METTL3-mediated m6A modification of has_circ_0007905 promotes age-related cataract progression through miR-6749-3p/EIF4EBP1

Data downloaded from the Gene Expression Omnibus (GEO) database

The circRNA-seq data and MeRIP-seq data were downloaded from the GEO database under accession number GSE153722 (six ARC vs. six normal). The differential expression analysis of circRNA-seq and MeRIP-seq was performed using the R package DESeq under the cut-off criteria: —log2FC—≥ 1. The m6A-enriched circRNA in the normal control and ARC samples were analyzed. The intersection of differentially expressed circRNAs and differentially methylated m6A-enriched circRNAs served as candidate circRNAs.

Additionally, the RNA-protein binding site between has_circ_0007905 and METTL3 was predicted using the RBPsuite software (http://www.csbio.sjtu.edu.cn/bioinf/RBPsuite/). The potential m6A modification sites of has_circ_0007905 were predicted using SRAMP software (http://www.cuilab.cn/sramp).

Sample collection of crystalline lens from ARC patients

Crystalline lens samples were obtained from conventional continuous curvilinear capsulorhexis in ARC patients (n = 10, each sample with three replicates), with no vascular contact or damage to the iris or other intraocular tissues. Patients with complex cataracts due to high myopia, trauma, uveitis, glaucoma, or other systemic diseases, such as hypertension and diabetes, were excluded from the study. Transparent lenses removed from normal subjects with shallow anterior chambers served as controls (n = 2, each sample with three replicates). The “shallow anterior chamber” is only a potential risk factor for glaucoma, but there is currently no related disease, and it also belongs to normal people. In this group, the clinical option was to deepen the anterior chamber through preventive lens surgery to remove the potential glaucoma risk. Signed informed consent was obtained from all patients. Detailed clinical data for each individual human subject are shown in Table S1. This study was approved by the Ethics Committee of Shanghai East Hospital, School of Medicine, Tongji University ([2021] Audit Research No. 79).

RT-qPCR analysis

Total RNA was extracted from crystalline lens samples and HLE-B3 cells using TRIzol (Invitrogen). The concentration and purity of total RNA were measured using a microspectrophotometer (Tiangen, Beijing, China). High-quality RNA was stored at −80 °C for subsequent RT-qPCR and RNA sequencing. Next, reverse transcription was conducted using a RevertAid™ First Strand cDNA Synthesis Kit (K16225; Thermo Fisher, Waltham, MA, USA). cDNA amplification was performed by 2 × PCR Master Mix (Roche, Basel, Switzerland) on the ABI Q6 Flex Real-time PCR system (Applied Biosystems, Foster City, CA, USA). Gene expression was calculated using the 2^−ΔΔCT method. All primers used in this study are shown in Table S2.

Cell culture and ultraviolet B (UVB) irradiation and transfection

The human lens epithelial cell line of HLE-B3 cells was purchased from BFB BLUEFBIO (BFN60805970; Bluebio (Yantai) Bio-Pharmaceutical Co., Ltd., Yantai, China) and cultured in high glucose DMEM (10-013-CVRC; Corning, Shanghai, China) supplemented with 10% FBS (10099, GIBCO) and 1% PS (E607011, Sangon). HLE-B3 cells were maintained in an incubator with a 5% CO₂ atmosphere at 37 °C. To construct ARC cell model, HLE-B3 cells were exposed to UVB light (from top to bottom) for 10 min, according to a previous study, with slight modifications in exposure time (Xiang et al., 2019).

For the knockdown of has_circ_0007905 and METLL3, siRNAs were designed and synthesized by GenePharma. The has-miR-6749-3p mimic and has-miR-6749-3p inhibitor were also synthesized by GenePharma to overexpress and knockdown the has-miR-6749-3p, respectively. For overexpression of EIF4EBP1, the full length of EIF4EBP1 was cloned into pCDNA3.1 vectors (OE-EIF4EBP1), and the blank vector served as NC.

Transfection was conducted using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Briefly, when the fusion rate of the cells reached 80–90%, the fresh medium was replaced. All siRNA, vectors, and Lipofectamine 2000 reagents were diluted with OPTI-MEM (5 µL:45 µL). Next, diluted Lipofectamine and RNA sequences were mixed for 20 min and added to the cell sample.

MeRIP-qPCR analysis

Isolated RNA was purified and fragmented into fragments with ∼200 bp using RNA Fragmentation Reagent (Invitrogen, Waltham, MA, USA). Around 10% of the volume of fragmented RNA was set aside as an input group. Magnetic beads A/G were mixed with anti-m6A antibodies and set aside. Next, fragmented RNA was incubated with the prepared anti-m6A antibody in an immunoprecipitation buffer, and anti-immunoglobulin G (IgG) served as a negative control. After incubation, RNA was eluted from the beads and subjected to RT-qPCR analysis.

CCK8 assay

The proliferation of HLE-B3 cells was assessed using a CCK-8 kit (C0037; Beyotime Biotechnology, Shanghai, China). Briefly, HLE-B3 cells were processed into single-cell suspensions with a density of 1 × 10⁴ cells/mL and were seeded into a 6-well plate with 1000 cells/well. Each sample set had six replicates. At the indicated times of 0, 24, 48, 72, and 96 h, 10 µL CCK-8 solution was added to each well. The absorbance value at 450 nm was detected after 1 h.

Flow cytometry

At 6 h post-transfection, the medium was replaced with fresh medium. The cells were cultured for 48 h for flow cytometry. The cells were harvested and gently washed with PBS. Cells were resuspended with 195 µL of 1 × Binding Buffer to a cell density of 2–5 × 10⁵ cells/mL. A total of 5 µL of Annexin V-FITC (C1062; Beyotime, Shanghai, China) was added to the cell resuspension and incubated for 15 min at room temperature in the dark. Next, the cells were washed with 200 µL 1 × Binding Buffer and centrifuged at 1000 rpm for 5 min to remove the supernatant. Finally, the cells were resuspended in 190 µL of 1 × Binding Buffer, and 10 µL of propidium iodide was added. A flow cytometry assay was performed within 4 h.

TUNEL analysis

The apoptosis of HLE-B3 cells was assessed with the One Step TUNEL Apoptosis Assay Kit (green fluorescence) (C1086; Beyotime Biotechnology, Shanghai, China). Briefly, HLE-B3 cells were fixed with 4% paraformaldehyde for 30 min and then washed with PBS, followed by permeabilization in PBS containing 0.3% Triton X-100 for 5 min. Afterward, 50 µL TUNEL solution was added to the HLE-B3 cells and incubated for 60 min in the dark. Lastly, HLE-B3 cells were sealed with antifluorescence quenching sealing tablets and photographed on a fluorescence microscope.

Transcriptome sequencing

RNA extracted from HLE-B3 cells after has_circ_0007905 knockdown (n = 3) or NC (n = 3) was used for transcriptome sequencing. RNA sequencing was performed by Yingbio Technology (Shanghai, China) on an Illumina HiSeq 2500 platform. The statistical power of this experimental design was calculated in RNASeqPower and was 0.896. The raw data were filtered using the FastQC method. Clean reads were subjected to screening of differentially expressed genes (DEGs) using the DEGSeq algorithm upon thresholds of Log2FC >1 or <-1 and FDR <0.05. GO and KEGG enrichment were analyzed on DEGs. The RNA-seq data generated in this study are available in the Sequence Read Archive under accession number PRJNA868911.

Western blot

HLE-B3 cells were harvested and lysed using a lysis buffer, and the protein concentration of cell extracts was quantified with a BSA kit. Next, appropriately 20 µg protein was loaded on 10 SDS-PAGE gels and transferred into PVDF membranes, followed by blocking with TBST solution containing 5% skim milk at 4 °C for 3 h. Membranes were incubated with primary antibodies at 4 °C overnight and then incubated with secondary antibodies of Goat Anti-Mouse IgG H&L (HRP) (1:1000, ab205719; Abcam, Cambridge, UK) and Goat Anti-Rabbit IgG H&L (HRP) (1:20000, ab6721; Abcam, Cambridge, UK). Finally, the protein bands were imaged using the Bio-Rad ChemiDoc XRS system. Primary antibodies, including GAPDH (1:2000, 60004-1-Lg; Proteintech, Rosemont, IL, USA), METTL3 (1:1000, 15073-I-AP; Proteintech), and EIF4EBP1 (1:2000, ab32024; Abcam, Cambridge, UK), were used.

RNA immunoprecipitation (RIP)

The RIP assay was performed in this study using the Magna RIP RNA-Binding Protein IP Kit (Millipore, Burlington, MA, USA) according to the product instruction. Cells were collected and washed twice with pre-cooled PBS and centrifuged at 1500 rpm for 5 min at 4 °C to discard supernatant, followed by adding RIP Lysis Buffer to lyse the cells on ice for 5 min. Diluted 50 µL of magnetic beads in 100 µL of RIP Wash Buffer was used. The sample was incubated with 5 µg of Argonaute-2 antibody or 1 µg IgG antibody for 30 min at room temperature with rotation. After light centrifugation, the supernatant was removed, and the precipitate was resuspended in 0.5 mL RIP Wash Buffer. After that, 900 µL of RIP Immunoprecipitation Buffer was added to the bead-antibody mixture and then incubated with 100 cell lysis products overnight at 4 °C with rotation. Finally, 150 µL proteinase K buffer was added to the complex product and incubated at 55 °C for 30 min, followed by RNA extraction for RT-qPCR.

Dual-luciferase activity assay

HLE-B3 cells were seeded into 96-well plates and co-transfected with has-miR-6749-3p mimic/NC and psiCHECK™-2 vectors containing has_circ_0007905 wild type (WT) or mutant (Mut) 3′ UTR sequence, and psiCHECK™-2 vectors containing EIF4EBP1 WT or Mut 3′ UTR sequence. At 48 h after transfection, the cells were lysed and incubated with firefly luciferase buffer to detect firefly luciferase activity, followed by incubation with Renilla luciferase to detect firefly luciferase activity (Promega, Madison, WI, USA).

Statistical analysis

Data analysis was executed using GraphPad Prism 9.0. The t-test was used to assess significant differences between two groups, and one-way analysis of variance (ANOVA) following Turkey’s test to assess significant differences among three groups. All data are displayed as mean ± standard deviation (mean ± SD), and a P-value lower than 0.05 was considered significant.

Identification of hypermethylated-upregulated expression of circRNAs

Data from GSE153722 were analyzed to screen differentially expressed circRNAs and differentially methylated circRNAs. According to GSE153722 circRNA-seq data, a total of 220 differentially expressed circRNAs were identified, including 117 upregulated and 103 downregulated in the ARC group compared to the NC group (Fig. 1A). According to GSE153722 MeRIP-seq data, a total of 220 differentially methylated circRNAs were identified, including 102 hypermethylation circRNAs and 118 hypomethylation circRNAs in the ARC group compared to the NC group (Fig. 1A). Subsequently, as shown in Fig. 1A, there were 50 overlapping circRNAs that satisfied the hypermethylation upregulated in the ARC group. These overlapping circRNAs have attracted our attention. To further narrow the scope of candidate hypermethylated-upregulated expression circRNAs, five circRNAs (has_circ_0007905, has_circ_0065244, has_circ_0003949, has_circ_0022997, and has_circ_0035228) with the most significantly changed were selected for validation. The distribution of m6A peaks of the five circRNAs is shown in Fig. 1B, and they all harbored more than three m6A peaks. We also examined the expression of five candidate circRNAs in ARC tissue using RT-qPCR. Compared with the NC group, only the expression of has_circ_0007905 (P = 0.00114) was upregulated in the ARC group, whereas the remaining four circRNA expressions were opposite (Fig. 1C). Next, we determined the expression pattern and m6A level of has_circ_0007905 in ARC cell model induced by UVB exposure. As expected, a significant increase of has_circ_0007905 expression in HLE-B3 cells after UVB exposure was observed (Fig. 1D; P = 0.00292). The total m6A level of has_circ_0007905 was also significantly elevated in the UVB treatment group compared with the control group (Fig. 1E; P = 0.00543). According to the annotation in UCSC Genome Browser on Human (GRCh37/hg19) (https://genome.mdc-berlin.de/index.html), has_circ_0007905 was formed by the back-splicing of exons 1 and 4 (Fig. 1F). The back-splicing site of has_circ_0007905 was confirmed with Sanger sequencing (Fig. 1G). Collectively, we obtained the dataset of hypermethylated-upregulated expression circRNAs in ARC tissue and focused on has_circ_0007905.

Silencing of has_circ_0007905 promotes proliferation and inhibits apoptosis of lens epithelial cells

To further investigate the function of has_circ_0007905 in ARC progression in vitro, we interfered with has_circ_0007905 expression in the HLE-B3 cell line using two siRNA sequences. We confirmed that has_circ_0007905 expression was significantly reduced upon the presence of siRNA, especially siRNA-1 (si-has_circ_0007905-1; P < 0.0001) (Fig. 2A). Meanwhile, CCK-8 assays indicated that silencing of has_circ_0007905 significantly boosted the proliferation of HLE-B3 cells (Fig. 2B; P < 0.0001). TUNEL staining demonstrated that apoptosis of HLE-B3 cells was significantly decreased after silencing of has_circ_0007905 compared with the NC group (Fig. 2C). These results highlight that has_circ_0007905 promotes the progression of ARC in vitro.

METTL3 mediates m6A modification of has_circ_0007905

As we all know, m6A modification is driven by the m6A writer composed of METTL3, METTL14, and WTAP, which are removed by m6A erasers, such as FTO and ALKBH5 (Deng et al., 2018). To further examine which enzyme is engaged in m6A modification of has_circ_0007905, we detected the expression of METTL3, METTL14, WTAP, FTO, and ALKBH5 in ARC tissues. Intriguingly, only METTL3 expression was significantly upregulated in ARC tissues compared to the NC group (Fig. 2D; P < 0.0001). Four RNA-protein binding sites between has_circ_0007905 and METTL3 (Fig. 2E) were predicted by the RBPsuite database (http://www.csbio.sjtu.edu.cn/bioinf/RBPsuite/). Seven potential m6A modification sites in has_circ_0007905 were also expected using SRAMP (http://www.cuilab.cn/sramp) (Fig. 2F). Moreover, the mRNA (P = 0.00882) and protein (P = 0.00162) expressions of METTL3 were significantly elevated in HLE-B3 cells upon UVB exposure (Figs. 2G and 2H). Thus, we initially guessed that the m6A modification of has_circ_0007905 was mediated by METTL3.

METTL3 inhibits proliferation and promotes apoptosis of lens epithelial cells via has_circ_0007905

Four siRNAs against METTL3 were synthesized to explore the functional role of METT L3 in ARC progression in vitro. It was shown in the results of RT-qPCR and western blot that the mRNA and protein expression of METTL3 was significantly decreased after transfection with siRNA-968. Thus, siRNA-968 was used for METTL3 knockdown experiments (Figs. 3A and 3B). Expectedly, METTL3 knockdown significantly diminished the expression (Fig. 3C; P = 0.012) and m6A modification level (Fig. 3D; P < 0.0001) of has_circ_0007905. In addition, METTL3 knockdown significantly facilitated proliferation (Fig. 3E; P = 0.0202) and inhibited apoptosis detected by flow cytometric assay (Fig. 3F; P = 0.010) (Fig. S1) and TUNEL (Fig. 3G) of HLE-B3 cells. These results suggested that knockdown of METTL3 attenuated ARC progression in vitro.

We conducted rescue experiments to further clarify whether the function of METTL3 in ARC progression is has_circ_0007905-dependent. CCK-8 assay demonstrated that the proliferation of HLE-B3 cells was significantly impaired (P < 0.0001) by the overexpression of METTL3. However, the proliferation was significantly restored by the simultaneous METTL3 overexpression and silencing of has_circ_0007905 (P < 0.0001) (Fig. 3H). In contrast, overexpression of METTL3 significantly accelerated apoptosis of HLE-B3 cells. Still, this phenotypic effect was prevented by has_circ_0007905 silencing, which was detected using flow cytometric assay (Fig. 3I; NC vs. OE-METTL3, P < 0.0001, OE-METTL3 vs. OE-METTL3+ si-circ_0007905, P = 0.0071) (Fig. S2) and TUNEL (Fig. 3J). Therefore, we concluded that METTL3 promotes ARC progression in vitro via has_circ_0007905 in an m6A-dependent manner.

Silencing of has_circ_0007905 leads to an alteration in transcriptome landscape in lens epithelial cells

One of the mechanisms of circRNA is to regulate the expression of target genes through the ceRNA mechanism. Thus, we mined the downstream target genes of has_circ_0007905 using transcriptome sequencing. We found that has_circ_0007905 interference resulted in 707 mRNAs downregulation and 2184 mRNAs upregulation compared with the NC group (Fig. 4A). These DEGs were clearly clustered into two branches (Fig. 4B). To investigate the physiological significance of DEGs in ARC progression, GO and KEGG pathway enrichment were performed for DEGs. GO enrichment revealed that these DEGs were mainly associated with the immune-related process, interestingly, 13 of the 20 high-ranking significantly enriched GO entries were related to immune processes, such as the immune system process, inflammatory response, immune response, and innate immune response (Fig. 4C). KEGG pathway enrichment showed that these DEGs were mainly involved in immune-related pathways, including cytokine-cytokine receptor interaction, complement and coagulation cascades, and proliferation-related pathways, such as the PI3K-Akt and NF-kappa B signaling pathways (Fig. 4D). These results implicated that has_circ_0007905 interference may be involved in the progression of ARC by affecting the immune response processes and proliferation-related pathways of HLE-B3 cells through DEGs.

To verify the reliability of the sequencing results, five down-regulated DEGs in the has_circ_0007905 interference group with high abundance and significant P-values were selected for RT-qPCR. Compared with the NC group, the expression of these five DEGs was significantly reduced in the has_circ_0007905 interference group (Fig. 4E). Given that ARC manifests as cellular senescence and loss and diminished proliferation, we focused on a target gene associated with proliferation and apoptosis (Yang et al., 2020). EIF4EBP1 has been reported to be associated with proliferation and apoptosis (Zhang & Su, 2022), so we focused on EIF4EBP1.

Has_circ_0007905 inhibits proliferation and promotes apoptosis via EIF4EBP1 in lens epithelial cells

To explore the function of EIF4EBP1 in ARC progression in vitro, we constructed plasmids that overexpressed EIF4EBP1 and transfected them into HLE-B3 cells. A significant increase in EIF4EBP1 expression at mRNA and protein levels was confirmed by RT-qPCR (Fig. 5A; P < 0.0001) and western blot (Fig. 5B; P = 0.00330) after overexpression of EIF4EBP1. Moreover, we observed that proliferation was significantly prevented (Fig. 5C; P < 0.0001) and that apoptosis was enhanced (Fig. 5D) in HLE-B3 cells after EIF4EBP1 overexpression, indicating that EIF4EBP1 contributes to ARC progression in vitro. EIF4EBP1 expression was significantly reduced by has_circ_0007905 interference (P = 0.0007) and this effect could be reversed with EIF4EBP1 overexpression (P = 0.0344) (Fig. 5E). The CCK8 results showed that the excessive proliferation caused by has_circ_0007905 interference was significantly attenuated by EIF4EBP1 overexpression (Fig. 5F; P < 0.0001). TUNEL staining proved that has_circ_0007905 interference resulted in the attenuation of apoptosis, which was significantly rescued by EIF4EBP1 overexpression (Fig. 5G). Taken together, these results supported that has_circ_0007905 inhibits proliferation and promotes apoptosis via EIF4EBP1 in HLE-B3 cells.

Has_circ_0007905 inhibits proliferation and promotes apoptosis via miR-6749-3 p/EIF4EBP1 in lens epithelial cells

To confirm the mode of interaction between has_circ_0007905 and EIF4EBP1, RIP-PCR and AGO2-dependent AGO2-RIP were performed, respectively. The results showed that the enrichment of EIF4EBP1 in the absence of an AGO2 antibody was not affected by the knockdown of has_circ_0007905 (Fig. 6A; P = 0.942). However, the enrichment of EIF4EBP1 was significantly reduced by has_circ_0007905 interference in the presence of an AGO2 antibody (Fig. 6A; P = 0.027). These results suggested that EIF4EBP1 is regulated by has_circ_0007905 through an AGO2-dependent ceRNA mechanism.

Given that has_circ_0007905 regulates the expression of EIF4EBP1 through the ceRNA mechanism, we next explored the miRNA that acts as a communication bridge between has_circ_0007905 and EIF4EBP1. According to the RNAhybrid and Miranda databases, has_circ_0007905 and EIF4EBP1 potentially bind 259 and 513 miRNAs, respectively. Among these, 103 miRNAs were shared by has_circ_0007905 and EIF4EBP1 (Fig. 6A). Five miRNAs with the highest binding energy were selected for RT-qPCR validation. It was found that only the expression of miR-6749-3p was significantly upregulated after the knockdown of has_circ_0007905 (Fig. 6B; P = 0.0272).

Furthermore, the WT binding sites and MUT binding sites of miR-6749-3p and has_circ_0007905/EIF4EBP1 displayed in Fig. 6C. As shown in Fig. 6D, miR-6749-3p mimic significantly decreased the luciferase activities of has_circ_0007905 in the cells carrying the WT plasmid rather than the MUT plasmid (P < 0.0001), also reduced the luciferase activities of EIF4EBP1-WT (P = 0.000561) (Fig. 6E). The decrease in EIF4EBP1 expression induced by has_circ_0007905 silencing (P = 0.0007) was significantly reversed by the miR-6749-3p inhibitor (P = 0.0103) (Fig. 6F). Importantly, has_circ_0007905 silencing conferred that the elevation in the proliferation of HLE-B3 cells was significantly mitigated by the miR-6749-3p inhibitor (Fig. 6G). The effects of has_circ_0007905 silencing on apoptosis were also significantly blocked by the miR-6749-3p inhibitor (Fig. 6H). In summary, these results demonstrated that has_circ_0007905 functions as a sponge for miR-6749-3p to regulate EIF4EBP1 expression to involve the proliferation and apoptosis of HLE-B3 cells.