Knockdown Of Methyltransferase-like 3 Research Articles

METTL3 promotes homologous recombination repair and modulates chemotherapeutic response in breast cancer by regulating the EGF/RAD51 axis.

Methyltransferase-like 3 (METTL3) and N6-methyladenosine (m6A) are involved in many types of biological and pathological processes, including DNA repair. However, the function and mechanism of METTL3 in DNA repair and chemotherapeutic response remain largely unknown. In present study, we identified that METTL3 participates in the regulation of homologous recombination repair (HR), which further influences chemotherapeutic response in both MCF-7 and MDA-MB-231 breast cancer (BC) cells. Knockdown of METTL3 sensitized these BC cells to Adriamycin (ADR; also named as doxorubicin) treatment and increased accumulation of DNA damage. Mechanically, we demonstrated that inhibition of METTL3 impaired HR efficiency and increased ADR-induced DNA damage by regulating m6A modification of EGF/RAD51 axis. METTL3 promoted EGF expression through m6A modification, which further upregulated RAD51 expression, resulting in enhanced HR activity. We further demonstrated that the m6A 'reader,' YTHDC1, bound to the m6A modified EGF transcript and promoted EGF synthesis, which enhanced HR and cell survival during ADR treatment in BC. Our findings reveal a pivotal mechanism of METTL3-mediated HR and chemotherapeutic drug response, which may contribute to cancer therapy.

METTL3 Enhances Hepatocellular Carcinoma Progression by Regulating Polycomb Repressive Complex 1 (PRC1) Components BMI1 and RNF2

N6‐methyladenosine (m6A) modification is the most abundant mRNA modification which plays important roles in the regulation of mRNA stability, splicing, translocation, and translation. Methyltransferase‐like 3 (METTL3), a primary RNA methyltransferase that catalyzes RNA m6A modification, is implicated in several human cancers including hepatocellular carcinoma (HCC), although its mechanisms of actions remain to be further defined. In the current study, we aimed to further determine the effect and mechanism of METTL3‐derived m6A in HCC. We have analyzed the TCGA database and observed that METTL3 expression is significantly upregulated in HCC patients which is associated with lower survival rate (P < 0.001). In a mouse model of HCC induced by hydrodynamic tail vein injection of hyperactive form of YAP and β‐catenin in conjunction with the sleeping beauty (SB) transposon system, we observed that the expression of METTL3 was notably high in YAP/β‐catenin‐induced HCC. In cultured human HCC cell lines (Huh7 and PLC/PRF/5), we observed that stable knockdown of METTL3 by shRNA significantly decreased tumor cell proliferation, colony formation and migration, in vitro. When Huh and PLC/PRF/5 cells with shRNA knockdown of METTL3 were inoculated into the livers of SCID mice, we found that METTL3 knockdown significantly inhibited xenograft tumor growth, in vivo. We next delivered METTL3 and YAP expression plasmids to the livers of wild type mice via hydrodynamic tail vein injection and observed that co‐expression of METTL3 plus YAP induced the development of HCC which involves almost the entire livers (20 weeks after tail vein injection). These findings provide important evidence for a tumor‐promoting role of METTL3 in HCC development. Through N6‐methyladenosine‐sequencing (m6A‐Seq) and RNA sequencing (RNA‐Seq), we identified BMI1 and RNF2, two key components of the polycomb repressive complex 1 (PRC1), as direct downstream targets of METTL3 in HCC. Our further analyses revealed that both BMI1 and RNF2 were significantly elevated in HCC patients and were associated with lower survival rate and that the expression of BMI1 and RNF2 were positively correlated with METTL3 in human HCC tissues. Consistent with the above results, our further data showed that knockdown of METTL3 in Huh7 and PLC/PRF/5 significantly decreased the expression of BMI1 and RNF2. Accordingly, treatment of Huh7 and PLC/PRF/5 cells with the METTL3 inhibitor, STM2457, significantly reduced the expression of BMI1 and RNF2 and decreased tumor cell proliferation and colony formation. Moreover, our data showed that inhibition of the m6A reader YTHDF1 by siRNA significantly decreased the expression of BMI1 and RNF2 in human HCC cells. Collectively, our study provides important evidence that METTL3 promotes HCC development and progression through m6A modification of BMI1 and RNF2 mRNAs which involve a YTHDF1 dependent mechanism. It is conceivable that the METTL3‐m6A‐BMI1/RNF2 axis may represent a promising target for HCC treatment.

Methyltransferase-Like 3 Rescues the Amyloid-beta protein-Induced Reduction of Activity-Regulated Cytoskeleton Associated Protein Expression via YTHDF1-Dependent N6-Methyladenosine Modification.

Activity-regulated cytoskeleton-associated protein (ARC) is activated by the induction of long-term potentiation and plays an important role in the synaptic plasticity of memory consolidation. Previous studies have shown that abnormal expression of ARC in the brains of patients with Alzheimer’s Disease (AD) leads to the disturbance of synaptic plasticity. ARC expression is mainly regulated by transcriptional and post-translational modification. However, it is unclear whether N6-methyladenosine (m6A) engages in the epigenetic modification of ARC. The AlzData database was used to analyze the brain of AD patients, and Aβ-induced cell models were used. We revealed that ARC expression was reduced in AD patients and Aβ-induced cell models. There were five m6A modification sites of ARC mRNA that were predicted by the SRAMP database, and ARC mRNA was confirmed as the target gene of methyltransferase-like 3 (METTL3) by MeRIP. Amyloid-beta protein (Aβ) repressed the m6A modification. Knockdown of METTL3 decreased ARC mRNA m6A modification and reduced ARC protein expression, while overexpression of METTL3 rescued ARC expression after Aβ treatment. Knockdown of YTH domain family, member 1 (YTHDF1) decreased ARC protein expression, while the overexpression of YTHDF1 could not rescue the loss of ARC protein expression after 3-deazaadenosine treatment or knockdown of METTL3. Our findings identify that METTL3 rescues the Aβ-induced reduction of ARC expression via YTHDF1-Dependent m6A modification, which suggests an important mechanism of epigenetic alteration in AD.

METTL14-mediated N6-methyladenosine modification of ITGB4 mRNA inhibits metastasis of clear cell renal cell carcinoma

BackgroundIntegrin β4 (ITGB4) participates in tumorigenesis and progression of several malignancies, but its role and related mechanisms in clear cell renal cell carcinoma (ccRCC) remain unclear.MethodsQuantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry were used to detect mRNA and protein levels of relevant genes. Biological functions of ITGB4 and methyltransferase-like 14 (METTL14) were determined by in vitro and in vivo experiments. The levels of N6-methyladenosine (m6A) in ccRCC tissues and adjacent normal tissues were calculated via total RNA m6A quantification assay. The m6A modification of ITGB4 was demonstrated via m6A RNA immunoprecipitation (MeRIP), RIP and luciferase reporter assays.ResultsITGB4 was significantly overexpressed in ccRCC tissues and high level of ITGB4 predicted poor prognosis as well as metastasis. Functionally, ITGB4 stimulated ccRCC cell migration and invasion in vitro and metastasis in vivo with epithelial–mesenchymal transition (EMT) strengthened. Mechanically, the total levels of m6A were reduced in ccRCC tissues. METTL14, a favorable factor for ccRCC patients’ prognosis, facilitated m6A modification on ITGB4 3′UTR and subsequently accelerated ITGB4 mRNA degradation, leading to its declined expression. Furthermore, the METTL14-mediated inhibition of ITGB4 expression was dependent on the YTH domain family protein 2 (YTHDF2), which acted as an m6A reader to bind to ITGB4 mRNA and to promote its decay. In addition, we demonstrated that knockdown of METTL14 promoted ccRCC cell migration, invasiveness and metastasis as well as stimulating the EMT process and the PI3K/AKT signal by overexpressing ITGB4.ConclusionOur study reveals that METTL14 inhibits ITGB4 expression via m6A modification to attenuate metastasis and EMT of ccRCC cells, suggesting the METTL14/ITGB4 axis as a potential prognostic biomarker and therapeutic target for ccRCC.DsMVtdrTRB4TieaKUTmiEpVideo

METTL14-mediated N6-methyladenosine modification of Pten mRNA inhibits tumour progression in clear-cell renal cell carcinoma.

Clear-cell renal-cell carcinoma (ccRCC) is one of the leading causes of tumour-related death worldwide. Methyltransferase-like 14 (METTL14) is reported to regulate m6A modification in cancers. The aim of this study is to investigate the biological function and molecular mechanism of METTL14 in the pathogenesis of ccRCC. Quantitative real-time PCR (qRT-PCR), western blot and immunohistochemical (IHC) assays were used to detect the expression of METTL14 and Pten. METTL14 overexpression or knockdown was used in the in vitro and in vivo studies to investigate the biological functions of METTL14. m6A-RNA immunoprecipitation and RNA immunoprecipitation were used to investigate the m6A modification mediated by METTL14. METTL14 expression was significantly down-regulated in ccRCC tissues. Functionally, upregulation of METTL14 inhibited ccRCC cells proliferation and migration in vitro. METTL14 overexpression significantly inhibited the activation of the phosphoinositide 3 kinase (PI3K)/AKT signalling pathway. Furthermore, phosphate and tension homology deleted on chromosome ten (Pten) is a target of METTL14. Overexpression of METTL14 increased the m6A enrichment of Pten, and promoted Pten expression. METTL14-enhanced Pten mRNA stability was dependent on YTHDF1. METTL14-mediated m6A modification of Pten mRNA inhibited tumour progression, suggesting that METTL14 might be a potential prognostic biomarker and effective therapeutic target for ccRCC.

Emodin relieves the inflammation and pyroptosis of lipopolysaccharide-treated 1321N1 cells by regulating methyltransferase-like 3 -mediated NLR family pyrin domain containing 3 expression

ABSTRACT Sepsis brain injury (SBI) is a major cause of death in critically ill patients. The present study aimed to investigate the role of emodin in SBI development. Human astrocyte 1321N1 cells were stimulated with 100 ng/mL lipopolysaccharide (LPS) to establish an SBI model in vitro. Flow cytometry was performed to measure the cell pyroptosis. The protein expression levels of syndecan-1 (SDC-1), NLR family pyrin domain containing 3 (NLRP3), Caspase-1, and the N-terminal fragment of gasdermin D (GSDMD-N) were measured using Western blotting. Interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor (TNF)-α levels in cells were measured using enzyme-linked immunosorbent assay kits. The N6-methyladenosine (m6A) modification was analyzed using the methylated RNA immunoprecipitation assay. NLRP3 activator, nigericin, was used to overexpress NLRP3. LPS treatment significantly enhanced the pyroptosis in 1321N1 cells, increased the levels of TNF-α, IL-1β, and IL-6, and decreased the levels of IL-10. The protein expression levels of NLRP3, SDC-1, GSDMD-N, and Caspase-1 were also increased. Emodin treatment decreased the levels of TNF-α, IL-1β, IL-6, NLRP3, SDC-1, GSDMD-N, and Caspase-1, while increasing the levels of IL-10 in LPS-treated 1321N1 cells. Nigericin reversed the effects of emodin. Furthermore, emodin upregulated m6A levels in NLRP3 by increasing the expression of methyltransferase-like 3 (METTL3). Meanwhile, knockdown of METTL3 reversed the effects of emodin on the mRNA expression and stability of NLRP3. Therefore, emodin inhibits the inflammation and pyroptosis of LPS-treated 1321N1 cells by inactivating METTL3-mediated NLRP3 expression.

The m6A RNA methyltransferase METTL3/METTL14 promotes leukemogenesis through the mdm2/p53 pathway in acute myeloid leukemia.

N6-methyladenosine (m6A) is the most abundant internal modification in mammalian mRNA and recent studies have highlighted the importance of m6A levels in tumor development. In this study, we investigated the expression of methyltransferase-like 3 (METTL3) and 14 (METTL14), components of the RNA m6A methyltransferase complex, in samples from 89 patients with acute myeloid leukemia (AML), and followed the survival of 75 of these patients. Our results show that METTL3 and METTL14 are highly expressed in most of the patients with AML (except those with APL), and high levels of METTL3 and/or METTL14 correlated to shorter survival in the patients. In leukemia cell lines K562 and kasumi-1, both METTL3 and METTL14 promote cell proliferation and cell cycle, and the knockdown of METTL3 and METTL14 inhibits proliferation, and induces apoptosis and differentiation. Notably, the knockdown of METTL3 and METTL14 in K562 cell line leads to several changes in the expression of p53 signal pathway, including the upregulation of p53, cyclin dependent kinase inhibitor 1A (CDKN1A/p21), and downregulation of mdm2. Importantly, the m6A level of mdm2 mRNA was significant lower after knock-down of METTL3 and METTL14 examined by m6A-RIP and mdm2 qPCR assay, and the half-life of mdm2 under actinomycin-D treatment became shorter. Taken together, our study demonstrates that the lower m6A levels of mdm2 mRNA mediated by the knockdown of METTL3 and METTL14 could lead to the low stability of mdm2 mRNA transcripts and low expression of MDM2, in the end, activate p53 signal pathway. Both METTL3 and METTL14 play an oncogenic role in AML by targeting mdm2/p53 signal pathway.

N6-methyladenosine-mediated LDHA induction potentiates chemoresistance of colorectal cancer cells through metabolic reprogramming.

Background: Chemoresistance to 5-fluorouracil (5-FU) is a major barrier to influence the treatment efficiency of colorectal cancer (CRC) patients, while the precise molecular mechanisms underlying 5-FU resistance remain to be fully elucidated.Methods: The metabolic profiles including ATP generation, glucose consumption, lactate generation, and oxygen consumption rate (OCR) in 5-FU resistant CRC cells were compared with those in their parental cells. Subsequently, a series of in vitro and in vivo experiments were carried out to investigate the mechanisms responsible for metabolic reprogramming of 5-FU resistant CRC cells.Results: We found that 5-FU resistant CRC cells showed increased levels of ATP generation, glucose consumption, lactate generation, and OCR as compared with those in their parental cells. Further, increased levels of mRNA N6-methyladenosine (m6A) and methyltransferase-like 3 (METTL3) were observed in 5-FU resistant CRC cells. Inhibition or knockdown of METTL3 can suppress glycolysis and restore chemosensitivity of 5-FU resistant CRC cells. Mechanistically, METTL3 enhances the expression of LDHA, which catalyzes the conversion of pyruvate to lactate, to trigger glycolysis and 5-FU resistance. METTL3 can increase the transcription of LDHA via stabilizing mRNA of hypoxia-inducible factor (HIF-1α), further, METTL3 also triggers the translation of LDHA mRNA via methylation of its CDS region and recruitment of YTH domain-containing family protein 1 (YTHDF1). Targeted inhibition of METTL3/LDHA axis can significantly increase the in vitro and in vivo 5-FU sensitivity of CRC cells.Conclusion: Our study indicates that METTL3/LDHA axis-induced glucose metabolism is a potential therapy target to overcome 5-FU resistance in CRC cells.

Targeting Ferroptosis as a Novel Approach to Alleviate Aortic Dissection.

A variety of programmed cell death types have been shown to participate in the loss of smooth muscle cells (SMCs) during the development of aortic dissection (AD), but it is still largely unclear whether ferroptosis is involved in the development of AD. In the present study, we found that the expression of key ferroptosis regulatory proteins, solute carrier family 7 member 11 (SLC7A11), ferroptosis suppressor protein 1 (FSP1) and glutathione peroxidase 4 (GPX4) were downregulated in aortas of Stanford type A AD (TAAD) patients, and liproxstatin-1, a specific inhibitor of ferroptosis, obviously abolished the β-aminopropionitrile (BAPN)-induced development and rupture of AD in mice. Furthermore, the expression of methyltransferase-like 3 (METTL3), a major methyltransferase of RNA m6A, was remarkably upregulated in the aortas of TAAD patients, and the protein levels of METTL3 were negatively correlated with SLC7A11 and FSP1 levels in human aortas. Overexpression of METTL3 in human aortic SMCs (HASMCs) inhibited, while METTL3 knockdown promoted SLC7A11 and FSP1 expression. More importantly, overexpression of METTL3 facilitated imidazole ketone erastin- and cystine deprivation-induced ferroptosis, while knockdown of METTL3 repressed ferroptosis of HASMCs. Overexpression of either SLC7A11 or FSP1 largely abrogated the effect of METTL3 on HASMC ferroptosis. Therefore, we have revealed that ferroptosis is a critical cause of AD in both humans and mice and that METTL3 promotes ferroptosis of HASMCs by inhibiting the expression of SLC7A11 and FSP1. Thus, targeting ferroptosis or m6A RNA methylation is a potential novel strategy for the treatment of AD.

Knockdown of METTL14 suppresses the malignant progression of non-small cell lung cancer by reducing Twist expression.

Non-small cell lung cancer (NSCLC) is one of the most malignant cancer types. N6-methyladenosine (m6A), an abundant eukaryotic mRNA modification, has been observed in multiple diseases, particularly cancer. Methyltransferase-like 14 (METTL14) is a central component of the m6A methyltransferase complex and has been reported to promote tumor development in several cancer types. The present study aimed to investigate the role of METTL14 in NSCLC. Relevant clinical and mRNA sequencing data for m6A-related genes were downloaded from The Cancer Genome Atlas database. R software was used to evaluate the expression of m6A regulators in NSCLC. The biological functions of METTL14 were evaluated using Cell Counting Kit-8, colony formation, Transwell migration and western blot analyses. The results demonstrated that METTL14 expression was upregulated in NSCLC tissues and cell lines, and its expression was high in cancer tissues from patients with NSCLC with all four stages (I, II, III and IV) of disease. METTL14 downregulation inhibited cell proliferation and migration in A549 and SK-MES-1 lung cancer cell lines. Knockdown of METTL14 in lung cancer cell lines increased E-cadherin expression and suppressed N-cadherin expression. Furthermore, METTL14 downregulation reduced the expression levels of the transcription factor Twist and the p-AKT/AKT ratio. In conclusion, the present findings revealed that silencing of METTL14 suppressed NSCLC malignancy by inhibiting Twist-mediated activation of AKT signaling. These data suggest that METTL14 may be a potential therapeutic target for NSCLC.

METTL3-Induced miR-222-3p Upregulation Inhibits STK4 and Promotes the Malignant Behaviors of Thyroid Carcinoma Cells.

Abnormally high expression of N6-methyladenosine (m6A) methyltransferase-like 3 (METTL3) has been implied to accompany thyroid carcinoma (TC) development. This study aimed to explore the protumorigenic role and downstream signaling axis of METTL3 in TC. This study was conducted at the Sun Yat-Sen Memorial Hospital Sun Yat-Sen University. METTL3 and miR-222-3p were overexpressed or downregulated in TC cells. Tumor and adjacent normal tissues were collected from 80 patients (19 men and 60 women, aged 30-70 years) with a pathological diagnosis of TC from January 2012 to January 2015. Cells were classified and subjected to different treatments. The expression of METTL3 was validated in TC tissues and cell lines. In functional studies, METTL3 and miR-222-3p were overexpressed or downregulated in TC cells to evaluate their effects on malignant behaviors, which were subsequently verified by xenografts in nude mice. The expression of METTL3 was elevated in TC, correlating with poor prognosis of TC patients. Heightened METTL3 expression accelerated malignant behaviors of TC cells. Mechanistically, METTL3 stimulated miR-222-3p expression by mediating the m6A modification of pri-miR-222-3p. miR-222-3p targeted and inversely regulated serine/threonine stress kinase 4 (STK4). Knockdown of METTL3 augmented STK4 expression by downregulating miR-222-3p, thereby suppressing the malignant behaviors of TC cells as well as tumor growth and lung metastasis in nude mice. Silencing METTL3 suppresses miR-222-3p expression and thus stimulates STK4 expression, thereby repressing the malignancy and metastasis of TC.

METTL3-m6A-Rubicon axis inhibits autophagy in nonalcoholic fatty liver disease

N6-methyladenosine (m6A) mRNA modification plays critical roles in various biological events and is involved in multiple complex diseases. However, the role of m6A modification in autophagy in nonalcoholic fatty liver disease (NAFLD) remains largely unknown. Here, we report that m6A modification was increased in livers of NAFLD mouse models and in free fatty acid (FFA)-treated hepatocytes, and the abnormal m6A modification was attributed to the upregulation of methyltransferase like 3 (METTL3) induced by lipotoxicity. Knockdown of METTL3 promoted hepatic autophagic flux and clearance of lipid droplets (LDs), while overexpression of METTL3 inhibited these processes. Mechanistically, METTL3 directly bound to Rubicon mRNA and mediated the m6A modification, while YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), as a partner of METTL3, interacted with the m6A-marked Rubicon mRNA and promoted its stability. Subsequently, RUBICON inhibited autophagosome-lysosome fusion and further blocked clearance of LDs. Taken together, our results showed a critical role of METTL3 and YTHDF1 in regulating lipid metabolism via the autophagy pathway and provided a novel insight into m6A mRNA methylation in NAFLD.

M6A modification impacts hepatic drug and lipid metabolism properties by regulating carboxylesterase 2

Methylation of adenosine at the N6 position to form N6-methyladenosine (m6A) is the most prevalent epitranscriptomic modification of mammalian mRNA. This modification is catalyzed by a methyltransferase-like 3 (METTL3)-METTL14 complex and is erased by demethylases such as fat mass and obesity-associated protein (FTO) or AlkB homolog 5 (ALKBH5). m6A modification regulates mRNA stability, nuclear export, splicing, and/or protein translation via recognition by reader proteins such as members of YT521-B homology (YTH) family. Carboxylesterase 2 (CES2) is a serine esterase responsible for the hydrolysis of drugs and endogenous substrates, such as triglycerides and diacylglycerides. Here, we examined the potential regulation of human CES2 expression by m6A modification. CES2 mRNA level was significantly increased by double knockdown of METTL3 and METTL14 but was decreased by knockdown of FTO or ALKBH5 in HepaRG and HepG2 cells, leading to changes in its protein level and hydrolase activity for 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11), suggesting that m6A modification negatively regulates CES2 expression. Consistent with the changes in CES2 expression, lipid accumulation in the cells was decreased by double knockdown of METTL3 and METTL14 but was increased by knockdown of FTO or ALKBH5. RNA immunoprecipitation assays using an anti-m6A antibody showed that adenosines in the 5′-untranslated region (UTR) and the last exon of CES2 are methylated. Luciferase assays revealed that YTHDC2, which degrades m6A-containing mRNA, downregulates CES2 expression by recognition of m6A in the 5′-UTR of CES2. Collectively, we demonstrated that m6A modification has a great impact on the regulation of CES2, affecting pharmacokinetics, drug response and lipid metabolism.

METTL3-mediated RNA m6A Hypermethylation Promotes Tumorigenesis and GH Secretion of Pituitary Somatotroph Adenomas.

Pituitary growth hormone-secreting (GH) pituitary adenomas (PAs) cause mass effects and dysregulated hypersecretion of GH. However, somatic mutation burden is low in PAs. While progress has been made in identifying the epigenetic changes involved in GH-PA initiation, the precise details of its tumorigenesis in GH-PA patients remains to be elucidated. As N6-methyladenosine (m6A) has been shown to often play a critical role in various tumors, it represents a possible initiation point for the tumorigenesis of pituitary adenomas. However, the role of RNA methylation in GH adenomas remains unclear. Protein expression of m6A regulators was measured by immunohistochemistry. Global levels and distribution of m6A methylation were separately analyzed by m6A enzyme-linked immunosorbent assay and m6A sequencing (m6A-seq). RNA interference and lentivirus knockdown system were used to investigate the role of methyltransferase-like 3 (METTL3) and its m6A- dependent regulatory mechanism in tumor progression and GH secretion. We show that both METTL3 messenger RNA and protein expression are elevated in GH-PA samples when compared with both normal pituitary tissue specimens and nonsecreting pituitary adenomas. Levels of m6A modification increased in GH-PAs, and hypermethylated RNAs are involved in hormone secretion and cell development. Knockdown of METTL3 in GH3 cell line resulted in decreased cell growth and GH secretion. Importantly, we found that GNAS and GADD45γ act as the downstream targets in this process. Our findings strongly suggest that m6A methyltransferase METTL3 promotes tumor growth and hormone secretion by increasing expression of GNAS and GADD45γ in a m6A-dependent manner. Thus, METTL3 and the methylated RNAs constitute suitable targets for clinical treatment of GH-PAs.

METTL3 promotes inflammation and cell apoptosis in a pediatric pneumonia model by regulating EZH2

In recent times, the medical science has developed by leaps and bounds, however, the molecular mechanism of pediatric pneumonia is still unclear. Although prior researches have shown that methyltransferase-like 3 (METTL3) is up-regulated in a variety of inflammatory diseases, its role and mechanism has been rarely studied in pediatric pneumonia, and need to be defined elaborately. In this study, the related molecular mechanism of METTL3 on inflammation and cell apoptosis in a pediatric pneumonia was investigated. Quantitative real-time polymerase chain reaction (qPCR) and western blot assays were employed to examine the mRNA and protein expression level of METTL3 and EZH2 in peripheral blood monocytes from pediatric pneumonia patients or cell model (WI-38). Then, qPCR and ELISA assay were applied to verify the inflammatory response in LPS-treated WI-38 cell lines after knockdown of METTL3. Besides, MTT cell viability assays, flow cytometry, and western blot assays were applied to examine the cell viability and cell apoptosis of LPS-treated WI-38 cell after knockdown of METTL3. Further, the western blot assays were employed to examine the protein expression levels of p-JAK2, JAK2, p-STAT3, STAT3, and EZH2 in LPS-treated WI-38 cell after knockdown of METTL3. Finally, ELISA and western blot were applied to verify the inflammatory response and cell apoptosis of LPS-treated WI-38 cell after knockdown of METTL3 and overexpression of EZH2. In this study, the results showed that METTL3 and EZH2 were highly expressed in pediatric pneumonia patients and cell models (WI-38), respectively. Besides, downregulation of METTL3 inhibited LPS-induced inflammatory response and cell apoptosis. Then, the fact that METTL3 regulates the JAK2/STAT3 signaling pathway through EZH was proved. Furthermore, downregulation of METTL3 inhibits inflammation and apoptosis through EZH2. This study found that METTL3 promotes inflammation and cell apoptosis in a pediatric pneumonia model by regulating EZH2.

METTL14 facilitates global genome repair and suppresses skin tumorigenesis

Global genome repair (GGR), a subpathway of nucleotide excision repair, corrects bulky helix-distorting DNA lesions across the whole genome and is essential for preventing mutagenesis and skin cancer. Here, we show that METTL14 (methyltransferase-like 14), a critical component of the N6-methyladenosine (m6A) RNA methyltransferase complex, promotes GGR through regulating m6A mRNA methylation-mediated DDB2 translation and suppresses ultraviolet B (UVB) radiation-induced skin tumorigenesis. UVB irradiation down-regulates METTL14 protein through NBR1-dependent selective autophagy. METTL14 knockdown decreases GGR and DDB2 abundance. Conversely, overexpression of wild-type METTL14 but not its enzymatically inactive mutant increases GGR and DDB2 abundance. METTL14 knockdown decreases m6A methylation and translation of the DDB2 transcripts. Adding DDB2 reverses the GGR repair defect in METTL14 knockdown cells, indicating that METTL14 facilitates GGR through regulating DDB2 m6A methylation and translation. Similarly, knockdown of YTHDF1, an m6A reader promoting translation of m6A-modified transcripts, decreases DDB2 protein levels. Both METTL14 and YTHDF1 bind to the DDB2 transcript. In mice, skin-specific heterozygous METTL14 deletion increases UVB-induced skin tumorigenesis. Furthermore, METTL14 as well as DDB2 is down-regulated in human and mouse skin tumors and by chronic UVB irradiation in mouse skin, and METTL14 level is associated with the DDB2 level, suggesting a tumor-suppressive role of METTL14 in UVB-associated skin tumorigenesis in association with DDB2 regulation. Taken together, these findings demonstrate that METTL14 is a target for selective autophagy and acts as a critical epitranscriptomic mechanism to regulate GGR and suppress UVB-induced skin tumorigenesis.

Abstract 653: Clinical value and oncogenic role of METTL3 expression in gastric cancer patients

Abstract Background: Gastric cancer (GC) is one of the most common cancers and leading cause of cancer-related deaths worldwide, primarily because of rapid disease progression to advanced stages and highly malignant potential. Methyltransferase-like 3 (METTL3) is a crucial component of the m6A methyltransferase complex, and plays pivotal roles in tumour progression. This study investigated the prognostic significance of METTL3 expression in gastric cancer (GC). Methods: We assessed expression levels of METTL3 by immunohistochemistry in formalin-fixed paraffin-embedded (FFPE) tissue specimens from one hundred fifty-eight GC patients. Propensity score matching (PSM) analysis was performed to clarify its prognostic potential. The METTL3 gene expression was also investigated in fresh frozen specimens from another independent cohort of fifty-seven GC patients to establish its clinical relevance. Knockdown of METTL3 by siRNA transfection was performed to evaluate its function in vitro. Results: METTL3 expression was significantly higher in cancerous tissues vs. corresponding normal mucosa (P<0.0001), and high METTL3 staining group was significantly associated with males (P=0.029), an advanced T stage (P=0.0002), the presence of venous invasion (P<0.0001), lymphatic vessel invasion (P=0.011), lymph node metastasis (P=0.004), distant metastasis (P=0.0004), and advanced TNM stage classification (P=0.0001) in the FFPE cohort of GC patients. Survival curve analysis showed that patients with increased expression of METTL3 showed poorer prognosis in terms of both overall survival (OS: P<0.0001, log-rank test) and disease-free survival (DFS: P=0.0005, log-rank test) compared to those with low expression. Multivariate analysis revealed that increased expression of METTL3 was an independent prognostic factor for overall survival (OS: hazard ratio (HR), 3.24; 95% confidence interval (CI), 1.57-6.68; p=0.001) and disease-free survival (DFS: HR, 2.4; 95% CI, 1.12-5.16; p=0.025) in the FFPE cohort of GC patients. PSM analysis revealed that elevated METTL3 expression was significantly associated with poor survival outcomes (OS: P=0.05; DFS: P=0.038; log-rank test), which was subsequently validated in another cohort of fresh frozen specimens (OS: p=0.007; DFS: p=0.029; log-rank test). The knockdown of METTL3 inhibited proliferation, invasion, migration, and anoikis resistance in GC cells. Conclusion: METTL3 expression may be used as a clinically feasible prognostic marker and could serve as a potential therapeutic target in GC patients. Citation Format: Yoshinaga Okugawa, Yuji Toiyama, Chengzeng Yin, Ma Ruiya, Akul Goel, Takashi Ichikawa, Hiroki Imaoka, Takahito Kitajima, Tadanobu Shimura, Mikio Kawamura, Hiromi Yasuda, Hiroyuki Fujikawa, Takeshi Yokoe, Ikuyo Mochiki, Masaki Ohi, Kaname Nakatani. Clinical value and oncogenic role of METTL3 expression in gastric cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 653.

METTL3‑mediated m6A modification of Bcl‑2 mRNA promotes non‑small cell lung cancer progression.

Methyltransferase-like 3 (METTL3) is an RNA methyltransferase that mediates modification of N6-methyladenosine (m6A), which serves as an oncogene in various types of cancer. The role of m6A modification in the onset and progression of cancer has attracted growing attention. However, the functional and regulatory mechanisms of METTL3 in non-small cell lung cancer (NSCLC) progression are still poorly understood. In the present study, METTL3 expression in NSCLC tissue was analyzed using the Gene Expression Profiling Interactive Analysis database. Western blotting and reverse transcription-quantitative PCR were performed to evaluate the expression of METTL3 in NSCLC tissue and cell lines. Here, knockdown and overexpression of METTL3 notably decreased NSCLC cell viability, apoptosis and migration in vitro and, as well as tumorigenicity in vivo. Expression of METTL3 was upregulated in NSCLC tissue. METTL3 overexpression promoted cell viability and migration in NSCLC, while knockdown of METTL3 yielded the opposite result in vivo and in vitro. METTL3 increased Bcl-2 translation via m6A modification, which increased viability and enhanced migration of NSCLC cells. METTL3 served as an oncogene in NSCLC via METTL3-mediated Bcl-2 mRNA m6A modification, which indicated that targeting METTL3 may be an effective therapeutic strategy for clinical management of NSCLC.

METTL3 regulates PM2.5-induced cell injury by targeting OSGIN1 in human airway epithelial cells

N6-methyladenosine (m6A) is implicated in alteration of cellular biological processes caused by exogenous environmental factors. However, little is known about the role of m6A in airborne fine particulate matter (PM2.5)-induced adverse effects. Thus, we investigated the role of m6A modification in PM2.5-induced airway epithelial cell injury. We observed a methyltransferase-like 3 (METTL3)-dependent induction of m6A modification after PM2.5 treatment in HBE and A549 cells. METTL3 knockdown attenuated PM2.5-induced apoptosis and arrest of cell cycle. mRNA sequencing and RNA N6-methyladenosine binding protein immunoprecipitation (Me-RIP) assay identified m6A-modified oxidative stress induced growth inhibitor 1 (OSGIN1) as the target gene of METTL3. Knockdown of METTL3 resulted a shorter mRNA half-life of OSGIN1 by catalyzing its m6A modification. Knockdown of METTL3 or OSGIN1 attenuated cell apoptosis, arrest of cell cycle and autophagy induced by PM2.5. In conclusion, METTL3 may mediate PM2.5-induced cell injury by targeting OSGIN1 in human airway epithelial cells. Our work uncovered a critical role of METTL3 in PM2.5-induced airway epithelial cell injury and provided insight into the vital role of m6A modification in PM2.5-induced human hazards.

N6-methyladenosine methyltransferase plays a role in hypoxic preconditioning partially through the interaction with lncRNA H19.

N6-methyladenosine (m6A), a methylation in the N6 position of adenosine especially in the mRNA, exerts diverse physiological and pathological functions. However, the precise role of m6A methylation in hypoxic preconditioning (HPC) is still unknown. Here, we observed that HPC treatment protected H9c2 cells against H2O2-induced injury, upregulated the m6A level in the total RNA and the expression of methyltransferase like 3 (METTL3), methyltransferase like 14 (METTL14), and long noncoding RNA (lncRNA) H19. Either knockdown of METTL3 or METTL14 notably reversed the HPC-induced enhancement of cell viability, anti-apoptosis ability, and H19 expression. Methylated RNA immunoprecipitation (IP) indicated that knockdown of METTL3 or METTL14 decreased m6A level in the lncRNA H19. Gain-of-function assay demonstrated that H19 overexpression could partially rescue the decreased protection mediated by METTL3 or METTL14 knockdown in HPC-treated H9c2 cells. RNA binding protein immunoprecipitation (RIP) assay showed that METTL3 and METTL14 could directly bind with H19. Our study identified a novel pattern of posttranscriptional regulation in HPC treatment. Since METTL3, METTL14, and lncRNA H19 were involved in HPC protection, they could be considered as potential biomarkers and therapeutic targets in HPC-derived cardiac rehabilitation and therapeutic approaches.