the miR-17-92 cluster, which is a polycistron encoding 6 mature miRNAs (miR-17, -20a, 18a, -19a, -19b-1 and -92a-1). The expression of all 6 members was decreased in senescent cells with a substantial fold change (8.0, 7.8, 6.4, 10.8, 11.1, and 4.1, respectively). MiR-17-92 is overexpressed in human cancers, and promotes tumorigenesis mainly by inhibiting oncogene-induced senescence 
. Given the anti-senescence activity of miR-17-92, our finding that all miR-17-92 members are sharply repressed with senescence suggests that its very low levels may initiate and/or sustain the senescence program. Furthermore, miR-19b was found to be downregulated in several human replicative and organismal aging models 
, and has been identified as an oncogene that activates the AKT/mTOR pathway, which modulates organismal life spans 
miRNA microarray studies have reported four upregulated miRNAs (miR-152, -410, -431, and -493) and four downregulated miRNAs (miR-15a, -20a, -25, and -155) in both replicative and stress-induced senescence 
. Decreased expression of one of these, miR-155, was observed in senescent BJ fibroblasts, aged primary human WI-38 fibroblasts, and peripheral blood mononuclear cells from older individuals 
. In our study, all of these eight miRNAs were regulated in the same way. Another study reported twelve miRNAs as senescence regulators 
. Four of twelve reported miRNAs (miR-217, -34a, -369-5p, and -20a) were regulated in the same way in our study; absence of the others in our study may reflect differences in cell types and senescence models. Furthermore, miR-217 has been reported to play a role in endothelial senescence and is implicated in the pathogenesis of atherosclerosis 
. The expression of miR-217 increases in aged endothelial cells and promotes senescence through inhibition of SirT1, which is known to promote longevity and mediates the beneficial effects of calorie restriction.
Our study identified many miRNAs that are reported here for the first time as differentially regulated by senescence, adding to the increasing evidence for miRNA regulation of the senescent program. Mir-432 is highly induced by senescence in our data as illustrated in , but was never reported in previous cell senescence microarray studies. It is mentioned in the literature only as abundant in the earliest stage of fetal development 
, and has a putative binding site in the 3′ UTR of some endothelial cell-restricted genes 
. Other miRNAs found in our study to be affected during senescence (), but not reported in previous senescence studies, include miR-1246, miR-584 and miR-323, which are implicated in certain cancers 
. Further investigation of these miRNAs may shed new light on the roles and mechanisms of miRNAs in cellular senescence.
To improve the identification of target genes of the miRNAs that changed expression during senescence, we adopted an approach based on co-analysis of changes in both miRNA and mRNA expressions. The mRNA transcripts that were detected by microarrays as differentially expressed during senescence, and simultaneously predicted in silico
as targets of differentially expressed miRNAs, were considered as potential target genes regulated by senescence-induced miRNA changes. Matching the in silico
predicted target genes with the differentially regulated mRNA transcripts derived from microarrays may minimize the false positives and negatives obtained from in silico
prediction. In total, 386 genes were predicted to be the targets of up-regulated miRNAs in senescent cells, while 131 genes were predicted as targets of down-regulated miRNAs (Tables S3
). We also included another fibroblast type, MRC5, in the matching analysis. We found 143 genes potentially downregulated by senescence-induced miRNA overexpression in both types of fibroblasts, IMR90 and MRC5, and 36 genes potentially upregulated by senescence-induced miRNA underexpression (Tables S5
). To establish the impact of these miRNA targets on senescence, we used functional annotation to identify the most relevant and meaningful biological processes and pathways associated with these potential target genes. Terms related to positive regulation of cell proliferation are by far the most highly represented and enriched among the target genes suppressed by miRNA upregulation during senescence. This is consistent with a pivotal role for miRNAs in senescence regulation, since inhibition of proliferation-promoting genes is the hallmark of senescence 
. In parallel to the inhibition of cell proliferation during senescence, we found that miRNAs tend to promote negative regulation of apoptosis; terms such as ‘negative regulation of apoptosis’ and ‘negative regulation of programmed cell death’ were significantly enriched among the target genes stimulated by miRNA downregulation during senescence. The cell commitment to senescence instead of apoptosis is pathologically relevant because, while apoptosis eliminates damaged or stressed cells, senescence arrests their growth and allows damaged cells to persist and acquire abnormalities that alter tissue microenvironment and promote aging and cancer. Our data suggests that miRNAs may sway the cellular decision to commit to senescence instead of apoptosis. Senescent fibroblasts are known to acquire resistance to apoptotic stimuli 
, however, it remains unclear what makes a cell undergo senescence or apoptosis 
. The miRNAs found here to be potential regulators of apoptosis resistance in senescent cells could be used to investigate the mechanisms responsible for committing cells to senescence instead of apoptosis.
Other enriched biological processes suppressed by miRNA upregulation during senescence include positive regulation of cellular metabolic and biosynthetic processes, indicating an inhibition of biosynthetic pathways, which may reflect the cessation of growth during senescence. However, GO terms related to protein metabolic processes and biopolymer modification were significantly enriched among the target genes stimulated by miRNA downregulation during senescence. The stimulation of protein metabolism and biopolymer modification is consistent with evidence that anabolic processes are enhanced during cellular senescence 
and could be part of metabolic alterations responsible for the increase in cell volume and mass resulting in the enlarged and flattened cell morphology that is typical of senescence. Furthermore, the KEGG pathway ‘actin cytoskeleton’, which is important for cell morphology, was also significantly enriched (Figure S1
); its dysregulation may be involved in rearranging cytoskeletal structures to give rise to the flattened cell morphology. We also found that terms related to positive regulation of cell adhesion were significantly enriched among the target genes stimulated by miRNA downregulation during senescence. Since the capability of cells to adhere to each other and to the extracellular matrix is linked to cytoskeleton organization and cellular shape, the changes in cell adhesion regulation may contribute to the morphological changes induced by senescence. In support of our result, a previous functional study of senescence-associated miRNA targets showed enrichment in pathways involved in cytoskeletal remodeling 
. Thus, miRNAs may regulate the morphological changes characteristic of the senescence phenotype.
In conclusion, our deep sequencing study has identified many known miRNAs that are differentially regulated with IMR90 cell senescence, and a number of novel miRNAs. Our results included known miRNAs that have not been previously reported as differentially regulated by senescence in microarray profiling studies, thus adding to the increasing evidence for miRNA contribution to senescence. Functional analysis of mRNA transcripts targeted by senescence-regulated miRNAs indicates that miRNAs may be key contributors to the cellular changes that make up the senescence phenotype. Hence, our findings could be used to generate new hypotheses to be tested in follow-up studies designed to further elucidate the role of miRNAs in senescence.