miR-377 induces senescence in human skin fibroblasts by targeting DNA methyltransferase 1

Abstract

Skin aging is a complicated physiological process and epigenetic feature, including microRNA-mediated regulation and DNA methylation, have been shown to contribute to this process. DNA methylation is regulated by DNA methyltransferase, of which DNA methyltransferase 1 (DNMT1) is the most abundantly known. But evidence supporting its role in skin aging remains scarce, and no report regards its specifical upstream-regulating molecules in the process of skin aging so far. Here, we found that DNMT1 expression was markedly higher in young human skin fibroblasts (HSFs) than that in passage-aged HSFs, and DNMT1 knockdown significantly induced the senescence phenotype in young HSFs. We predicted the upstream miRNAs which could regulate DNMT1 with miRNA databases and found miR-377 had high homology with a sequence in the 3'-UTR of human DNMT1 mRNA. We confirmed that miR-377 was a potential regulator of DNMT1 by luciferase reporter assays. miR-377 expression in passage-aged HSFs was markedly higher than that in the young HSFs. miR-377 overexpression promoted senescence in young HSFs, and inhibition of miR-377 reduced senescence in passage-aged HSFs. Moreover, these functions were mediated by targeting DNMT1. Microfluidic PCR and next-generation bisulfite sequencing of 24 senescent-associated genes' promoters revealed alterations of the promoter methylation levels of FoxD3, p53, and UTF1 in HSFs treated with miR-377 mimics or inhibitors. We also verified that the miR-377-mediated changes in p53 expression could be reversed by regulation of DNMT1 in HSFs. Similarly, there was a negative correlation between miR-377 and DNMT1 expression in young and photoaged HSFs, HSFs, or skin tissues from UV-unexposed areas of different aged donors. Our results highlight a novel role for miR-377-DNMT1-p53 axis in HSF senescence. These findings shed new light on the mechanisms of skin aging and identify future opportunities for its therapeutic prevention.

Figure 1

The expression and role of DNMT1 in HSFs senescence. (a) DNMT1 mRNA in passage-aged (PD>50) and young (PD<10) HSFs was detected by RT-qPCR (Data represented as the mean±S.E.M. n=10, *P<0.05). Representative data was shown. (b) DNMT1 and p16 protein levels in passage-aged (PD>50) and young (PD<10) HSFs were detected by western blot (n=10, *P<0.05). Representative data was shown. (c) SA-β-gal-positive cells in young HSFs (PD<10) transfected with control shRNA or DNMT1-shRNA were detected by using kit (left). The SA-β-gal-positive ratio was shown (right; Data represented as the mean±S.E.M. n=3, *P<0.05). (d) DNMT1, p16, and Rb protein expressions and phosphorylation of Rb in young HSFs (PD<10) transfected with control shRNA or DNMT1-shRNA were detected by western blot (*P<0.05). Representative data was shown. (e) Absorbance at 490 nm in young HSFs (PD<10) transfected with control shRNA or DNMT1-shRNA was detected by MTS assays (Data represented as the mean±S.E.M. n=3 at each time point, *P<0.05). (f) SA-β-gal-positive cells in the passage-aged HSFs (PD>50) transfected with control cDNA or DNMT1 cDNA were detected by using kit (left). The SA-β-gal-positive ratio was shown (right; data represented as the mean±S.E.M. n=3, *P<0.05). (g) DNMT1, p16, and Rb protein expressions and phosphorylation of Rb were detected by western blot in the passage-aged HSFs (PD>50) after being transfected with control cDNA or DNMT1 cDNA (*P<0.05). Representative data was shown. (h) Absorbance at 490 nm was detected in the passage-aged HSFs (PD>50) after being transfected with control cDNA or DNMT1 cDNA by MTS assays. (Data represented as the mean±S.E.M. n=3 at each time point, *P<0.05)

Figure 2

miR-377 could regulate DNMT1 expression by directly targeting DNMT1 in HSFs. (a) Though bioinformatics prediction, the sequence of the miR-377 binding site in the 3′-UTR of DNMT1 was shown at the upper site. Mutated residues were shown at the lower site. (b) Luciferase activity change of the wild-type 3′-UTR reporters and the mutant 3′-UTR reporters in 293T cells treated with control mimics or miR-377 mimics (left) and 293T cells treated with control inhibitors or miR-377 inhibitors (right) was shown, respectively (Data represented as the mean±S.E.M. n=3, *P<0.05, respectively). (c) miR-377 level in young HSFs (PD<10) treated with control mimics or miR-377 mimics (left) and in passage-aged HSFs (PD>50) treated with control inhibitors or miR-377 inhibitors (right) was respectively detected by RT-qPCR (Data represented as the mean±S.E.M. n=3, *P<0.05, respectively). (d) DNMT1 mRNA and protein expression in the young HSFs (PD<10) treated with control mimics or miR-377 mimics was detected by RT-qPCR and western blot, respectively (Data represent the mean±S.E.M. n=3, *P<0.05). (e) DNMT1 mRNA and protein expression in the passage-aged HSFs (PD>50) treated with control inhibitors or miR-377 inhibitors was detected by RT-qPCR and western blot, respectively (Data represent the mean±S.E.M. n=3, *P<0.05)

Figure 3

miR-377 mediated senescence in HSFs. (a) miR-377 level in the young (PD<10) and passage-aged (PD>50) HSFs was detected by RT-qPCR (Data represented as the mean±S.E.M. n=10, *P<0.05). (b) SA-β-gal-positive cells in young HSFs (PD<10) treated with control mimics or miR-377 mimics were detected by using kit (left). The SA-β-gal-positive ratio was shown (right; data represented as the mean±S.E.M. n=3, *P<0.05). (c) SA-β-gal-positive cells in passage-aged HSFs (PD>50) treated with control inhibitors or miR-377 inhibitors were detected by using kit (left). The SA-β-gal-positive ratio was shown (right; data represented as the mean±S.E.M. n=3, *P<0.05). (d) p16 protein expression was respectively detected by western blot in passage-aged HSFs (PD>50) treated with control inhibitors or miR-377 inhibitors (left) and in young HSFs treated with control mimics or miR-377 mimics (right; n=3, *P<0.05). Representative data was shown. (e) Absorbance at 490 nm was respectively detected by MTS assays in young HSFs (PD<10) treated with control mimics or miR-377 mimics (left) and in passage-aged HSFs treated with control inhibitors or miR-377 inhibitors (right; data represented as the mean±S.E.M. n=3 at each time point, *P<0.05)

Figure 4

miR-377 promoted senescence by suppressing DNMT1 expression in HSFs. (a) Cellular senescence was detected by evaluating SA-β-gal-positive cells in young HSFs (PD<10) treated with miR-377 mimics together with control cDNA or DNMT1 cDNA as indicated (left). The positive cell quantification was shown (right; data represent the mean±S.E.M. n=3, *P<0.05). (b) DNMT1, p16, and Rb expressions and phosphorylation level of Rb were detected by western blot in young HSFs (PD<10) treated with miR-377 mimics together with control cDNA or DNMT1 cDNA as indicated (*P<0.05). Representative data was shown. (c) Absorbance at 490 nm was detected by MTS assays in young HSFs (PD<10) treated with miR-377 mimics together with control cDNA or DNMT1 cDNA as indicated (data represented as the mean±S.E.M. n=3 at each time point, *P<0.05). (d) Cellular senescence was detected by evaluating SA-β-gal-positive cells in passage-aged HSFs (PD>50) treated with miR-377 inhibitors together with control shRNA or DNMT1 shRNA as indicated (left). The SA-β-gal-positive ratio was shown (right; data represented as the mean±S.E.M. n=3, *P<0.05). (e) DNMT1, p16, and Rb expressions and phosphorylation level of Rb were detected by western blot in passage-aged HSFs (PD>50) treated with miR-377 inhibitors together with control cDNA or DNMT1 cDNA as indicated (*P<0.05). Representative data was shown. (f) Absorbance at 490 nm was detected by MTS assays in passage-aged HSFs (PD>50) treated with miR-377 inhibitors together with control cDNA or DNMT1 cDNA as indicated (data represented as the mean±S.E.M. n=3 at each time point, *P<0.05)

Figure 5

Role of miR-377 in modulating promoter methylation levels of senescent-associated genes in HSFs. (a) The promoter methylation levels of FoxD3, p53 and UTF1 were analyzed in young HSFs (PD<10) transfected with control mimics or miR-377 mimics through microfluidic PCR and next-generation bisulfite sequencing (data represented as the mean±S.E.M. *P<0.05). (b) The promoter methylation levels of FoxD3, p53 and UTF1 were analyzed in passage-aged HSFs (PD>50) transfected with control inhibitors or miR-377 inhibitors through microfluidic PCR and next-generation bisulfite sequencing (data represented as the mean±S.E.M. *P<0.05). (c) p53 mRNA was detected by RT-qPCR in young HSFs (PD<10) treated with miR-377 mimics together with control cDNA or DNMT1 cDNA as indicated (data represented as the mean±S.E.M. n=3, *P<0.05). (d) DNMT1, p53, and Rb expressions and phosphorylation of Rb were detected by western blot in young HSFs (PD<10) treated with miR-377 mimics together with control cDNA or DNMT1 cDNA as indicated (*P<0.05). Representative data was shown. (e) p53 mRNA was detected by RT-qPCR in passage-aged HSFs (PD>50) treated with miR-377 inhibitors together with control cDNA or DNMT1 cDNA as indicated (data represented as the mean±S.E.M. n=3, *P<0.05). (f) DNMT1, p53, and Rb expressions and phosphorylation of Rb were detected by western blot in passage-aged HSFs (PD>50) treated with miR-377 inhibitors together with control shRNA or DNMT1 shRNA as indicated (*P<0.05). Representative data was shown

Figure 6

miR-377 and DNMT1 expression in vivo and photoaged HSFs. (a) miR-377 level was detected by RT-qPCR in the young and the old skin tissues. (Data represented as the mean±S.E.M. n=10, *P<0.05). (b) miR-377 level was detected by RT-qPCR in HSFs from the young and the old skin tissues (Data represented as the mean±S.E.M. n=10, *P<0.05). (c) DNMT1 and p16 expressions were detected by western blot in the young and the old skin tissues (n=10, *P<0.05). Representative data was shown. (d) DNMT1 and p16 expressions were detected by western blot in HSFs from the young and the old skin tissues (n=10, *P<0.05). Representative data was shown. (e and f) The correlations between miR-377 and DNMT1 levels in different couples of young and old skin tissues and in HSFs from young and old skin tissues were shown respectively (n=10, R²=0.482,0.450, respectively, *P<0.05). (g) miR-377 level was detected in control and photoaged HSFs by RT-qPCR (Data represented as the mean±S.E.M. n=3, *P<0.05). (h) DNMT1 and p16 expression levels were detected by western blot in control and photoaged HSFs (*P<0.05). Representative data was shown. (i) The correlation between miR-377 and DNMT1 levels was analyzed in several couples of UVA-untreated and UVA-treated HSFs (n=10, R²=0.471, *P<0.05)