Title: A miR-34a-SIRT6 axis in the squamous cell differentiation network
Abstract: Article16 July 2013free access Source Data A miR-34a-SIRT6 axis in the squamous cell differentiation network Karine Lefort Corresponding Author Karine Lefort Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Department of Dermatology, University Hospital CHUV, Lausanne, Switzerland Search for more papers by this author Yang Brooks Yang Brooks Cutaneous Biology Research Center Massachusetts General Hospital, Charlestown, MA, USA Search for more papers by this author Paola Ostano Paola Ostano Laboratory of Cancer Genomics, Fondazione Edo ed Elvo Tempia Valenta, Biella, Italy Search for more papers by this author Muriel Cario-André Muriel Cario-André Inserm U876 and National Reference Centre for Rare Skin Diseases, Bordeaux University Hospitals, Bordeaux, France Search for more papers by this author Valérie Calpini Valérie Calpini Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Juan Guinea-Viniegra Juan Guinea-Viniegra Fundación Banco Bilbao Vizcaya (F-BBVA) - CNIO Cancer Cell Biology Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain Search for more papers by this author Andrea Albinger-Hegyi Andrea Albinger-Hegyi HNO Praxis, HNO Zuerich Fraumünster, Zürich, Switzerland Search for more papers by this author Wolfram Hoetzenecker Wolfram Hoetzenecker Cutaneous Biology Research Center Massachusetts General Hospital, Charlestown, MA, USA Search for more papers by this author Ingrid Kolfschoten Ingrid Kolfschoten Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Erwin F Wagner Erwin F Wagner Fundación Banco Bilbao Vizcaya (F-BBVA) - CNIO Cancer Cell Biology Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain Search for more papers by this author Sabine Werner Sabine Werner Institute of Cell Biology, ETH Zürich, Zürich, Switzerland Search for more papers by this author Gian Paolo Dotto Corresponding Author Gian Paolo Dotto Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Cutaneous Biology Research Center Massachusetts General Hospital, Charlestown, MA, USA Search for more papers by this author Karine Lefort Corresponding Author Karine Lefort Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Department of Dermatology, University Hospital CHUV, Lausanne, Switzerland Search for more papers by this author Yang Brooks Yang Brooks Cutaneous Biology Research Center Massachusetts General Hospital, Charlestown, MA, USA Search for more papers by this author Paola Ostano Paola Ostano Laboratory of Cancer Genomics, Fondazione Edo ed Elvo Tempia Valenta, Biella, Italy Search for more papers by this author Muriel Cario-André Muriel Cario-André Inserm U876 and National Reference Centre for Rare Skin Diseases, Bordeaux University Hospitals, Bordeaux, France Search for more papers by this author Valérie Calpini Valérie Calpini Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Juan Guinea-Viniegra Juan Guinea-Viniegra Fundación Banco Bilbao Vizcaya (F-BBVA) - CNIO Cancer Cell Biology Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain Search for more papers by this author Andrea Albinger-Hegyi Andrea Albinger-Hegyi HNO Praxis, HNO Zuerich Fraumünster, Zürich, Switzerland Search for more papers by this author Wolfram Hoetzenecker Wolfram Hoetzenecker Cutaneous Biology Research Center Massachusetts General Hospital, Charlestown, MA, USA Search for more papers by this author Ingrid Kolfschoten Ingrid Kolfschoten Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Erwin F Wagner Erwin F Wagner Fundación Banco Bilbao Vizcaya (F-BBVA) - CNIO Cancer Cell Biology Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain Search for more papers by this author Sabine Werner Sabine Werner Institute of Cell Biology, ETH Zürich, Zürich, Switzerland Search for more papers by this author Gian Paolo Dotto Corresponding Author Gian Paolo Dotto Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Cutaneous Biology Research Center Massachusetts General Hospital, Charlestown, MA, USA Search for more papers by this author Author Information Karine Lefort 1,2, Yang Brooks3, Paola Ostano4, Muriel Cario-André5, Valérie Calpini1, Juan Guinea-Viniegra6, Andrea Albinger-Hegyi7, Wolfram Hoetzenecker3, Ingrid Kolfschoten1, Erwin F Wagner6, Sabine Werner8 and Gian Paolo Dotto 1,3 1Department of Biochemistry, University of Lausanne, Epalinges, Switzerland 2Department of Dermatology, University Hospital CHUV, Lausanne, Switzerland 3Cutaneous Biology Research Center Massachusetts General Hospital, Charlestown, MA, USA 4Laboratory of Cancer Genomics, Fondazione Edo ed Elvo Tempia Valenta, Biella, Italy 5Inserm U876 and National Reference Centre for Rare Skin Diseases, Bordeaux University Hospitals, Bordeaux, France 6Fundación Banco Bilbao Vizcaya (F-BBVA) - CNIO Cancer Cell Biology Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain 7HNO Praxis, HNO Zuerich Fraumünster, Zürich, Switzerland 8Institute of Cell Biology, ETH Zürich, Zürich, Switzerland *Corresponding authors. Department of Biochemistry, University of Lausanne, Chemin des Boveresses 155, Epalinges 1066, Switzerland. Tel.:+41 21 692 5720; Fax:+41 21 692 5705; E-mail: [email protected] or Tel.:+41 21 692 5730; Fax:+41 21 692 5705; E-mail: [email protected] The EMBO Journal (2013)32:2248-2263https://doi.org/10.1038/emboj.2013.156 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Squamous cell carcinomas (SCCs) are highly heterogeneous tumours, resulting from deranged expression of genes involved in squamous cell differentiation. Here we report that microRNA-34a (miR-34a) functions as a novel node in the squamous cell differentiation network, with SIRT6 as a critical target. miR-34a expression increases with keratinocyte differentiation, while it is suppressed in skin and oral SCCs, SCC cell lines, and aberrantly differentiating primary human keratinocytes (HKCs). Expression of this miRNA is restored in SCC cells, in parallel with differentiation, by reversion of genomic DNA methylation or wild-type p53 expression. In normal HKCs, the pro-differentiation effects of increased p53 activity or UVB exposure are miR-34a-dependent, and increased miR-34a levels are sufficient to induce differentiation of these cells both in vitro and in vivo. SIRT6, a sirtuin family member not previously connected with miR-34a function, is a direct target of this miRNA in HKCs, and SIRT6 down-modulation is sufficient to reproduce the miR-34a pro-differentiation effects. The findings are of likely biological significance, as SIRT6 is oppositely expressed to miR-34a in normal keratinocytes and keratinocyte-derived tumours. Introduction Squamous cell carcinoma (SCC) is the most frequent type of solid human tumours and a main cause of cancer-related death, occurring in many internal organs as well as skin. A network of squamous cell differentiation connected genes has emerged that is either deregulated or mutated in SCC, and plays a demonstrated or likely driver role (Agrawal et al, 2011; Stransky et al, 2011; Wang et al, 2011b). The skin provides an excellent model system to understand integrated control of squamous cell differentiation under normal conditions, in response to external insults and in tumourigenesis (Lefort and Dotto, 2004). Complex biological systems such as the skin appear to be intrinsically 'robust', that is, organized as scale-free networks, with most connections being made to critical 'hubs' (or signalling centres). Several 'hubs' that integrate and maintain skin homeostasis have been identified, including the transcriptional regulators p53 and Notch1 (Dotto, 2009). A main role of p53 is that of a sensor of acute or chronic alterations in normal cellular physiology and, more specifically, DNA and chromosomal integrity (Riley et al, 2008; Olivier et al, 2009). However, while initial analysis of p53 knockout mice suggested that this gene is dispensable for development (Donehower et al, 1992), subsequent studies pointed to its involvement in differentiation of several tissues (Dotto, 2009; Olivier et al, 2009). Importantly, p53 has also been implicated as a negative regulator of stem cell potential with clear implications for cancer development (Jerry et al, 2008; Zhang et al, 2008; Zheng et al, 2008). In keratinocytes of the proliferative compartment, increased p53 activity caused by UVB exposure or inhibition of EGFR signalling triggers a pro-differentiation transcriptional response connected with increased Notch1 expression and activity (Kolev et al, 2008; Mandinova et al, 2008; Guinea-Viniegra et al, 2012). Down-regulation of Notch1 transcription in keratinocyte-derived tumours can also be explained, at least in part, by compromised p53 function: in primary human keratinocytes, endogenous p53 binds to the Notch1 promoter and p53 knockdown results in down-modulation of Notch1 expression, whereas increased p53 levels lead to Notch1 upregulation (Lefort et al, 2007; Yugawa et al, 2007; Mandinova et al, 2008). In concert with protein-encoding genes, microRNAs (miRNAs) play a key role in integrating multiple signalling inputs and coordinating the overall gene expression response of cells and tissues. miRNAs are often expressed in a lineage- and time-specific fashion and can control cell fate decisions as well as tumour development (reviewed in Lee and Dutta, 2009). In the skin, miRNAs appear to play an important role in control of epidermal and hair follicle development and function (Andl et al, 2006; Yi et al, 2006; Lena et al, 2008; Zhang et al, 2011). Keratinocyte-specific deletion of the Dicer, DGCR8, or Ago 1 and 2 genes, encoding essential miRNA processing enzymes, results in severely altered hair follicle morphogenesis and in their degeneration (Yi et al, 2006, 2009; Teta et al, 2012; Wang et al, 2012). miRNAs have also been implicated in epidermal homeostasis (Botchkareva, 2012; Rivetti di Val Cervo et al, 2012), wound healing (Banerjee et al, 2011; Pastar et al, 2012), various skin disorders (Bostjancic and Glavac, 2008; Schneider, 2012), epidermal cell cycle control and carcinogenesis (Antonini et al, 2010; Dziunycz et al, 2010; Darido et al, 2011; Sand et al, 2012; Xu et al, 2012). miR-34 family members (a, b, and c) are among the most highly studied miRNAs (Hermeking, 2010). They are best understood as mediators of p53 action on growth arrest, senescence, and apoptosis (Hermeking, 2007; Raver-Shapira et al, 2007), as well as inhibition of epithelial–mesenchymal transition (EMT) (Siemens et al, 2011). miR-34a is the most prevalent form, except in lung and testis where miR-34b and miR-34c are more abundant (Bouhallier et al, 2010; Hermeking, 2010). Expression of miR-34a is down-modulated in a variety of cancers, including melanoma, prostate, pancreatic, colorectal, and non-small-cell lung cancers and neuroblastoma (Hermeking, 2010). miR-34a maps to the 1p36 genomic region that is frequently deleted in human cancers. In cancers where this region is intact, increased methylation of the miR-34a promoter region has been found (Hermeking, 2010). Besides its role as downstream mediator of p53, miR-34a can enhance p53 activity through a mechanism involving decreased deacetylation via down-modulation of SIRT1 expression (Yamakuchi and Lowenstein, 2009). The sirtuin family comprises seven members, SIRT1 to −7. They are NAD+-dependent protein deacetylases and/or mono-[ADP-ribosyl] transferases. These proteins diverge in localization and functions, with SIRT1, −2, −6, and −7 acting as critical modulators of epigenetic modifications, while others, SIRT3, −4 and −5, functioning mostly in the mitochondria. Through one or more of these mechanisms, sirtuins are emerging as key players in development, cell differentiation, and ageing (Bosch-Presegue and Vaquero, 2011; Carafa et al, 2012). Here, as part of a study of miRNAs that are aberrantly deregulated in keratinocyte-derived cancer, we have uncovered miR-34a as a novel node in the squamous cell differentiation network, with SIRT6 as critical target. Results miR-34a expression is suppressed in skin and oral SCCs and in keratinocytes with a compromised differentiation programme Keratinocyte differentiation and tumour development are inversely related events. We hypothesized that specific miRNAs participate in keratinocyte tumour suppression through a mechanism linked with differentiation. As an initial test of this possibility, the total pattern of microRNA expression, as assessed by microarray hybridization, was compared in exponentially growing primary human keratinocytes (HKCs) versus the keratinocyte-derived SCC13 cell line (Rheinwald and Beckett, 1980) using two different platforms (LC Sciences and Agilent Technologies). A number of miRNAs showed opposite expression in the two cell types, with miR-34a and miR-203 being the most significantly downregulated ones in the SCC cells (Figure 1A). While the role of miR-203 in keratinocyte differentiation is well established (Lena et al, 2008; Yi et al, 2008), a role of miR-34a has been only studied in the context of the cell cycle (Antonini et al, 2010). Of the three isoforms (miR-34a, b, and c), miR-34a is the one mainly expressed in HKCs, while miR-34b and c are present only at low levels (Supplementary Figure S1A). qRT–PCR analysis showed strong upregulation of miR-34a but not miR-34b and c in differentiating primary human keratinocytes (HKCs) (Figure 1B and C and Supplementary Figure S1B), and significant down-modulation in several keratinocyte-derived SCC cell lines (Figure 1D). Consistent with these results, in situ hybridization revealed more pronounced expression of miR-34a in the suprabasal than basal layers of normal epidermis and drastic suppression in a skin SCC of the same patient (Figure 1E). Several human skin samples of in situ squamous cell carcinoma lesions (actinic keratoses) excised together with flanking normal epidermis were utilized for laser capture microdissection (LCM) followed by qRT–PCR analysis. As shown in Figure 1F, miR-34a expression was found to be significantly downregulated in the neoplastic versus normal epidermis areas (P-value=0.0086), in parallel with down-modulation of the keratin 1 differentiation marker in all samples except one (Figure 1F). MiR-34a expression was also reduced in a set of oral SCCs versus normal mucosa from the same patients (Figure 1G). Figure 1.miR-34a expression is downregulated in keratinocyte-derived SCC cell lines and tumours, while it is induced with differentiation. (A) Differential miRNA expression in human skin SCC13 cells versus normal human primary keratinocytes (HKCs) was analysed using both the LC Sciences (white bars) and Agilent Technologies miRNA microarray platforms (grey bars). Results are expressed as fold change of miRNA levels in SCC13 cells versus HKCs. Shown are only miRNAs with fold change >2 or <−2 and P-values<0.01 in both platforms. (B, C) HKCs were induced to differentiate either by culture in suspension (susp.) for the indicated time periods (B) or at confluency for 7 days (C). Levels of mature (miR-34a) were measured by qRT–PCR using specific Taqman probes using the snRNA Z30 for normalization and levels of precursor miR-34a (pre-miR-34a) were measured by conventional qRT–PCR using 36β4 for normalization. (D) Mature miR-34a expression was assessed in HKCs and in different skin- (SCC12 and SCC13) and oral mucosa- (SCCO11, SCCO12, SCCO22, and SCCO28) derived SCC cell lines by qRT–PCR as previously described (Raymond et al, 2005) with 5S RNA for normalization. (E) Human skin (upper panels) as well as skin SCC (lower panels) from the same patient were probed with double digoxigenin (DIG)-miR-34a probes for in situ hybridization. Bars, 50 μm. (e refers to epidermis, d to dermis, and t to tumour). (F) Excised skin samples (S1-S8), containing, in each case, a field of normal epidermis (NE) well separated from one with actinic keratosis (AK) lesions were utilized for laser capture microdissection (LCM) of the AK epithelium versus epidermis further away (as shown for one of the cases—S7, by the histological image after LCM), followed by qRT–PCR analysis of precursor miR-34a and keratin 1 marker expression, using β-actin for normalization. Values are shown as matched pairs of AK versus normal epidermis for each of the samples. Statistical significance of the differences between the AK versus normal epidermis groups was calculated by Student's paired t-test. (G) Levels of precursor miR-34a (pre-miR-34a) were measured by qRT–PCR in oral SCC versus matched normal oral mucosa from the same patient with 36β4 for normalization. Statistical significance of the differences between tumour and oral mucosa values was calculated by Student's paired t-test. Download figure Download PowerPoint Loss or mutations of p53 is an important mechanism underlying altered differentiation of keratinocyte-derived cancer cells (Lefort et al, 2007; Kolev et al, 2008). In HKCs with either p53 knockdown or expression of a mutant p53 protein, differentiation-induced levels of miR-34a and differentiation markers keratin 1 (K1) and involucrin were concomitantly reduced (Figure 2A and B). Even in the absence of any exogenous manipulations, there is a basal level of p53 activity as well as spontaneously occurring differentiation in keratinocyte cultures (Mandinova et al, 2008), and, even under these conditions, knockdown of p53 resulted in a parallel down-modulation of differentiation markers and miR-34a expression (Figure 2A and Supplementary Figure S2A). Conversely, miR-34a and involucrin as well as p21WAF1/Cip1 were induced in SCC cells upon expression of wild-type p53, while no such effects were elicited by expression of different p53 mutant forms (Figure 2C). Similar results were obtained by analysis of a mouse genetic model for p53 function in skin carcinogenesis. The model is based on mice with a homozygous knock-in replacement of the p53 gene with a gene encoding a p53ER-TAM fusion protein, whose activity can be induced by treatment with tamoxifen (Christophorou et al, 2005; Guinea-Viniegra et al, 2012). In chemically-induced papillomas of these mice with silent p53, restoration of p53 activity by tamoxifen treatment resulted in a concomitant induction of K1 differentiation marker expression and miR-34a levels (Figure 2D). Parallel induction of K1 and miR-34a was also observed in mouse keratinocytes with wild-type p53 or a tamoxifen-induced p53ER-TAM knock-in gene upon oncogenic H-rasV12 expression (Figure 2E). Figure 2.Control of miR-34a expression in keratinocyte differentiation and SCC cells. (A) HKCs stably transduced with a shRNA retrovirus against p53 (+) or empty vector control (−) were kept under proliferating conditions (−) or induced to differentiate in suspension (+) for 24 h. Levels of the indicated transcripts were measured by qRT–PCR with 36β4 for normalization. (B) HKCs stably transduced with a lentiviral vector for the doxycycline-inducible expression of p53 mutant R248W (p53 mt) were either untreated (−) or treated with doxycycline (+) for 5 days. Cells were kept under proliferating conditions (−) or induced to differentiate in suspension (+) for 6 h. Left panel: Levels of the indicated transcripts were measured by qRT–PCR with 36β4 for normalization. Right panel: Parallel cultures were examined by immunoblotting for p53 protein expression. (C) SCC13 cells were transduced for 5 days with retroviruses expressing either p53 wild type (WT), three different p53 mutants harbouring the indicated single amino-acid substitutions (R175H, R248W, and V143A) or empty vector control (ctrl). Levels of mature miR-34a were measured by qRT–PCR as previously described in (Raymond et al, 2005) with 5S RNA for normalization. Levels of p21WAF1/CIP1 and involucrin were measured as in A. (D) p53KI/KI mice with chemically-induced papillomas were treated for 15 days with tamoxifen or vehicle (black and white bars respectively) as reported (Guinea-Viniegra et al, 2012). Levels of mature miR-34a were measured in papillomas from four mice per group by qRT–PCR with Taqman probes and U6 RNA for normalization, with parallel assessment of keratin 1 mRNA levels. (E) Cultured primary keratinocytes from wild-type (p53+/+) and p53KI/KI (p53KI/KI) mice were transduced for 6 days with a H-RasV12 expressing retrovirus (+) or empty vector control (−), followed by treatment with 1 μM 4-OH tamoxifen for 48 h as previously described (Guinea-Viniegra et al, 2012). Levels of mature miR-34a were measured by qRT–PCR, with 5S RNA for normalization, in parallel with keratin 1 mRNA. (F) DNA was extracted from the indicated cells and subjected to bisulphite conversion. Methylation of miR-34a was assayed by PCR using primers specific for unmethylated (U) and methylated (M) miR-34a promoter sequences (primer sequences are given in Supplementary Table 3). (G, H) SCC13 cells were treated with the indicated concentrations (μM) of 5-aza-2′deoxycytidine (5-aza-dC) for 4 days. Levels of mature (miR-34a), primary (pri-34a), and precursor (pre-34a) miR-34a as well as indicated gene expression were determined by qRT–PCR (G) and immunoblotting for Keratin 10 and involucrin (with γ-tubulin for equal loading control) (H).Source data for this figure is available on the online supplementary information page. Source Data for Figure 2 [embj2013156-sup-0001-SourceData-S1.pdf] Download figure Download PowerPoint It has been previously shown that, in mouse keratinocytes, under proliferative conditions, miR-34a expression is under negative control of p63 (Antonini et al, 2010). Given its frequent deregulation in SCCs (Perez and Pietenpol, 2007), we tested whether p63 was also involved in control of miR-34a expression in HKC differentiation and/or SCC cells. Consistent with the previous report (Antonini et al, 2010), sustained ΔNp63α expression via retroviral vector infection reduced miR-34a levels in HKCs under basal conditions, but did not prevent its induction with differentiation, and even slightly enhanced it (Supplementary Figure S2B–D). As a control, FGF21, a known p63 target (Vigano et al, 2006), was upregulated in p63 overexpressing HKCs under both growing and differentiating conditions (Supplementary Figure S2C). In converse experiments, p63 knockdown in human keratinocytes caused only a slight upregulation of miR-34a expression (in one experiment, with no upregulation in another) that could also be explained by a concomitant upregulation of p53 (Supplementary Figure S2E). This is at variance with the strong upregulation of miR-34a reported for mouse keratinocytes upon ΔNp63α knockdown (Antonini et al, 2010), possibly due to species-specific differences in regulation of this miRNA and/or different culture conditions. p63 knockdown had also no consistent effects on miR-34a expression in SCC cells (Supplementary Figure S2F). Increased promoter DNA methylation could contribute to the low miR-34a expression in SCC cells, as it has been reported for other cancer types (Lodygin et al, 2008; Chim et al, 2010). Bisulphite DNA conversion followed by PCR analysis with methylated- versus unmethylated-specific primers revealed much greater methylation at the miR-34a promoter in SCC cells than either growing or differentiating HKCs (Figure 2F). Treatment of SCC cells with 5-aza-2′-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, resulted in the concomitant induction of miR-34a and differentiation marker expression (Figure 2G and H). miR-34a has pro-differentiation functions To assess whether the pro-differentiation effects of increased p53 activity are linked to miR-34a, HKCs were transfected with specific antagomiRs, causing >10-fold down-modulation of this miRNA (Figure 3A). As previously reported, for increased p53 activity in proliferating keratinocytes (Yugawa et al, 2007; Kolev et al, 2008; Guinea-Viniegra et al, 2012), nutlin-3a treatment resulted in increased involucrin differentiation marker expression, under conditions that resulted in little or no change in apoptosis (Supplementary Figure S2G) in accordance with a previous report on nutlin-3a-treated keratinocytes (Kranz and Dobbelstein, 2006). The nutlin-3a effects on differentiation were paralleled by increased miR-34a expression (Supplementary Figure S2H) and counteracted by antagomiR-mediated silencing of this miRNA (Figure 3B). Basal levels of differentiation marker expression were also suppressed by the miR-34a antagomiRs (Figure 3B). Similar results were observed upon UVB exposure of HKCs plus/minus miR-34a silencing (Figure 3C and Supplementary Figure S2I and J). Figure 3.miR-34a is a positive determinant of keratinocyte differentiation. (A) Proliferating HKCs were transfected for 3 days with miR-34a-specific antagomiRs (α34a) (50 nM) or control scrambled antagomiR (αscr) and levels of mature miR-34a were measured by qRT–PCR using specific Taqman probes. (B) HKCs were transfected as in A and, 24 h later, treated or not with nutlin-3a (10 μM) for additional 48 h. Involucrin and p53 protein levels were analysed by immunoblotting with β-actin as equal loading control. Numbers correspond to folds of induction of involucrin expression relative to untreated control, calculated after densitometric scanning of the autoradiographs and normalization for β-actin expression. (C) HKCs transfected with antagomiR against miR-34a (α34a) or control (αscr) as in A for a total of 3 days were UVB irradiated (50 mJ/cm2) at the indicated times from the end of the experiment, followed by immunoblot analysis for involucrin expression and data quantification by densitometric analysis as in the previous panel. Pattern of p53 protein expression is shown in Supplementary Figure S2J. (D) HKCs transfected for 3 days with miR-34a precursor (+) (25 nM) or scrambled control oligonucleotides (−) were examined for involucrin (inv), keratin 1 (K1), integrin α6 (itga6) and ΔNp63α expression by qRT–PCR with 36β4 for normalization. (E) HKCs at 3 and 5 days after transfection as in the previous panels were analysed by immunoblotting for expression of the indicated proteins. p21WAF1, cyclin E2 and β-actin blots were performed by sequential blotting of the same membrane without stripping, while ΔNp63α and involucrin expression was assessed by a parallel gel/blot with β-actin giving the same pattern of expression. (F) HKCs were stably infected with a lentivirus for doxycycline-inducible expression of p21WAF1/Cip1. Cells were either untreated (−) or treated with doxycycline (500 ng/ml) for 5 days (+) for sustained p21WAF1/Cip1 expression and associated cell cycle arrest. Cells were transfected with miR-34a mimics (25 nM) or scrambled controls for the last 3 days of the experiment. Expression of the indicated genes was assessed by qRT–PCR with 36β4 for normalization. (G, H) HKCs were transfected for 2 days with miR-34a precursor at low concentrations (5 and 10 nM) with scrambled controls used to keep equal total amounts of transfected oligonucleotides. Cells were examined for expression of the indicated differentiation marker (G) and cell cycle and senescence genes (H), by qRT–PCR with 36β4 for normalization. (I) HKCs were stably infected with a lentivirus for doxycycline-inducible expression of miR-34a. Cells were either untreated or treated with doxycycline at the indicated concentrations for 2 days followed by determination of mature miR-34a levels in parallel with expression of the indicated genes by qRT–PCR with 36β4 for normalization. (J) HKCs stably infected with a lentivirus for doxycycline-inducible expression of miR-34a (pTRIPZ-34a) or empty vector control (pTRIPZ) were treated with doxycycline (50 ng/ml) for 4 days, followed by measurement of mature miR-34a levels in parallel with expression of the indicated genes by qRT–PCR as in I.Source data for this figure is available on the online supplementary information page. Source Data for Figure 3 [embj2013156-sup-0002-SourceData-S2.pdf] Download figure Download PowerPoint To assess the more direct consequences of increased miR-34a expression, HKCs were transfected with miR-34a precursor oligonucleotides in parallel with scrambled controls. Transfection of miR-34a mimics at commonly used doses (25 nM) resulted in significant morphological changes associated with growth arrest and senescence rather than apoptosis (Supplementary Figure S3 A–D). miR-34a is part of a positive feedback mechanism that reinforces p53 activity through down-modulation of SIRT1 deacetylation (Yamakuchi and Lowenstein, 2009). Consistent with this, p53 protein levels were found to oscillate in keratinocytes with miR-34a over-expression (Supplementary Figure S3D), as expected from increased p53 activity causing destabilization of the p53 protein through induction of MDM2 (Wu et al, 1993). In parallel, miR-34a overexpression caused increased expression of terminal differentiation markers like involucrin and keratin 1, and down-modulation of genes associated with the proliferative compartment, like the genes encoding integrin α6 and ΔNp63α (Figure 3D and E). To assess whether th