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METTL3-dependent N⁶-methyladenosine RNA modification mediates the atherogenic inflammatory cascades in vascular endothelium.

ABSTRACT: Atherosclerosis is characterized by the plaque formation that restricts intraarterial blood flow. The disturbed blood flow with the associated oscillatory stress (OS) at the arterial curvatures and branch points can trigger endothelial activation and is one of the risk factors of atherosclerosis. Many studies reported the mechanotransduction related to OS and atherogenesis; however, the transcriptional and posttranscriptional regulatory mechanisms of atherosclerosis remain unclear. Herein, we investigated the role of N⁶-methyladenosine (m⁶A) RNA methylation in mechanotransduction in endothelial cells (ECs) because of its important role in epitranscriptome regulation. We have identified m⁶A methyltransferase METTL3 as a responsive hub to hemodynamic forces and atherogenic stimuli in ECs. OS led to an up-regulation of METTL3 expression, accompanied by m⁶A RNA hypermethylation, increased NF-κB p65 Ser⁵³⁶ phosphorylation, and enhanced monocyte adhesion. Knockdown of METTL3 abrogated this OS-induced m⁶A RNA hypermethylation and other manifestations, while METTL3 overexpression led to changes resembling the OS effects. RNA-sequencing and m⁶A-enhanced cross-linking and immunoprecipitation (eCLIP) experiments revealed NLRP1 and KLF4 as two hemodynamics-related downstream targets of METTL3-mediated hypermethylation. The METTL3-mediated RNA hypermethylation up-regulated NLRP1 transcript and down-regulated KLF4 transcript through YTHDF1 and YTHDF2 m⁶A reader proteins, respectively. In the in vivo atherosclerosis model, partial ligation of the carotid artery led to plaque formation and up-regulation of METTL3 and NLRP1, with down-regulation of KLF4; knockdown of METTL3 via repetitive shRNA administration prevented the atherogenic process, NLRP3 up-regulation, and KLF4 down-regulation. Collectively, we have demonstrated that METTL3 serves a central role in the atherogenesis induced by OS and disturbed blood flow.

SUBMITTER: Chien CS

PROVIDER: S-EPMC7896299 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:As one of the most frequent chemical modifications in eukaryotic mRNAs, N6-methyladenosine (m6A) modification exerts important effects on mRNA stability, splicing, and translation. Recently, the regulatory role of m6A in tumorigenesis has been increasingly recognized. However, dysregulation of m6A and its functions in tumor epithelial-mesenchymal transition (EMT) and metastasis remain obscure. METHODS:qRT-PCR and immunohistochemistry were used to evaluate the expression of methyltransferase-like 3 (METTL3) in gastric cancer (GC). The effects of METTL3 on GC metastasis were investigated through in vitro and in vivo assays. The mechanism of METTL3 action was explored through transcriptome-sequencing, m6A-sequencing, m6A methylated RNA immunoprecipitation quantitative reverse transcription polymerase chain reaction (MeRIP qRT-PCR), confocal immunofluorescent assay, luciferase reporter assay, co-immunoprecipitation, RNA immunoprecipitation and chromatin immunoprecipitation assay. RESULTS:Here, we show that METTL3, a major RNA N6-adenosine methyltransferase, was upregulated in GC. Clinically, elevated METTL3 level was predictive of poor prognosis. Functionally, we found that METTL3 was required for the EMT process in vitro and for metastasis in vivo. Mechanistically, we unveiled the METTL3-mediated m6A modification profile in GC cells for the first time and identified zinc finger MYM-type containing 1 (ZMYM1) as a bona fide m6A target of METTL3. The m6A modification of ZMYM1 mRNA by METTL3 enhanced its stability relying on the "reader" protein HuR (also known as ELAVL1) dependent pathway. In addition, ZMYM1 bound to and mediated the repression of E-cadherin promoter by recruiting the CtBP/LSD1/CoREST complex, thus facilitating the EMT program and metastasis. CONCLUSIONS:Collectively, our findings indicate the critical role of m6A modification in GC and uncover METTL3/ZMYM1/E-cadherin signaling as a potential therapeutic target in anti-metastatic strategy against GC.

Project description:Gastric cancer (GC) is the fifth most common cancer and the third deadliest cancer in the world, and the occurrence and development of GC are influenced by epigenetics. Methyltransferase-like 3 (METTL3) is a prominent RNA n6-adenosine methyltransferase (m6A) that plays an important role in tumor growth by controlling the work of RNA. This study aimed to reveal the biological function and molecular mechanism of METTL3 in GC. The expression level of METTL3 in GC tissues and cells was detected by qPCR, Western blot and immunohistochemistry, and the expression level and prognosis of METTL3 were predicted in public databases. CCK-8, colony formation, transwell and wound healing assays were used to study the effect of METTL3 on GC cell proliferation and migration. In addition, the enrichment effect of METTL3 on DEK mRNA was detected by the RIP experiment, the m6A modification effect of METTL3 on DEK was verified by the MeRIP experiment and the mRNA half-life of DEK when METTL3 was overexpressed was detected. The dot blot assay detects m6A modification at the mRNA level. The effect of METTL3 on cell migration ability in vivo was examined by tail vein injection of luciferase-labeled cells. The experimental results showed that METTL3 was highly expressed in GC tissues and cells, and the high expression of METTL3 was associated with a poor prognosis. In addition, the m6A modification level of mRNA was higher in GC tissues and GC cell lines. Overexpression of METTL3 in MGC80-3 cells and AGS promoted cell proliferation and migration, while the knockdown of METTL3 inhibited cell proliferation and migration. The results of in vitro rescue experiments showed that the knockdown of DEK reversed the promoting effects of METTL3 on cell proliferation and migration. In vivo experiments showed that the knockdown of DEK reversed the increase in lung metastases caused by the overexpression of METTL3 in mice. Mechanistically, the results of the RIP experiment showed that METTL3 could enrich DEK mRNA, and the results of the MePIP and RNA half-life experiments indicated that METTL3 binds to the 3'UTR of DEK, participates in the m6A modification of DEK and promotes the stability of DEK mRNA. Ultimately, we concluded that METTL3 promotes GC cell proliferation and migration by stabilizing DEK mRNA expression. Therefore, METTL3 is a potential biomarker for GC prognosis and a therapeutic target.

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METTL3-dependent N⁶-methyladenosine RNA modification mediates the atherogenic inflammatory cascades in vascular endothelium.

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OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

METTL3-dependent N6-methyladenosine RNA modification mediates the atherogenic inflammatory cascades in vascular endothelium.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

METTL3-dependent N⁶-methyladenosine RNA modification mediates the atherogenic inflammatory cascades in vascular endothelium.