Methyltransferase-like 3 Expression Research Articles

The m6A Methyltransferase METTL3-Mediated N6-Methyladenosine Modification of DEK mRNA to Promote Gastric Cancer Cell Growth and Metastasis.

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.

P-250 A novel small molecule inhibits enzyme associated with colorectal cancer

Colorectal cancer (CRC) represents one of the most common malignant tumor with high morbidity worldwide. N6-methyladenosine (m6A) methylation, is the most frequently observed RNA modifications that tremendously contribute to cancer initiation, progression, and resistance. METTL3 (Methyltransferase like 3), is a predominant catalytic enzyme in the m6A methyltransferase system. METTL3 has been implicated in carcinogenesis in various cancers including colon cancer by stabilizing oncogenic mRNAs in an m6A-dependent manner which is why it is considered as an attractive target for cancer therapy. Here, we identified STM2457, as a selective small-molecule METTL3 inhibitor that exerts promising anticancer activity in CRC. We detected the mRNA and protein expression of METTL3 and its target oncogenes in human colorectal cancer cell line panel. The anti-tumor activities of STM2457 in human colorectal cancer cell line panel were investigated by MTT, wound healing, colony formation, western blot assays, and 3D spheroid model. m6A level of mRNA was measured by an m6A-RNA methylation quantification kit. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) was used to screen the target genes of METTL3. We discovered that STM2457 treatment exerted potential anticancer effects in CRC cells. Pharmacological inhibition of METTL3 by STM2457 reduced cellular proliferation, invasion, colony formation, and tumorsphere formation of CRC cells. STM2457 treatment reduced m6A level in CRC cells. Mechanistically, MeRIP-seq illustrated that SLC2A1 (Solute Carrier Family 2 Member 1) as a target of METTL3. The TCGA data analysis showed that SLC2A1 mRNA and protein were highly expressed in colorectal cancer tissues. In addition, the highly expressed SLC2A1 mRNA associated with metastasis and poor survival in CRC patients. Thus, the METTL3/m6A/SLC2A1 axis accelerated colorectal carcinogenesis. The data show that the blockade of METTL3/m6A/SLC2A1 axis by a potent and novel small-molecule inhibitor, STM2457 is a promising approach for treatment of CRC.

Selective inhibition of METTL3 enhanced anticancer activity via targeting the c-Myc pathway.

e15089 Background: Methyltransferase-like 3 (METTL3) is a master regulator enzyme in the N6-methyladenosine (m6A) methyltransferase system, that not only plays a crucial role in cancer but has become a promising therapeutic target for the same. This study was aimed to discover molecular mechanisms underlying the potential anticancer effects of a novel and selective METTL3 inhibitor named STM2457 against oral squamous cell carcinoma (OSCC). Methods: OSCC patient-derived organoids (PDOs) and the corresponding 2D cell lines were established from the patients who have increased expression of METTL3 and c-Myc. The pharmacological activity of STM2457 was extensively studied with organoids and primary OSCC cells. Cell proliferation, wound healing and colony formation assays were used to detect STM2457 anticancer activity in vitro. Patient-Derived tumor Xenograft (PDX) model of OSCC was used to detect STM2457 anticancer activity in vivo. m6A level of mRNA was measured by an m6A-RNA methylation quantification kit. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) was used to screen the target genes of METTL3. RNA stability assay was used to detect the half-lives of METTL3 target genes. Real-time quantitative PCR was used for gene expression and western blot was used for protein expression analysis. Results: We found that STM2457 treatment exerted potential anticancer activity in PDOs and primary OSCC cells. MeRIP-seq revealed that c-Myc was the downstream gene of METTL3. Functionally, knockdown or pharmacological inhibition of METTL3 by STM2457 reduced stability and expression of c-Myc. This eventually, results in significant attenuation of migration, invasion, tumorsphere formation, and colony formation abilities of OSCC cells. Moreover, STM2457-treated OSCC cells showed reduced tumorigenic activity in vivo. Furthermore, STM2457 treatment reduced m6A level in PDOs and OSCC cells by blocking METTL3 activity thereby elucidating the importance of personalized approach for effective cancer treatment. Conclusions: Altogether, the revelation of anticancer effect of STM2457 exerted by blocking of METTL3-c-Myc signaling pathway opens novel avenues into the field of epitranscriptomics.

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.

METTL3 Is Associated With the Malignancy of Esophageal Squamous Cell Carcinoma and Serves as a Potential Immunotherapy Biomarker.

Methyltransferase-like 3 (METTL3) is an RNA methyltransferase mediating N6 methyladenosine (m6A) modification. Its role in cancer pathogenesis and progression has attracted increasing attention. However, the immunological role, possible immune mechanism, and clinical significance of METTL3 in esophageal squamous cell carcinoma (ESCC) remain to be confirmed. The Tumor Genome Atlas (TCGA) provided clinical and transcriptome sequencing data for this study (162 tumor tissue samples and 11 normal tissue samples), while the Immunology Database and Analysis Portal (immport, https://www.immport.org/home) provided 2483 immune-related genes. METTL3 was substantially expressed in ESCC and linked to poor prognosis in ESCC, according to the findings. Functional analysis revealed that METTL3 is mainly involved in chromosomal homologous recombination and DNA mismatch repair processes, which could be potential mechanisms for tumor disease development and progression. Analysis on the TISIDB website shows that effector memory CD8 T cells, NK cells, neutrophils and other cells are highly correlated with METTL3 expression. We screened immune genes associated with METTL3 by Spearman’s analysis and performed functional analysis. These immune genes were mostly linked with immune processes, such as cytokine receptors, the MAPK signaling pathway, and natural killer cell-mediated cytotoxicity, indicating that METTL3 is a key molecule in the immune regulation of esophageal cancer. In addition, based on METTL3-related immune genes, we separated the patients into several subgroups and constructed a prognostic prediction model consisting of six immune genes. As an independent prognostic indicator for ESCC, the risk score of this model can be employed. A nomogram was also developed to accurately evaluate individual prognoses based on clinical indicators and risk scores. In summary, this study suggests that METTL3 is not only a potential pathogenic molecule for esophageal carcinogenesis and progression but also a potential biological marker for forecasting ESCC patient prognosis and could serve as a basis for clinical decision making.

Hsa_circ_0000523/miR-let-7b/METTL3 axis regulates proliferation, apoptosis and metastasis in the HCT116 human colorectal cancer cell line.

Circular RNAs (circRNAs/circs) have gained attention as a class of potential biomarkers for the early detection of multiple cancers. However, the functions and mechanisms of circRNAs in the oncogenesis of human colorectal cancer (CRC) remain to be elucidated. The present study aimed to investigate the roles of hsa_circ_0000523 and its parental gene methyltransferase-like 3 (METTL3) in regulating cell proliferation, apoptosis and invasion in the HCT116 human CRC cell line. To uncover the regulated function of hsa_circ_0000523 in HCT116 cells, a dual-luciferase reporter assay, flow cytometry, reverse transcription-quantitative PCR, Cell Counting Kit-8 assay, cell invasion and western blot assay were used. In HCT116 cells, hsa_circ_0000523 indirectly regulated METTL3 expression by suppressing the transcription of microRNA (miR)-let-7b. The expression of METTL3 promoted cell proliferation and suppressed apoptosis. In the present study, it was found that miR-let-7b promoted cell viability and inhibited apoptosis and invasion, while circ_0000523 exerted the opposite effects. Higher levels of METTL3 expression were associated with more aggressive tumor invasion. The present results suggest that circRNAs and METTL3 may be applied for highly sensitive diagnosis of CRC and for predicting prognosis in patients who have undergone therapy.

Exenatide ameliorates hydrogen peroxide-induced pancreatic β-cell apoptosis through regulation of METTL3-mediated m6A methylation

Exenatide, a glucagon-like peptide-1 (GLP-1) receptor agonist, is a commonly used hypoglycemic agent in clinical practice; it inhibits reactive oxygen species-induced pancreatic β-cell apoptosis. N6-methyladenosine (m6A) is produced by the methylation of RNA N6 residues and has recently been shown to play a crucial role in the regulation of islet β-cell growth and development. However, the involvement of m6A methylation in the β-cell protective effects of exenatide has not been clarified. In this study, the m6A-methylated RNA content and methyltransferase-like 3 (METTL3) expression levels in NIT-1 cells and primary mouse islets were found to significantly decrease following treatment with hydrogen peroxide (H2O2). Treatment with exenatide induced an increase in m6A content and METTL3 expression in the H2O2-treated NIT-1 cells and islets. Moreover, METTL3 silencing resulted in NIT-1 cell apoptosis under normal culture conditions. METTL3 upregulation significantly ameliorated H2O2-induced apoptosis in NIT-1 cells and primary islets. Furthermore, the anti-apoptotic effects of exenatide were obviously reversed by METTL3 knockdown. In conclusion, these findings suggest that exenatide elicits its anti-apoptotic effects in pancreatic β-cells by promoting m6A methylation through the upregulation METTL3 expression.

METTL3 stabilizes HDAC5 mRNA in an m6A-dependent manner to facilitate malignant proliferation of osteosarcoma cells

Osteosarcoma (OS) is a prevalent primary bone sarcoma. Methyltransferase-like 3 (METTL3) is dysregulated in human malignancies. This study explored the mechanism of METTL3 in OS cell proliferation. Our results demonstrated that METTL3 was highly expressed in OS, and correlated with the tumor size, clinical stage, and distant metastasis of OS patients. Higher METTL3 expression indicated poorer prognosis. METTL3 silencing inhibited the malignant proliferation of OS cells, while METTL3 overexpression led to an opposite trend. METTL3 upregulated histone deacetylase 5 (HDAC5) expression in OS cells by increasing the m6A level. HDAC5 reduced the enrichment of H3K9/K14ac on miR-142 promoter, thus suppressing miR-142-5p expression and upregulating armadillo-repeat-containing 8 (ARMC8) level. HDAC5 overexpression or miR-142-5p silencing attenuated the inhibitory effect of METTL3 silencing on OS cell proliferation. Xenograft tumor experiment in nude mice confirmed that METTL3 silencing repressed OS cell proliferation in vivo via the HDAC5/miR-142-5p/ARMC8 axis. Collectively, METTL3-mediated m6A modification facilitated OS cell proliferation via the HDAC5/miR-142-5p/ARMC8 axis.

Methyltransferase-like 3 gene (METTL3) expression and prognostic impact in acute myeloid leukemia patients

BackgroundDNA methylation is involved in pathogenesis of acute myeloid leukemia (AML). N6-methyladenosine (m6A) modification of mRNA, mediated by methyltransferase-like 3 (METTL3), is one of the well-identified mRNA modifiers associated with the pathogenesis of AML. High level of METTL3 mRNA is detected in AML cells, thus can be a potential target therapy for AML. This is a preliminary study that aimed at measuring METTL3 mRNA expression level in de novo AML patients and correlating it with clinicopathological, laboratory and prognostic markers. METTL3 expression was analyzed by quantitative reverse transcription polymerase chain reaction in 40 newly diagnosed AML adults and was re-measured in the 2nd month of chemotherapy. Patients were followed up for periods up to 6 months post-induction therapy.ResultsMETTL3 expression was found to be significantly upregulated in AML patients compared to control subjects (p < 0.001). METTL3 gene was significantly expressed among non-responders compared to responders (p < 0.001). A cutoff value was assigned for normalized METTL3 values to categorize AML patients according to response to therapy. Statistically significant association was observed between high pretreatment normalized METTL3 gene level and failure to attain complete remission at 2nd, 4th and 6th month following therapy (p = 0.01, 0.02 and 0.003, respectively). However, insignificant correlation was found between pretreatment normalized METTL3 gene level and event free survival or clinicopathological prognostic factors.ConclusionMETTL3 is overexpressed in AML patients and is associated with adverse prognostic effect and failure to attain hematological remission within 6 months post-induction therapy.

JNK Signaling Promotes Bladder Cancer Immune Escape by Regulating METTL3-Mediated m6A Modification of PD-L1 mRNA.

The identification of a novel m6A-dependent mechanism underlying immune system evasion by bladder cancer cells reveals JNK signaling as a potential target for bladder cancer immunotherapy.

METTL3 regulates LPS-induced inflammatory response via the NOD1 signaling pathway

N6-methyladenosine (m6A) is a prevalent mRNA modification that plays a crucial function in multiple biological processes. Methyltransferase-like 3 (METTL3), an m6A methyltransferase, is essential for the m6A modification. Recently, the effect of METTL3 on the immune response has been reported. However, the effect is unclear, and the results are contradictory. In the present study, the total m6A and the expression of METTL3 decreased in LPS-stimulated macrophages. METTL3 knockdown significantly upregulated expression of proinflammatory cytokines, including TNF-α, IL-6 and NO. RNA sequencing analysis showed that the upregulated genes were enriched in inflammation-related signaling pathways and that the NOD-like receptor signaling pathway might be the target molecules of METTL3. METTL3 depletion resulted in upregulation of the NOD1 pathway without impacting NOD2. Moreover, the increase in proinflammatory cytokines induced by METTL3 knockdown was reversed by blocking the NOD1 pathway using specific inhibitors. Mechanistically, METTL3 knockdown promoted the mRNA expression and stability of NOD1 and RIPK2, and the same results were detected in m6A-binding protein YTHDF1- or YTHDF2-silenced cells. All findings suggested that METTL3 depletion inhibits the degradation of NOD1 and RIPK2 mRNA mediated by YTHDF1 and YTHDF2, which upregulate the NOD1 pathway and subsequently promote the LPS-induced inflammatory response in macrophages.

Methyltransferase-like 3 upregulation is involved in the chemoresistance of non-small cell lung cancer.

BackgroundTreatments for non-small cell lung cancer (NSCLC) have improved tremendously, but therapeutic resistance is a common and major clinical challenge in treatment. Methyltransferase-like 3 (METTL3) is a ribonucleic acid (RNA) methyltransferase that has crucial functions in the development and progression of cancers, including drug resistance, by regulating N6-methyladenosine (m6A) modification. However, the role of METTL3 in the progression and drug resistance of NSCLC is poorly understood.MethodsThe expression levels of METTL3 and AKT serine/threonine kinase 1 (AKT1) in NSCLC tissues were detected using quantitative real-time PCR (RT-qPCR), Western blots, and immunohistochemical assays. The m6A levels of AKT1 messenger RNA (mRNA) in NSCLC tissues were detected using m6A methylated RNA immunoprecipitation–quantitative polymerase chain reaction.ResultsThe expression levels of METTL3 and the AKT1 protein were significantly increased in NSCLC tissues, and m6A expression levels of AKT1 mRNA were dramatically upregulated in NSCLC tissues. Additionally, METTL3, AKT1 protein, and m6A levels of AKT1 mRNA were overexpressed in chemoresistant NSCLC samples, and high expression levels of METTL3 and AKT1 were correlated with poor patient survival, especially in chemoresistant NSCLC patients. Further, AKT1 protein expression and m6A levels of AKT1 mRNA were positively correlated with METTL3 expression, and AKT1 protein expression was positively correlated with m6A levels of AKT1 mRNA. Moreover, METTL3 and AKT1 protein expression levels were significantly associated with cisplatin susceptibility, tumor, node, metastasis stage, and lymph node metastasis.ConclusionsTaken together, our results indicate that METTL3 contributes to the progression and chemoresistance of NSCLC by promoting AKT1 protein expression through regulating AKT1 mRNA m6A levels, and may provide an efficient therapeutic intervention target for overcoming chemoresistance in NSCLC.

METTL3 Contributes to Osteosarcoma Progression by Increasing DANCR mRNA Stability via m6A Modification.

Background: Osteosarcoma (OS) is the most prevalent bone cancer among children and adolescents, with relatively high mortality rates. RNA N6-methyladenosine (m6A) is the most common human mRNA modification with diverse functions in a variety of biological processes. Previous studies indicated that methyltransferase-like 3 (METTL3), the first methyltransferase to be identified, acted as an oncogene or tumor suppressor in multiple human cancers. However, its functions and underlying mechanisms in OS progression remain unclear; therefore, we explored these processes. Methods: We used real-time quantitative PCR (RT-qPCR) and Western blot assays to explore METTL3 expression in OS tumor tissues and five OS cell lines to assess its clinical significance. To further examine the functional role of METTL3 during OS progression, CCK-8 analyses, transwell assays, and xenograft model studies were conducted after silencing METTL3. Additionally, underlying mechanisms were also explored using RIP-seq and RIP-qPCR approaches. Results: METTL3 was upregulated in OS tumor tissues and cell lines and was associated with a worse prognosis. Moreover, METTL3 silencing suppressed OS cell proliferation, migration, and invasion. Also, in vivo METTL3 oncogenic functions were confirmed in the xenograft model. Comprehensive mechanistic analyses identified long non-coding RNA (lncRNA) DANCR as a potential target of METTL3, as indicated by reduced DANCR levels after METTL3 silencing. Also, lncRNA DANCR knockdown repressed OS cell proliferation, migration, and invasion. Furthermore, both METTL3 and lncRNA DANCR silencing significantly suppressed OS growth and metastasis. Finally, we hypothesized that METTL3 regulated DANCR expression via m6A modification-mediated DANCR mRNA stability. Conclusion: METTL3 contributes to OS progression by increasing DANCR mRNA stability via m6A modification, meaning that METTL3 may be a promising therapeutic target for OS treatment.

N(6)-methyladenosine RNA methyltransferase like 3 inhibits extracellular matrix synthesis of endplate chondrocytes by downregulating sex-determining region Y-Box transcription factor 9 expression under tension

Tension stimulation is an important inducer of endplate cartilage degeneration, but the specific regulatory mechanism remains unclear. This study was the first to reveal the mechanism by which methyltransferase-like 3 (METTL3)-mediated N(6)-methyladenosine (m6A) modification affected the extracellular matrix anabolism by tension-induced endplate chondrocytes. We examined the differences in METTL3 expression and m6A methylation levels in human endplate chondrocytes and human cartilage endplate tissues under in vitro tension. The effect on endplate cartilage degeneration was evaluated by manipulating m6A methylation mediated by METTL3 in vivo and in vitro. The effect of METTL3-mediated m6A methylation on the stability of sex-determining region Y-box transcription factor 9 (SOX9) gene expression was determined experimentally. METTL3 expression and m6A methylation levels were significantly increased in degenerative human endplate cartilage tissue. Similarly, tension stimulation inhibited the ability of human endplate chondrocytes to synthesize extracellular matrix, which was accompanied by an increase in METTL3-mediated m6A methylation. The ability of endplate chondrocytes to resist tension was significantly enhanced by inhibiting METTL3 expression and subsequently downregulating m6A methylation in vitro and in vivo, thereby reducing intervertebral disc degeneration. Furthermore, METTL3 mediated SOX9 RNA methylation and disrupted SOX9 mRNA stability, thereby inhibiting the gene expression of the downstream collagen type II alpha 1 chain. Tension stimulation downregulated SOX9 expression through METTL3-mediated m6A methylation, thereby inhibiting the synthesis of extracellular matrix in endplate chondrocytes.

METTL3 promotes lung adenocarcinoma tumor growth and inhibits ferroptosis by stabilizing SLC7A11 m6A modification

BackgroundN6-methyladenosine (m6A) has emerged as a significant regulator of the progress of various cancers. However, its role in lung adenocarcinoma (LUAD) remains unclear. Here, we explored the biological function and underlying mechanism of methyltransferase-like 3 (METTL3), the main catalyst of m6A, in LUAD progression.MethodsThe expression of m6A, METTL3, YTHDF1 and SLC7A11 were detected by immunochemistry or/and online datasets in LUAD patients. The effects of METTL3 on LUAD cell proliferation, apoptosis and ferroptosis were assessed through in vitro loss-and gain-of-function experiments. The in vivo effect on tumorigenesis of METTL3 was evaluated using the LUAD cell xenograft mouse model. MeRIP-seq, RNA immunoprecipitation and RNA stability assay were conducted to explore the molecular mechanism of METTL3 in LUAD.ResultsThe results showed that the m6A level, as well as the methylase METTL3 were both significantly elevated in LUAD patients and lung cancer cells. Functionally, we found that METTL3 could promote proliferation and inhibit ferroptosis in different LUAD cell models, while METTL3 knockdown suppressed LUAD growth in cell-derived xenografts. Mechanistically, solute carrier 7A11 (SLC7A11), the subunit of system Xc−, was identified as the direct target of METTL3 by mRNA-seq and MeRIP-seq. METTL3-mediated m6A modification could stabilize SLC7A11 mRNA and promote its translation, thus promoting LUAD cell proliferation and inhibiting cell ferroptosis, a novel form of programmed cell death. Additionally, we demonstrated that YTHDF1, a m6A reader, was recruited by METTL3 to enhance SLC7A11 m6A modification. Moreover, the expression of YTHDF1 and SLC7A11 were positively correlated with METTL3 and m6A in LUAD tissues.ConclusionsThese findings reinforced the oncogenic role of METTL3 in LUAD progression and revealed its underlying correlation with cancer cell ferroptosis; these findings also indicate that METTL3 is a promising novel target in LUAD diagnosis and therapy.

METTL3 inhibition ameliorates liver damage in mouse with hepatitis B virus-associated acute-on-chronic liver failure by regulating miR-146a-5p maturation

Hepatitis B virus (HBV)-associated acute-on-chronic liver failure (ACLF) is a clinical syndrome of severe liver damage. HBV infection is affected by N6-methyladenosine (m6A) RNA modification. Here, we investigated whether methyltransferase-like 3 (METTL3)-mediated m6A methylation can affect ACLF. Human hepatic cells (THLE-2) were treated with lipopolysaccharide (LPS) to induce cell damage. Proliferation, apoptosis and m6A modification were measured by MTT assay, flow cytometry and Dot blot assay. Our results showed that HBV infection significantly enhanced the levels of m6A modification and elevated the expression of METTL3 and mature-miR-146a-5p in THLE-2 cells, which was repressed by cycloleucine (m6A inhibitor). METTL3 overexpression enhanced m6A modification and promoted mature-miR-146a-5p expression. METTL3 overexpression promoted HBV replication and apoptosis, enhanced the levels of pro-inflammatory cytokines, hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg), and repressed cell proliferation in THLE-2 cells, which attributed to repress miR-146a-5p maturation. Moreover, a severe liver failure mouse model was established by HBV infection to verify the impact of METTL3 knockdown on liver damage in vivo. HBV-infection led to a severe liver damage and increase of apoptosis in hepatic tissues of mice, which was abolished by METTL3 knockdown. METTL3 knockdown reduced METTL3 expression and impeded miR-146a-5p maturation in HBV-infected mice. In conclusion, this work demonstrates that METTL3 inhibition ameliorates liver damage in mouse with HBV-associated ACLF, which contributes to repress miR-146a-5p maturation. Thus, this article suggests a novel therapeutic avenue to prevent and treat HBV-associated ACLF.

METTL3 improves cardiomyocyte proliferation upon myocardial infarction via upregulating miR-17-3p in a DGCR8-dependent manner

Myocardial infarction (MI), one of the most severe types of heart attack, exerts a strong negative effect on heart muscle by causing a massive and rapid loss of cardiomyocytes. However, the existing therapies do little to improve cardiac regeneration. Due to the role of methyltransferase-like 3 (METTL3) in the physiological proliferation of cardiomyocytes, we aimed to determine whether METTL3 could also promote cardiomyocyte proliferation under pathological conditions and to elucidate the underlying mechanism. The effects of METTL3 on cardiomyocyte proliferation and apoptosis were investigated in an in vivo rat model of MI and in an in vitro model of neonatal rat cardiomyocytes (NRCMs) exposed to hypoxia. We found that METTL3 expression was downregulated in hypoxia-exposed NRCMs and MI-induced rats. Furthermore, METTL3 pretreatment enhanced cardiomyocyte proliferation and inhibited cardiomyocyte apoptosis under hypoxic or MI conditions, and silencing METTL3 had the opposite effects. Additionally, METTL3 overexpression upregulated miR-17-3p expression. The miR-17-3p agomir mimicked the pro-proliferative and antiapoptotic effects of METTL3 in hypoxia-exposed cells or rats with MI, while the miR-17-3p antagomir blocked these effects. Additionally, pretreatment with the RNA-binding protein DGCR8 also hampered the protective role of METTL3 in hypoxia-exposed cells. Overall, the current study indicated that METTL3 could improve cardiomyocyte proliferation and subsequently ameliorate MI in rats by upregulating proliferation-related miR-17-3p in a DGCR8-dependent pri-miRNA-processing manner.

PiRNA-36741 regulates BMP2-mediated osteoblast differentiation via METTL3 controlled m6A modification.

The osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is essential for bone formation, and its imbalance can lead to bone diseases such as osteoporosis. It is reported that PIWI-interacting RNA-36741 (piR-36741) is up-regulated during the osteogenic differentiation, but its role in regulating osteogenic differentiation remains unclear. Here, the primary human BMSCs were used to induce osteogenic differentiation, and the expression of piR-36741 and METTL3 (methyltransferase like 3) was up-regulated during the osteogenic differentiation of BMSCs. Moreover, interference with piR-36741 or METTL3 markedly hindered the osteogenic differentiation of BMSCs, which was manifested by a reduction in osteoblast marker expression (including RUNX2, COL1A1, OPN and OCN), osteogenic phenotype and matrix mineralization. Mechanistically, the piR-36741-PIWIL4 complex directly interacted with METTL3 and prevented METTL3-mediated m6A modification of BMP2 mRNA transcripts, thereby promoting BMP2 expression. And overexpression of BMP2 reversed the inhibitory effect of piR-36741 silence on osteogenic differentiation and the Smad pathway activity. In addition, administration with piR-36741 mimic alleviated ovariectomy-induced osteoporosis in mice. In conclusion, piRNA-36741 overexpression promoted osteogenic differentiation of BMSCs and mitigated ovariectomy-induced osteoporosis through METTL3-mediated m6A methylation of BMP2 transcripts.

An integrated model of FTO and METTL3 expression that predicts prognosis in lung squamous cell carcinoma patients.

BackgroundLung squamous cell carcinoma (LUSC) approximately accounts for a third of lung cancers. However, the role of N6-methyladenosine (m6A) in LUSC remains largely unknown according to previous studies.MethodsIn this study, we investigated the mutations, copy number variants (CNVs), expression of 20 m6A RNA methylation regulators, and clinical data from The Cancer Genome Atlas-LUSC (TCGA-LUSC). These data were used for the training cohort of screening potential biomarkers. The prognostic model of m6A RNA methylation regulators was constructed. A receiver operating characteristic (ROC) analysis was undertaken to determine the area under the curves (AUCs) (for 3- and 5-year survival) for the model. Additionally, the accuracy of the two-gene model was confirmed with external data verifications. Combined two-gene model and clinincal information were performed to construct a nomogram to predict patient’s prognostic risk assessment.ResultsFat mass- and obesity-associated protein (FTO) and methyltransferase-like 3 (METTL3) were identified as potential prognostic biomarkers to evaluate benign and malignant tumors and prognosticate. The following prognostic model of m6A RNA methylation regulators was constructed: risk score = 0.162 × FTO − 0.069 × METTL3. Patients in low-risk group [median overall survival (mOS), 43.4 months] had longer survival than those with high-risk (mOS, 67.3 months) with P=0.0023. The smoking grade and risk score could be independent prognostic factors (P=0.00098 and P=0.0014, respectively). Ultimately, a nomogram was developed to assist clinicians to predict clinical outcomes.ConclusionsFTO and METTL3 are potential prognostic biomarkers of LUSC. The two-gene model’s use of prognostic risk scores may provide guidance in the selection of therapeutic strategies.

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.