Senescence-associated microRNAs target cell cycle regulatory genes in normal human lung fibroblasts

Highlights

• Identification of a small set of senescence-associated microRNAs (SA-miRs).

• SA-miRs target genes regulating cell cycle progression and telomere maintenance.

• MiR-221 is up-regulated during replicative and oxidative stress induced senescence

• MiR-221/222 targets cell cycle effectors.

Abstract

Senescence recapitulates the ageing process at the cell level. A senescent cell stops dividing and exits the cell cycle. MicroRNAs (miRNAs) acting as master regulators of transcription, have been implicated in senescence. In the current study we investigated and compared the expression of miRNAs in young versus senescent human fibroblasts (HDFs), and analysed the role of mRNAs expressed in replicative senescent HFL-1 HDFs. Cell cycle analysis confirmed that HDFs accumulated in G₁/S cell cycle phase. Nanostring analysis of isolated miRNAs from young and senescent HFL-1 showed that a distinct set of 15 miRNAs were significantly up-regulated in senescent cells including hsa-let-7d-5p, hsa-let-7e-5p, hsa-miR-23a-3p, hsa-miR-34a-5p, hsa-miR-122-5p, hsa-miR-125a-3p, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-181a-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-503-5p, hsa-miR-574-3p, hsa-miR-574-5p and hsa-miR-4454. Importantly, pathway analysis of miRNA target genes down-regulated during replicative senescence in a public RNA-seq data set revealed a significant high number of genes regulating cell cycle progression, both G₁/S and G₂/M cell cycle phase transitions and telomere maintenance. The reduced expression of selected miRNA targets, upon replicative and oxidative-stress induced senescence, such as the cell cycle effectors E2F1, CcnE, Cdc6, CcnB1 and Cdc25C was verified at the protein and/or RNA levels. Induction of G1/S cell cycle phase arrest and down-regulation of cell cycle effectors correlated with the up-regulation of miR-221 upon both replicative and oxidative stress-induced senescence. Transient expression of miR-221/222 in HDFs promoted the accumulation of HDFs in G1/S cell cycle phase. We propose that miRNAs up-regulated during replicative senescence may act in concert to induce cell cycle phase arrest and telomere erosion, establishing a senescent phenotype.

Introduction

Cellular senescence is a state of stable and irreversible cell cycle arrest with active metabolism that normal cells undergo after a finite number of divisions, the Hayflick limit. Senescence can be triggered by intrinsic and extrinsic stimuli including telomere shortening at the end of the cell's lifespan (telomere-initiated senescence) and in response to oxidative, genotoxic or oncogenic stresses (stress-induced premature senescence; SIPS). Several effector mechanisms have been proposed to explain the manner in which diploid cells senesce, including the DNA damage response (DDR), epigenetic regulation, the secretion of a complex mixture of extracellular matrix and inflammatory cytokines generating an inflammatory microenvironment, referred to as the senescence-associated secretory phenotype (SASP), and autophagy. Senescent cells display senescence-associated β-galactosidase (SA-β-Gal) activity and undergo chromatin remodeling resulting in senescence-associated heterochromatin foci (SAHF) and DNA damage foci (SDFs). SAHFs are characterised by the presence of heterochromatin-associated histone modifications (H3K9me) and heterochromatin protein-1 (HP1), and contain E2F target genes, which SAHFs are thought to silence. Senescence is established by de-repression of the INK4/ARF locus, acting as sensor to link stress with pRb and p53 tumor suppressors (Campisi and d'Adda di Fagagna, 2007, d'Adda di Fagagna, 2008, Fumagalli and d'Adda di Fagagna, 2009, Rodier and Campisi, 2011).

Several studies on the mechanisms of senescence have identified a class of non-coding RNAs (ncRNAs), microRNAs (miRNAs, miRs) and long ncRNAs as important regulators of cell senescence associated gene expression programs (Bischof and Martinez-Zamudio, 2015, Gorospe and Abdelmohsen, 2011, Grillari and Grillari-Voglauer, 2010, Lanceta et al., 2010, Olivieri et al., 2013).

MiRNAs are small, non-coding, endogenous single-stranded RNA molecules of ~ 22 nucleotides (nt) length that regulate gene expression in many cellular processes at both the transcriptional and post-transcriptional levels. MiRNAs act mostly as negative regulators of mRNA translation and/or stability by binding to complementary sequences in the 3′ untranslated regions (3′ UTR) of their target mRNAs, including those genes that mediate cell cycle regulation, stress responses, differentiation, senescence, apoptosis, inflammation and have also emerged as important regulators of tumourigenesis acting as tumor promoters (oncomirs) or suppressors (Croce, 2009, d'Adda di Fagagna, 2014, Esquela-Kerscher and Slack, 2006, Esteller, 2011, Gartel and Kandel, 2008, Gorospe and Abdelmohsen, 2011, Hammond, 2007, Iorio and Croce, 2012, Leung and Sharp, 2010, Lujambio and Lowe, 2012, Ventura and Jacks, 2009, Voorhoeve, 2010, Voorhoeve and Agami, 2007). Some miRNAs regulate specific individual targets, while others can function as master regulators of key processes (Ambros, 2004, Ameres and Zamore, 2013, Bueno and Malumbres, 2011, Cech and Steitz, 2014, Gurtan and Sharp, 2013, Inui et al., 2010, Leung and Sharp, 2010).

MicroRNAs have emerged as mediators of senescence and the ageing process (Harries, 2014). Previous studies showed that the expression of several miRs is altered in senescent human lung and skin fibroblasts (HDFs) (Bischof and Martinez-Zamudio, 2015, Gorospe and Abdelmohsen, 2011, Hackl et al., 2010, Holly et al., 2015, Li et al., 2009a, Maes et al., 2009, Marthandan et al., 2016, Marthandan et al., 2015, Wang et al., 2011), in senescent endothelial cells and other cell types (Dellago et al., 2013, Di Bernardini et al., 2014, Greussing et al., 2013, Hackl et al., 2010, Napolitano et al., 2014, Zhu et al., 2013). Importantly, senescent cells of different lineages demonstrate tissue-specificity in miRNA profiles (Holly et al., 2015).

Many miRNAs act as inducers of senescence, also known as senescence-associated miRNAs (SA-miRNAs or SA-miRs) (Bischof and Martinez-Zamudio, 2015, Gorospe and Abdelmohsen, 2011). In was shown that in senescent human WI-38 and MRC-5 HDFs a group of 15 miRNAs were down-regulated and these miRNAs represented three clusters including miRs-106b/93/25, the miR-17-92 polycistron and the miR-106a-92 polycistron. Overexpression of representative miRNAs of this group promoted cell proliferation and delayed senescence (Brosh et al., 2008). Interestingly, p53 which is induced in senescence, down-regulates the expression of oncomiR-17-92 and induces the transcription of SA-miRs, miR-34a/b/c which repress a number of target genes to promote growth arrest and apoptosis (He et al., 2007, Hermeking, 2010). In senescing primary mouse and human fibroblasts the histone demethylase KDM2B is epigenetically silenced leading to the induction of the tumor suppressor miRNAs let-7b (Benhamed et al., 2012, Johnson et al., 2007, Toledano, 2013). Several other miRs, including miR-34a and miR-146 have been implicated in both replicative and oncogene-induced senescence (Bhaumik et al., 2009, Bonifacio and Jarstfer, 2010, Christoffersen et al., 2010, Maes et al., 2009, Tazawa et al., 2007). In contrast to SA-miRs, functional genetic screens have identified miRNAs that rescued HDFs from oncogene-induced senescence (Borgdorff et al., 2010) including the oncomirs, miR-372 and miR-373 (Voorhoeve et al., 2006), and miR-378 (Kooistra et al., 2014).

Here, we investigated the expression of miRNAs in human lung diploid fibroblasts (HDFs), HFL-1, undergoing replicative senescence (RS) and in their young counterparts, and analysed the targets of the SA-miRs exclusively expressed in senescent HFL-1 and MRC-5 HDFs. Comparisons and bioinformatics analysis revealed a significant high number of down-regulated genes regulating cell cycle progression, both G₁/S and G₂/M cell cycle transitions, including CcnE, E2F1, Cdc6, CcnB1 and Cdc25C and telomere maintenance, both of which govern senescence and cancer. Moreover, transient overexpression of one of the SA-miRs, miR-221/222 retarded cell proliferation by targeting cell cycle effector gene expression.