N6-methyladenosine (m6A) writer METTL3 accelerates the apoptosis of vascular endothelial cells in high glucose

doi:10.21203/rs.3.rs-1981358/v1

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N6-methyladenosine (m6A) writer METTL3 accelerates the apoptosis of vascular endothelial cells in high glucose

https://doi.org/10.21203/rs.3.rs-1981358/v1

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published 28 Feb, 2023

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Recent studies have shown that N⁶-methyladenosine (m⁶A) methylation, one of the most prevalent epigenetic modifications, is involved in the diabetes mellitus. However, whether m⁶A plays roles in diabetic vascular endothelium injury is still elusive. Present research aimed to investigate the regulation and mechanism of m⁶A on vascular endothelium injury. Upregulation of METTL3 was observed in the high glucose (HG)-induced human umbilical vein endothelial cells (HUVECs), followed by upregulation of m⁶A methylation level. Functionally, METTL3 silencing repressed the apoptosis and recovered the proliferation of HUVECs disposed by HG. Moreover, HG exposure upregulated the expression of suppressor of cytokine signalling 3 (SOCS3). Mechanistically, METTL3 targeted the m⁶A modified site on SOCS3 genomic, which positively regulated the mRNA stability of SOCS3 mRNA. In conclusion, METTL3 silencing attenuated the HG-induced vascular endothelium cell injury via promoting SOCS3 expression. Our research expands the understanding of m⁶A on vasculopathy in diabetes mellitus and provides a potential strategy for the protection of vascular endothelial injury.

6-methyladenosine

HUVECs

high glucose

METTL3

SOCS3.

In the pathophysiology of diabetes mellitus, hyperglycemia could lead to a series of pathophysiological changes that impair vascular endothelial function^{1, 2}. These vascular damages might ultimately lead to atherosclerosis or contribute to other cardiovascular events³. Endothelial dysfunction frequently occurs in type 2 diabetes⁴. Metabolic imbalance caused by hyperglycemia and other factors (e.g. insulin deficiency, dyslipidemia and other confounding factors) has a negative vascular effect, resulting in endothelial permeability increasing, impaired vasodilation, impaired vascular compliance.

Recent studies have revealed the function of N⁶-methyladenosine (m⁶A) modification in pathophysiology, including cardiovascular and cerebrovascular diseases^{5, 6}. For instance, m⁶A methyltransferase Mettl3 was observed to be increased in vivo and in vitro, and significantly hypermethylated and downregulated mRNAs were discovered by MeRIP-seq and RNA-seq⁷. In atherosclerosis, METTL3 is highly expressed and silencing of METTL3 alleviates the atherosclerosis progression and stabilizes the atherosclerosis plaques in mice, which facilitates the phenotypic transformation of vascular smooth muscle cells⁸. Thus, the potential functions of m⁶A modification are of more and more research significance.

Here, present study was designed to explore the mechanism underlying METTL3-dependent m⁶A modification during the development of diabetes mellitus vascular endothelium. Our team hypothesized that METTL3 might regulate the high glucose‑induced endothelial cell injury. High glucose (HG) exposure upregulated the expression of METTL3 in the HUVECs, mechanistically, METTL3 positively regulated the mRNA stability of suppressor of cytokine signalling 3 (SOCS3).

2.1. Cells and administration

The human umbilical vascular endothelium cells (HUVEC) were provided from KeyGen BioTech Corpor., Ltd (Nanjing, China) and cultured in DMEM (Thermo Fisher Scientific, San Jose, USA, cat. 11995065) with 5% CO₂ at 37℃. The model of high glucose endothelial cell injury was constructed by exposing to 30 mmol/L d-glucose (high glucose group, HG). For controls (normal glucose, NG), HUVECs were exposed to 5.6 mmol/L d-glucos and 24.4 mmol/L mannitol.

2.2. Transfection

HUVECs were transfected at 60–70% confluence with shRNA targeting METTL3 as per the manufacturer’s protocol. Scramble shRNA (shNC) for METTL3 was utilized as negative controls. The interfering RNA sequences were listed in Table S1.

2.3. Quantitative realtime PCR (qRT‑PCR)

The total RNA of HUVECs were extracted with RNAiso Reagent (TaKaRa, China) and then re-transcribed TO cDNA. qRT-PCR was performed with SYBR Green PCR kit (TaKaRa, Dalian, China) on Applied Biosystems 7300 to identified METTL3 mRNA and SOCS3 mRNA. Actin was used as endogenous control. The RNA primer sequences were listed in Table S1.

2.4. Western blot assay

As previously described, the total protein was collected from HUVEC cells with RIPA lysis buffer (Solarbio, Beijing, China) after the treatment transfection for 24 h and then quantified with BCA protein assay kit (Solarbio)⁹. Total extracted protein was determined using a radio immunoprecipitation assay (RIPA, R0010, Solarbio Science and Technology Ltd, Beijing, China) buffer. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed and then protein was transferred to PVDF membrane. After non-fat milk blockage for 1 h, membrane was incubated with the primary antibody and then with secondary antibody for at room temperature for 1 h. Lastly, the protein was immunoluminescent with ECL kit (cat. CW0048M, CoWin Biotech Co. Ltd, Beijing, China). Primary antibodies were purchased from Abcam (Cambridge, UK) (anti-METTL3, ab195352. 1:1000).

2.5. Flow cytometry apoptosis analysis

Cellular apoptosis was analyzed by flow cytometry as described previously¹⁰. In briefly, transfected HUVEC cells were harvested by trypsinization and double stained with propidium iodide (PI) and fluorescein isothiocyanate (FITC)-annexin V and performed by the FITC Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer’s recommendations. The relative ratio of early apoptotic cells and apoptotic cells was calculated for each experiment.

2.6. Wound healing assay

The wound-healing assay for HUVECs’ migration was performed as previously described. In brief, confluent cell monolayer was sterilely scratched using 1 ml pipette tip and then washed twice by PBS and cultured for 24 in medium. Images of the same areas were immediately taken using an Olympus digital camera. The wounding scratches width at different time-points was measured for relative percentage of initial distance (set as 100%). All experiments were performed in triplicates.

2.7. m⁶A content analysis

TRIzol reagent (Qiagen, Hilden, Germany) was used to extract total RNA from HUVECs. Then, GenElute mRNA Miniprep Kit (cat. MRN10, Sigma, Louis, MO, USA) was used to purified the Poly(A) + RNA. In brief, 200 ng of sample RNA and 80 µL of binding solution were added into each well and incubated at 37°C. The m⁶A content was detected using the m⁶A RNA Methylation Assay Kit (cat. ab185912, Abcam, Hercules, CA, USA). Lastly, the wells were incubated with solution in the dark at 25°C. The solution was determined using a microplate reader at 450 nm wavelength.

2.8. m⁶A RNA immunoprecipitation PCR (RIP‑qPCR)

The m⁶A-RIP test was carried out by Magna MeRIP m⁶A Kit (Merck Millipore Darmstadt, Germany) according to the manufacturer’s instruction. Total RNA was extracted or fragmented by ultrasound, and then incubated with Magnetic beads of anti-m⁶A antibody. After washing by buffer, the binding RNA was eluted and then purified by the RNA purification kit (Qiagen, USA). The purified RNA was identified by PCR detection. The relative fold expression was calculated using 2^−ΔΔCt methods.

2.9. Quantification of MeRIP-PCR

The m⁶A-modified SOCS3 mRNA levels was identified by methylated RNA immunoprecipitation was performed¹¹. Total RNA was isolated from HUVEC cells by Trizol. Anti-m6A antibody (3 µg, cat. ABE572, Millipore) or anti-IgG (Cell Signaling Technology, cat. #5946) was conjugated to protein A/G magnetic beads in IP buffer (140 mM NaCl, 20 mM Tris pH 7.5, 1% NP-40, 2 mM EDTA) for overnight at 4°C. Total RNA (100 µg) was then incubated with these antibodies in IP buffer with RNase /protease inhibitor. Then, the RNA was eluted from beads at 4 ºC. For qRT-PCR analysis, input total RNA (10 ng) was reverse-transcribed with Superscript III Synthesis system (Invitrogen, US). The enrichment of m⁶A-containing transcripts was calculated by analyzing the 2^−ΔΔCt of eluate RNA relative to the input sample.

2.10. mRNA stability analysis

HUVEC cells were seeded into six-well plates at 80% confluence. Cells were transfected with knockdown of METTL3 or controls were treated with 8 µg/ml actinomycin D (Act D) at 0, 3, 6 h. RNA was extracted and reversely transcribed for qRT-PCR. The relative quantification was determined by 2^−ΔΔCt method and normalized to β-actin. The half-time of SOCS3 mRNA was analyzed by GraphPad software.

2.11. Statistical analysis

All statistical analysis was performed using the SPSS software v 18.0 (SPSS Inc., USA) and GraphPad Prism v8.0 (GraphPad Software Inc., La Jolla, CA, USA). Data were produced as mean ± SD. Un-paired two-tailed Student’s t tests and multiple group was analyzed by one-way variance (ANOVA) analysis. p-value < 0.05 was considered significant.

3.1. METTL3 was highly expressed in high glucose‑induced HUVECs

High glucose endothelial cell injury was constructed by exposing to 30 mmol/L d-glucose (high glucose group, HG). In the high glucose‑induced HUVECs, different concentrations of glucose was administrated to trigger the hyperglycaemia. Firstly, we detected the m⁶A modification content in the HG‑induced HUVECs, and results showed that m⁶A modified content increased upon glucose exposure (Fig. 1A). Then, we detected the expression of several m⁶A methyltransferase writers (METTL3, METTL14, WTAP, KIAA1429) in HG‑induced HUVECs (Fig. 1B). Among these candidate writers, METTL3 exhibited higher level upon glucose exposure. In the gradient concentration change of glucose, METTL3 expression level increased, including mRNA (Fig. 1C) and proteins (Fig. 1D). Overall, these data suggested that METTL3 was highly expressed in high glucose‑induced HUVECs.

3.2. METTL3 high glucose‑induced endothelial cell injury

To detect the role of METTL3 on the endothelial cells, silencing transfection targeting METTL3 was performed in high glucose‑induced HUVECs. The transfection efficient was detected by RT-PCR (Fig. 2A) and western blot (Fig. 2B). The apoptosis of HUVECs was measured and results indicated that high glucose exposure up-regulated the apoptotic rate of HUVECs, while the knockdown of METTL3 repressed the apoptosis (Fig. 2C, 2D). Migration analysis found that high glucose exposure inhibited the migrative ration of HUVECs, while the knockdown of METTL3 increased the migration (Fig. 2E, 2F). Overall, these data suggested that METTL3 high glucose‑induced endothelial cell injury.

3.3. SOCS3 acted as the target of METTL3 in high glucose‑induced endothelial cells

To detect the downstream target of METTL3, the online analysis was performed to discovery the potential mRNA in high glucose‑induced HUVECs. The m⁶A modified motif on SOCS3 mRNA towards METTL3 was AUGGACU (Fig. 3A). The genomic sequences were analyzed to discovery the m⁶A site on the 3’-UTR of SOCS3 mRNA near stop codon (Fig. 3B). A sequence-based m⁶A modification site predictor SRAMP was performed to screen the m⁶A modified sites on SOCS3 mRNA (Fig. 3C). The interaction within SOCS3 and METTL3 was identified by RIP-PCR assay, and results indicated that METTL3 significantly combined with SOCS3 in high glucose‑induced HUVECs (Fig. 3D). To detect the m⁶A modified content on SOCS3 mRNA, MeRIP-PCR analysis was performed and results showed that high glucose exposure elevated the m⁶A level, while METTL3 knockdown repressed the m⁶A modified level (Fig. 3E). Overall, these data suggested that SOCS3 acted as the target of METTL3 in high glucose‑induced endothelial cells.

3.4. METTL3 enhanced the stability of SOCS3 mRNA in m⁶A-dependent manner

Given that the SOCS3 acted as the target of METTL3, further experiments were needed to find out whether METTL3 could regulate SOCS3 metabolism. Firstly, RNA decay analysis was performed to detect the stability of SOCS3 mRNA. Results suggested that METTL3 knockdown repressed the remaining level of SOCS3 mRNA upon Act D treatment (Fig. 4A, 4B). In the high glucose exposed HUVECs, SOCS3 mRNA level increased and knockdown of METTL3 repressed the SOCS3 mRNA, as well as SOCS3 protein (Fig. 4C, 4D). Overall, these data suggested that METTL3 enhanced the stability of SOCS3 mRNA in m⁶A-dependent manner.

M⁶A modification has been proposed to be participated in cardiovascular physiological and pathological processes^12–14. However, the vital roles of m⁶A writer METTL3 in high glucose exposure vascular endothelial cells are unknown^{15, 16}. Here, present research was performed to investigate the potential regulation and mechanism in hyperglycemia induced vascular endothelial cells (Fig. 5).

In this work, we discovered that the high content of m⁶A modification was inspired by hyperglycemia in human umbilical vein endothelial cells (HUVECs)¹⁷. The high glucose administration could up-regulate the level of m⁶A and METTL3 in high glucose induced HUVECs. Based on this result, we found a possible evidence that METTL3 might regulate the HUVECs apoptosis and proliferation via m⁶A-dependent manner.

In the pathological features of HUVECs in cardiovascular disease, the role of m⁶A modification is attracting more and more attention. In atherosclerosis of TNF-α-treated HUVECs, the ALKBH5 expression is significantly decreased and the ALKBH5 overexpression promotes the proliferation and inhibits the apoptosis of HUVECs⁹. In the atherosclerosis of ox-LDL-induced dysregulated HUVECs, METTL3 is highly expressed and METTL3 knockdown inhibits the cell proliferation, migration, tube formation of HUVECs¹⁸. In high-fat diet APOE-/- mice and ox-LDL treated HUVECs, METTL14 and METTL3 significantly upregulates and METTL14 reduces cell viability and promotes the apoptosis of HUVECs via m6A modification of p65¹⁹. In conclusion, these data indicate that m⁶A modification could regulate the pathophysiological process of HUVECs in diversified pathologies.

Previous works have demonstrated that m⁶A methylation is significantly associated with the response to environmental stimuli, e.g. inflammation, oxidative stress or malignant tumors^{20, 21}. In this study, we firstly discovered that the methyltransferases METTL3 was up-regulated in HG-induced HUVECs, therefore the critical m⁶A writer METTL3 might participate in the diabetic vasculopathy. Moreover, HG exposure upregulated the expression of suppressor of cytokine signalling 3 (SOCS3). Mechanistically, METTL3 targeted the m⁶A modified site on SOCS3 genomic, which positively regulated the mRNA stability of SOCS3 mRNA.

SOCS3 is a critical element in the vascular complications of diabetes mellitus. For instance, in the presence of high glucose and high lipid in HUVECs, SOCS3-mediated degradation of ubiquitinated AdipoR1 could accelerate diabetic endothelial dysfunction²². Moreover, SOCS3 promoted the HG-induced vascular endothelial injury through MALAT1/miR-361-3p/SOCS3 axis²³. Vascular endothelial injury is the important pathophysiologic manifestations of patients with diabetes mellitus.

In conclusion, METTL3 silencing attenuated the HG-induced vascular endothelium cell injury via promoting SOCS3 expression. METTL3 acts as a potential target for endothelial injury therapy for diabetes mellitus. Our research expands the understanding of m⁶A on vasculopathy in diabetes mellitus and provides a potential strategy for the protection of vascular endothelial injury.

Conflict of interest

All authors declare no conflicts of interest

Acknowledgement

No.

Data Availability Statement

No research data shared.

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No competing interests reported.

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Download PDF

Journal Publication

published 28 Feb, 2023

Read the published version in Heliyon →

Version 1

posted

You are reading this latest preprint version

N6-methyladenosine (m6A) writer METTL3 accelerates the apoptosis of vascular endothelial cells in high glucose

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Cells and administration

2.2. Transfection

2.3. Quantitative realtime PCR (qRT‑PCR)

2.4. Western blot assay

2.5. Flow cytometry apoptosis analysis

2.6. Wound healing assay

2.7. m⁶A content analysis

2.8. m⁶A RNA immunoprecipitation PCR (RIP‑qPCR)

2.9. Quantification of MeRIP-PCR

2.10. mRNA stability analysis

3. Results

3.1. METTL3 was highly expressed in high glucose‑induced HUVECs

3.2. METTL3 high glucose‑induced endothelial cell injury

3.3. SOCS3 acted as the target of METTL3 in high glucose‑induced endothelial cells

3.4. METTL3 enhanced the stability of SOCS3 mRNA in m⁶A-dependent manner

4. Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

N6-methyladenosine (m6A) writer METTL3 accelerates the apoptosis of vascular endothelial cells in high glucose

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Cells and administration

2.2. Transfection

2.3. Quantitative realtime PCR (qRT‑PCR)

2.4. Western blot assay

2.5. Flow cytometry apoptosis analysis

2.6. Wound healing assay

2.7. m6A content analysis

2.8. m6A RNA immunoprecipitation PCR (RIP‑qPCR)

2.9. Quantification of MeRIP-PCR

2.10. mRNA stability analysis

3. Results

3.1. METTL3 was highly expressed in high glucose‑induced HUVECs

3.2. METTL3 high glucose‑induced endothelial cell injury

3.3. SOCS3 acted as the target of METTL3 in high glucose‑induced endothelial cells

3.4. METTL3 enhanced the stability of SOCS3 mRNA in m6A-dependent manner

4. Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

2.7. m⁶A content analysis

2.8. m⁶A RNA immunoprecipitation PCR (RIP‑qPCR)

3.4. METTL3 enhanced the stability of SOCS3 mRNA in m⁶A-dependent manner