Abstract

   RNA N6-methylation (m^6A) modification is common in eukaryotic mRNA and
   has been linked to various physiological disorders, including
   UVB-induced photoaging. To identify biological differences among
   photoaging. Three pairs of normal and photoaged skin tissues were
   collected for m^6A RNA sequencing assay. Transcriptome profiles showed
   differential m^6A methylation modifications in 1365 mRNAs in photoaging
   skin tissues. Pathway analysis revealed the involvement of cellular
   stress response and regulation of cell cycle G2/M phase transition in
   m^6A-mRNAs. Further experiments validated the differential expression
   of m^6A methyltransferases (METTL3 and METTL14) and hypermethylation
   modification in mRNAs (CENPE, PPM1B and TPM1). In vitro studies
   demonstrated that increased METTL3 and METTL14 levels promoted m^6A
   methylation of CENPE, PPM1B and TPM1 in UVB-induced photoaging cells,
   and further experiments on mice showed that downregulation of METTL3
   and METTL14 reduced m^6A modifications in CENPE, PPM1B and TPM1,
   leading to the delayed appearance of photoaging phenotypes, suggesting
   that these genes could serve as potential therapeutic targets for
   treating photoaging. Our study characterized key transcriptome changes
   in photoaging and identified the role of METTL3 and METTL14 in
   mediating m^6A modification, resulting in the upregulation of CENPE,
   PPM1B and TPM1 expression, which may be crucial in UVB-induced
   photoaging.

   Keywords: N6-methylation, Methyltransferases, METTL3, METTL14,
   Photoaging

Graphical abstract

   [39]Image 1
   [40]Open in a new tab

Highlights

     * •
       Multiple differentially expressed genes and pathways in UVB-induced
       photoaging.
     * •
       A whole-transcriptome atlas of the sun-protected and sun-exposed
       skins is lacking.
     * •
       The roles of m6A methyltransferases in UVB-induced photoaging.
     * •
       Three DEGs not previously reported in photoaging are reported in
       this study.
     * •
       Three DEGs can potentially be used as therapeutic targets for
       treating photoaging.

1. Introduction

   Skin aging can be classified into two main categories: endogenous
   aging, which occurs naturally with age, and exogenous aging, which is
   primarily caused by external factors such as exposure to ultraviolet
   (UV) radiation. The cumulative of UV light on the skin leading to
   various detrimental effects, including the production of reactive
   oxygen species (ROS) [[41]1,[42]2], DNA damage and collagen degradation
   [[43]3,[44]4], which can lead to various conditions such as melasma,
   solar keratosis and even skin tumors [[45]5,[46]6]. Despite the evident
   impact of UV-induced photoaging, the underlying mechanisms are not yet
   fully understood. Thus, further research on posttranscriptional gene
   regulation can deepen our understanding of the molecular basis of skin
   photoaging, which may facilitate the development of comprehensive
   anti-aging strategies and interventions aimed at delaying age-related
   skin phenotypes.

   The loss of structural integrity and changes in phenotypic and
   physiological traits in the skin are influenced by both intrinsic and
   extrinsic factors [[47]7]. Among extrinsic factors, UVB radiation
   primarily affects the epidermis and gradually induces photoaging,
   causing sunburn more prominently than UVA radiation [[48]8]. Previous
   studies suggest that UVB exposure leads to the generation of ROS within
   cells, triggering the activation of the mitogen-activated protein
   kinase (MAPK) signaling pathway [[49]9], which, in turn, increases the
   expression of matrix metalloproteinases (MMPs), resulting in the
   degradation of collagen and lipids in the skin and the manifestation of
   aging-related characteristics (i.e., skin aging phenotypes) [[50][10],
   [51][11], [52][12]]. Additionally, UVB radiation can induce DNA damage
   and expedite skin aging by activating internal aging factors [[53][13],
   [54][14], [55][15]], and N6-methyladenosine (m^6A) methylation has been
   reported to play a unique role in repairing critical DNA segments
   [[56][16], [57][17], [58][18]]. However, the precise regulation of m^6A
   methylation during the aging process remains to be elucidated.

   m^6A modification refers to the addition of a methyl group to the
   nitrogen-6 position of adenosine nucleotides [[59]19]. This
   modification is the most prevalent internal alteration found in
   mammalian cells' mRNA and plays a critical role in regulating various
   processes, such as precursor mRNA maturation, translation, and
   degradation, which are associated with different disorders
   [[60]20,[61]21]. Three primary proteins are involved in m^6A
   modifications: methyltransferases, demethylases, and proteins that
   specifically recognize m^6A-methylated transcripts [[62][22], [63][23],
   [64][24], [65][25]]. Some studies have reported that METTL3 and METTL14
   methylation may be involved in repairing DNA damage caused by UV
   radiation [[66]26]. Additionally, it has been demonstrated that METTL3
   regulates global genome repair and protects against UVB-induced
   photoaging through selective autophagy. Moreover, the m^6A-modified RNA
   profile can be influenced by advanced glycosylation end products and
   METTL14 overexpression in UVB-induced photoaging human dermal
   fibroblasts, further promote fibroblast proliferation. These findings
   suggest that METTL3 and METTL14 may have complex regulatory effects on
   UVB-induced photoaging depending on specific circumstances [[67]27].

   To further explore the pathogenesis of photoaging, we conducted a
   comprehensive whole-transcriptome analysis to identify the mRNA and
   long noncoding RNA (lncRNA) molecules that exhibit differential
   expression in photoaged skin compared to normal skin. Our findings
   highlight the significantly elevated levels of METTL3 and METTL14 in
   photoaged skin, and we conducted follow-up in vitro and in vivo
   experiments to determine and validate the roles of the identified genes
   and pathways in photoaging pathogenesis.

2. Materials and methods

2.1. Patient samples

   Skin tissue samples were collected from both The normal and photoaging
   patients at the Third Affiliated Hospital of Central South University,
   following approval by the Hospital Institutional Review Board. The skin
   specimens were obtained from two distinct areas: sun-protected regions
   (15 cases from the back or buttocks) and sun-exposed regions (15 cases
   from the face) of subjects with normal areas surrounding benign
   lesions.

2.2. Cell culture and UVB-induced photoaging model

   HaCaT and Primary keratinocytes, obtained from the ATCC (Type Culture
   Collection) in the USA and certified, were cultured in Dulbecco's
   Modified Eagle Medium (Corning, USA) supplemented with 15 % fetal
   bovine serum (Gibco, USA) and antibiotics (Gibco, USA). To simulate UVB
   radiation exposure, keratinocytes were subjected to varying intensities
   of UVB radiation (range, 0–120 mJ/cm^2) three times within a 72-h
   timeframe, and the optimal exposure intensity was determined.

2.3. UVB-induced photoaged mouse modelell culture

   The minimum erythema dose (MED) was determined using the following
   procedure: Eight Balb/c mice were selected, and the hair on their backs
   was shaved. Four different irradiation time points were established,
   consisting of 10 min, 15 min, 20 min, and 30 min, with two mice
   assigned to each time point. On the second day, mice exposed to 10 or
   15 min of irradiation did not exhibit noticeable erythema, whereas
   those exposed to 30 min showed evident erythema. Therefore, an
   irradiation time of 30 min was selected as the MED. The calculation of
   the MED was performed as follows: 1 MED =
   (0.134 μW/cm^2) × 30 min = 4.02 mJ/cm^2, with an irradiation height of
   30 cm. Based on preliminary experiments, the mice underwent an adaptive
   feeding period and were exposed to 1 MED for eight weeks.

2.4. SAH-induced mouse model

   SAH is an inhibitor for METTL3-METTL14 heterodimer complex (METTL3-14)
   with an IC50 of 0.9 μM. Balb/c mice back hairs was shaved and then
   injected with SAH (5 μM) 1 time for 2 weeks. Collected their skins and
   detected METTL3 and METTL14 levels to convince the SAH-induced mouse
   model was constructed successfully.

2.5. Small interfering (si) RNA and adenovirus transfection of cells

   Keratinocytes were seeded onto 6 cm dishes at a density of
   1 × 106 cells and incubated in a medium containing 10 % fetal bovine
   serum. The cells were then treated with siMETTL3, siMETTL14, AAV-METTL3
   and AAV-METTL14 for 48 h, following which images of the cells were
   captured using a Zeiss LSM710 confocal microscope system (Carl Zeiss,
   Oberkochen, Germany), and image analysis was performed using Adobe
   Photoshop R@ZEN Lite 2012 (Adobe Systems, CA, USA).

2.6. Quantification of global m^6A levels

   The EpiQuikTM m6A RNA Methylation Quantification Kit (colorimetric
   method; EpigenTek, NY, USA) was used according to the manufacturer's
   protocol. Briefly, 200 ng of RNA isolated from skin tissues was applied
   to different assay wells. Subsequently, the capture antibody solution,
   detection anti-m6A antibody solution, and enhancer solution were
   sequentially added to the detection wells at appropriate dilution
   concentrations. The levels of m6A were quantified by measuring the
   absorbance of each well at 450 nm and calculating the values based on a
   standard curve. All experiments were repeated three times.

2.7. Methylated RNA immunoprecipitation (MeRIP)-qPCR

   To isolate 40 μg of total RNA was used for IP with an anti-m6A
   antibody. Briefly, 300 μL of IP buffer was added to a mixture of total
   RNA and m6A pegged controls, followed by incubation at 4 °C for 2 h.
   The mixture was then washed and incubated with sheep anti-rabbit IgG.
   Afterward, three washes with wash buffer for 1 h. Finally, the enriched
   RNA was purified using the RNeasy Mini Kit (Qiagen, MD, USA) for
   subsequent analysis. To validate the data obtained from the microarray
   analysis, the IP-RNA from the samples (with a ratio of 3:3 between the
   control group and experimental group) was subjected to MeRIP-qPCR using
   real-time fluorescence quantitative polymerase chain reaction.

2.8. m^6A-mRNA & lncRNA Epitranscriptomic microarray analysis and
bioinformatics analysis

   Microarray hybridization was performed following the standard protocol
   provided by ArrayStar. Briefly, the cRNAs were labeled with cy5, then
   fragmented before being hybridized to transcribed microarrays (860K,
   Arraystar) specifically designed for human m^6A-mRNA and lncRNA
   analysis. Following the hybridization process, the slides were rinsed,
   and the hybridization arrays were scanned using an Agilent G2505C
   scanner (Agilent, Beijing, China) in a two-color channel, and the data
   were analyzed using Agilent feature extraction software (version
   11.0.1.1). To identify differentially methylated m^6A RNA between the
   two groups, a fold-change threshold of ≥2 and a p-value threshold of
   <0.05 were used. All microarray analyses were conducted by Kangchen
   Biotechnology (Shanghai, China). The differentially expressed
   m6A-methylated mRNA transcription products were assigned to various
   Gene Ontology (GO) databases ([68]http://geneontology.org/).
   Additionally, the Kyoto Encyclopedia of Genes and Genomes (KEGG)
   database ([69]https://www.kegg.jp/) was used to further analyze the
   functional pathways associated with these mRNAs.

2.9. Statistical analysis

   The results are representative of at least three independent
   experiments. The significance of the microarray analysis and MeRIP-qPCR
   results was assessed using Student's t-test. Fisher's exact test was
   utilized to determine the significance of the GO and KEGG analyses. All
   data are expressed as means ± standard deviations. Statistically
   significant p-values were set at < 0.05.

3. Results

3.1. m6A modification profiles of mRNAs in sun-protected and sun-exposed skin
tissues

   We performed a transcriptional microarray study on mRNA and lncRNA to
   identify specific m^6A methylation modifications in photoaged skin. By
   analyzing the m^6A modification profiles of three pairs of healthy
   human sun-protected and sun-exposed skin tissues, we identified a total
   of 42,993 transcripts, including 34,906 mRNAs and 8087 lncRNAs
   ([70]Figs. S1A and B). Hierarchical clustering analysis revealed
   significant expression pattern differences between the sun-protected
   and sun-exposed regions ([71]Fig. 1A). We further screened genes with
   significant changes (fold change ≥2) and found that 340 (1.91 %) mRNA
   genes were downregulated and 703 (3.95 %) mRNA genes were upregulated
   ([72]Fig. 1B). In addition, 983 (5.52 %) genes were hypermethylated,
   and 56 (0.31 %) genes were hypomethylated ([73]Fig. 1C). Association
   analysis of mRNAs and m^6A-mRNAs was performed on the differentially
   expressed genes, revealing that 133 (6.97 %) genes exhibited
   upregulated mRNA expression accompanied by m^6A hypermethylation, while
   41 (2.15 %) genes displayed downregulated mRNA expression and m^6A
   hypermethylation simultaneously ([74]Fig. 1D). Furthermore, we
   identified 43 (2.78 %) biological processes (BP) that showed
   simultaneous m^6A hypermethylation modification (BP-Hypo) and
   downregulation of mRNA expression (BP-Down), as well as 159 (10.28 %)
   BP pathways that exhibited simultaneous m^6A hypermethylation
   modification (BP-Hyper) ([75]Fig. 1E and F). KEGG pathway enrichment
   analysis revealed that mRNAs with high m^6A methylation levels were
   primarily associated with the "cellular response to stress" and
   "regulation of cell cycle G2/M phase transition" pathways, while mRNAs
   with low m^6A levels were mainly involved in the "regulation of
   apoptotic process" and "glucose metabolic process" pathways ([76]Fig.
   1G). Notably, the top 19 differentially elevated mRNAs and m^6A -mRNAs,
   as well as the top 5 downregulated mRNAs and m^6A-mRNAs, were
   identified ([77]Fig. 1H–S1C, D). Overall, these findings strongly
   suggest that increased methylation levels and m^6A modification of
   mRNAs have an impact on skin photoaging.

Fig. 1.

   [78]Fig. 1
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   Epitope transcription microarray analysis of mRNAs and m^6A-mRNAs in
   photoaging. (A) Principal component analysis (PCA) plot showing
   significant methylation differences between sun-protected and
   sun-exposed skins (n = 3). (B, C) Volcano plot showing the
   differentially expressed mRNAs and m^6A-mRNAs between sun-protected and
   sun-exposed skins (upregulation: red; downregulation: purple). (D, E,
   F) Venn diagram showing the overlap of differentially expressed genes
   (DEGs), m^6A-modified genes, biological processes (BPs), and the hsa
   pathways between sun-exposed and non-exposed skins. (G) Enrichment
   analysis of 16 pathways involved by GO and KEGG. (H) Enrichment
   analysis of DEGs and m^6A-modified genes involved by GO and KEGG. GO:
   Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes. P values
   are indicated by different colors. The significance was defined by Fold
   change ≥2, FDR <0.05 and P < 0.05.

3.2. METTL3 and METTL14 overexpression induced m6A modification of CENPE,
PPM1B, and TPM1 in vitro

   In this study, we utilized UVB-induced HaCaT keratinocytes to
   investigate the impact of differentially expressed genes (DEGs) during
   photoaging in an in vitro setting ([80]Fig. 2A). Firstly, we observed
   that UVB-treated HaCaT keratinocytes exhibited senescence, as indicated
   by β-galactosidase staining and higher levels of intracellular reactive
   oxygen species (ROS) compared to the control group ([81]Fig. 2B and C).
   The increased expression of P16 and P21, along with elevated levels of
   senescence-associated secretory phenotypes (SASP) such as IL-6, IL-1β,
   and TNF-α, further confirmed the aging phenotype of UVB-induced
   photoaging cells ([82]Fig. 2D). Additionally, we observed a higher
   global level of m^6A modification and significantly increased
   expression levels of METTL3 and METTL14 in UVB-induced cells compared
   to control cells ([83]Fig. 2E). This led us to hypothesize that the
   elevated m^6A levels of DEGs in photoaging may be attributed to the
   upregulation of the m^6A methyltransferases METTL3 and METTL14.
   Moreover, the mRNA expression levels of CENPE, PPM1B and TPM1, which
   are known as m^6A-modified DEGs, were significantly elevated in the
   photoaging group ([84]Fig. 2F), suggesting their involvement in
   photoaging. Subsequently, we confirmed that the protein levels of the
   two m^6A methyltransferases, METTL3 and METTL14, as well as the three
   indicated m^6A-modified DEGs, CENPE, PPM1B, and TPM1, were upregulated
   in UVB-induced photoaging cells compared to the control group ([85]Fig.
   2G). Furthermore, MeRIP-qPCR analysis performed on UVB-induced HaCaT
   cells revealed significant regulation of m^6A levels in CENPE, PPM1B,
   and TPM1 ([86]Fig. 2H). Taken together, the findings suggest that UVB
   exposure leads to the upregulation of METTL3, METTL14, and three DEGs,
   resulting in increased overall m^6A methylation levels and elevated
   m^6A levels, specifically in CENPE, PPM1B and TPM1.

Fig. 2.

   [87]Fig. 2
   [88]Open in a new tab

   The m^6A levels and m^6A-related DEGs elevated by UVB induced in vitro.
   (A) Schematic diagram of the overall design process of the experiment.
   (B) Representative image of β-Gal staining of control and UVB-photoaged
   Primary cells. (C) Representative image of ROS fluorescence staining of
   control and UVB-photoaged Primary cells. (D) The mRNA expression levels
   of SASP factors (P16, P21, IL-6, IL-1β, TNF-α and VEGF) in control and
   UVB-photoaged Primary cells (n = 6). (E) The global level of m^6A and
   m^6A modification enzymes mRNA levels in control and UVB-photoaged
   Primary cells (n = 5). (F) qPCR analysis of m^6A-related genes in
   control and UVB-photoaged Primary cells (n = 3). (G) Immunoblot
   analysis of the indicated proteins (METTL3, METTL14, CENPE, PPM1B and
   TPM1) in control and UVB-photoaged Primary cells (n = 3). Gray values
   (measured by Image J) indicate significant differences. (H) MeRIP-qPCR
   analysis showing the m^6A modification levels of three indicated DEGs
   (CENPE, PPM1B and TPM1) in control and UVB-photoaged Primary cells
   (n = 5). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

3.3. Identification of METTL3 and METTL14 mediated m6A modification of CENPE,
PPM1B, and TPM1 in sun-exposed skin tissues

   To further validate the findings on the changes in METTL3, METTL14,
   CENPE, PPM1B and TPM1, we performed additional experiments using
   sun-protected and sun-exposed skin samples obtained from 12 pairs of
   age-matched human subjects. Histological analysis through HE and Masson
   staining revealed a significant increase in inflammatory cells,
   particularly neutrophils, in sun-exposed skins compared to
   sun-protected skins ([89]Fig. 3A). Additionally, collagen synthesis was
   reduced, and structural disruptions were observed in sun-exposed skins,
   indicating signs of photoaging ([90]Fig. 3B). Moreover, we found that
   the overall m^6A levels were upregulated in sun-exposed skins compared
   to sun-protected skins ([91]Fig. 3C). Furthermore, the expression
   levels of METTL3, METTL14, CENPE, PPM1B, and TPM1 were significantly
   elevated in sun-exposed skins compared to sun-protected skins ([92]Fig.
   3D and E). Immunohistochemistry staining confirmed the higher
   expression levels of METTL3, METTL14, CENPE, PPM1B, and TPM1 in
   sun-exposed skins compared to sun-protected skins, supporting that
   significant positive correlations between the increased expression of
   METTL3 and METTL14 and higher expression levels of CENPE, PPM1B, and
   TPM1 in sun-exposed samples ([93]Fig. 3F and G). Additionally, the m^6A
   levels of CENPE, PPM1B, and TPM1 were also found to be upregulated in
   sun-exposed skins ([94]Fig. 3H). Collectively, these findings support
   the notion that the elevated expression of METTL3 and METTL14 mediates
   higher m^6A modification in sun-exposed skins, leading to increased
   expression of CENPE, PPM1B, and TPM1, which are implicated in
   photoaging.

Fig. 3.

   [95]Fig. 3
   [96]Open in a new tab

   The m^6A levels and three indicated DEGs increased in sun-exposed skin
   tissues. (A, B) HE and Masson staining analysis of sun-protected and
   sun-exposed skin tissues (n = 8). (C) The global level of m^6A in
   sun-protected and sun-exposed skin tissues (n = 7). (D) The mRNA
   expression levels of METTL3, METTL14, CENPE, PPM1B and TPM1 in
   sun-protected and sun-exposed skin tissues (n = 12). (E) Immunoblot
   analysis of METTL3, METTL14, CENPE, PPM1B and TPM1 in sun-protected and
   sun-exposed skin tissues (n = 8). Gray values (measured by Image J)
   indicate significant differences. (F, G) Immunohistochemical staining
   of METTL3, METTL14, CENPE, PPM1B and TPM1 in sun-protected and
   sun-exposed skin tissues (n = 8). (H) MeRIP-qPCR analysis showing the
   m^6A modification levels of CENPE, PPM1B and TPM1 in sun-protected and
   sun-exposed skin tissues (n = 5). Scale bar = 50 μm. The data represent
   means ± SEMs. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

3.4. UVB-induced photoaging depends on METTL3 and METTL14 m6A modified three
indicated DEGs

   To confirm the causal role of METTL3 and METTL14 in regulating the
   expression of the three candidate DEGs in photoaging, we employed siRNA
   knockdown of METTL3 and METTL14, as well as adeno-associated virus
   (AAV) overexpression of METTL3 and METTL14 in primary keratinocytes
   ([97]Fig. S2A). Initially, we screened for small RNA sequences and
   viruses with the most effective knockdown and overexpression effects.
   Subsequently, we observed significant downregulation of CENPE, PPM1B
   and TPM1 expression in primary keratinocytes with stable knockdown of
   METTL3 ([98]Fig. 4A and B). Similarly, the knockdown of METTL14 also
   led to a significant reduction in the expression of CENPE, PPM1B and
   TPM1 ([99]Fig. 4C and D). Conversely, the overexpression of METTL3 and
   METTL14 substantially increased the expression of CENPE, PPM1B and TPM1
   in primary keratinocytes ([100]Fig. 4E, F, G, H). These findings
   suggest that the depletion of METTL3 and METTL14 results in
   downregulating CENPE, PPM1B and TPM1 expression and reduced methylation
   of CENPE, PPM1B, and TPM1. This supports the mechanism whereby the
   CENPE, PPM1B, and TPM1 genes may function as downstream targets of
   METTL3 and METTL14-mediated m^6A modifications, contributing to the
   initiation and progression of UVB-induced photoaging.

Fig. 4.

   [101]Fig. 4
   [102]Open in a new tab

   Validation of three indicated DEGs expression in primary keratinocytes
   after intervening with the expression of METTL3 and METTL14. (A, B)
   qPCR and immunoblot analysis of METTL3, CENPE, PPM1B and TPM1
   expression levels in primary keratinocytes post knockdown of METTL3.
   (C, D) qPCR and immunoblot analysis of METTL14, CENPE, PPM1B and TPM1
   expression levels in primary keratinocytes with knockdown of METTL14.
   (E, F) qPCR and immunoblot analysis of METTL3, CENPE, PPM1B and TPM1
   expression levels in primary keratinocytes post overexpressed METTL3.
   (G, H) qPCR and immunoblot analysis of METTL14, CENPE, PPM1B and TPM1
   expression levels in primary keratinocytes with overexpressed METTL14.
   Gray values (measured by Image J) indicate significant differences. The
   data represent means ± SEMs. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

3.5. METTL3 and METTL14 regulate the m^6A modification of three candidate
DEGs in vivo

   Next, we investigated the in vivo changes in m^6A methylation genes in
   mice subjected to UVB-induced photoaging ([103]Fig. 5A). Compared to
   the control group, the mice in the model group exhibited noticeable
   wrinkles, irregular thickening, and pigmentation on their back skin
   after eight weeks. Photoaging scoring confirmed a significant increase
   in the score of the UVB model group ([104]Fig. 5B). HE staining
   revealed characteristic features of photoaging skin in the model group,
   including excessive keratosis of the stratum corneum, irregular
   thickening of the epidermis, and significant infiltration of
   inflammatory cells in the dermis, as opposed to healthy skin ([105]Fig.
   5C). Furthermore, Masson staining demonstrated a decrease in the
   content of collagen fibers in the dermis of the UVB model group, with
   an irregular arrangement and the presence of broken and sparse fibers
   ([106]Fig. 5D). Additionally, we measured the levels of malondialdehyde
   (MDA), a lipid peroxide product associated with aging, which
   significantly increased in the UVB-induced photoaging mouse model,
   indicating the successful induction of the photoaging phenotype
   ([107]Fig. 5E). Furthermore, we observed a greater increase in the
   global m^6A level in the skin of the UVB mouse model compared to the
   control group ([108]Fig. 5F). Based on the photoaging standard, the
   model group exhibited significantly higher levels of IL-1β, IL-6,
   COL1A1 and MMP13 than the control group ([109]Fig. 5G). Moreover, we
   observed the mRNA expression levels of METTL3, METTL14, CENPE, PPM1B
   and TPM1 significant increase in following UVB treatment ([110]Fig.
   5H). These findings suggest that m^6A-related modifying enzymes are
   upregulated to varying degrees in the skin of the photoaging model
   mice.

Fig. 5.

   [111]Fig. 5
   [112]Open in a new tab

   Validation of METTL3, METTL14 and three indicated DEGs expression in
   vivo. (A) Schematic diagram of UVB-induced photoaging model mice. (B)
   Observation of back skin and skin macroscopic score in the control
   group and the model group at 0, 2, 4, 6 and 8 weeks after modeling. (C,
   D) HE and Masson staining analysis of control and model group skin
   tissues (n = 8). (E) ELISA analysis of MDA levels in control and model
   group skin tissues. (F) The global level of m^6A in the skin tissues
   (n = 3) of the control and the model group. (G) qPCR analysis of IL-6,
   IL-1β, COL1A1 and MMP13 mRNA levels in the skin tissues (n = 3) of the
   control and the model group. (H) qPCR analysis of METTL3, METTL14,
   CENPE, PPM1B and TPM1 expression levels in the skin tissues (n = 3) of
   the control and the model group. The data represent means± SEMs.
   ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

3.6. METTL3 and METTL14 mediated three indicated DEGs methylation facilitates
UVB-induced photoaging

   To investigate whether METTL3 and METTL14 can affect the m^6A
   modification of the three candidate DEGs, we subcutaneously injected
   SAH (a METTL3 and METTL14 inhibitor) into the UVB-induced photoaging
   model mice. The macroscopic appearance of the back skin in each group
   of mice is shown in the figure ([113]Fig. 6A). Compared to the control
   group, the UVB group displayed typical skin photoaging features,
   including wrinkles, erythema, edema, and pigmentation. However, the SAH
   group exhibited a significant reduction in wrinkles and improved color
   sedimentation compared to the UVB group. The content of MDA
   significantly increased in the UVB group, and there was a slight
   elevation in the UVB + SAH group compared to the UVB + Vehicle group
   ([114]Fig. 6B). HE sections revealed a significant decrease in
   epidermal thickness in the UVB + SAH group compared to the UVB group
   and the UVB + Vehicle group ([115]Fig. 6C). Masson staining showed
   fewer collagen fiber breaks and reduced infiltration of inflammatory
   cells in the UVB + SAH group compared to the UVB + Vehicle group
   ([116]Fig. 6D). Moreover, IHC analysis demonstrated that the expression
   of METTL3 and METTL14 significantly increased in the UVB model group
   and UVB + Vehicle group compared to the control group, while it was
   lower in the UVB + SAH group ([117]Fig. 6E and F). Consistently, we
   confirmed that the mRNA and protein expression levels of METTL3 and
   METTL14 were significantly higher in the UVB + Vehicle group and the
   UVB model group compared to the control group, while they were lower in
   the UVB + SAH group ([118]Fig. 6G). The mRNA expression levels of IL-6,
   IL-1β, COL1A1, and MMP13 increased in the UVB model group and
   UVB + Vehicle group after UVB induction and significantly decreased in
   the UVB + SAH group following UVB + SAH intervention ([119]Fig. 6H). In
   summary, these findings demonstrate that the genes regulated by the
   m^6A methyltransferases METTL3 and METTL14 play a role in the
   initiation and progression of UVB-induced photoaging.

Fig. 6.

   [120]Fig. 6
   [121]Open in a new tab

   Validation of METTL3 and METTL14 mediated three indicated DEGs
   methylation facilitates UVB-induced photoaging. (A) Schematic diagram
   and phenotypic changes of back lesions in UVB-induced photoaging model
   mice treated with SAH (METTL3 and METTL14 inhibitor). (B) Skin
   macroscopic score and MDA levels in mice groups after modeling. Dermal
   infiltrating cells and the dermal collagen volume fraction are
   quantified by Image J. (C, D) HE and Masson staining analysis of mice
   skin tissues. (E, F) Immunohistochemical staining of METTL3 and METTL14
   in mice skin tissues. (G) qPCR and immunoblot analysis of METTL3 and
   METTL14 expression levels. (H) qPCR analysis of IL-6, IL-1β, COL1A1,
   and MMP13 mRNA levels in mice skin tissues (n = 3). The data represent
   means± SEMs. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

4. Discussion

   Photoaging is a harmful skin condition caused by prolonged exposure to
   external ultraviolet radiation. Apart from visible signs of aging like
   wrinkles and pigmentation, it also compromises the skin's protective
   barrier, increases susceptibility to infections, and even raises the
   risk of skin cancer [[122]28]. The early pathological changes in
   photoaging involve DNA damage, inflammatory responses, increased
   production of ROS and enhanced secretion of senescence-associated
   secretory phenotypes (SASP), resulting in premature senescence of major
   skin cells [[123]29,[124]30]. Senescent cells release molecules
   associated with aging, including IL-6 and TNF-α, which further
   accelerate skin aging [[125]31,[126]32]. Recently, m^6A, the most
   prevalent posttranscriptional mRNA modification in eukaryotes, has
   emerged as a significant regulator of aging [[127]33]. Therefore, we
   aimed to investigate the expression patterns of m^6A regulatory
   molecules in photoaging and explore the methylation modification
   pathways that contribute to the distinct epigenetics of photoaging. Our
   findings indicate that METTL3 and METTL14 levels increase and m6A total
   methylation levels are up-regulated, which can regulate the
   hypermethylation modification of the mRNA of CENPE, PPM1B and TPM1
   molecules, thus promoting the inflammatory pathway related to cell
   aging and positively regulating the initiation and progression of
   photoaging.

   The molecular and cellular mechanisms underlying UVB-induced photoaging
   are diverse. Several studies have highlighted the involvement of
   epigenetic changes in aging processes across different systems. For
   instance, the impact of m^6A modification on AGO2 mRNA levels during
   human aging has been observed to regulate the expression of
   aging-related miRNAs [[128]34]. It has also been demonstrated that UV
   exposure leads to increased m^6A mRNA levels and localization at DNA
   damage sites, and this process is positively regulated by METTL3 and
   METTL14 [[129]18]. AGEs have been shown to elevate overall m^6A
   modification levels in photodamaged skin in vivo and in skin
   fibroblasts in vitro [[130]35]. In line with these findings, our study
   through sequencing of exposed and non-exposed skin tissues, UVB-induced
   photoaging cell models, and cellular molecular biological methods, we
   found that the expression of METTL3 and METTL14 was upregulated and the
   hypermethylated genes CENPE, PPM1B, TPM1 and pathway were upregulated
   in exposed skin tissues. Overexpression of METTL3 and METTL14
   significantly increased the expression of CENPE, PPM1B, TPM1 and
   senescence related secretory molecules in UVB-induced KC cells. These
   results indicated that METTL3 and METTL14 could regulate the m^6A
   modification of CENPE, PPM1B and TPM1 to promote the photoaging
   phenotype induced by UVB. This reveals that the upregulation of METTL3
   and METTL14 in UVB-induced photoaging promotes the high expression of
   CENPE, PPM1B and TPM1 and the accumulation of m^6A mRNA levels, which
   subsequently contributes to the release of aging-related secretory
   molecules and accelerates the skin photoaging process.

   CENPE is a microtubule kinesin protein involved in cell cycle
   regulation [[131]36]. It has been observed that CENPE DNA methylation
   is upregulated in certain cancers, and inhibiting its expression can
   regulate transcription and promote tumor cell growth [[132]37]. PPM1B
   is a member of the PP2C family of serine/threonine protein phosphatases
   involved in cell cycle regulation through the dephosphorylation of
   cyclin-dependent kinases (CDKs) [[133]38]. The overexpression of PPM1B
   can induce cell growth arrest or apoptosis, and it is known to be
   involved in cellular senescence [[134]39]. PPM1B has been shown to
   control senescence and cell proliferation in public databases
   [[135]40]. TPM1 is an actin-binding protein associated with cellular
   senescence and inflammatory responses [[136]41], which can promote the
   LPS-induced microglial inflammatory response and peripheral neuronal
   death via the PKA/CREB pathway [[137]42]. According to our study, the
   expression and methylation of CENPE, PPM1B, and TPM1 were highly
   coexpressed in UVB-induced photoaging cells and sun-exposed skins,
   indicating that these enzymes may be involved in the pathological
   modifications associated with photoaging. The increase in transcription
   of CENPE, PPM1B, and TPM1 induced by m^6A methylation suggests their
   potential involvement in the onset and progression of photoaging.

   In vivo experiment, we inhibited the expression of METTL3 and METTL14
   in the epidermis of mice by SAH (METTL3 and METTL14 inhibitors), and
   found that after UVB irradiation, the skin wrinkles, erythema, edema
   and pigmentation of mice in SAH group were significantly reduced.
   Additionally, the SAH-treated group had a thinner epidermis and more
   organized collagen fiber bundles compared to the photoaging model
   group. SAH administration effectively delayed the progression of skin
   photoaging, promoting the repair of skin structure and regeneration of
   collagen fibers. SAH injection resulted in reduced expression of
   inflammation factors IL-6, IL-1β, COL1A1, and MMP13 mRNA, as well as
   decreased levels of MDA in the photoaging skin, indicating that SAH
   inhibited the aging process. Moreover, SAH treatment decreased the
   expression of m^6A modifying enzymes METTL3 and METTL14, along with
   related markers in the UVB-induced model, resulting in lower m^6A
   levels and suppression of photoaging. Thus, our findings indicate that
   inhibition of METTL3 and METTL14 can regulate the m^6A modification of
   photoaging related molecules and delay the UVB-induced skin photoaging
   phenotype.

   In our analysis, external UVB up-regulated the expression of METTL3 and
   METTL14 in the skin, thus promoting the hypermethylation of CENPE,
   PPM1B and TPM1, activating the cellular inflammatory pathway and cell
   aging, resulting in skin photoaging. However, the specific regulatory
   mechanisms of how METTL3 and METTL14 are added or removed from mRNA and
   how to regulate the specific target of mRNA still need to be further
   studied. The follow-up demonstration can be carried out in gene
   knockout mice with low expression of METTL3 and METTL14 in the
   epidermis of photoaging mice to further demonstrate our results. In
   addition, the sequencing results showed that 133 mRNA expressions of
   differentially expressed genes in exposed skin were up-regulated and
   m6A hypermethylated, 41 mRNA expressions were down-regulated and m^6A
   hypomethylated, and 159 BP pathways showed m^6A hypermethylated
   modification. We can further study the differentially expressed genes
   and pathways to find other potential therapeutic targets.

   Our study highlights the important role of m^6A methylation in
   regulating gene expression in UVB-induced photoaging. We found that the
   upregulation of METTL3 and METTL14 in UVB-induced photoaging cells and
   sun-exposed skin tissues contribute to the upregulation of CENPE, PPM1B
   and TPM1 expression through m^6A methylation, which may promote the
   development of photoaging. Targeting METTL3 and METTL14, m^6A
   methylation modifications of CENPE, PPM1B and TPM1 in keratinocytes and
   fibroblasts can be reduced, thereby reducing the expression of aging
   related molecules and inflammatory factors, and slowing down the
   UVB-induced cellular aging process. It is a promising strategy for
   preventing and treating UVB-induced photoaging. Further investigations
   are warranted to validate these findings and elucidate the underlying
   mechanisms.

5. Conclusions (4–5 lines - Optional)

   In summary, our study sheds light on the molecular pathways and
   signatures involved in the development of age-related phenotypes, with
   a specific focus on the role of RNAs methylation in photoaging,
   findings RNAs m^6A modification facilitates UVB-induced photoaging,
   which has broad implications for the diagnosis and targeted treatment
   of photoaging and the improvement of the aging state.

Ethical approval statement

   Ethics approval was granted by The IRB of Third Xiangya Hospital Ethics
   Committee (No. 2022-S171) and the Institutional Experimental Animal
   Committee of Central South University (No.XMSB-2022-0167).

CRediT authorship contribution statement

   Shuping Zhang: Writing – original draft, Software, Methodology,
   Conceptualization. Meng Wu: Data curation. Tingting Lu: Visualization.
   Xiaoying Tian: Visualization. Lihua Gao: Investigation. Siyu Yan:
   Supervision. Dan Wang: Software. Jinrong Zeng: Validation. Lina Tan:
   Writing – review & editing.

Data availability statement

   Data associated with study not been deposited into a publicly available
   repository. Data will be made available on request.

Funding

   This work was supported by grants from the Wisdom Accumulation and
   Talent Cultivation Project of The Third Xiangya Hospital of Central
   South University (No. YX202214), the National Natural Science
   Foundation of China (No. 82273508) and China Postdoctoral Science
   Foundation funded project (No. 2022M713537).

Declaration of competing interest

   The authors declare that they have no known competing financial
   interests or personal relationships that could have appeared to
   influence the work reported in this paper.

Acknowledgements