Skip to main content
Download PDF

METTL3-mediated m6A modification of SIRT1 mRNA affects the progression of diabetic cataracts through cellular autophagy and senescence

Journal of Translational Medicine volume 22, Article number: 865 (2024) Cite this article

Abstract

Background

The increasing incidence of diabetes mellitus has established diabetic cataracts (DC) as a significant worldwide public health issue. The mechanisms underlying DC remain unknown, and effective prevention and treatment strategies are lacking. Accordingly, we aimed to explore the role and mechanism behind N6-methyladenosine (m6A) in DC progression.

Methods

Methyltransferase-like 3 (METTL3), p21, Beclin1, LC3, and p62 expression levels were measured in human tissues. This study assessed total m6A levels and common m6A-regulated biomarkers in both in vitro and in vivo DC models. Autophagy flux was detected in vitro through Ad-mCherry-GFP-LC3B and Monodansylcadaverine (MDC) staining. Cellular senescence was assessed utilizing the senescence-associated β-galactosidase (SA-β-Gal) assay. Furthermore, the effect of METTL3 on SIRT1 mRNA modification was demonstrated, and its mechanism was elucidated using RT-qPCR, western blot, RNA stability assays, and RIP analysis.

Results

METTL3, p21, and p62 expression levels were elevated in lens epithelial cells (LECs) from DC patients, while Beclin1 and LC3 levels were reduced. Silencing METTL3-mediated m6A modifications restored high-glucose-induced autophagy inhibition and prevented premature senescence in LECs. Notably, SIRT1720 and Metformin significantly enhanced autophagosome generation and delayed cellular senescence. The m6A-reading protein YTHDF2 bound to m6A modifications, and YTHDF2 silencing significantly reduced METTL3-mediated SIRT1 inactivation.

Conclusions

METTL3 induces senescence in DC by destabilizing SIRT1 mRNA in an m6A-YTHDF2-dependent manner. The METTL3-YTHDF2-SIRT1 axis is a key target and potential pathogenic mechanism in DC.

Introduction

The rapidly growing prevalence of obesity is accompanied by a corresponding rise in the incidence of diabetic mellitus (DM). Approximately 463 million individuals have DM, which is projected to be 693 million by 2045, making it a globally considerable public health issue [1,2,3]. In individuals with diabetes mellitus, the occurrence of cataracts, which are the primary cause of blindness, is 2–5 times greater than in those without diabetes [4]. Up to 20% of cataract surgeries are performed on diabetic patients [5]. Surgery is the only treatment for cataracts, but diabetic patients are significantly more likely to experience intraoperative and postoperative complications [6]. Preventing diabetic cataracts (DC) has become a significant concern in ophthalmology.

N6-methyladenosine (m6A), a ubiquitous, dynamic, and reversible post-transcriptional modification in eukaryotic mRNAs, was first identified in 1974 [7, 8]. The biological process of m6A modification involves the coordinated participation of methyltransferase (writer), demethylase (eraser), and reader proteins [9]. The methyltransferase METTL3 is the core catalytic component of the m6A modification. Additionally, m6A-modified RNA can be recognized by m6A reader proteins, such as YTHDC1/2 and YTHDF1/2/3 [10,11,12,13]. Dysregulated m6A modifications and their associated regulatory proteins influence the development of various diseases by affecting RNA metabolism, transport, and translation [14, 15]. It has been found that m6A methylation modifications are widely involved in developing ocular diseases. Recently, researchers have demonstrated differential expression of m6A-related regulatory proteins in DM-related eye diseases [16,17,18,19]. Notably, the accumulation of methyltransferase-like 3 (METTL3) in DC impeded lens epithelial cells (LECs) proliferation while promoting apoptosis [19]. Nonetheless, the mechanisms behind the m6A modification in LECs under a high glucose (HG) environment remain to be fully elucidated.

Cellular senescence has been elucidated to diminish normal function and hasten disease progression. Evidence suggests that DM can accelerate cellular senescence through macromolecular dysfunction (loss of proteostasis, DNA damage, autophagy, and mitochondria) and sterile inflammation [20, 21]. Therefore, we hypothesized that m6A could influence the senescence of LECs under an HG environment. Autophagy is a catabolic process essential for maintaining the homeostasis of intracellular proteins and organelles. Numerous studies have found that autophagic activity declines with age, and a compromised autophagy system may be linked to expedited senescence. Inducing autophagy prolongs longevity and decelerates senescence [22,23,24]. Autophagy defects in LECs were found to cause cataracts in animal models [25, 26]. Beclin1 may be associated with autophagosome synthesis, and a decrease in autophagosomes may contribute to cataract formation [27]. Nevertheless, the influence of m6A on cellular autophagy and senescence in LECs requires further investigation.

Herein, we aimed to clarify the involvement and possible mechanisms behind METTL3 in DC. Our experiments observed in vitro and in vivo that METTL3 expression was upregulated in DC and correlated with autophagy inhibition and senescence. Mechanistically, METTL3 downregulates SIRT1 expression by decreasing downstream SIRT1 mRNA stability in an m6A-YTHDF2-dependent manner. Our results indicate that METTL3, YTHDF2, and SIRT1 are potential target genes in DC for future research.

Materials and methods

Study participants and clinical samples

This study enrolled 12 DC and 12 age-related cataracts (ARC) patients from the Second Affiliated Hospital of Harbin Medical University. Four age-matched controls from donor eyes of eye bank with Lens Opacities Classification System III (LOCS III) scores of ≤ C1, ≤N1 or ≤ P1 were enrolled. Each participant underwent a comprehensive ophthalmologic assessment and was evaluated by an independent examiner using the LOCS III [28].

Inclusion criteria for ARC were: (1) age over 50 years; (2) lens scores of C2-C4, N2-N3, or P2-P3; and (3) absence of DM. Inclusion criteria for DC were: (1) age over 50 years; (2) lens scores of C2-C4, N2-N3, or P2-P3; (3) diagnosis of DM for 2 years or more; and (4) diagnosis of severe NPDR or PDR per the International Clinical Diabetic Retinopathy (ICDR) severity scale [29]. The exclusion criteria for all participants included: (1) the diagnosis of other types of cataracts, (2) ocular diseases such as high myopia and uveitis, (3) a history of intraocular surgery, and (4) other systemic diseases. Table S1 show the demographic characteristics of participants.

During cataract surgery, tissue samples of the anterior lens capsular membrane were obtained from the lens central polar region by continuous circular capsulotomy (CCC) performed by the same ophthalmologist. The CCC diameter was about 5.5–6.0 mm. Subsequently, the tissue samples were immediately frozen in liquid nitrogen and stored at − 80 °C or fixed in 4% paraformaldehyde. This study adhered to the Declaration of Helsinki and was authorized by the Ethics Review Committee of the Second Affiliated Hospital of Harbin Medical University, with each participant signing informed consent (YJSKY2023-477).

Animals

Male C57BL/6 mice (22–25 g; Animal Center of the Second Affiliated Hospital of Harbin Medical University) were housed under a 12-h light/dark cycle at a temperature of 22–24 °C, humidity between 50 ± 10%, and with ad libitum access to food and water. The mice were acclimatized for at least 1 week before the experiments. Type 1 DM was induced by intraperitoneally injecting streptozotocin (STZ) at a dose of 55 mg/kg (Beijing Solarbio Science & Technology Co., Ltd., China) for 5 days consecutively. Mice that received STZ injections were considered diabetic when their blood glucose levels consistently surpassed 16.7 mmol/L (300 mg/dL) for two days consecutively. Diabetic mice were used 4 months post-last STZ injection, with blood glucose levels above 16.7 mmol/L. Any mice that did not exhibit DM development were eliminated from the experiment. The control group of mice (CON) was administered a comparable dosage of citric acid buffer [30].

Four weeks after successful STZ induction, AAV2sh-METTL3 (2 μL; 1 × 1013 vg/mL; WZ Biosciences Inc., China) was injected into the vitreous humor of the right eyes of these mice using a 30-gauge needle and a microinjector (Hamilto Sigma-Aldrich, St.Louis, MO, USA), forming the ‘STZ + AAV2sh-METTL3’ group. Mice injected with AAV2sh-NC were designated as the ‘STZ + AAV2sh-NC’ group. At the same time, the left eye was administered 2μL saline and was designated as the ‘STZ’ group. The eyes of control mice were administered the same saline dosage and were designated as the ‘Control-sham’ group. Mice were euthanized at 4 months, and their eyes were removed. The Second Affiliated Hospital Ethics Committee of Harbin Medical University authorized the animal experiments (YJSDW2023-088).

Cell culture and treatment

The human LEC line HLEB3 (ATCC, RRID: CVCL_6367) was cultured in Eagle’s medium (MEM, HyClone) that contained 20% FBS (Gibco) and 1% penicillin/streptomycin and incubated at 37 °C and 5% CO2 (normal glucose, NG). This study used cells with fewer than 30 passages. Following the previous experimental method, the experimental group was treated with 60 mM D-glucose (Sigma-Aldrich; Merck KGaA) for 72 h (HG) [31].

Small interfering RNAs (siRNAs) and overexpression plasmids were transfected per the protocols. Gene-silencing siRNAs targeting METTL3 (siMETTL3), YTHDF1/2/3 (siYTHDF1/2/3), and negative control (si-NC) were procured from GenePharma (Shanghai, China). Sangon Biotech (Shanghai, China) constructed METTL3 (OE-METTL3), YTHDF2 (OE-YTHDF2), and control vector on overexpression plasmids. The transfection of siRNAs or plasmids was conducted with Lipofectamine™ 2000 Transfection Reagent for 12 h, and the medium was aspirated off for further experimental steps. After transfection, HLEB3 cells were cultured in an HG environment supplemented with 1 mM SRT1720 (SC0267, Beyotime, China) and 50 μM Metformin (MET) (Sigma-Aldrich, St. Louis, MO, USA) for 72 h [31]. Table S2 lists siRNA sequences.

m6A colorimetric quantitative assay

Relative m6A levels in total RNA were assessed using the m6A RNA Methylation Quantification Kit (P-9005, Epigentek, USA) with a colorimetric assay. Total RNA extraction was performed utilizing TRIzol Extraction Reagent (Invitrogen, USA), followed by adding 200 μg/μL into each well and incubating the microplate at 37 °C for 90 min per the manufacturer’s instructions. Subsequently, capture and detection antibodies were added, measuring m6A levels at 450 nm absorbance (Bio-Tek, USA). Three experimental replicates were conducted for each reaction to ensure quantitative accuracy.

m6A dot blot assay

Total RNA was extracted using the procedure mentioned above. The RNA was subjected to denaturation by heating at 95 °C for 5 min and ice-cooling. Next, 2 μL mRNA samples were blotted onto a nitrocellulose membrane (NC, FFN10, Beyotime, China), followed by UV cross-linking and blocking with 5% skimmed milk. The membranes were then incubated overnight at 4 °C with an m6A-specific antibody (202003, Synaptic Systems). After washing, membranes were immersed in goat anti-rabbit IgG antibody (ZB-2306, zsbio) for 1 h at room temperature with gentle shaking. After additional washes, samples were treated with an ECL detection reagent (MA0186, Meilunbio), thereby using a Biochem System (BIO-RAD, USA) for visualization.

Quantitative real‑time polymerase chain reaction (RT‑qPCR)

Total RNA extraction from lens capsule membrane samples or cultured cells was conducted through TRIzol reagent, followed by assessying RNA concentration and purity. cDNA was subjected to reverse transcription from the obtained RNA through All-in-One First-Strand cDNA Synthesis SuperMix (TransGen Biotech, China) per the protocols. RT-qPCR was conducted with a SYBR Green Supermax kit (Roche, Switzerland) for RT-qPCR. Data in this experiment were measured by a classical 2−ΔΔCt and normalized to β-actin expression housekeeping genes. Table S3 summarizes primer sequences for each gene.

Western blot

After PBS wash, samples were completely lysed with RIPA buffer (R0010, Solarbio, China), quantifying protein concentrations using the BCA Protein Assay Kit (P0010, Beyotime, China). Total protein extracts were separated by SDS-PAGE and transferred to PVDF membranes (Roche, Switzerland) blocked with 5% skimmed milk. Memebrates were subjected to overnight incubation with primary antibodies against METTL3 (ab195352, Abcam), p21 (ab109199, Abcam; ab188224, Abcam), p53 (ab131442, Abcam), Beclin1(11306-1-AP, Proteintech), LC3 (12741, Cell Signaling Technology), SQSTM1/p62 (ab314504, Abcam), SIRT1 (ab189494, Abcam), YTHDF2 (24744-1-AP, Proteintech), and β-actin (AC026 ABclonal). Afterward, the bands were incubated with a secondary antibody (ZB-2306, zsbio) for 1 h. Immunoreactive protein detection was performed using a chemiluminescent HRP substrate (MA0186, Meilunbio). As an internal protein control, β-actin was employed. The bands were visualized using the Biochem System and quantified with ImageJ software. Relative protein levels were expressed as fold expression.

Monodansylcadaverine (MDC) staining

MDC staining was deployed as an autophagosome tracer to detect autophagy. Staining revealed the presence of positive cells in the perinuclear area, and autophagy was recognized in all acidic vesicles. After treating the cells in groups, MDC staining solution (C3018, Beyotime, China) was added to each group. The cells were incubated at 37 °C for 30 min, protected from light, and received three wash cycles with PBS. Blue fluorescence was detected at 358 nm excitation light using a fluorescence microscope (Leica, Mannheim, Germany).

Determination of Ad-mCherry-GFP-LC3B

After culturing HLEB3 cells to 50% confluence using confocal dishes, they were transfected with Ad-mCherry-GFP-LC3B (C3012, Beyotime, China) at a multiplicity of infections for 24 h. Following the indicated treatments, autophagy was detected under a confocal microscope (LSM980, ZEISS, Germany).

Senescence-associated β-galactosidase (SA-β-Gal) assay

A senescence assay was conducted utilizing the SA-β-gal staining kit (C0602, Beyotime, China). After treatments, cells were rinsed with phosphate-buffered saline and fixed with fixative for 15 min. Subsequently, they were stained with SA-β-Gal staining for a whole night at 37 °C without CO2. Observations were conducted using light microscopy.

RNA immunoprecipitation (RIP)

Following the instructions, RIP experiments were conducted using the RIP Kit (P0102, Geneseed, China). HLEB3 cells were lysed in a buffer and centrifuged at low temperatures. A portion of the supernatant was collected as the Input group, followed by adding magnetic beads to the remaining supernatant, and corresponding samples were obtained using METTL3 antibody, YTHDF2 antibody, and IgG antibody. Following the washing process, the lysates were subjected to digestion with protease and RNase inhibitors to achieve purification. Ultimately, RT-qPCR was conducted to quantify the target RNA levels.

Hematoxylin and eosin (HE) staining

Fresh lens anterior capsules (human) and lenses (human or mouse) were immediately fixed in 4% paraformaldehyde and embedded in paraffin. The sections were sliced into 4 μm slices for histologic examination and stained with HE. The results were examined using an optical microscope.

Immunohistochemical (IHC) staining

Tissue sections were prepared as previously described. After incubation at 60 °C for 2 h, the samples were dewaxed using a series of xylene and graded alcohols. Subsequently, antigen retrieval was performed by boiling the samples in 0.1 M citric acid (pH 6.1) for 30 min, then cooling to room temperature. Post-three washes with PBS, the samples were soaked in 0.3% H2O2 to inhibit endogenous peroxidase activity. Next, the sections were blocked at 4 °C with the primary antibody (METTL3 (ab195352, Abcam), p21 (ab109199, Abcam; ab188224, Abcam), Beclin1(11306-1-AP, Proteintech), SQSTM1/p62 (ab314504, Abcam), LC3 (12741, Cell Signaling Technology), SIRT1 (ab189494, Abcam)) for a night. After washing, they were incubated with the corresponding secondary antibody at 37 °C for 1 h. The sections were then incubated in DAB until the desired staining intensity developed. Five fields were observed for each section. Scoring was conducted according to the immunoreactive score (IRS) standard, as previously reported [32].

RNA stability assay

After transfection, actinomycin D (Act D, HY-17559, 10 μg/mL, MCE) was introduced to HLEB3 cells and incubated for 0, 3, and 6 h to assess the half-life of SIRT1 mRNA. Subsequently, total RNA was harvested at the indicated times, and the remaining SIRT1 mRNA was analyzed using RT-qPCR.

Statistical analysis

GraphPad Prism 9 was employed for statistical analysis. Data were tested for normality using the Shapiro-Wilk test. Datasets that conform to a normal distribution are presented as mean ± standard deviation (SD). Data from experiments involving two treatments or conditions were analyzed using a two-tailed Student’s t-test for normally distributed data or a nonparametric test for non-normally distributed data. Multiple group comparisons were conducted using ANOVA for normally distributed data or the Kruskal-Wallis test for non-normally distributed data. P < 0.05 was considered statistically significant.

Results

HG environment elevates m6A modification levels in vitro and in vivo, with METTL3 as a significant regulatory factor

An in vitro HG model was created using established experimental methods with a glucose concentration of 60 mM. m6A expression levels were examined after various induction times (Fig. 1A). An HG induction time of 72 h was selected for subsequent experiments due to its higher m6A expression level (Fig. 1B). Common m6A regulators were also detected, including writers (METTL3, METTL14 and WTAP) and erasers (FTO and ALKBH5) in vitro. RT-qPCR results demonstrated that METTL3 was significantly elevated in HG-induced HLEB3 cells. At the same time, the levels of METTL14, WTAP, FTO, and ALKBH5 were not significantly different between the two groups (Fig. 1C). Similarly, western blot analysis detected elevated METTL3 expression in the HG environment in vitro (Fig. 1D, E). Consequently, the STZ mouse model was created, and m6A expression levels were measured after different induction times (Fig. 1F). STZ mice modeled for 16 weeks were selected for subsequent experiments (Fig. 1G). Compared to the control, the lenses of STZ mice appeared unevenly turbid (Fig. 1H). RT-qPCR and western blot results confirmed a significantly overexpressed METTL3 in vitro (Fig. 1I-K). HE staining revealed that control LECs were round, regularly arranged, and densely packed, whereas LECs from STZ mice appeared flattened and sparse. Additionally, IHC staining showed increased expression of METTL3 in LECs from STZ mice compared to control (Fig. 1L, M).

Fig. 1
figure 1

Elevated m6A methylation levels in HG induced HLEB3 and LECs of STZ mice. (A-B) m6A dot blot assay with anti-m6A antibody (A) and m6A ELISA kit (B), showing the level of m6A in HLEB3 cells induced by HG over different periods (n = 3). (C) RT-qPCR analysis of the relative mRNA levels of m6A-related methylase and demethylase after 72 h of HG induction in HLEB3 cells (n = 3). (D-E) Relative METTL3 levels in HLEB3 cells under NG and HG environment (n = 3). (F-G) m6A dot blot assay (F) with anti-m6A antibody and m6A ELISA kit (G), showing the level of m6A in the lens capsule of STZ mice after induction for different times (n = 3). (H) Representative images of the lens morphology in control mice (left) and 16-week STZ mice (right). (I) Relative m6A-related methylase and demethylase mRNA levels in lens capsules of STZ mice induced for 16 weeks (n = 3). (J-K) Relative METTL3 levels in the lens capsule of control mice and 16-week STZ mice (n = 3). (L) Representative images of HE staining and IHC staining of METTL3 in lens tissues of control mice (left) and STZ mice (right). Scale bar = 100 μm. (M) IHC score of METTL3 in lens tissues (n = 5). Statistical significance was determined by a two-sided Student’s t-test as appropriate

Full size image

Clinical samples were collected to investigate METTL3 expression in LECs from patients with DC and ARC compared to control LECs. RT-qPCR results indicated elevated METTL3 expression in DC samples and reduced METTL3 expression in ARC samples compared to non-cataract patients (Fig. 2A). HE staining revealed morphological differences, showing flattened and sparse LECs in DC and ARC samples compared to controls. Additionally, IHC staining demonstrated significant differences in METTL3 expression among DC, ARC, and control samples (Fig. 2B, C).

Fig. 2
figure 2

Pathological characteristics of the capsule membrane in patients with non-cataract, DC, and ARC. (A) Relative METTL3 mRNA levels in the capsule of non-cataract, DC, and ARC participants (n = 4 for non-cataract, n = 12 for DC, n = 12 for ARC). (B) Representative images of HE staining and IHC staining of METTL3 in the capsule of non-cataract, DC, and ARC patients. Scale bar = 50 μm. (C) IHC score of METTL3 in human lens tissues. Data were quantitatively analyzed using randomized fields of view from 5 independent experiments. Statistical significance was determined using the Kruskal-Wallis test (A) with Dunn’s multiple comparisons and ANOVA (C) with Bonferroni’s multiple comparisons

Full size image

Biological evidence indicates that METTL3 inhibits cellular autophagy and accelerates senescence under the HG environment in vitro

Control lenses and DC and ARC capsule membranes were collected to explore the relationship between cataract autophagy and senescence. Overexpression of p21 (a senescence-associated marker) and p62 (an autophagy substrate marker) were observed (Fig. 3A, C), along with decreased expression of Beclin1 (an autophagosome marker) in the lens capsule membranes of DC and ARC patients compared to controls (Fig. 3B). Furthermore, LC3 (an autophagosome marker) was reduced in DC. Simultaneously, LC3 was elevated in ARC (Fig. 3D).

Fig. 3
figure 3

Relative mRNA levels in the lens capsule membranes of patients with non-cataract, DC, and ARC. (A-D) RT-qPCR: p21, Beclin1, LC3, and p62 (n = 4 for non-cataract, n = 12 for DC, n = 12 for ARC). Statistical significance was determined by the Kruskal-Wallis test, followed by Dunn’s multiple comparisons

Full size image

Accordingly, it was hypothesized that METTL3 is pivotal in DC development. To investigate whether METTL3 affects cataract progression by regulating autophagy and senescence, METTL3 expression was reduced in HLEB3 cells through siMETTL3 transfection. Transfection efficiency was evaluated (Fig. 4A-C), along with the total m6A level, using RT-qPCR and western blot (Fig. 4D). Next, we selected the most efficient siMETTL3-#2 for further experiments. Autophagy-related biomarker expression was then detected by western blot, and the number of autophagosomes and autolysosomes was measured using the Ad-mCherry-GFP-LC3B reporter. In the confocal fluorescence merged images, yellow puncta represent autophagosomes, while autophagic lysosomes are depicted as red puncta. Compared to the control group, HG treatment significantly down-regulated Beclin1 and LC3II/I and increased p62 in HLEB3 cells (Fig. 4E, F). Furthermore, HG-treated cells exhibited fewer red and yellow puncta, indicating impaired autophagosome formation and autophagosome and lysosome fusion in HLEB3 cells (Fig. 4G, H). MDC is an eosinophilic fluorescent probe that specifically labels autophagosomes. Figure 4I shows that HG attenuated fluorescence, whereas METTL3 knockdown enhanced fluorescence intensity. The above results suggest that HG inhibits the early conversion of LC3-I to LC3-II in HLEB3 cells. Western blot and SA-β-gal staining depicted that knocking down METTL3 effectively rescued HG-provoked senescence in HLEB3 cells (Fig. 4J-M). Additionally, m6A, particularly METTL3, was overexpressed in HLEB3 cells (Fig. 5A-C), elevating total m6A levels (Fig. 5D), which further reduced Beclin1 and LC3II/I levels while increasing p62 levels (Fig. 5E, F). Confocal and MDC fluorescence showed further decreased autophagosome production (Fig. 5G-I). The increased expression of p53 and p21 and a higher proportion of SA-β-gal-positive cells indicated a significant trend toward cell senescence (Fig. 5J-M).

Fig. 4
figure 4

HG-induced impaired autophagic flux and accelerated senescence in HLEB3 cells, and knockdown of METTL3 restored autophagic flux and delayed senescence. (A-C) Assessment of METTL3 silencing effectiveness in siMETTL3-transfected HLEB3 cells at mRNA level using RT-qPCR and protein level using western blot analysis (n = 3). (D) m6A level in siMETTL3-transfected HLEB3 cells with an anti-m6A antibody. (E-F) HLEB3 cells were cultured in HG for 72 h post-transfection. Autophagy expression following various treatments using western blot (n = 3). (G-H) Ad-mCherry-GFP-LC3B transfection into HLEB3 cells post-various treatments. Representative confocal microscopy images and quantification of fluorescent puncta per cell are shown. Data were quantitatively analyzed using randomized fields of view from 3 independent experiments. Scale bar = 10 μm. (I) Representative images of autophagosomes visualized via the MDC method; Scale bar = 50 μm. (J-K) Senescence biomarkers levels in HLEB3 cells subjected to different treatments using western blot (n = 3). (L-M) SA-β-Gal staining was used to detect the senescence levels in differently treated HLEB3 cells. Data were quantitatively analyzed using randomized fields of view from 3 independent experiments. Scale bar = 100 μm. Statistical significance was determined by two-sided Student’s t-test or one-way ANOVA with Bonferroni’s multiple comparisons, as appropriate

Full size image
Fig. 5
figure 5

Overexpression of METTL3 further impairs HG-induced autophagic flux in HLEB3 cells and accelerates senescence. (A-C) Assessment of METTL3 overexpressing effectiveness in OE-METTL3-transfected HLEB3 cells at mRNA level using RT-qPCR and protein level using western blot analysis (n = 3). (D) m6A level in OE-METTL3-transfected HLEB3 cells with an anti-m6A antibody. (E-F) Autophagy-related biomarker expression in HLEB3 cells under different treatments using western blot (n = 3). (G-H) Ad-mCherry-GFP-LC3B was transfected into HLEB3 cells, followed by various treatments. Representative confocal microscopy images and quantification of fluorescent puncta per cell are shown. Data were quantitatively analyzed using randomized fields of view from 3 independent experiments (n = 3). Scale bar = 10 μm. (I) Representative images of autophagosomes visualized via the MDC method. Scale bar = 50 μm. (J-K) Senescence biomarkers levels in HLEB3 cells subjected to different treatments using western blot (n = 3). (L-M) SA-β-Gal staining was used to detect the senescence levels in differently treated HLEB3 cells. Data were quantitatively analyzed using randomized fields of view from 3 independent experiments. Scale bar = 100 μm. Statistical significance was determined by two-sided Student’s t-test or one-way ANOVA with Bonferroni’s multiple comparisons, as appropriate

Full size image

Biological evidence that METTL3 influences DC development by modulating cellular autophagy and senescence in vivo

The AAV2sh-METTL3-transduced region was identified by the green fluorescent protein (GFP) signal before staining (Fig. 6A). As shown in Fig. 6B-C, METTL3 expression was reduced in LECs of AAV2sh-METTL3 mice. Additionally, western blot and IHC analyses confirmed that Beclin1 and LC3II/I levels were increased, while p62 levels were decreased in LECs following vitreous injection of AAV2-sh-METTL3 compared to the NC group (Fig. 6D, E, H and I), indicating enhanced autophagy. Markers of cellular senescence (p53 and p21) were significantly reduced in LECs of 3AAV2-sh-METTL3 mice, suggesting delayed senescence (Fig. 6F-I).

Fig. 6
figure 6

Knocking down METTL3 in vivo increases the autophagic flux of HLEB3 cells and decelerates senescence. (A) Fluorescent representative images of GFP expression in the anterior capsule membrane of mice lens transfected with AAV2. Scale bar = 100 μm. (B-C) METTL3 expression changes following AAV2-shMETTL3 transfection using western blot analysis (n = 3). (D-E) Autophagy-related biomarker expression, and (F-G) senescence-related biomarker expression in LECs of control, STZ-AAV2-sh-NC, and STZ-AAV2-sh-METTL3 mice (n = 3). (H-I) HE staining, IHC images and IHC score in lens tissues of mice: Control, STZ-AAV2 sh-NC, and STZ-AAV2 sh-METTL3. Data were quantitatively analyzed using randomized fields of view from 5 independent experiments. Scale bar = 50 μm. Statistical significance was determined by two-sided Student’s t-test or one-way ANOVA with Bonferroni’s multiple comparisons, as appropriate

Full size image

METTL3 negatively governs SIRT1 expression by influencing m6A modifications both in vitro and in vivo

Subsequently, the downstream targets of METTL3 were explored. Our previous study indicated that overexpressed SIRT1 accelerated DC progression [31]. To determine whether m6A modifications regulate SIRT1, the SRAMP database was used to predict m6A binding sites on SIRT1 mRNA in humans (Fig. 7A) and mice (Fig. 7B). The results revealed the presence of high-confidence m6A modification sites on the mRNA of SIRT1 in both species. IHC staining of mice lenses revealed a significant SIRT1 overexpression in LECs from the sh-METTL3 group compared to the NC group (Fig. 7C, D). Similarly, SIRT1 levels were further reduced after overexpressing METTL3 in vitro (Fig. 7E, F). Negative regulation between METTL3 and SIRT1 may be involved in cataract progression. Next, the role of METTL3 in regulating SIRT1 was explored. Quantitative RIP experiments demonstrated a direct interaction between SIRT1 mRNA and METTL3 in HLEB3 cells. Upon METTL3 knockdown, the enrichment of SIRT1 mRNA was significantly reduced (Fig. 7G).

Fig. 7
figure 7

SIRT1, a downstream target, negatively governed by METTL3-mediated m6A modification in vitro and in vivo. (A-B) SRAMP bioinformatics (https://www.cuilab.cn/sramp/) analysis was used to predict m6A methylation sites on SIRT1 mRNA in humans and mice. (C-D) Representative images of IHC staining and IHC score of SIRT1 in control mice, STZ-AAV2 sh-NC mice, and STZ-AAV2 sh-METTL3 mice (n = 5). (E-F) METTL3 and SIRT1 protein levels in HLEB3 cells when subjected to NG, HG, HG + vector, and HG + OE-METTL3 using western blot (n = 3). (G) Detecting the SIRT1 enrichment level in HLEB3 cells after METTL3 knockdown using RIP-qPCR (n = 3). Scale bar = 50 μm. Statistical significance was determined by two-sided Student’s t-test or one-way ANOVA with Bonferroni’s multiple comparisons, as appropriate

Full size image

Activation of SIRT1 promotes autophagy and rescues senescence in HLEB3 cells overexpressing METTL3

Here, the SIRT1 activator SIRT1720 and the anti-senescence medication MET were used to assess the impacts of SIRT1 and METTL3 on autophagy and senescence. We found that SIRT1720 and MET could promote autophagy, leading to a substantial increase in Beclin1 and LC3II/I and a significant reduction in p62 (Fig. 8A, B). Administration of SIRT1720 and MET increased the number of autophagosomes and restored autophagic flux compared to the METTL3 overexpression group (Fig. 8C, D). As shown in our results, SIRT1720 and MET could significantly decrease the expression of senescence-related biomarkers (Fig. 8E, F) and significantly decrease SA-β-gal positive cell proportion (Fig. 8G, H) upon overexpression of METTL3.

Fig. 8
figure 8

The activation of SIRT1 increases autophagy and delays the progression of senescence. (A-B) Detecting the SIRT1 and autophagy-related biomarker expression levels following various treatments using western blot (n = 3). (C-D) Ad-mCherry-GFP-LC3B transfection into HLEB3 cells, followed by various treatments. Representative confocal microscopy images and quantification of fluorescent puncta per cell are shown. Data were quantitatively analyzed using randomized fields of view from 3 independent experiments. Scale bar = 10 μm. (E-F) Detecting the senescence biomarkers in HLEB3 cells under various treatments using western blot (n = 3). (G-H) SA-β-Gal staining was used to detect the senescence levels in differently treated HLEB3 cells. Data were quantitatively analyzed using randomized fields of view from 3 independent experiments. Scale bar = 100 μm. Statistical significance was determined by two-sided Student’s t-test or one-way ANOVA with Bonferroni’s multiple comparisons, as appropriate

Full size image

METTL3-induced SIRT1 mRNA decay is dependent on m6A-YTHDF2

Herein, relative SIRT1 mRNA levels were assessed by RT-qPCR after individually knocking down members of the YTHDF family in HG-induced HLEB3 cells. It was found that only the knockdown of YTHDF2 significantly impacted SIRT1 expression levels (Fig. 9A). After assessing the transfection efficiency of OE-YTHDF2 and siYTHDF2 (Fig. 9B, C), a quantitative RIP assay was conducted to validate the interaction between SIRT1 mRNA and YTHDF2. The results indicated that the enrichment of SIRT1 was dramatically reduced after YTHDF2 knockdown, suggesting that SIRT1 is a target gene of YTHDF2 (Fig. 9D). However, overexpression of YTHDF2 resulted in down-regulated SIRT1, which could be partially elevated by knocking down METTL3 (Fig. 9E, F). YTHDF2 overexpression significantly decreased SIRT1 mRNA stability in HLEB3 cells (Fig. 9G). Conversely, the knockdown of YTHDF2 partially rescued the reduced SIRT1 expression caused by the overexpression of METTL3 (Fig. 9H, I). Our results suggest that METTL3-mediated modification of m6A can affect the translational stability of SIRT1 mRNA in a YTHDF2-dependent manner. Eventually, we propose a model illustrating the role of the m6A (METTL3)-SIRT1-YTHDF2 pathway in the progression of DC (Fig. 9J).

Fig. 9
figure 9

METTL3 reduces SIRT1 mRNA stability in a YTHDF2-dependent manner. (A) Relative SIRT1 mRNA levels following the knockdown of the YTHDF family (n = 3). YTHDF2 mRNA expression in OE-YTHDF2 (B) and siYTHDF2 (C), both in transfected HLEB3 cells (n = 3). (D) Detecting the SIRT1 enrichment level in HLEB3 cells after YTHDF2 knockdown using RIP-qPCR (n = 3). (E-F) YTHDF2 and SIRT1 protein levels in HG-induced HLEB3 cells transfected with si-NC + vector, siMETTL3, OE-YTHDF2, siMETTL3 + OE-YTHDF2 (n = 3). (G) SIRT1 mRNA stability at different time points after Act D (10 μg/ml) treatment (n = 3). (H-I) YTHDF2 and SIRT1 protein levels in HG-induced HLEB3 cells transfected with si-NC + vector, OE-METTL3, siYTHDF2 and OE-METTL3 + siYTHDF2 (n = 3). (J) Schematic representation of METTL3-mediated regulation of SIRT1 in accelerating the progression of diabetes-associated cataracts via an m6A-dependent mechanism. Statistical significance was determined by two-sided Student’s t-test or one-way ANOVA with Bonferroni’s multiple comparisons, as appropriate

Full size image

Discussion

Untreated cataracts are the leading cause of blindness worldwide, and surgery is the only treatment option [33]. Compared to patients with ARC, those with DC have a higher rate of complications during and after cataract surgery, as well as poorer visual outcomes [6]. This study demonstrated that METTL3-mediated m6A modification in LECs exposed to HG inhibits autophagy, accelerates senescence, and induces cataract progression. Intervening in METTL3 expression or using SIRT1 activators and MET, plays a significant role in delaying cataract progression.

In eukaryotic mRNAs, m6A is the most abundant post-transcriptional RNA modification and is associated with the progression of many ocular diseases [34]. In a review by Umari et al. [35], m6A was shown to regulate the progression of diabetic retinopathy. Additionally, Wen et al. [36], through transcriptome-wide m6A methylome sequencing of the anterior lens capsule in cases of high myopia, discovered that m6A modifications can influence the composition of the extracellular matrix, thereby altering the structure of the fundus. In conclusion, m6A modifications are crucial in various ocular disease pathogenesis. Notably, recent studies in LECs from ARC models have identified the involvement of the m6A-associated regulatory proteins METTL3 and ALKBH5 in cataract progression [37, 38]. One study found that elevated expression of the methylase METTL3 in DC inhibited proliferation and promoted apoptosis in LECs [19]. In this study, we examined the levels of common m6A-associated regulators in vitro and in vivo and selected METTL3 for subsequent mechanistic studies due to its relatively stable expression. We observed a similar alteration trend in our induced models, as reported previously. Nonetheless, the m6A regulatory role in DC remains largely unclear. Assessing the epigenetics role and the molecular mechanisms behind DC could help identify advantageous targets for DC prevention.

In all eukaryotic cells, autophagy constitutes the major degradation pathway for damaged organelles and aggregated proteins. Ample evidence indicates that autophagy and lysosomes play a crucial role in protecting cells from oxidative stress and maintaining intracellular mass balance [39, 40]. Nevertheless, its role in DC progression remains unclear. Our study found that in LECs of DC models induced both in vitro and in vivo, Beclin1 and LC3II/I levels were reduced, while p62 levels were elevated. Additionally, we also found that fluorescent spots representing autophagosomes were diminished in the in vitro models. During autophagy, p62 functions as a receptor for transporting cargo and binds to the autophagosome membrane protein LC3II through the LC3 interaction region. In cells deficient in autophagy, p62 protein typically accumulates. Therefore, LC3 and p62 are frequently employed markers to assess autophagic activity. Researchers have found increased LC3 and p62 levels in H2O2-treated HLEB3 cells in vitro and in ARC in vivo, suggesting impaired binding of autophagosomes to lysosomes and resulting in impaired autophagic flux. This pathological process promotes the development of ARC [26, 41, 42]. The Beclin1 gene is a well-known regulator of autophagy, playing a key role in autophagosome formation and serving as an important indicator of autophagy initiation and levels [43]. Our experimental phenomenon demonstrates that the early LC3-I conversion to LC3-II may be blocked, and autophagosome formation is reduced in DC [44, 45]. Furthermore, although both ARC and DC present with cataracts, differences in the most prevalent type of lens turbidity and trends in autophagy-related biomarker expression suggest distinct pathogenetic mechanisms between the two conditions.

Epigenetics is well-known to be extensively contributing to cellular senescence. Oxidative stress, inflammation, decreased availability of nitric oxide, and accumulation of reactive oxygen species (ROS) due to hyperglycemia accelerate tissue and cellular senescence [22]. Herein, both in vitro and in vivo observations demonstrated that an HG environment induces senescence in LECs, consistent with previous reports [31, 46]. In exploring the downstream targets of METTL3-mediated m6A modification in DC, SIRT1 was selected due to its established role as a classical regulator of autophagy, inflammation, and cellular senescence [47]. Our study demonstrated that SIRT1 was significantly down-regulated in response to HG induction. Surprisingly, even in cases of METTL3 overexpression, the reduced expression trend of SIRT1 could still be reversed by the SIRT1 activator SIRT1720 and the anti-senescence medicines MET. Therefore, SIRT1720 and MET can be considered effective medicine for alleviating LECs senescence. Our previous study also demonstrated that MET acts as a potent SIRT1 activator to ameliorate HG-induced autophagy inhibition and senescence in HLEB3 cells [31]. Thus, despite the mechanisms involved may be multifaceted, SIRT1 be an effective target for counteracting METTL3-induced autophagy inhibition and senescence.

The m6A reader regulator YTHDF2 is a key component in m6A modification and is known to promote mRNA degradation [48,49,50]. YTHDF2 has been reported to be essential for senescence [51, 52]. Nevertheless, there is limited evidence regarding the role of YTHDF2 in DC. Combining literature and RT-qPCR results, we hypothesized that the reader protein YTHDF2 is a key factor participating in SIRT1 down-regulation. Subsequently, YTHDF2 was either knocked down or overexpressed in HLEB3 cells. Fortunately, our results indicated that overexpression of YTHDF2 led to a reduction in SIRT1 mRNA levels compared to controls. Additionally, RIP experiments demonstrated a direct interaction between YTHDF2 and SIRT1 mRNA. These results confirm that METTL3 inactivates SIRT1 mRNA in an m6A-YTHDF2-dependent manner, highlighting m6A modification as a pivotal link in this axis.

This study is limited by the following factors: First, the study focused on exploring the mechanism of action METTL3 in DC, while the role of METTL3 in ARC requires further confirmation through in vitro and in vivo experiments. Second, while autophagy and senescence expression trends in DC models were identified, their mutual influence and interaction sites have not yet been explored. Further experiments are needed to clarify these interactions. Additionally, applying medications and targets to delay cataract progression within these pathways deserves greater attention.

Conclusions

In conclusion, our results unveil a new epigenetic mechanism underlying DC progression and present convincing evidence. METTL3 expression was upregulated in an HG environment and associated with autophagy inhibition and accelerated senescence. The in vitro and in vivo experiments elucidated that METTL3 knockdown restored autophagosome production and delayed senescence. Furthermore, in vitro experiments mechanistically revealed that m6A modification reduced SIRT1 mRNA stability via YTHDF2, ultimately decreasing SIRT1 expression levels. The anti-senescence medicines SIRT1720 and MET could rescue this pathological process. As far as we know, this study is the first to uncover the entire mechanism by which m6A-mediated modifications impact the progression of DC.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

References

  1. Teo ZL, Tham YC, Yu M, et al. Global prevalence of diabetic retinopathy and projection of burden through 2045: systematic review and meta-analysis. Ophthalmology. 2021;128(11):1580–91.

    Article  PubMed  Google Scholar 

  2. Cole JB, Florez JC. Genetics of diabetes mellitus and diabetes complications. Nat Rev Nephrol. 2020;16(7):377–90.

    Article  PubMed  PubMed Central  Google Scholar 

  3. International Diabetes Federation (IDF). Diabetes atlas. 7th Edition, International Diabetes Federation, Brussels, Belgium. 2015. [Last accessed on 2015 Nov 06]. http://www.diabetesatlas.org

  4. Kelkar A, Kelkar J, Mehta H, et al. Cataract surgery in diabetes mellitus: a systematic review. Indian J Ophthalmol. 2018;66(10):1401–10.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Grauslund J, Green A, Sjølie AK. Cataract surgery in a population-based cohort of patients with type 1 diabetes: long-term incidence and risk factors. Acta Ophthalmol. 2011;89(1):25–9.

    Article  PubMed  Google Scholar 

  6. Rossi T, Panozzo G, Della Mura G, et al. Diabetes and diabetic retinopathy in patients undergoing cataract surgery: a prevalence study-DiCat study report #2. Acta Diabetol. 2020;57(6):645–50.

    Article  CAS  PubMed  Google Scholar 

  7. Roundtree IA, Evans ME, Pan T, et al. Dynamic RNA modifications in gene expression regulation. Cell. 2017;169(7):1187–200.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Desrosiers R, Friderici K, Rottman F. Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells. Proc Natl Acad Sci U S A. 1974;71(10):3971–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wang X, Lu Z, Gomez A, et al. N6-methyladenosine-dependent regulation of messenger RNA stability. Nature. 2014;505(7481):117–20.

    Article  PubMed  Google Scholar 

  10. Shi H, Wang J, Cheng Y, et al. Ythdc2 is an N6-methyladenosine binding protein that regulates mammalian spermatogenesis. Cell Res. 2017;27(9):1115–27.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Xiao W, Adhikari S, Dahal U, et al. Nuclear m(6)a reader YTHDC1 regulates mRNA splicing. Mol Cell. 2016;61(4):507–19.

    Article  CAS  PubMed  Google Scholar 

  12. Shi H, Wang X, Lu Z, et al. YTHDF3 facilitates translation and decay of N6-methyladenosine-modified RNA. Cell Res. 2017;27(3):315–28.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wang X, Zhao BS, Roundtree IA, et al. N(6)-methyladenosine modulates messenger RNA translation efficiency. Cell. 2015;161(6):1388–99.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Dominissini D, Moshitch-Moshkovitz S, Schwartz S, et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature. 2012;485(7397):201–6.

    Article  CAS  PubMed  Google Scholar 

  15. Jiang X, Liu B, Nie Z, et al. The role of m6A modification in the biological functions and diseases. Signal Transduct Target Ther. 2021;6(1):74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Li X, Ma B, Zhang W, et al. The essential role of N6-methyladenosine RNA methylation in complex eye diseases. Genes Dis. 2022;10(2):505–20.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Wang Y, Chen Y, Liang J et al. METTL3-mediated m6A modification of HMGA2 mRNA promotes subretinal fibrosis and epithelial-mesenchymal transition. J Mol Cell Biol. 2023;15(3).

  18. Suo L, Liu C, Zhang QY, et al. METTL3-mediated N(6)-methyladenosine modification governs pericyte dysfunction during diabetes-induced retinal vascular complication. Theranostics. 2022;12(1):277–89.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Yang J, Liu J, Zhao S, Tian F. N(6)-methyladenosine METTL3 modulates the proliferation and apoptosis of lens epithelial cells in diabetic cataract. Mol Ther Nucleic Acids. 2020;20:111–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Varela-Eirín M, Demaria M. Cellular senescence. Curr Biol. 2022;32(10):R448-R452.

  21. Roger L, Tomas F, Gire V. Mechanisms and regulation of cellular senescence. Int J Mol Sci. 2021;22(23).

  22. Palmer AK, Gustafson B, Kirkland JL, et al. Cellular senescence: at the nexus between ageing and diabetes. Diabetologia. 2019;62(10):1835–41.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Zhang Y, Huang W, Zheng Z, et al. Cigarette smoke-inactivated SIRT1 promotes autophagy-dependent senescence of alveolar epithelial type 2 cells to induce pulmonary fibrosis. Free Radic Biol Med. 2021;166:116–27.

    Article  CAS  PubMed  Google Scholar 

  24. Wong SQ, Kumar AV, Mills J, Lapierre LR. Autophagy in aging and longevity. Hum Genet. 2020;139(3):277–90.

    Article  PubMed  Google Scholar 

  25. Makley LN, McMenimen KA, DeVree BT, et al. Pharmacological chaperone for α-crystallin partially restores transparency in cataract models. Science. 2015;350(6261):674–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Chen M, Fu Y, Wang X, et al. Metformin protects lens epithelial cells against senescence in a naturally aged mouse model. Death Discov. 2022;8(1):8.

    Article  Google Scholar 

  27. Hill SM, Wrobel L, Ashkenazi A, et al. VCP/p97 regulates beclin-1-dependent autophagy initiation. Nat Chem Biol. 2021;17(4):448–55.

    Article  CAS  PubMed  Google Scholar 

  28. Grewal DS, Brar GS, Grewal SP. Correlation of nuclear cataract lens density using scheimpflug images with lens opacities classification system III and visual function. Ophthalmology. 2009;116(8):1436–43.

    Article  PubMed  Google Scholar 

  29. Wilkinson CP, Ferris FL 3rd, Klein RE, et al. Proposed international clinical diabetic retinopathy and diabetic macular edema disease severity scales. Ophthalmology. 2003;110(9):1677–82.

    Article  CAS  PubMed  Google Scholar 

  30. Sergeys J, Etienne I, Van Hove I, et al. Longitudinal in vivo characterization of the streptozotocin-induced diabetic mouse model: focus on early inner retinal responses. Invest Ophthalmol Vis Sci. 2019;60(2):807–22.

    Article  CAS  PubMed  Google Scholar 

  31. Fu Y, Wu R, Dong S, et al. Metformin protects human lens epithelial cells from high glucose-induced senescence and autophagy inhibition by upregulating SIRT1. Graefes Arch Clin Exp Ophthalmol. 2024;262(2):477–85.

    Article  CAS  PubMed  Google Scholar 

  32. Zhang J, Yang C, Wang C, et al. AGE-induced keratinocyte MMP-9 expression is linked to TET2-mediated CpG demethylation. Wound Repair Regen. 2016;24(3):489–500.

    Article  PubMed  Google Scholar 

  33. Thompson J, Lakhani N. Cataracts. Prim Care. 2015;42(3):409–23.

    Article  PubMed  Google Scholar 

  34. Tang Y, Chen K, Song B, et al. m6A-Atlas: a comprehensive knowledgebase for unraveling the N6-methyladenosine (m6A) epitranscriptome. Acids Res. 2021;49(D1):D134–43.

    Article  CAS  Google Scholar 

  35. Kumari N, Karmakar A, Ahamad Khan MM, et al. The potential role of m6A RNA methylation in diabetic retinopathy. Exp Eye Res. 2021;208:108616.

    Article  CAS  PubMed  Google Scholar 

  36. Wen K, Zhang Y, Li Y, et al. Comprehensive analysis of transcriptome-wide m(6)a methylome in the anterior capsule of the lens of high myopia patients. Epigenetics. 2021;16(9):955–68.

    Article  PubMed  Google Scholar 

  37. Li P, Yu H, Zhang G, et al. Identification and characterization of N6-methyladenosine CircRNAs and methyltransferases in the lens epithelium cells from age-related cataract. Invest Ophthalmol Vis Sci. 2020;61(10):13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Li R, Zhu H, Li Q, et al. METTL3-mediated m6A modification of has_circ_0007905 promotes age-related cataract progression through miR-6749-3p/EIF4EBP1. PeerJ. 2023;11:e14863.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues. Cell. 2011;147(4):728–41.

    Article  CAS  PubMed  Google Scholar 

  40. Zhang L, Ding WY, Wang ZH, et al. Early administration of trimetazidine attenuates diabetic cardiomyopathy in rats by alleviating fibrosis, reducing apoptosis and enhancing autophagy. J Transl Med. 2016;14(1):109.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Wang L, Lou W, Zhang Y, et al. HO-1-mediated autophagic restoration protects lens epithelial cells against oxidative stress and cellular senescence. Invest Ophthalmol Vis Sci. 2023;64(15):6.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Chen M, Zhang C, Zhou N, et al. Metformin alleviates oxidative stress-induced senescence of human lens epithelial cells via AMPK activation and autophagic flux restoration. J Cell Mol Med. 2021;25(17):8376–89.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Mizushima N, Yoshimori T, Ohsumi Y. The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol. 2011;27:107–32.

    Article  CAS  PubMed  Google Scholar 

  44. Liu C, Zhou X, Lu J, et al. Autophagy mediates 2-methoxyestradiol-inhibited scleroderma collagen synthesis and endothelial-to-mesenchymal transition induced by hypoxia. Rheumatology (Oxford). 2019;58(11):1966–75.

    Article  CAS  PubMed  Google Scholar 

  45. Xue JF, Shi ZM, Zou J, et al. Inhibition of PI3K/AKT/mTOR signaling pathway promotes autophagy of articular chondrocytes and attenuates inflammatory response in rats with osteoarthritis. Biomed Pharmacother. 2017;89:1252–61.

    Article  CAS  PubMed  Google Scholar 

  46. Orimo M, Minamino T, Miyauchi H, et al. Protective role of SIRT1 in diabetic vascular dysfunction. Arterioscler Thromb Vasc Biol. 2009;29(6):889–94.

    Article  CAS  PubMed  Google Scholar 

  47. Hwang JW, Yao H, Caito S, et al. Redox regulation of SIRT1 in inflammation and cellular senescence. Free Radic Biol Med. 2013;61:95–110.

    Article  CAS  PubMed  Google Scholar 

  48. Li Z, Fang F, Zafar MI, et al. RNA m(6)a modification regulates L1 retrotransposons in human spermatogonial stem cell differentiation in vitro and in vivo. Cell Mol Life Sci. 2024;81(1):92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Ren M, Pan H, Zhou X, et al. KIAA1429 promotes gastric cancer progression by destabilizing RASD1 mRNA in an m(6)A-YTHDF2-dependent manner. J Transl Med. 2024;22(1):584.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Meng L, Lin H, Huang X, et al. METTL14 suppresses pyroptosis and diabetic cardiomyopathy by downregulating TINCR lncRNA. Cell Death Dis. 2022;13(1):38.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Chen X, Gong W, Shao X, et al. METTL3-mediated m6A modification of ATG7 regulates autophagy-GATA4 axis to promote cellular senescence and osteoarthritis progression. Ann Rheum Dis. 2022;81(1):87–99.

    Article  CAS  PubMed  Google Scholar 

  52. Fan T, Du Y, Zhang M, et al. Senolytics cocktail dasatinib and quercetin alleviate human umbilical vein endothelial cell senescence via the TRAF6-MAPK-NF-κB axis in a YTHDF2-dependent manner. Gerontology. 2022;68(8):920–34.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank for the Future Medical Laboratory of the Second Affiliated Hospital of Harbin Medical University (Harbin, China).

Funding

This work was supported by the Natural Science Foundation of Heilongjiang Province of China (LH2023H041, NZ) and China Primary Health Care Foundation ([2022] 005, NZ).

Author information

Authors and Affiliations

  1. Department of Ophthalmology, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150086, China

    Su Dong, Jiajia Zhang, Yushan Fu, Gege Tang, Dawei Sun, Yanhua Qi & Nan Zhou

  2. Laboratory Animal Center, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150086, China

    Jianfeng Chen

Authors
  1. Su Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jiajia Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yushan Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gege Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jianfeng Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dawei Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yanhua Qi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Nan Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

SD, YQ and NZ vised the study design, main conceptual ideas, and project outline; DS provided critical suggestions and discussions throughout the entire study; SD and YF contributed to cell experiments; JZ and JC contributed to animal experiments; SD and GT processed and analyzed the data; GT, DS, YQ and NZ procured human samples; DS draw mechanism diagrams; SD and NZ wrote the manuscript.

Corresponding authors

Correspondence to Dawei Sun, Yanhua Qi or Nan Zhou.

Ethics declarations

Ethics approval and consent to participate

All experiments involving animals were approved by the Ethics Committee of The Second Affiliated Hospital of Harbin Medical University (YJSDW2023-088). The studies involving human participants were approved by the Ethics Committee of The Second Affiliated Hospital of Harbin Medical University (YJSKY2023-477). Written informed consent was obtained from all patients before collection.

Consent for publication

Not applicable.

Conflict of interest

The authors declare no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Supplementary Material 2

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dong, S., Zhang, J., Fu, Y. et al. METTL3-mediated m6A modification of SIRT1 mRNA affects the progression of diabetic cataracts through cellular autophagy and senescence. J Transl Med 22, 865 (2024). https://doi.org/10.1186/s12967-024-05691-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12967-024-05691-w

Keywords

Journal of Translational Medicine

ISSN: 1479-5876

Contact us