N6-Methyladenosine METTL3 Modulates the Proliferation and Apoptosis of Lens Epithelial Cells in Diabetic Cataract

Jun Yang; Jingshu Liu; Shaozhen Zhao; Fang Tian

doi:10.1016/j.omtn.2020.02.002

. 2020 Feb 11;20:111–116. doi: 10.1016/j.omtn.2020.02.002

N⁶-Methyladenosine METTL3 Modulates the Proliferation and Apoptosis of Lens Epithelial Cells in Diabetic Cataract

Jun Yang ¹, Jingshu Liu ², Shaozhen Zhao ^1,^∗, Fang Tian ^1,^∗∗

PMCID: PMC7066033 PMID: 32163892

Abstract

N⁶-methyladenosine (m⁶A) is the most prevalent eukaryotic messenger RNA modification. Diabetic cataract (DC) is caused by high glucose (HG) in diabetes mellitus. However, the regulatory mechanism of m⁶A in the DC pathogenesis is poorly understood. In present research, we performed the m⁶A-RNA immunoprecipitation sequencing (MeRIP-Seq) analysis and detected the m⁶A modification profile in the HG- or normal glucose (NG)-induced human lens epithelial cells (HLECs). Results revealed that methyltransferase-like 3 (METTL3) was upregulated in the DC tissue specimens and HG-induced HLECs. Besides, total m⁶A modification level was higher in the HG-induced HLECs. Functionally, METTL3 knockdown promoted the proliferation and repressed the apoptosis of HLECs induced by HG. MeRIP-Seq analysis revealed that ICAM-1 might act as the target of METTL3. Mechanistically, METTL3 targets the 3′ UTR of ICAM-1 to stabilize mRNA stability. In conclusion, this research identified the regulation of METTL3 in the HG-induced HLECs, providing a potential insight of the m⁶A modification for DC.

Keywords: N⁶-methyladenosine, human lens epithelial cells, diabetic cataract, METTL3, ICAM-1

Introduction

Diabetic cataract (DC) is characterized by the disorder and nubecula of human lens epithelial cells (HLECs) caused by abnormal blood supply of terminal circulation in diabetes mellitus.¹^,² In addition, other complications may be aroused by the diabetes mellitus, such as microcirculation abnormity and metabolic disorders.³ In the DC pathogenesis, high glucose (HG) could give rise to the lens apoptosis and metabolism disorder.

N⁶-methyladenosine (m⁶A) is the most common internal modification of eukaryotic mRNA, participating in the protein-coding transcripts, exports, translation, and decay.⁴^,⁵ For the m⁶A installation, methyltransferase protein complex could deposit the methyl to the adenosine, including methyltransferase-like 3 (METTL3), METTL14, Wilms’ tumor 1-associating protein (WTAP), and Virilizer homolog (KIAA1429).⁵ For the uninstallation, demethylases remove the methyl from adenosine, including AlkB homolog 5 (ALKBH5) and fat mass and obesity-associated (FTO). Besides, the reader proteins are responsible for the recognition, including YTH family domain of YT521-B homology (YTHDF1-3, YTHDC1-2).⁶

Approximately, 0.1%–0.4% of adenosines in total RNAs are modified by m⁶A methylation.⁷ The consensus motif of m⁶A is identified as RRACH (R: G, A, U; R: G, A; H: U, A, C).⁸ According to the high-throughput m⁶A profiling, results revealed that m⁶A sites are mainly enriched in 3′ untranslated regions (3′ UTRs) and near stop codons. METTL3 is a critical m⁶A writer and functions as an essential initiating factor in multiple pathogenesis. For instance, METTL3 associates with ribosomes and enhances mRNA translation through an interaction with the translation initiation machinery to promote the translation in the cytoplasm.⁹

In present research, we performed the m⁶A-RNA immunoprecipitation sequencing (MeRIP-Seq) analysis and found that the m⁶A peaks distribution was distributed near the stop codon, covering the coding sequence (CDS) and 3′ UTR. METTL3 silencing could promote the proliferation and repress the apoptosis of HLECs induced by HG. Further experiments revealed that METTL3 targets the 3′ UTR of ICAM-1 to stabilize its protein expression. In conclusion, this research identified the regulation of METTL3 in the HG-induced HLECs, providing a potential insight of the m⁶A modification for DC.

Results

MeRIP-Seq Analysis Reveals the m⁶A Modification in HLECs

In order to investigate the m⁶A modification of DC, MeRIP-Seq analysis was performed in the HG-induced HLECs. Density distribution of m⁶A peaks across the mRNA transcripts revealed that the main components of m⁶A peaks were concentrated on the stop codon, covering CDS and 3′ UTR (Figure 1A). Proportion of m⁶A peaks distribution illustrated that m⁶A peaks were distributed in 3′ UTR, 5′ UTR, exon, intron, downstream region, and distal intergenic region (Figure 1B). Based on the MeRIP-Seq analysis, several potential consensus m⁶A motifs were identified (Figure 1C). Among these candidate motifs, the consensus sequence “GGAC” occupied a significant portion, which is in accord with the previous reports.¹⁰^,¹¹ Volcano plot of the MeRIP-Seq analysis showed the expression difference in the m⁶A-tagged or un-tagged transcripts (Figure 1D). The Integrative Genomics Viewer (IGV) tool revealed the m⁶A peaks distribution in several candidate target genes, including transforming growth factor-β1 (TGF-β1), ICAM-1, SIRT1, and DUSP5 (Figure 1E). Overall, these data revealed the m⁶A modification in the HLECs detected by MeRIP-Seq analysis.

METTL3 Was Upregulated in DC Tissue and HG-Induced HLECs

In the enrolled DC tissue samples and lens anterior capsules samples, RT-PCR revealed that METTL3 expression was upregulated in the DC tissue samples (Figure 2A). Western blot analysis revealed that METTL3 protein was upregulated in the DC tissue samples (Figure 2B). In the HG-induced HLECs, RT-PCR revealed that METTL3 mRNA was upregulated as comparing to the normal glucose (NG) induced HLECs (Figure 2C). Moreover, western blot analysis showed that the METTL3 protein was upregulated in HG-induced HLECs (Figure 2D). The m⁶A quantitative analysis unveiled that the percentage of m⁶A content in the total RNA was increased in the HG-induced HLECs (Figure 2E). Overall, these findings concluded that METTL3 was upregulated in DC tissue and HG-induced HLECs, which was also correlated with the m⁶A content.

METTL3 Regulated the HG-Induced HLECs Proliferation and Apoptosis

In order to investigate the biological role of METTL3, short hairpin RNA (shRNA) for METTL3 was constructed and transfected into HLECs to silence the METTL3 mRNA (Figure 3A). Western blot analysis revealed that the METTL3 protein was decreased after the sh-METTL3 transfection (Figure 3B). Cell counting kit-8 (CCK-8) assay revealed that the HG administration reduced the proliferation of HLECs, and sh-METTL3 transfection rescued the proliferation (Figure 3C). Flow cytometry revealed that the HG administration promoted the apoptosis and HG administration alleviated the apoptosis of HLECs (Figure 3D). 5-Ethynyl-2′-deoxyuridine (EdU) assay illustrated that HG administration repressed the proliferation of HLE B-3 cells and sh-METTL3 transfection promoted the proliferation (Figure 3E). These data indicated that METTL3 participated in the HG-induced HLECs proliferation and apoptosis.

ICAM-1 Acted as a Target Protein of METTL3 in the HLECs

The IGV tool revealed that, comparing with the Input group, the m⁶A peaks distribution IP group was located in the 5′ UTR and 3′ UTR (Figure 4A). Moreover, we found that the major m⁶A peaks were distributed at the 3′ UTR. With the help of online bioinformatics tools (http://www.cuilab.cn/sramp), we found that the METTL3 motif in the HLECs is the consensus sequence (Figure 4B). Dot blot analysis revealed that the m⁶A quantity is upregulated in the HG-induced HLECs, while the quantity is reduced after the METTL3 silencing (Figure 4C). Analogously, m⁶A quantitative analysis revealed that METTL3 silencing could reduce the m⁶A level as comparing to the control group (Figure 4D). RIP-qPCR showed that the METTL3 silencing remarkedly decreased the ICAM-1 mRNA enrichment precipitated by antibody (Figure 4E). Western blot illustrated that the HG administration could enhance the ICAM-1 protein expression as comparing to NG, however, the METTL3 silencing repressed the ICAM-1 protein expression (Figure 4F). RNA stability assay showed that METTL3 silencing decreased the mRNA half-life (t_1/2) as compared to the control (Figure 4G). In conclusion, these data supported that ICAM-1 acted as a target protein of METTL3 in the HLECs.

Discussion

The m⁶A modification for RNA is a critical regulation in the epigenetic regulation catching more and more researchers’ attention. In multiple human pathophysiological processes, including cancer and embryonic development, m⁶A has been identified to modify extensive RNA transcription and protein generation.¹²^,¹³ However, the roles of m⁶A in the DC pathogenesis are still unclear.

The m⁶A for RNA is a dynamic and reversible process catalyzed by methyltransferase complex, including METTL3, METTL14, and WTAP. In our present research, we performed the MeRIP-Seq and found that the m⁶A peaks distribution was located near the stop codon, covering CDS and 3′ UTR. Moreover, the MeRIP-Seq analysis unveiled several potential consensus m⁶A motifs. Volcano plot of the MeRIP-Seq analysis showed the ectopically expressed m⁶A-tagged or un-tagged transcripts within HG-induced or NG-induced HLECs. Besides, we selected several candidate target genes with remarkable m⁶A peaks, including ICAM-1, TGF-β1, and DUSP5.

In present research, we established the DC and control models using the HG-induced or NG-induced HLECs. In the HG-induced HLECs, m⁶A methyltransferase METTL3 was upregulated as comparing to the NG-induced HLECs. Meanwhile, the m⁶A level was upregulated in the HG-induced HLECs, indicating that the HG could stimulate the m⁶A level in the HLECs. These data supported that HG stimulation could enhance the methylation level in total transcripts, illustrating that the RNA methylation might regulate the DC pathogenesis. Therefore, the investigation of m⁶A modification in the DC could shed light on its underlying mechanism.

In the functional experiments, we found that METTL3 silencing promoted the proliferation and alleviated the apoptosis of HG-induced HLECs. Therefore, the finding clued that METTL3 indeed could regulate the biological phenotype of HLECs. The roles of METTL3 in the human diseases have been wildly illustrated. For example, in the colorectal carcinoma, METTL3 specifically methylated the SOX2 transcripts via targeting the CDS regions through specific m6A “reader” IGF2BP2.¹⁴ In bladder cancer, the increased METTL3 induced the upregulation of ITGA6, and METTL3 could increase m⁶A methylations of the ITGA6 mRNA 3′ UTR to promote the translation of ITGA6 mRNA via readers (YTHDF1, YTHDF3).¹⁵

In present research, we found that METTL3 specifically targeted the ICAM-1 3′ UTR and promoted its protein expression. The m⁶A peak targeted by the METTL3 is found to be located in the 3′ UTR, which is approximate to the stop codon. These findings in our research are in accord with previous literature that m⁶A sites are intensively distributed near the stop codon.⁶^,¹⁶ In our previous research, we investigated the roles of long noncoding RNAs (lncRNAs) in the HLECs and identified the functions of lncRNAs.¹⁷^,¹⁸ In further research, we would perform more investigation to unveil the interaction within lncRNA and m⁶A.19, 20, 21

In conclusion, present research found that METTL3 was upregulated in the HG-induced HLECs, and METTL3 installed the higher methylation level. Mechanistically, METTL3 targets the 3′ UTR of ICAM-1 to stabilize its protein expression. Overall, the finding could provide a novel insight for the m⁶A modification for DC.

Materials and Methods

Tissue Samples Collection

Anterior lens capsule tissues were collected from DC patients and normal patient individuals without diabetes mellitus. The clinical assay was approved by the Ethics Committee of Eye Hospital of Tianjin Medical University. Informed consents were signed by every volunteer before the surgery.

Cells and Culture

Human LEC line (HLE B-3) was purchased from ATCC (CRL-11421) and cultured in the DMEM medium supplemented with 10% FBS (fetal bovine serum) and 15 mM HEPES as previously described,²² and then maintained at 37°C with 5% CO₂.

MeRIP-Seq

Total RNA was extracted from the HLEC cells administrated with HG and NG. MeRIP-Seq analysis was performed by Jiayin Biotechnology (Shanghai, China).

Oligonucleotides Transfection

The shRNA and blank control targeting METTL3 were synthesized by the GENEWIZ (Suzhou, China). The transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s specifications.

RNA Isolation and qRT-PCR

Total RNA was isolated from HLECs and DC tissue specimens using QIAGEN RNeasy Mini kit (Hilden, Germany). cDNA was reverse-transcribed using the iScript cDNA Synthesis Kit (BioRad) according to the manufacturer’s protocol. qRT-PCR was performed using the SYBR Premix Ex Taq (Takara, Otsu, Japan) using Applied Biosystems StepOne Plus Real-Time Thermal Cycling. GAPDH was used as an internal control for mRNA. The relative level of RNA was calculated using the 2^−ΔΔCt method. The primer sequences were shown in Table S1.

Western Blot Analysis

Protein was extracted from HLECs and DC tissue specimens using radioimmunoprecipitation assay (RIPA) lysis buffer with 1% protease inhibitor (Solarbio, Beijing, China). Proteins were separated by sodium dodecylsulphate-polyacrylamide gel electrophoresis (10% SDS-PAGE) and transferred to polyvinylidene difluoride member (PVDF, Millipore, MA, USA). Primary antibody (anti-METTL3, Abcam, ab195352, 1:1,000, anti-ICAM-1, ab53013, 1:2,000) was incubated with PVDF member overnight at 4°C. Then, the PVDF members were incubated with secondary antibodies (anti-β-actin, 1:1,000). The signals were visualized using enhanced chemiluminescence (Thermo Scientific, Rockford, IL, USA) and graphed using Gel Doc 2000 imaging scanner (Bio-Rad).

m⁶A Quantification

Total RNA was isolated using TRIzol (Invitrogen, CA) according to the manufacturer’s instruction. After the quality of RNA validation by NanoDrop, the m⁶A content in the total RNAs was assessed using m⁶A RNA methylation quantification kit (ab185912, Abcam).

HLECs Proliferative Assay

The proliferative ability was performed using CCK-8 and EdU assay. For the CCK-8 assay, HLECs were seeded in 96-well culture plates and administrated with 10 μL of CCK-8 assay kit (Dojindo Japan). 24 h later, cells were measured for the absorbance at 450 nm. For the EdU assay, HLECs were seeded in 24-well plates at 2 × 10⁴ cells using the EdU labeling/detection kit (Ribobio, Guangzhou, China). EdU labeling medium was added and fixed in 4% paraformaldehyde (pH 7.4) for 30 min. After counterstaining with 250 mL DAPI (Invitrogen, Molecular Probes, Eugene, OR, USA) for 25 min, EdU-positive cells were imaged under a fluorescence microscope (Nikon Corporation, Tokyo, Japan).

Flow Cytometry

The apoptosis of HLECs were detected using flow cytometry assay. In brief, HLECs were washed with PBS and resuspended in 100 μL of 1 × binding buffer. Fluorescein isothiocyanate (FITC) Annexin V (5 μL) and propidium iodide (PI, 5 μL) were added for 15 min at room temperature in the dark. The cells were analyzed by FACS Canto II flow cytometry (BD Biosciences).

Dot Blot Assay

Dot blots were performed as previously described.²³ RNA (400 ng) was spotted onto a nylon membrane (GE Healthcare, Fairfield, CT, USA). Nylon membranes were UV crosslinked and then incubated with m⁶A antibody (anti-m6A, ab208577, Abcam, 1:100) and horseradish peroxidase (HRP) conjugate anti-rabbit immunoglobulin G (IgG). The final images were incubated by HRP-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology was added to the blots for 1 h at room temperature. The same RNA (400 ng) were spotted on the nylon membrane, stained with 0.02% methylene blue in 0.3 M sodium acetate (pH 5.2) for 2 h, and washed with ribonuclease-free water for 5 h.

RNA Stability

The ICAM-1 mRNA stability was detected as previously described.²⁴ RNA was extracted using Trizol reagent (Invitrogen, Grand Island, NY, USA) from HLECs at different time points, mRNAs were treated with actinomycin D (1 μg/mL). Reverse transcription was performed using oligo (dT) primers and ICAM-1 mRNA level was detected using qRT-PCR.

RNA Immunoprecipitation PCR (RIP-qPCR)

Total RNA was isolated from HLECs using Trizol. Anti-METTL3 antibody (Abcam, ab195352) and anti-IgG (Abcam, ab172730) were conjugated to protein A/G magnetic beads in IP buffer (140 mM NaCl, 1% NP-40, 2 mM EDTA, 20 mM Tris pH 7.5) for overnight at 4°C. The total RNA was incubated with the antibody in IP buffer and then precipitated RNA was eluted from the beads. Finally, the precipitated RNA and input total RNA were eluated and reverse-transcribed for qRT-PCR. The relative fold enrichment was calculated using 2^-ΔΔCt methods.

Statistical Analysis

All statistical analysis was calculated by SPSS software version 19.0 (Chicago, IL, USA) and the GraphPad Prism 5.0 software (La Jolla, CA, USA). Data are displayed as mean ± SD. Group pairs were calculated using Student’s t test. p < 0.05 was considered statistically significant.

Author Contributions

Jun Yang acts as the major performer. Jingshu Liu acts as the assitance. Shaozhen Zhao and Fang Tian act as the project designer.

Conflicts of Interest

The authors declare no competing interests.

Acknowledgments

This research was supported by the Tianjin Clinical Key Discipline Project (grant number: TJLCZDXKQ007 and TJLCZDXKM003), Science & Technology Development Fund of Tianjin Education Commission for Higher Education (grant number: 2019YD06).

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.omtn.2020.02.002.

Contributor Information

Shaozhen Zhao, Email: zhaosz1997@sina.com.

Fang Tian, Email: tianfang06@tmu.edu.cn.

Supplemental Information

Document S1. Table S1

mmc1.pdf^{(23.8KB, pdf)}

Document S2. Article plus Supplemental Information

mmc2.pdf^{(1.5MB, pdf)}

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