miR-376a-3p and miR-376b-3p overexpression in Hutchinson-Gilford progeria fibroblasts inhibits cell proliferation and induces premature senescence

Summary Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder, in which an abnormal and toxic protein called progerin, accumulates in cell nuclei, leading to major cellular defects. Among them, chromatin remodeling drives gene expression changes, including miRNA dysregulation. In our study, we evaluated miRNA expression profiles in HGPS and control fibroblasts. We identified an enrichment of overexpressed miRNAs belonging to the 14q32.2-14q32.3 miRNA cluster. Using 3D FISH, we demonstrated that overexpression of these miRNAs is associated with chromatin remodeling at this specific locus in HGPS fibroblasts. We then focused on miR-376b-3p and miR-376a-3p, both overexpressed in HGPS fibroblasts. We demonstrated that their induced overexpression in control fibroblasts decreases cell proliferation and increases senescence, whereas their inhibition in HGPS fibroblasts rescues proliferation defects and senescence and decreases progerin accumulation. By targeting these major processes linked to premature aging, these two miRNAs may play a pivotal role in the pathophysiology of HGPS.

miR-376a and miR-376b overexpression decreases cell proliferation and increases senescence

INTRODUCTION
Hutchinson-Gilford progeria syndrome (HGPS; OMIM #176670) is a rare genetic disease affecting approximately one in 8-10 million children. Children with HGPS appear healthy at birth and progressively develop clinical features of premature and accelerated aging within the first years of life. Myocardial infarction due to systemic arterial alterations and stiffness is the most frequent cause of death, which occurs at a mean age of 14.6 years (Gordon et al., 2014). Classic HGPS is primarily caused by a de novo mutation (c.1824C > T, p.G608G) in exon 11 of the LMNA gene, encoding nuclear A-type lamins (De Sandre-Giovannoli et al., 2003;Eriksson et al., 2003). This mutation leads to the production of a 50 amino acid internally truncated, farnesylated prelamin A called progerin. This toxic protein affects the structure and functions of the nucleus and triggers multiple deleterious effects in HGPS cells. For instance, progerin induces mechanical defects, slows proliferation and the cell cycle, alters chromatin organization, and delays DNA repair, enhancing senescence (Cau et al., 2014). Moreover, progerin accumulates in cells, likely due to defective clearance, which might be linked with autophagy (Cenni et al., 2011;DuBose et al., 2018).
Identification in 2012 of the major role of the miR-9 microRNA (miRNA) in HGPS led to a better understanding of the disease (Jung et al., 2012;Nissan et al., 2012). MiRNAs are small noncoding RNAs (19-25 nucleotides) that control gene expression by pairing in most cases, to the 3 0 UTR of messenger RNAs (mRNAs), leading to translational repression or mRNA degradation (Kim and Nam, 2006). MiR-9, which is highly expressed in neurons, binds to the 3 0 UTR of wild-type prelamin A and progerin transcripts, triggering their degradation, thus participates to the protection of neural cells from progerin accumulation in affected children (Jung et al., 2012;Nissan et al., 2012). Several other miRNAs have been identified as dysregulated in laminopathies and are associated with deleterious effects (Frankel et al., 2018). Using Zmpste24 À/À progeria mouse model, only four miRNAs have been described (Mariñ o et al., 2010;Ugalde et al., 2011;Xiong et al., 2015;Zhang et al., 2017). To our knowledge, no global miRNA profiling has been reported in human HGPS fibroblasts.
To bridge this gap, we performed global miRNA profiling of 375 miRNAs in five HGPS and five control (wildtype) fibroblasts. We identified eight overexpressed miRNAs, among which seven belonged to the 14q32.2-14q32.3 miRNA gene cluster. At this locus, expression changes correlate with chromatin modifications. By focusing on miR-376b-3p and miR-376a-3p, two miRNAs from this cluster belonging to the same family, we discovered that they may play a role in cell proliferation and progerin clearance defects, ultimately leading to the premature senescence, which participates to HGPS pathophysiology.

Profiling miRNA expression in HGPS fibroblasts
We profiled the expression of 375 miRNAs using a quantitative RT-PCR (RT-qPCR) approach in five HGPS compared with five wild-type (controls) fibroblasts at passage 12 G 2 (P12 G 2). To evaluate miRNA expression evolution in the context of ''cellular aging'' (Benson et al., 2010), we also included a later passage (P20 G 1) for two controls (C1 and C5) and three HGPS fibroblasts (HGPS2, HGPS3 and HGPS4) (Table  S1). Of note, for HGPS1 and HGPS5, fibroblasts stopped proliferating before P20 due to premature senescence. Based on recorded quantification cycle (Cq) values, 188 miRNAs out of the 375 tested were considered expressed (see STAR Methods section) in our samples and were retained for further analysis (https:// doi.org/10.6084/m9.figshare.9642803.v1).
Among the 188 miRNAs expressed, statistical analyses led to the identification of 14 miRNAs that were differentially expressed between HGPS and control cells (p< 0.05, t-test, see STAR Methods section). Unsupervised hierarchical clustering of these 14 differentially expressed miRNAs separated samples into two different groups ( Figure 1A). The first group included three HGPS fibroblasts at late passage P20 G 1 and two HGPS fibroblasts at early passage P12 G 2, whose proliferation ceased before P20. The second group included all controls and the three other HGPS samples at P12 G 2. This clustering suggests that, depending on the patient, miRNA profiling of HGPS fibroblasts at early passages (P12 G 2) could be similar either to controls or alternatively to other HGPS fibroblasts at later passages (P20 G 1). Among the 14 differentially expressed miRNAs, almost all upregulated miRNAs (7 out of 8) and none of the downregulated miR-NAs (0 out of 6) belonged to a large gene cluster located in the 14q32.2-14q32.3 region ( Figure 1B, p< 0.05, hypergeometric test), suggesting a global dysregulation of this cluster. iScience Article miRNA dysregulation in the 14q32 region is associated with epigenetic changes A-type lamins are involved in chromatin organization, and contact-repressed genomic regions are called lamin-associated domains (LADs) (Guelen et al., 2008;Robin and Magdinier, 2016). Because the 14q32.2-14q32.3 miRNA gene cluster (hereafter called the 14q32 cluster) is localized in the vicinity of one of these LADs (Rao et al., 2014) ( Figure S1A), we wondered whether this dysregulation might result from local chromatin remodeling. Therefore, we investigated the topology of the locus by three-dimensional fluorescence in situ hybridization (3D-FISH) using a DNA probe spanning the region of interest (RP11-123M6) and a probe targeting the most unique telomeric region of chromosome 14q (subtelomeric probe, Figure 2A). The 14q32 cluster is localized 5 Mb from the telomeres. The subtelomeric probe (<1 Mb from telomeres) is used to evaluate changes in the position of the 14q32 cluster relative to the telomeric region and to its position to the nuclear periphery (Wood et al., 2014).
We performed 3D-FISH in four HGPS fibroblasts (HGPS2, HGPS4, HGPS5, and HGPS6) and in three control fibroblasts (C1, C2, and C5) ( Figure 2B and Figure S1B). In control fibroblasts, we showed that the 14q32 cluster is localized close to the nuclear envelope. The probe spanning the 14q32 cluster and the subtelomeric probe were colocalized in 95.6% of nuclei ( Figure 2C). In HGPS fibroblasts, the distance between the two probes was increased, with only 5.75% of nuclei presenting colocalized probes ( Figures 2B and 2C, p< 0.0001, Fisher's test; Figure S1C, Mann Whitney test), as was the distance between the nuclear rim and the 14q32 probe ( Figure 2D, p< 0.05, Mann-Whitney test).
To determine whether the presence of progerin is the cause of this cluster delocalization, we first transfected HGPS5 fibroblasts with antisense morpholinos (AON) targeting exons 10 and 11 of the LMNA gene, known to reduce progerin production . As expected, only 32% of HGPS5 nuclei expressed progerin after transduction of AON, as opposed to 86% and 90% under Endoporter and scramble conditions, respectively ( Figure S1D). In HGPS cells, AON transfection restored colocalization between the two probes and their proximity to the nuclear envelope compared with control conditions (scramble and Endoporter, Figures 2E-2G), highlighting the direct link between progerin production and changes in the topology of this locus.
We challenged these results by reciprocally overexpressing progerin in control fibroblasts to evaluate the impact on chromatin conformation. Control fibroblasts (C2) were transfected with various constructs, namely, a plasmid expressing GFP-progerin, GFP-wild-type lamin A, or GFP alone, and the chromatin structure of the 14q32 locus was evaluated by 3D-FISH ( Figure 2H). As anticipated, 100%, 97% and 98% of nuclei showed colocalized signals in the control conditions (nontransfected, transfected with GFP, and transfected with GFP-wild-type lamin A, respectively), whereas we observed only 72% of colocalized signals in control C2 fibroblasts transfected with the GFP-progerin plasmid ( Figure 2I, p< 0.001, Fisher's test). Moreover, the distance between the 14q32 probe and the nuclear envelope was increased under GFP-progerin conditions compared with control conditions ( Figure 2J).
To assess the specificity of the chromatin remodeling observed in 14q32 locus, we investigated a subtelomeric control region, at the 1p36 locus (a region with LADs located around 5Mb from the telomeres) in C2 fibroblasts transfected with GFP-progerin, GFP-wild-type lamin A, or GFP alone plasmids. We used two probes on the 1p36 separated by approximately 1.4 Mb, as in the 14q32 experiments ( Figure S2A). For this other subtelomeric region, we did not find any difference in the distance of the probes to the nuclear envelope, nor to the number of separated and colocalized probes, between the conditions GFP-progerin and GFP-wild-type lamin A ( Figures S2B-S2D). These results suggest that, in our hands, the progerin modifies specifically the miRNA cluster at the 14q32 locus and seems to not have a global effect (at the LADs levels).
Finally, we assessed the local chromatin structure in C5, HGPS5, and HGPS6 fibroblasts by chromatin immunoprecipitation combined with quantification of enrichment by droplet digital PCR (ChiP-ddPCR) for different sites across the cluster, i.e., miR-376a (miR-376a-1 and miR-376a-2), miR-376b, and other miRNAs (Table S2), using antibodies against CTCF and H3K36me3 shown to be enriched at this locus (UCSC genome browser [Casper et al., 2018]). We observed significant changes in CTCF and H3K36me3 enrichment in HGPS fibroblasts compared with the control ( Figures S2E and S2F), reinforcing the results obtained by 3D-FISH and again, confirming the chromatin changes in this cluster.
Altogether, these observations highlight the conformational change of the 14q32 locus in HGPS fibroblasts containing genes of the overexpressed miRNAs and the role of progerin in this reorganization.
Altogether, these results confirm the overexpression of these two miRNAs in both in vitro and in vivo models of HGPS, suggesting their possible involvement in pathophysiology of this syndrome.
To validate our hypothesis, we first aimed to reproduce the overexpression identified in HGPS fibroblasts by transfecting these two miRNAs (independently) or a control miRNA (miR-control) into control fibroblasts, and we evaluated consequences on cell proliferation, cell cycle, and senescence.

OPEN ACCESS
iScience 25, 103757, February 18, 2022 7 iScience Article reduced CDK2 protein level, whereas the mutant mimics had no effect ( Figures 4D and 4E), confirming the specific effect of these two miRNAs on CDK2 mRNA in our model.
Altogether, these results indicate that the overexpression of miR-376a-3p or miR-376b-3p in fibroblasts reduces cell proliferation associated with CDK2 inhibition and induces premature senescence, which could participate in HGPS pathophysiology.
As autophagy is involved in progerin degradation, we decided to explore if autophagy could be modulated in HGPS fibroblasts due to miR-376a-3p and miR-376b-3p overexpression.
In both assays, we observed a lower level of autophagic markers in HGPS when compared with control fibroblasts, as reported by lower LC3BII levels ( Figures S4A and S4B, p= 0.0052, unpaired t-test) and lower median fluorescence intensity (MFI) ( Figure S4C, p = 0.047, unpaired t-test). Under induction of autophagy with rapamycin at the same concentration, LC3BII levels were increased in control fibroblasts, whereas they remain unmodified in HGPS samples ( Figures S4D and S4E). In addition, treatment of HGPS fibroblasts with bafilomycin blocked autophagic flow. Altogether, these results suggest reduced basal autophagic activity in HGPS fibroblasts, with decreased activation or sensitivity to rapamycin treatment under the experimental conditions tested. (C) DNA replication of C5 fibroblasts measured with BrdU relative expression after transfection of miR-376a-3p, miR-376b-3p, or miR-control (unpaired t-test, n = 4).
(K and L) Western blot analysis on HGPS fibroblasts whole-cell lysates, representative experiment. Immunodetection of lamin A/C, progerin, and GAPDH, after antimiR-376a-3p or antimiR-376b-3p transfection compared with antimiR-control (unpaired t-test, n = 3 on HGPS1 and HGPS5). Data are represented as mean G SEM (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). iScience Article To further assess the impact of miR-376a-3p and miR-376b-3p on autophagy in HGPS fibroblasts, and evaluate autophagic flux, we used ptfLC3, an mRFP-GFP tandem fluorescent-tagged LC3 plasmid (ptfLC3) (Kimura et al., 2007). This plasmid allows discrimination of autophagosomes, which exhibit GFP and mRFP signals (corresponding to yellow punctua) before fusion with lysosomes, from autolysosomes, which only exhibit the mRFP signal. We then starved cells in HBSS medium to induce autophagy. Alternatively, we treated cells with chloroquine, an autophagic inhibitor that alters the acidic pH of lysosomes, to inhibit the fusion between autophagosomes and lysosomes, thus leading to autophagosomes accumulation. Regardless of the condition of autophagy stimulation, the number of autophagosomes increased in HGPS fibroblasts 48 h after antimiR-376a-3p or antimiR-376b-3p transfection (i.e., 24 h after ptfLC3 plasmid transfection) compared with antimiR-control transfection ( Figures S4F  and S4G, ANOVA test). HBSS did not significantly increase the number of autophagosomes per cell compared with the untreated condition. This result confirmed the decrease of sensitivity of HGPS fibroblasts to usual autophagy inducers, already observed after rapamycin treatment. On the other hand, and as expected, the treatment with chloroquine induced an increase of autophagosomes compared with the untreated condition. Lysosome labeling was not observed under any of the conditions tested. We confirmed these results by flow cytometry, showing an increase of the MFI after antimiR-376a-3p or antimiR-376b-3p transfection compared with antimiR-control ( Figure S4H, p< 0.0001 and p = 0.0006 and, unpaired t-test) and by western blot on HGPS6 fibroblasts treated with rapamycin and bafilomycin ( Figure S4I).
Altogether, these results suggest that overexpression of miR-376a-3p or miR376b-3p could participate in progerin accumulation in HGPS fibroblasts due to a decrease of its degradation by autophagy inhibition.

DISCUSSION
This study is the first to present the results of miRNA expression profiling in HGPS human fibroblasts. To our knowledge, only two other studies have reported miRNA expression profiling in a progeroid context, either in mouse embryonic fibroblasts derived from the murine Zmpste24 À/À model, phenotypically close to HGPS patients ( We identified 14 differentially expressed miRNAs in HGPS fibroblasts compared with controls, with seven out of the eight overexpressed belonging to the same cluster at the 14q32 region. This region is an imprinted locus that contains both maternally imprinted noncoding RNAs (MEG3, MEG8, and AS-RTL1) and paternally imprinted genes (DLK1, DIO3, and RTL1). The 14q32 miRNAs are generated from a polycistronic transcript that contains the whole cluster under a coordinated regulation with MEG3 (Glazov et al., 2008;Seitz et al., 2004). Dysregulation of miRNAs belonging to this region has been described in several pathologies, mostly in cancers (Benetatos et al., 2013;Merkerova et al., 2018). Nadal et al. found a correlation between the expression of DLK1, MEG3, and the 14q32 miRNAs cluster and hypothesized a regulation by DNA methylation (Nadal et al., 2014). Among the miRNAs tested in our study belonging to the 14q32 cluster, some of them were not overexpressed. Other studies in different pathological contexts also described an heterogeneity of expression of the 14q32 cluster suggesting other regulating mechanisms (Jishnu et al., 2020;Krokker et al., 2021).
In HGPS cells, progerin accumulates with passages and induces chromatin conformational changes (Harhouri et al., 2017;Kö hler et al., 2020;Lionetti et al., 2020;Robin and Magdinier, 2016;Sebestyé n et al., 2020;Shumaker et al., 2006). We hypothesized that the enrichment of overexpressed miRNAs from the same region could be secondary to chromatin remodeling in HGPS fibroblasts, as supported by other studies at different loci in other contexts (McCord et al., 2013;Merkerova et al., 2018;Oldenburg et al., 2017;Wood et al., 2014). We observed that this locus is in the vicinity of the inner nuclear membrane in control fibroblasts, but distant in HGPS fibroblasts, suggesting loss of its anchoring to the nuclear lamina. Finally, we demonstrated that the decrease in progerin induced by AON transfection relocates the miRNA gene cluster at the nuclear envelope in HGPS fibroblasts, and consistently, overexpression of progerin in control fibroblasts localizes the miRNA gene cluster away from the nuclear envelope. Therefore, we confirmed the major role of A-type lamins in the topological organization of chromosomal territories and the deleterious effect of progerin on chromatin organization in HGPS fibroblasts nuclei. Very importantly, these results also iScience Article indicate the possibility of restoring the nuclear chromosome topology by direct and targeted reduction of progerin. Indeed, the decrease of progerin should have an impact on other regions than the 14q32 region.
Among the deregulated miRNAs pointed by our study, we identified the overexpressed miR-376a-3p and miR-376b-3p as potential actors in HGPS pathophysiology, for several reasons.
First, we demonstrated that, in control fibroblasts, miR-376a-3p or miR-376b-3p ectopic overexpression decreases cell proliferation and CDK2 expression as described in hematopoietic progenitor cells (Wang et al., 2011). Consistently, in HGPS cells, the inhibition of miR-376a-3p results in an increase of cell proliferation. MiR-376a-3p is described to decrease proteins involved in cell-cycle regulation, in addition to CDK2, such as CHK1, Cyclin D2, and Cyclin A (Martinho et al., 1998;Sheng et al., 2013;Yao et al., 2018). We further demonstrated that miR-376a-3p or miR-376b-3p overexpression increases cellular senescence, therefore contributing to the accelerated aging phenotype, whereas their inhibition delays the transition into senescent state. The miR-376a-3p is already described to participate in senescence, even if its effect seems cell-type-dependent (Ye et al., 2020;Yentrapalli et al., 2015).
Moreover, we demonstrated that the inhibition of miR-376a-3p or miR-376b-3p in HGPS fibroblasts moderately but significantly decreases progerin, and that autophagy could participate in this process. Autophagy is a mechanism of degradation and recycling of cellular components, including misfolded, mutant, or abnormal proteins. Our results suggest that, under basal conditions, autophagy is decreased in HGPS fibroblasts compared with controls and is not induced after treatment with conventional simulators (starvation or rapamycin) at the incubation time and concentration tested, contrary to control fibroblasts. Our results are not inconsistent with other studies that showed an activation of autophagy using rapamycin in HGPS fibroblasts, as rapamycin was incubated for a much longer time (from 6 to 150 days) (Cao et al., 2011;Cenni et al., 2011). Therefore, we hypothesized that the incubation time is a major criteria to activate autophagy in HGPS fibroblasts, as these cells seem less sensitive to the usual autophagy activation conditions used in control fibroblasts. Using different techniques, we confirmed that miR-376a-3p or miR-376b-3p inhibition increases autophagy in HGPS fibroblasts, possibly linked to the direct targeting of mRNAs encoding major autophagic proteins, e.g., ATG4c, Beclin-1, and ATG5 (Korkmaz et al., 2012(Korkmaz et al., , 2013Yang et al., 2018).
Finally, we also observed that miR-376a-3p and miR-376b-3p are overexpressed in vivo in aorta (aortic arch and thoracic aorta) from Lmna G609G/G609G mice and are expressed in primary VSMCs from this tissue, whereas the VSMCs from wild-type mice do not express these miRNAs. These cells are deeply depleted in the medial layer of the aortic arch of Lmna G609G/G609G mice Zaghini et al., 2020), a region subjected to high hemodynamic stress. These results emphasize the potential role of these miRNAs in in vivo vascular alteration, representing a major event conditioning the vital prognosis in this disease, which could be very interesting to explore.
Overall, our results further illustrate the complexity of HGPS pathophysiology. We propose a pathophysiological model in which the accumulation of progerin leads to epigenetic modifications associated with the overexpression of several miRNA-encoding genes located in the 14q32 cluster. Among them, miR-376a-3p and miR-376b-3p participate in cell-cycle modulation, leading to decreased proliferation and premature entry into senescence that contribute to accelerated aging at the organism level. Moreover, miR-376a-3p and miR-376b-3p overexpression amplified progerin accumulation in HGPS cells possibly by targeting transcripts encoding proteins involved in the autophagy process, inducing a vicious deleterious cycle.

Limitations of the study
In the study presented here, we provide evidence for the decrease of progerin expression linked to miR-376a-3p or miR-376b-3p inhibition in HGPS fibroblasts. Our results suggest that autophagy is involved in this mechanism and that autophagy is different in HGPS fibroblasts compared with control. A limitation of the study is that, although we showed that miR-376a-3p or miR-376b-3p inhibition leads to autophagy activation in HGPS fibroblasts, we did not precisely dissect all the mechanisms, as autophagy process is very complex. Further experiments are required to fully understand which step is modified in HGPS fibroblasts and how miR-376a-3p and miR-376b-3p are involved. This approach would highly benefit from further improvements including mRNAs and proteins quantification of autophagic actors, including targets of miR-376a-3p and miR-376b-3p linked to this process.
We performed t-tests on the normalized data (DCq), comparing the expression level of all miRNAs in HGPS vs control fibroblasts. We considered a miRNA as significantly differentially expressed if the resulting p value was <0.05. To generate the heatmaps, we also used the normalized data (DCq). We calculated the Euclidian distance between samples and used Ward.D2 as the agglomeration method for hierarchical clustering.

Quantification of individual miRNAs by quantitative RT-PCR
Expression levels of specific miRNAs were obtained using the miRCURY LNA SYBRâ Green PCR Kit (Qiagen). Quantitative PCR amplifications were performed in triplicate using the primers miRCURY LNA miRNA PCR Assay for hsa-miR-376a-3p, hsa-miR-376b-3p, mmu-miR-376a-3p and mmu-miR-376b-3p (Qiagen) on a QuantStudio 5 Real-Time PCR System (Thermo Fisher). The threshold cycle (Cq) was used to calculate relative miRNA expression by the 2 ÀDDCT method normalizing to hsa-SNORD49A expression (Qiagen), which were defined using GeNorm and NormFinder in Exiqon GenExâ software as the optimal reference for fibroblasts in our miRNome analysis. mmu-SNORD110 (Qiagen) was used for the normalization of mice tissues and VSMCs. For graphical representation and statistical analysis, we used a value of 41 for the Cq of WT VSMC mice although the qPCR presented no value (>40 cycles).
Events were ranked as separated or colocalized after reconstruction of signals using IMARIS (as previously described in (Robin et al., 2014)). Images were average 3 times to improve the signal to noise ratio. Generated.lsm files with a voxel size of 0.1 mm 3 0.1 mm 3 0.24 mm were processed using the IMARIS software (Bitplane, AG). After 3D reconstruction we retrieved the distances between adjacent probes using their associated intensity center as set point. Colocalization was materialized by an overlap of the two reconstructed probes. Notably, this translates into a mean average of 0.5 mm for colocalization signals.
For each nucleus 3D objects (x10) were created having the same center defined as the center of the cuboid in which the object (nucleus) is inscribed. The 3D created objects correspond to the nuclear rim of the nucleus and its reduced-dimension copies (generated by IMARIS). Reduction of the object is created until the FISH probe matches the reduced envelope. The reduced dimension object was mathematically turned into equivalent sphere volume in order to obtain a distribution of the probe within the nuclear volume for each experimental condition.
In brief, for the position of probes regarding the nuclear envelope, we generated (IMARIS) series of 10 concentric zones of equal volume per nucleus (each zone corresponding roughly to 1 mm) and compared the mean volume ratio of nuclei of probes. After 3D reconstruction, at least 30 nuclei (60 alleles) were examined per condition, and the volume of the probes, distances between their gravity centers, distances to the nuclear envelope and volume of nuclei were calculated and used for statistical analysis.
Values were compared using a non-parametric Kruskal-Wallis test; populations of separated or colocalized signals were compared to the reference condition using a Chi-square test.

Chromatin immunoprecipitation (ChiP)
ChIP for H3, CTCF and H3K36me3 (Millipore) was performed according to the manufacturer's instructions (TruChIP, Covaris; and Magna ChIPA/G, Millipore). ChIPs were processed using a 4 C overnight incubation (concentration of antibodies at 1.5 mg) with 1 mL of each preparation: IP, IgG, rabbit nonimmune serum, no ll OPEN ACCESS iScience 25, 103757, February 18, 2022 iScience Article crosslink control, no antibody control and 1% input were used as controls for digital droplet PCR analysis (Ludlow et al., 2014). Primers were designed for each miRNA and surrounding regions enriched with histone marks using the UCSC genome browser tools (Table S2). Each PCR primer pair was tested on genomic DNA to verify specificity and efficiency. The results were normalized to inputs and to H3.

Proliferation and toxicity assays
Control fibroblasts were seeded at 5,000 cells per well in 96-well plates supplemented with 100 mL DMEM. Toxicity and proliferation rates were determined 96 h post transfection using CellToxâ Green Cytotoxicity Assay and CellTiter-Gloâ, respectively (Promega, Madison, WI, USA), according to the manufacturer's instructions. Fluorescence and luminescence were measured using a GloMaxâ Microplate Reader (Promega).
Proliferation/replication was also evaluated 96 hours after miRNA transfection using the BrdU Cell Proliferation ELISA Kit (ab126556, Abcam, Cambridge, UK) according to the manufacturer's instructions. In 3 wells, cells or BrdU were omitted to serve as negative controls for nonspecific binding. Absorbance was measured at 450 nm on a GloMaxâ Microplate Reader (Promega).
Proliferation of HGPS fibroblasts has been assessed with trypan blue and counted on kovaslide (Labelians, Nemours, France) using a microscope (Leica, Wetzlar, Germany), after 15 days of culture.

Cell cycle analysis
The cell cycle was analyzed in control fibroblasts using FxCycle TM Far Red Stain (Thermo Fisher Scientific) 48 h post transfection. After trypsinization and centrifugation, cells were washed in PBS, centrifuged, and fixed with a fixation buffer (Biolegend, San Diego, USA) for 20 min at room temperature. Cells were washed with Intracellular Staining Permeabilization Wash Buffer (1/10, Biolegend), centrifuged and permeabilized using the same solution for 20 min at RT. After the last centrifugation, cells were resuspended in 1 mL PBS containing 1 mL FX Cycle Far Red and 5 mL Ribonuclease A (Sigma) and incubated for 30 min at RT. Cell cycle distribution was analyzed on an Attune cytometer (Thermo Fisher). Results were analyzed using FlowJo software (FlowJo software, Oregon, USA).

Senescence associated b-galactosidase staining
Senescence was evaluated using a Senescence b-Galactosidase Staining Kit (Cell Signaling Technology Leiden, The Netherlands) according to the manufacturer's instructions. The percentage of stained cells was evaluated on a microscope (Leica, Wetzlar, Germany) by two independent observers using manual blind counting. At least 100 fibroblasts were randomly selected for each condition. Results are expressed graphically as the mean percentage of senescent cells.

Autophagy level quantification by flow cytometry
Autophagy was measured 48 h post transfection using the Autophagy Assay Kit (ab139484, Abcam) according to the manufacturer's instructions. Sytox (1/2,000 dilution, AAdvanced TM Dead Cell Stain Kit, Thermo Fisher Scientific) was added at the last step to evaluate the percentage of dead cells and measure autophagy levels specifically in living cells. Samples were assessed using an Attune cytometer (Thermo Fisher).

Western blot analysis
Total fibroblast proteins were extracted in a lysis buffer containing 62.5 mM Tris-HCl, 2.3% SDS, 10% glycerol, 1 mM PMSF and bromophenol blue and then sonicated three times (30 s each). Protein concentration was evaluated with a Pierceä BCA Protein Assay Kit according to the manufacturer's instructions (Thermo Fisher Scientific). The absorbance was measured using a UV-Visible Absorbance Module, 540-590 nm bandpass, on a GloMaxâ Microplate Reader (Promega).