Overexpression Of METTL3 Research Articles

METTL3-mediated Hsa_circ_0000390 downregulation enhances the proliferation, migration, and invasion of colorectal cancer cells by increasing Notch1 expression.

Circular RNAs (circRNAs), as noncoding RNAs, have gained widespread attention in cancers. Circ_0000390 has been verified to be downregulated in gastric cancer, while its function and regulatory mechanism in cancer is largely unknown. The purpose of this study is to investigate the expression, functions, and potential mechanism of circ_0000390 in colorectal cancer (CRC). Circ_0000390 expression in CRC tissues was first identified with RT-qPCR. Besides, the function of circ_0000390 was assessed through gain-of-function and rescued experiments in CRC cells and mouse xenograft models. Our results showed that circ_0000390 was lowly expressed in CRC tissues, and circ_0000390 could downregulate Notch1 and be downregulated by METTL3. Functionally, results showed circ_0000390 overexpression suppressed the proliferation, cell migration, and invasion of CRC cells, which also could be reversed by Notch1 overexpression. Additionally, METTL3 overexpression could accelerate the proliferation, cell migration, and invasion of CRC cells, which also was weakened by circ_0000390 overexpression in CRC cells in vitro and in vivo. This study suggested that circ_0000390 might be anti-tumor factor in CRC and METTL3/Notch1 might be a therapeutic targets for CRC.

KDM1A-mediated upregulation of METTL3 ameliorates Alzheimer's disease via enhancing autophagic clearance of p-Tau through m6A-dependent regulation of STUB1

BackgroundAlzheimer's disease (AD) is a severe neurodegenerative disorder that progressively destroys cognitive skills. Exploring the mechanism underlying autophagic clearance of phosphorylated tau (p-Tau) contributes to developing novel therapeutic strategies for AD. MethodsSH-SY5Y and HT22 cells were treated with Aβ1-42 to establish an in vitro model of AD. Cell viability was examined using CCK-8. TUNEL staining was applied to evaluate cell apoptosis. LC3 puncta was examined by IF staining. m6A modification level was evaluated through MeRIP. RNA pull-down and RIP assays were used for analyzing the interaction between IGF2BP1 and STUB1 transcripts. The binding of KDM1A to the promoter of METTL3 was confirmed by ChIP assays. APP/PS1 transgenic mice were used as an in vivo model of AD. Cognitive skills of mice were evaluated with the Morris water maze. Hippocampal damage and Aβ deposition were detected through H&E and IHC staining. ResultsDysregulated levels of autophagy, p-Tau and m6A was observed in an in vitro model of AD. Overexpression of METTL3 or STUB1 enhanced autophagy but reduced p-Tau level in Aβ1-42-treated cells. METTL3 stabilized STUB1 mRNA through the m6A-IGF2BP1-dependent mechanism and naturally promoted STUB1 expression, thereby enhancing autophagic p-Tau clearance in Aβ1-42-treated cells. Overexpression of KDM1A enhanced autophagy, m6A modification and autophagic p-Tau clearance in Aβ1-42-treated cells. KDM1A-mediated upregulation of METTL3 promoted autophagic p-Tau clearance and ameliorated Alzheimer's disease both in vitro and in vivo. ConclusionKDM1A-mediated upregulation of METTL3 enhances autophagic clearance of p-Tau through m6A-dependent regulation of STUB1, thus ameliorating Alzheimer's disease. Our study provides novel mechanistic insights into AD pathogenesis and potential drug targets for AD.

METTL3-Mediated lncSNHG7 m6A Modification in the Osteogenic/Odontogenic Differentiation of Human Dental Stem Cells

Background: Human dental pulp stem cells (hDPSCs) play an important role in endodontic regeneration. N6-methyladenosine (m6A) is the most common RNA modification, and noncoding RNAs have also been demonstrated to have regulatory roles in the expression of m6A regulatory proteins. However, the study on m6A modification in hDPSCs has not yet been conducted. Methods: Single base site PCR (MazF) was used to detect the m6A modification site of lncSNHG7 before and after mineralization of hDPSCs to screen the target m6A modification protein, and bioinformatics analysis was used to analyze the related pathways rich in lncSNHG7. After knockdown and overexpression of lncSNHG7 and METTL3, the osteogenic/odontogenic ability was detected. After METTL3 knockdown, the m6A modification level and its expression of lncSNHG7 were detected by MazF, and their binding was confirmed. Finally, the effects of lncSNHG7 and METTL3 on the Wnt/β-catenin pathway were detected. Results: MazF experiments revealed that lncSNHG7 had a m6A modification before and after mineralization of hDPSCs, and the occurrence site was 2081. METTL3 was most significantly upregulated after mineralization of hDPSCs. Knockdown/ overexpression of lncSNHG7 and METTL3 inhibited/promoted the osteogenic/odontogenic differentiation of hDPSCs. The m6A modification and expression of lncSNHG7 were both regulated by METTL3. Subsequently, lncSNHG7 and METTL3 were found to regulate the Wnt/β-catenin signaling pathway. Conclusion: These results revealed that METTL3 can activate the Wnt/β-catenin signaling pathway by regulating the m6A modification and expression of lncSNHG7 in hDPSCs to enhance the osteogenic/odontogenic differentiation of hDPSCs. Our study provides new insight into stem cell-based tissue engineering.

METTL3 promotes glycolysis and cholangiocarcinoma progression by mediating the m6A modification of AKR1B10

ObjectiveN6-methyladenosine (m6A) RNA methylation is involved in governing the mechanism of tumor progression. We aimed to excavate the biological role and mechanism of the m6A methyltransferase METTL3 in cholangiocarcinoma (CCA).MethodsMETTL3 expression was determined by database and tissue microarray analyses. The role of METTL3 in CCA was explored by loss- and gain-of-function experiments. The m6A target of METTL3 was detected by RNA sequencing. The role of AKR1B10 in CCA was explored, and the association between METTL3 and AKR1B10 was confirmed by rescue experiments.ResultMETTL3 expression was upregulated in CCA tissue, and higher METTL3 expression was implicated in poor prognoses in CCA patients. Overexpression of METTL3 facilitated proliferation, migration, invasion, glucose uptake, and lactate production in CCA cells, whereas knockdown of METTL3 had the opposite effects. We further found that METTL3 deficiency inhibited CCA tumor growth in vivo. RNA sequencing and MeRIP-qPCR confirmed that METTL3 enhanced AKR1B10 expression and m6A modification levels. Furthermore, METTL3 directly binds with AKR1B10 at an m6A modification site. A CCA tissue microarray showed that AKR1B10 expression was upregulated in CCA tissue and that silencing AKR1B10 suppressed the malignant phenotype mentioned above in CCA. Notably, knockdown of AKR1B10 rescued the tumor-promoting effects induced by METTL3 overexpression.ConclusionElevated METTL3 expression promotes tumor growth and glycolysis in CCA through m6A modification of AKR1B10, indicating that METTL3 is a potential target for blocking glycolysis for application in CCA therapy.

Mettl3-mediated m6A modification of Fgf16 restricts cardiomyocyte proliferation during heart regeneration.

Cardiovascular disease is the leading cause of death worldwide due to the inability of adult heart to regenerate after injury. N6-methyladenosine (m6A) methylation catalyzed by the enzyme methyltransferase-like 3 (Mettl3) plays an important role in various physiological and pathological bioprocesses. However, the role of m6A in heart regeneration remains largely unclear. To study m6A function in heart regeneration, we modulated Mettl3 expression in vitro and in vivo. Knockdown of Mettl3 significantly increased the proliferation of cardiomyocytes and accelerated heart regeneration following heart injury in neonatal and adult mice. However, Mettl3 overexpression decreased cardiomyocyte proliferation and suppressed heart regeneration in postnatal mice. Conjoint analysis of methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq identified Fgf16 as a downstream target of Mettl3-mediated m6A modification during postnatal heart regeneration. RIP-qPCR and luciferase reporter assays revealed that Mettl3 negatively regulates Fgf16 mRNA expression in an m6A-Ythdf2-dependent manner. The silencing of Fgf16 suppressed the proliferation of cardiomyocytes. However, the overexpression of ΔFgf16, in which the m6A consensus sequence was mutated, significantly increased cardiomyocyte proliferation and accelerated heart regeneration in postnatal mice compared with wild-type Fgf16. Our data demonstrate that Mettl3 post-transcriptionally reduces Fgf16 mRNA levels through an m6A-Ythdf2-dependen pathway, thereby controlling cardiomyocyte proliferation and heart regeneration.

Interactive regulation of DNA demethylase gene TET1 and m6A methyltransferase gene METTL3 in myoblast differentiation

DNA methylation (5mC) and mRNA N6-methyladenosine (m6A) play an essential role in gene transcriptional regulation. DNA methylation has been well established to be involved in skeletal muscle development. Interacting regulatory mechanisms between DNA methylation and mRNA m6A modification have been identified in a variety of biological processes. However, the effect of m6A on skeletal muscle differentiation and the underlying mechanisms are still unclear. It is also unknown whether there is an interaction between DNA methylation and mRNA m6A modification in skeletal myogenesis. In the present study, we used m6A-IP-qPCR, LC–MS/MS and dot blot assays to determine that the DNA demethylase gene, TET1, exhibited increased m6A levels and decreased mRNA expression during bovine skeletal myoblast differentiation. Dual-luciferase reporter assays and RIP experiments demonstrated that METTL3 suppressed TET1 expression by regulating TET1 mRNA stability in a m6A-YTHDF2-dependent manner. Furthermore, TET1 mediated DNA demethylation of itself, MYOD1 and MYOG, thereby stimulating their expression to promote myogenic differentiation. Ectopic expression of TET1 rescued the effect of METTL3 knockdown on reduced myotubes. In contrast, TET1 knockdown impaired the myogenic differentiation promoted by METTL3 overexpression. Moreover, ChIP experiments found that TET1 could bind and demethylate METTL3 DNA, which enhanced METTL3 expression. In addition, TET1 knockdown decreased m6A levels. ChIP assays also showed that TET1 knockdown contributed to the binding of H3K4me3 and H3K27me3 to METTL3 DNA. Our results revealed a negative feedback regulatory loop between TET1 and METTL3 in myoblast differentiation, which unveiled the interplay among DNA methylation, RNA methylation and histone methylation in skeletal myogenesis.

METTL3 promotes colorectal cancer metastasis by promoting the maturation of pri-microRNA-196b

PurposeMethyltransferase-like 3 (METTL3), a key member of the m6A methyltransferase complex, is upregulated in multiple human malignancies and plays a role in regulating tumor migration. This study aimed to reveal the underlying mechanism by which METTL3 in regulates the metastasis of colorectal cancer (CRC).MethodsWe compared METTL3 expression levels in CRC tumor tissues and adjacent nontumor tissues by immunohistochemistry (IHC). The functional roles of METTL3 in CRC were assessed by real-time cell migration assays, wound-healing assays and Transwell assays. miRNA sequencing (miRNA-seq), RNA-binding protein immunoprecipitation (RIP) assays and N6-methyladenosine immunoprecipitation (MeRIP) assays were performed to confirm the molecular mechanism underlying the involvement of METTL3 in CRC cell metastasis.ResultsWe found that METTL3 was overexpressed in CRC tissues. METTL3 knockdown significantly inhibited CRC cell migration and invasion, while METTL3 overexpression had the opposite effects. Furthermore, we demonstrated that METTL3 regulates miR-196b expression via an N6-methyladenosine (m6A)-pri-miR-196b-dependent mechanism and thereby promotes CRC metastasis.ConclusionThis study shows the important role of METTL3 in CRC metastasis and provides novel insight into m6A modification in CRC metastasis.

Prognostic and clinicopathological value of m6A regulators in human cancers: a meta-analysis.

N6-methyladenosine (m6A) is the most abundant epigenetic modification. Although the dysregulation of m6A regulators has been associated with cancer progression in several studies, its relationship with cancer prognosis and clinicopathology is still controversial. Therefore, we evaluated the prognostic and clinicopathological value of m6A regulators in cancers by performing a comprehensive meta-analysis. The PubMed, Cochrane Library, Web of Science, and Embase databases were searched up to April 2022. Hazard ratios were used to analyze the association between m6A with prognosis. We also analyze the relationship between m6A and clinicopathology using odds ratios. METTL3 overexpression predicted poor overall survival and disease-free survival in cancer patients (p < 0.001) such as gastric cancer (p < 0.001), esophageal squamous cell carcinoma (p < 0.001), oral squamous cell carcinoma (p = 0.002) and so on. Additionally, METTL3 overexpression was associated with poor pT stage (p < 0.001), pN stage (p < 0.001), TNM stage (p < 0.001), tumor size >5 cm (p < 0.001) and vascular invasion (p = 0.024). Conversely, METTL14 overexpression was positively associated with better OS (p < 0.001), negatively with poor pT stage (p = 0.001), pM stage (p = 0.002), pN stage (p = 0.011) and TNM stage (p < 0.001). Moreover, KIAA1429 overexpression was associated with poor OS (p = 0.001). YTHDF1 overexpression was also associated with advanced pM stage (p < 0.001) and tumor size >5 cm (p < 0.001). However, ALKBH5 overexpression was negatively associated with vascular invasion (p = 0.032). High expression of METTL3 predicted poor outcome. In contrast, high expression of METTL14 was associated with better outcome. Thus, we suggest that among all the m6A regulators, METTL3 and METTL14 could be potential prognostic markers in cancers.

Methyltransferase like 3-mediated N6-methylatidin methylation inhibits vascular smooth muscle cells phenotype switching via promoting phosphatidylinositol 3-kinase mRNA decay.

N6-methylatidine (m6A) is involved in post-transcriptional metabolism and a variety of pathological processes. However, little is known about the role of m6A in vascular proliferative diseases, particularly in vascular smooth muscle cells (VSMCs) phenotype switching-induced neointimal hyperplasia. In the current study, we discovered that methyltransferase like 3 (METTL3) is a critical candidate for catalyzing a global increase in m6A in response to carotid artery injury and various VSMCs phenotype switching. The inhibited neointimal hyperplasia was obtained after in vivo gene transfer to knock-down Mettl3. In vitro overexpression of Mettl3 resulted in increased VSMC proliferation, migration, and reduced contractile gene expression with a global elevation of m6A modification. In contrast, Mettl3 knockdown reversed this facilitated phenotypic switch in VSMCs, as demonstrated by downregulated m6A, decreased proliferation, migration, and increased expression of contractile genes. Mechanistically, Mettl3 knock-down was found to promote higher phosphatidylinositol 3-kinase (Pi3k) mRNA decay thus inactivating the PI3K/AKT signal to inhibit VSMCs phenotype switching. Overall, our findings highlight the importance of METTL3-mediated m6A in VSMCs phenotype switching and offer a novel perspective on targeting METTL3 as a therapeutic option for VSMCs phenotype switching modulated pathogenesis, including atherosclerosis and restenosis.

N6-methyladenosine modification in trophoblasts promotes circSETD2 expression, inhibits miR-181a-5p, and elevates MCL1 transcription to reduce apoptosis of trophoblasts.

Preeclampsia (PE) is an obstetric disorder. N6-methyladenosine (m6A) modification is related to PE trophoblast biological behaviors. This study explored the mechanism of m6A-modified circSETD2 in trophoblast biological behaviors. Chorionic trophoblast apoptosis and circSETD2 expression in PE rat models were detected. HTR8/SVneo cells were induced by CoCl2 to establish PE trophoblast models. circSETD2 was silenced or overexpressed to evaluate its effect on cell proliferation, invasion, and apoptosis. m6A level of circSETD2 in trophoblasts was changed by pcDNA3.1-METTL3 and pcDNA3.1-FTO. The targeting relations among miR-181a-5p, circSETD2, and MCL1 were verified by dual-luciferase assay. miR-181a-5p and MCL1 expressions were interfered with to confirm the effect of m6A-modified circSETD2. m6A methylation level was changed in PE rats for in vivo validation. PE rats showed diminished circSETD2 expression and increased apoptosis index. circSETD2 overexpression promoted trophoblast proliferation and invasion, and reduced apoptosis. METTL3 overexpression increased total m6A, circSETD2 m6A, and circSETD2 levels. m6A modification mediated circSETD2 upregulation. circSETD2 was a sponge of miR-181a-5p to elevate MCL1 transcription. miR-181a-5p overexpression or MCL1 silencing annulled the role of m6A-modified circSETD2. circSETD2 inhibition negated suppression of METTL3 overexpression on chorionic trophoblast apoptosis in vivo. Collectively, m6A modification of circSETD2 suppressed miR-181a-5p and increased MCL1 transcription, thus regulating trophoblasts.

WTAP regulates postnatal development of brown adipose tissue by stabilizing METTL3 in mice

Abstract Brown adipocyte maturation during postnatal development is essential for brown adipose tissue (BAT) to protect animals against cold. Impaired maturation of brown adipocytes leads to cold intolerance. However, the molecular mechanisms that determine the maturation of brown adipocytes during postnatal development are not fully understood. Here, we identify Wilms’ tumor 1-associating protein (WTAP) as an essential regulator in the postnatal development and maturation of BAT. BAT-specific knockout of Wtap (Wtap-BKO) severely impairs maturation of BAT in vivo by decreasing the expression of BAT-selective genes, leading to the whitening of interscapular BAT (iBAT). Single nucleus RNA-sequencing analysis shows the dynamic changes of cell heterogeneity in iBAT of Wtap-BKO mice. Adult mice with WTAP deficiency in BAT display hypothermic and succumb to acute cold challenge. Mechanistically, WTAP deficiency decreases m6A mRNA modification by reducing the protein stability of METTL3. BAT-specific overexpression of Mettl3 partially rescues the phenotypes observed in Wtap-BKO mice. These data demonstrate that WTAP/METTL3 plays an essential role in iBAT postnatal development and thermogenesis.

METTL3 upregulates COPS5 expression in osteosarcoma in an m6A-related manner to promote osteosarcoma progression

N6-methyladenosine (m6A) is the most abundant and well-studied internal modification of messenger RNAs (mRNAs). Although m6A mRNA modification has been frequently observed in osteosarcoma, the roles and underlying mechanisms of m6A modification are not yet fully elucidated. In this study, an m6A regulator, METTL3, showed to be dramatically up-regulated within osteosarcoma tissues and cells than non-cancerous healthy samples and human normal osteoblasts, respectively. In vitro, knockdown of METTL3 suppressed the viability of osteosarcomas, and their abilities to migrate and invade; in vivo, knockdown of METTL3 repressed tumor growth within xenotransplant tumor model. METTL3 upregulates COPS5 expression may be through promoting COPS5 methylation to stabilize COPS5 mRNA. The expression level of COPS5 also showed to be up-regulated within osteosarcoma tissue samples and cells. COPS5 knockdown caused no changes in METTL3 effects on METTL3 expression but partially eliminated METTL3 effects on COPS5 expression. METTL3 overexpression promoted, whereas COPS5 knockdown inhibited the malignant behaviors of osteosarcoma cells; COPS5 knockdown partially eliminated the effects of METTL3 overexpression on osteosarcoma cells. Conclusively, METTL3 and COPS5 serve as oncogenic regulators in osteosarcoma. METTL3 upregulates COPS5 expression in osteosarcoma in an m6A-related manner.

METTL3 promotes non-small cell lung cancer (NSCLC) cell proliferation and colony formation in a m6A-YTHDF1 dependent way

BackgroundN6-methyladenosine (m6A) is the most common RNA modification, which plays a pivotal role in tumor development and progression. In this study, we assessed the role of m6A methyltransferase METTL3 in FRAS1-involved cell proliferation and colony formation of non-small cell lung cancer (NSCLC) cell lines.MethodsCell viability was analyzed by Cell Counting Kit (CCK-8) and colony formation. M6A RNA immunoprecipitation (IP), Ribosomal immunoprecipitation, RNA immunoprecipitation (RIP) were performed to verify the relationship between METTL3, FRAS1 and YTHDF1. Rescue experiments to confirm the regulatory mechanism of METTL3-FRAS1 promoted NSCLC cell proliferation through CDON by cooperating YTHDF1.ResultsWe found that FRAS1 was correlated with poor prognosis in NSCLC patients, of which the transcript undergoes m6A modification regulated by METTL3. METTL3 silence reduced cell viability of NSCLC cells HCC827 and NCI-H1975, which could be restored by FRAS1 overexpression. The m6A modification of FRAS1 could be recognized by YTHDF1. FRAS1 silence or YTHDF1 silence could rescue the elevated NSCLC cell proliferation, colony formation, and tumor growth induced by METTL3 overexpression in vitro and in vivo.ConclusionsOur study reveals that METTL3-FRAS1 plays a crucial role in NSCLC cell proliferation, colony formation, and tumor growth through the regulation of CDON by cooperating YTHDF1.

Mettl14-driven senescence-associated secretory phenotype facilitates somatic cell reprogramming

SummaryThe METTL3-METTL14 complex, the “writer” of N6-methyladenosine (m6A), plays an important role in many biological processes. Previous studies have shown that Mettl3 overexpression can increase the level of m6A and promote somatic cell reprogramming. Here, we demonstrate that Mettl14, another component of the methyltransferase complex, can significantly enhance the generation of induced pluripotent stem cells (iPSCs) in an m6A-independent manner. In cooperation with Oct4, Sox2, Klf4, and c-Myc, overexpressed Mettl14 transiently promoted senescence-associated secretory phenotype (SASP) gene expression in non-reprogrammed cells in the late stage of reprogramming. Subsequently, we demonstrated that interleukin-6 (IL-6), a component of the SASP, significantly enhanced somatic cell reprogramming. In contrast, blocking the SASP using a senolytic agent or a nuclear factor κB (NF-κB) inhibitor impaired the effect of Mettl14 on reprogramming. Our results highlight the m6A-independent function of Mettl14 in reprogramming and provide new insight into the interplay between senescence and reprogramming in vitro.

Identification and Validation of METTL3-Related Molecules for Predicting Prognosis and Efficacy of Immunotherapy in Gastric Cancer Based on m6A Methylome and Transcriptome Sequencing Analysis.

Abnormal N6-methyladenosine (m6A) modification levels caused by METTL3 have been identified to be a critical regulator in human cancers, and its roles in the immune microenvironment and the relationship between targeted therapy and immunotherapy sensitivity in gastric cancer (GC) remain poorly understood. In this study, we assessed the transcriptome-wide m6A methylation profile after METTL3 overexpression by m6A sequencing and RNA sequencing in BGC-823 cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to analyze the function of core targets of METTL3. Eighteen methylation core molecules were identified in GC patients by combining transcriptome and methylome sequencing. GC patients can be separated into two subtypes based on the expression of 18 methylation core molecules. Furthermore, subgroup analysis showed that patients with different subtypes had a different OS, PFS, stage, grade, and TMB. Gene set enrichment analysis (GSEA) showed that immune-related pathways were enriched among subtype A. The ESTIMATE analysis suggested that the extent of infiltration of immune cells was different in two subtypes of GC patients. Tumor Immune Dysfunction and Exclusion (TIDE) and The Cancer Immunome Atlas (TCIA) database also showed that there were significant differences in the efficacy of immunotherapy among different types of GC patients. Altogether, our results reveal that METTL3-mediated m6A methylation modification is associated with the immune microenvironment and the effects of immunotherapy in GC patients. Our findings provide novel insights for clinicians in the diagnosis and optimal treatment of GC patients.

Mmu-lncRNA 121686/hsa-lncRNA 520657 induced by METTL3 drive the progression of AKI by targeting miR-328-5p/HtrA3 signaling axis

The pathogenesis of acute kidney injury (AKI) is still not fullyunderstood, and effective interventions are lacking. Here, we explored whether methyltransferase 3 (METTL3) was involved in the progression of AKI via regulation of cell death. We reported that PT（proximal tubule）-METTL3-knockout (KO) noticeably suppressed ischemic-induced AKI via inhibition of renal cell apoptosis. Furthermore, we also found that the expression of mmu-long non-coding RNA (lncRNA) 121686 was upregulated in antimycin-treated Boston University mouse proximal tubule (BUMPT) cells and a mouse ischemia-reperfusion (I/R)-induced AKI model. Functionally, mmu-lncRNA 121686 could promote I/R-induced mouse renal cell apoptosis. Mechanistically, mmu-lncRNA 121686 acted as a competing endogenous RNA (ceRNA) to prevent microRNA miR-328-5p-mediated downregulation of high-temperature requirement factor A 3 (Htra3). PT-mmu-lncRNA 121686-KO mice significantly ameliorated the ischemic-induced AKI via the miR-328-5p/HtrA3 axis. In addition, hsa-lncRNA 520657, homologous with lncRNA 121686, sponged miR-328-5p andupregulated Htra3 to promote I/R-induced human renal cell apoptosis. Interestingly, we found that mmu-lncRNA 121686/hsa-lncRNA 520657 upregulation were dependent onMETTL3 via N6-methyladenosine (m6A) modification. The mmu-lncRNA 121686/miR-328-5p or hsa-lncRNA 520657/miR-328-5p /HtrA3 axis was induced invitro by METTL3 overexpression; in contrast, this effect was attenuated by METTL3 small interfering RNA (siRNA). Furthermore, we found that PT-METTL3-KO or METTL3 siRNA significantly suppressed ischemic, septic, and vancomycin-induced AKI via downregulation of the mmu-lncRNA 121686/miR-328-5p/HtrA3 axis. Taken together, our data indicate that the METTL3/mmu-lncRNA 121686/hsa-lncRNA 520657/miR-328-5p/HtrA3 axis potentially acts as a therapeutic target for AKI.

Mettl3-mediated m6 A modification of Lrp2 facilitates neurogenesis through Ythdc2 and elicits antidepressant-like effects.

N6 -methyladenosine (m6 A) is the most abundant mRNA modification affecting diverse biological processes. However, the functions and precise mechanisms of m6 A signaling in adult hippocampal neurogenesis and neurogenesis-related depression remain largely enigmatic. We found that depletion of Mettl3 or Mettl14 in neural stem cells (NSCs) dramatically reduced m6 A abundance, proliferation, and neuronal genesis, coupled with enhanced glial differentiation. Conversely, overexpressing Mettl3 promoted proliferation and neuronal differentiation. Mechanistically, the m6 A modification of Lrp2mRNA by Mettl3 enhanced its stability and translation efficiency relying on the reader protein Ythdc2, which in turn promoted neurogenesis. Importantly, mice lacking Mettl3manifested reduced hippocampalneurogenesis, which could contribute to spatial memory decline, and depression-like behaviors. We found that these defective behaviors were notably reversed by Lrp2 overexpression. Moreover, Mettl3 overexpression in the hippocampus of depressive mice rescues behavioral defects. Our findings uncover the biological role of m6 A modification in Lrp2-mediated neurogenesis via m6 A-binding protein Ythdc2, andproposearationale that targeting Mettl3-Ythdc2-Lrp2 axis regulation of neurogenesis might serve as a promising antidepressant strategy.

METTL3-Mediated m6A RNA Methylation of ZBTB4 Interferes With Trophoblast Invasion and Maybe Involved in RSA.

N6-methyladenosine (m6A) was the most abundant modification of mRNA and lncRNA in mammalian cells and played an important role in many biological processes. However, whether m6A modification was associated with recurrent spontaneous abortion (RSA) and its roles were still unclear. Methods: Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) was used to study the global m6A modification pattern in RSAs and controls. RNA sequencing (RNA-Seq) was used to study the level of global mRNA in two groups. Real-time quantitative PCR (RT-qPCR) was used to verify the level of mRNA of METTL3 and ZBTB4. MeRIP–qPCR was conducted to test the level of ZBTB4 m6A modification in two groups. In order to further explore whether ZBTB4 was the substrate of METTL3, the HTR-8/SVneo (HTR-8) cell line was selected for the knockdown and overexpression of METTL3. To study whether METTL3 regulated the ZBTB4 expression by recognizing ZBTB4 mRNA m6A motifs in coding sequences (CDS), dual-luciferase reporter assay was conducted. RNA stability assays using actinomycin D were conducted to study the RNA stability of the HTR-8 cell line with METTL3 overexpression and knockdown. To illustrate the role of METTL3 in the invasion of trophoblast, matrigel invasion assays and transwell migration assays were conducted using the HTR-8 cell line with METTL3 overexpression and knockdown. Results: A total of 65 genes were found with significant differences both in m6A modification and mRNA expression. We found m6A methyltransferase METTL3 was significantly down-regulated in the RSA group. Through gene function analysis, RT-qPCR, MeRIP–qPCR validation experiment, knockdown, and overexpression of METTL3 in the HTR-8 cell line, ZBTB4 was selected as one target of METTL3. Furthermore, we clarified that METTL3 regulated the expression of ZBTB4 by recognizing ZBTB4 mRNA m6A motifs in the CDS using the dual-luciferase reporter assay and METTL3 regulated the invasion of trophoblast by altering the stability and expression of ZBTB4 by RNA stability, matrigel invasion, and transwell migration assays. Conclusion: Our study revealed the mechanism by which METTL3 regulated the stability and expression of ZBTB4 and the trophoblast migration ability of RSA. A new perspective was provided for exploring the mechanism of embryonic development in RSA patients.

Molecular mechanism of m6A methylation of circDLC1 mediated by RNA methyltransferase METTL3 in the malignant proliferation of glioma cells

Glioma is an intracranial malignant tumor and remains largely incurable. Circular RNAs are prominent modulators in glioma progression. This study investigated the function of circular RNA DLC1 (circDLC1) in the malignant proliferation of glioma cells. circDLC1 expression in glioma tissues and cells was determined using RT-qPCR. The effect of circDLC1 on the malignant proliferation of glioma cells was analyzed using CCK-8, colony formation, and EdU staining assays. METTL3, miR-671-5p, and CTNNBIP1 expressions were determined. N6 methyladenosine (m6A) level of circDLC1 was analyzed using MeRIP. The binding relationship between miR-671-5p and circDLC1 or CTNNBIP1 was verified using RNA pull-down and dual-luciferase assays. A xenograft tumor model was established in nude mice to verify the effect of METTL3-mediated circDLC1 on glioma in vivo. circDLC1 was poorly expressed in glioma. circDLC1 overexpression suppressed glioma cell proliferation. Mechanically, METTL3-mediated m6A modification enhanced circDLC1 stability and upregulated circDLC1 expression in glioma. circDLC1 upregulated CTNNBIP1 transcription by competitively binding to miR-671-5p. METTL3 overexpression repressed the malignant proliferation of glioma via circDLC1/miR-671-5p/CTNNBIP1 in vivo. Collectively, METTL3-mediated m6A modification upregulated circDLC1 expression, and circDLC1 promoted CTNNBIP1 transcription by sponging miR-671-5p, thus repressing the malignant proliferation of glioma.

METTL3-m6 A methylase regulates the osteogenic potential of bone marrow mesenchymal stem cells in osteoporotic rats via the Wnt signalling pathway.

ObjectivesBone marrow mesenchymal stem cells (BMSCs) hold a high osteogenic differentiation potential, but the mechanisms that control the osteogenic ability of BMSCs from osteoporosis (OP‐BMSCs) need further research. The purpose of this experiment is to discuss the osteogenic effect of Mettl3 on OP‐BMSCs and explore new therapeutic target that can enhance the bone formation ability of OP‐BMSCs.Materials and MethodsThe bilateral ovariectomy (OVX) method was used to establish the SD rat OP model. Dot blots were used to reveal the different methylation levels of BMSCs and OP‐BMSCs. Lentiviral‐mediated overexpression of Mettl3 was applied in OP‐BMSCs. QPCR and WB detected the molecular changes of osteogenic‐related factors and Wnt signalling pathway in vitro experiment. The staining of calcium nodules and alkaline phosphatase detected the osteogenic ability of OP‐BMSCs. Micro‐CT and histological examination evaluated the osteogenesis of Mettl3 in OP rats in vivo.ResultsThe OP rat model was successfully established by OVX. Methylation levels and osteogenic potential of OP‐BMSCs were decreased in OP‐BMSCs. In vitro experiment, overexpression of Mettl3 could upregulate the osteogenic‐related factors and activate the Wnt signalling pathway in OP‐BMSCs. However, osteogenesis of OP‐BMSCs was weakened by treatment with the canonical Wnt inhibitor Dickkopf‐1. Micro‐CT showed that the Mettl3(+) group had an increased amount of new bone formation at 8 weeks. Moreover, the results of histological staining were the same as the micro‐CT results.ConclusionsTaken together, the methylation levels and osteogenic potential of OP‐BMSCs were decreased in OP‐BMSCs. In vitro and in vivo studies, overexpression of Mettl3 could partially rescue the decreased bone formation ability of OP‐BMSCs by the canonical Wnt signalling pathway. Therefore, Mettl3 may be a key targeted gene for bone generation and therapy of bone defects in OP patients.