METTL3 Knockdown Research Articles

METTL3 regulates the proliferation, metastasis and EMT progression of bladder cancer through P3H4

Bladder cancer, the most common malignant tumor in the urinary system, exhibits significantly up-regulated expression of P3H4, which is associated with pathological factors. The objective of this study was to elucidate the underlying mechanism of P3H4 in bladder cancer. Initially, we analyzed P3H4 gene expression using the TCGA database and evaluated P3H4 levels in clinical samples and various bladder cell lines. P3H4 was found to be markedly overexpressed in bladder cancer samples. Subsequently, bladder cancer cells were transfected with shRNA targeting P3H4 (sh-P3H4), sh-METTL3, and P3H4 overexpression vectors (P3H4 OE). Viability, migration, and invasion of bladder cancer cells were assessed using CCK-8, wound healing, and transwell assays. Western blot analysis was performed to determine the levels of EMT-associated proteins, while RNA stability assays determined the half-life of P3H4. Knockdown of P3H4 resulted in inhibition of bladder cancer cell proliferation, migration, invasion, and EMT progression. Mechanistically, METTL3 was found to regulate the mRNA stability of P3H4 in bladder cancer. Moreover, overexpression of P3H4 reversed the inhibitory effects of METTL3 knockdown on bladder cancer cell behaviors. Stable cell lines were established by infecting EJ cells with lentiviral vectors containing sh-METTL3 or P3H4 OE. These cells were then implanted into the skin of BALB/c nude mice, and IHC analysis was used to analyze the expression levels of EMT-associated proteins. In vivo studies demonstrated that inhibition of METTL3 suppressed bladder cancer growth and EMT through P3H4. In conclusion, our findings suggest that METTL3 regulates the proliferation, metastasis, and EMT progression of bladder cancer through P3H4, highlighting its potential as a therapeutic target.

METTL3-mediated lncRNA HOXD-AS1 stability regulates inflammation, and the migration and invasion of trophoblast cells via the miR-135a/ β-TRCP axis

BackgroundPreeclampsia (PE) is a serious pregnancy-specific syndrome associated with the inadequate invasion of trophoblast cells and inflammation of the uterus. A previous study found that lncRNA HOXD-AS1 promotes PE. However, its regulatory network requires additional exploration. MethodsHOXD-AS1-targeted miRNAs and genes were predicted by different databases in a bioinformatics analysis. The expression HOXD-AS1 and its potential m6A methylase (METTL3) were detected in placentas from healthy female patients with PE. The targeting relationship and role of the HOXD-AS1/miR-135a/β-TRCP axis in trophoblast cells were demonstrated by overexpression/knockdown assays. The levels of β-TRCP downstream pathway proteins IκBα, NF-κB, and p65 were measured. The levels of inflammatory factors in cell supernatants were detected by ELISA. To verify the molecular mechanism of β-TRCP in PE, IκBα was co-overexpressed in β-TRCP in trophoblast cells. ResultsThe levels of METTL3, HOXD-AS1, and β-TRCP were elevated in PE placental tissues, while miR-135a levels were reduced. MiR-135a was found to be targeted by HOXD-AS1, and HOXD-AS1 expression was maintained at a high level by METTL3-mediated m6A methylation. Overexpression of METTL3, HOXD-AS1, and β-TRCP, and knockdown of miR-135a in HTR-8/SVneo cells all inhibited cell invasion and migration, and promoted apoptosis and the secretion of inflammatory factors. Knockdown of METTL3, HOXD-AS1, and β-TRCP, and overexpression of miR-135a had the opposite effects. Furthermore, IκBα expression was negatively associated with β-TRCP expression, and the levels of NF-κB, p65, and NLRP3 were positively regulated by β-TRCP. A high level of β-TRCP expression attenuated the effect of HOXD-AS1 knockdown in trophoblast cells. ConclusionMETTL3 functioned to maintain a high level of HOXD-AS1 expression in PE, which influenced inflammation and the migration and invasion of trophoblast cells via the miR-135a/β-TRCP axis and NF-κB pathway.

Mettl3/Ythdf2 regulate macrophage inflammation and ROS generation by controlling Pyk2 mRNA stability

As one of the most prevalent modifications on RNA, N6-methyladenosine (m6A) has been recently found implicated in various pathological processes. Emerging studies have demonstrated the role of m6A and its writer Mettl3 in fine-tuning the immune response, which now becomes a research hotspot owing to its potential therapeutic value. However, the results are inconsistent and even contradictory, suggesting that there might be multiple Mettl3 target genes involved in different pathways. To delve deeper into the function of Mettl3 in the cellular inflammatory response, we first conducted bioinformatics analysis using RNA-seq in Mettl3 ablation macrophages, and found that Mettl3 might attenuate LPS-induced proinflammatory pathways and reactive oxygen species (ROS) generation process. Mettl3 knockdown significantly increased the LPS-induced IL-6, TNF-α, NOXs (Nox1, Nox2, Ncf1, and Ncf2) expression, ROS generation, and the phosphorylation of MAPKs and AKT signaling. Combining the results of RNA-seq and m6A mapping, we found that Pyk2 might be the target gene of Mettl3 affecting the inflammatory response. Mettl3 and Ythdf2 depletion increased the expression and mRNA stability of Pyk2, and RIP-PCR showed that Ythdf2 directly targeting Pyk2 was Mettl3 dependent. Moreover, the upregulated expression of TNF-α, IL-6, NOXs, ROS generation, and the phosphorylation of MAPKs and AKT signaling were downregulated by Pyk2 inhibitor in Mettl3 knockdown cells. All of these results suggest that Mettl3 regulates the mRNA stability and expression of Pyk2 in a Ythdf2-dependent way, which consequently triggers the activation of MAPKs and AKT signaling and upregulation of NOXs, thus promoting the generation of proinflammatory cytokines and ROS.

M6A regulates breast cancer proliferation and migration through stage-dependent changes in Epithelial to Mesenchymal Transition gene expression.

While many factors have been implicated in breast cancer progression, effective treatments are still lacking. In recent years, it has become clear that posttranscriptional regulation plays a key role in the aberrant gene expression underlying malignancy and metastasis. For example, the mRNA modification N6-methyladenosine (m6A) is involved in numerous post-transcriptional regulation processes and has been implicated in many cancer types, including breast cancer. Despite intense study, even within a single type of cancer, there is little consensus, and often conflicting results, as to the role of m6A, suggesting other factors must influence the process. The goal of this study was to determine if the effects of m6A manipulation on proliferation and migration differed based on the stage of disease progression. Using the MCF10 model of breast cancer, we reduced m6A levels by targeting METTL3, the main cellular m6A RNA methyltransferase. Knocking down Mettl3 at different stages of breast cancer progression indeed shows unique effects at each stage. The early-stage breast cancer line showed a more proliferative phenotype with the knockdown of Mettl3 while the transformed breast cancer line showed a more migratory phenotype. Interestingly, the metastasized breast cancer cell line showed almost no effect on phenotype with the knockdown of Mettl3. Furthermore, transcriptome wide analysis revealed EMT as the probable pathway influencing the phenotypic changes. The results of this study may begin to address the controversy of m6A's role in cancer and suggest that m6A may have a dynamic role in cancer that depends on the stage of progression.

METTL3-mediated m6A methylation of C1qA regulates the Rituximab resistance of diffuse large B-cell lymphoma cells

Rituximab has been incorporated into the standard treatment regimen for diffuse large B-cell lymphoma (DLBCL), and induces the death of tumor cells via complement-dependent cytotoxicity (CDC). Unfortunately, the resistance of DLBCL cells to Rituximab limits its clinical usefulness. It remains unclear whether the complement system is related to Rituximab resistance in DLBCL. A Rituximab-resistant DLBCL cell line (Farage/R) was generated under the stress of Rituximab. Constituent proteins of the complement system in wild-type Farage cells (Farage/S) and Farage/R cells were analyzed by qPCR, western blotting, and immunofluorescence. In vitro and in vivo knockdown and overexpression studies confirmed that the complement 1Q subcomponent A chain (C1qA) was a regulator of Rituximab resistance. Finally, the mechanism by which C1qA is regulated by m6A methylation was explored. The reader and writer were identified by pull-down studies and RIP-qPCR. Activity of the complement system in Farage/R cells was suppressed. C1qA expression was reduced in Farage/R cells due to post-transcriptional regulation. Furthermore, in vitro and in vivo results showed that C1qA knockdown in Farage/S cells decreased their sensitivity to Rituximab, and C1qA overexpression in Farage/R cells attenuated the Rituximab resistance of those cells. Moreover, METTL3 and YTHDF2 were proven to be the reader and writer for m6A methylation of C1qA, respectively. Knockdown of METTL3 or YTHDF2 in Farage/R cells up-regulated C1qA expression and reduced their resistance to Rituximab. In summary, the aberrant downregulation of C1qA was related to Rituximab resistance in DLBCL cells, and C1qA was found to be regulated by METTL3- and YTHDF2-mediated m6A methylation. Enhancing the response of the complement system via regulation of C1qA might be an effective strategy for inhibiting Rituximab resistance in DLBCL.

METTL14-mediated N6-methyladenosine modification of Col17a1/Itgα6/Itgβ4 governs epidermal homeostasis

BackgroundN6-methyladenosine (m6A) is the most abundant and reversible modification occurring in eukaryotic mRNAs, however, its functions in mammalian epidermal development are still not fully elucidated. ObjectiveTo explore the role of METTL14 (Methyltransferase like 14), one of the m6A methyltransferases, in maintaining epidermal homeostasis. MethodsWe constructed mice with Mettl14-inactivation in the epidermal basal cells. The phenotype was explored by H&E staining and immunofluorescence staining. To explore the underlying mechanisms, we performed RNA-seq, Ribosome profiling and MeRIP-seq on wild-type and Mettl14-inactivation epidermal keratinocytes. Moreover, HaCaT cells were used for in vitro validation. ResultsInactivation of Mettl14 in murine epidermis led to transient thicker epidermis and exhaustion of the epidermal stem cell pool. Interestingly, we found that the mRNA of type XVII collagen (Col17a1), integrin β4 (Itgβ4) and α6 (Itgα6) had m6A modifications, and the proteins expression were decreased in Mettl14-inactivated epidermis. Furthermore, in epidermis-specific Mettl4-inactivated mice, the epidermis was detached from the dermis and presented a phenotype similar to junctional epidermolysis bullosa (JEB), which may result from hemidesmosomes damage (decrease of COL17A1, ITGB4 and ITGA6). Knockdown of Mettl14 in HaCaT cells impaired the self-renewal and decreased the protein level of COL17A1, ITGB4 and ITGA6 and Itgβ4 knockdown inhibited colony formation. ConclusionOur study highlighted the role of METTL14 in the maintenance of epidermal homeostasis and identified its critical role through m6A-mediated translational inhibition of Col17a1, Itgβ4 and Itgα6. Our study suggested that METTL14 may be a potential therapeutic target for the treatment of hemidesmosomes-deficient diseases, such as JEB.

Juvenile hormone regulates silk gene expression by m6A RNA methylation.

Juvenile hormone (JH) is an indispensable insect hormone that is critical in regulating insect development and physiology. N6-methyladenosine (m6A) is the most abundant modification of RNA that regulates RNA fate in eukaryotic organisms. However, the relationship between m6A and JH remains largely unknown. Here, we found that the application of a Juvenile hormone analog (JHA) extended the larval period of Bombyxmori and increased the weight and thickness of the cocoon. Interestingly, global transcriptional patterns revealed that m6A-related genes are specifically regulated by JHA in the posterior silk gland (PSG) that synthesizes the major component of cocoon silk. By transcriptome and m6A sequencing data conjointly, we discovered that JHA significantly regulated the m6A modification in the PSG of B. mori and many m6A-containing genes are related to nucleic acid binding, nucleus, and nucleobase-containing compound metabolism. Notably, 547 genes were significantly regulated by JHA at both the m6A modification and expression levels, especially 16 silk-associated genes, including sericin2, seroin1, Serine protease inhibitors 4 (BmSPI4), Serine protease inhibitors 5 (BmSPI5), and LIM domain-binding protein 2 (Ldb). Among them, 11 silk associated genes were significantly affected by METTL3 knockdown, validating that these genes are targets of m6A modification. Furthermore, we confirm that JHA directly regulates the expression of BmSPI4 and BmSPI5 through m6A modification of CDS regions. These results demonstrate the essential role of m6A methylation regulated by JH in PSG, and elucidate a novel mechanism by which JH affects silk gland development via m6A methylation. This study uncoversthatm6A modification is a critical factor mediating the effect of JH in insects.

Low RNA stability signifies strong expression regulatability of tumor suppressors.

RNA expression of a gene is determined by not only transcriptional regulation, but also post-transcriptional regulation of RNA decay. The precise regulation of RNA stability in the cell plays an important role in normal development. Dysregulation of RNA stability can lead to diseases such as cancer. Here we found tumor suppressor RNAs tended to decay fast in normal cell types when compared with other RNAs. Consistent with a negative effect of m6A modification on RNA stability, we observed preferential deposition of m6A on tumor suppressor RNAs. Moreover, abundant m6A and fast decay of tumor suppressor RNAs both tended to be further enhanced in prostate cancer cells relative to normal prostate epithelial cells. Further, knockdown of m6A methyltransferase METTL3 and reader YTHDF2 in prostate cancer cells both posed stronger effect on tumor suppressor RNAs than on other RNAs. These results indicated a strong post transcriptional expression regulatability mediated by abundant m6A modification on tumor suppressor RNAs.

Influence of METTL3 knockdown on PDLSC osteogenesis in E. coli LPS-induced inflammation.

This study aimed to investigate the effect of METTL3 knockdown on osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) in the weak inflammation microenvironments, as well as the underlying mechanisms. PDLSCs were stimulated by lipopolysaccharide from Escherichia coli (E. coli LPS), followed by quantification of METTL3. METTL3 expression was assessed using RT-qPCR and Western blot analysis in periodontitis. METTL3 knockdown PDLSCs were stimulated with or without E. coli LPS. The evaluation included proinflammatory cytokines, osteogenic markers, ALP activity, and mineralized nodules. Bioinformatics analysis and Western blot determined the association between METTL3 and the PI3K/Akt pathway. METTL3 was overexpressed in periodontitis. METTL3 knockdown in PDLSCs reduced proinflammatory cytokines, osteogenic markers, ALP activity, and mineralized nodules in both environments. Bioinformatics analysis suggested a link between METTL3 and the PI3K/Akt pathway. METTL3 knockdown inhibited PI3K/Akt signaling pathway activation. METTL3 knockdown might inhibit osteogenesis in PDLSCs through the inactivation of PI3K/Akt signaling pathway. Concomitant findings might shed novel light on the roles and potential mechanisms of METTL3 in the LPS-stimulated inflammatory microenvironments of PDLSCs.

METTL3 and METTL14 regulate IL-6 expression via RNA m6A modification of zinc transporter SLC39A9 and DNA methylation of IL-6 in periodontal ligament cells

The inflammatory response is a key process in periodontitis. The N6-methyladenosine (m6A) modification has been proven to be involved in various physiological and pathological processes. This study aims to investigate the role and downstream mechanism of N6-adenosine-enzyme subunits methyltransferase (METTL) 3 and 14 in the inflammatory response of periodontal ligament cells (PDLCs). The total m6A content and the expression of METTL3 and METTL14 were upregulated in lipopolysaccharide (LPS)-stimulated PDLCs. Knockdown of METTL3 or METTL14 suppressed the LPS-induced interleukin (IL)-6 expression, as shown by quantitative polymerase chain reaction (qPCR) and enzyme linked immunosorbent assay (ELISA). Mechanistically, conjoint analysis of m6A sequencing of METTL3-knockdown and METTL14-knockdown PDLCs revealed that the expression of solute carrier family 39 member 9 (SLC39A9) was mediated in a m6A-dependent manner. The suppression of LPS-induced IL-6 by METTL3 or METTL14 knockdown was partially counteracted by SLC39A9 knockdown, which induced downregulation of intracellular zinc via immunofluorescence staining. Amplicon bisulfite sequencing (AmpBS) demonstrated that METTL3/14 knockdown increased the methylation at one position of the IL-6 promoter, while SLC39A9 knockdown decreased it, which was basically consistent with the intracellular zinc concentration and negatively associated with IL-6 expression. Moreover, METTL3 or METTL14 knockdown attenuated the LPS-induced phosphorylation of p38 and JNK mitogen-activated protein kinase (MAPK), which was partially counteracted by SLC39A9 knockdown. These results revealed the “LPS-METTL3/14-SLC39A9-zinc-IL-6” axis and involvement of p38 and JNK MAPK signaling pathway in the inflammatory responses of PDLCs.

METTL3 exerts synergistic effects on m6A methylation and histone modification to regulate the function of VGF in lung adenocarcinoma

BackgroundMultiple genetic and epigenetic regulatory mechanisms play a vital role in tumorigenesis and development. Understanding the interplay between different epigenetic modifications and its contribution to transcriptional regulation in cancer is essential for precision medicine. Here, we aimed to investigate the interplay between N6-methyladenosine (m6A) modifications and histone modifications in lung adenocarcinoma (LUAD).ResultsBased on the data from public databases, including chromatin property data (ATAC-seq, DNase-seq), methylated RNA immunoprecipitation sequencing (MeRIP-seq), and gene expression data (RNA-seq), a m6A-related differentially expressed gene nerve growth factor inducible (VGF) was identified between LUAD tissues and normal lung tissues. VGF was significantly highly expressed in LUAD tissues and cells, and was associated with a worse prognosis for LUAD, silencing of VGF inhibited the malignant phenotype of LUAD cells by inactivating the PI3K/AKT/mTOR pathway. Through the weighted correlation network analysis (WGCNA) and integration of TCGA-LUAD RNA-seq and m6A methyltransferase METTL3-knockdown RNA-seq data, a significant positive correlation between METTL3 and VGF was observed. By using the MeRIP-qPCR and dual-luciferase reporter assays, we demonstrated that METTL3 knockdown decreased m6A modification level of VGF coding sequences in LUAD cells, the colorimetric m6A quantification assay also showed that METTL3 knockdown significantly decreased global m6A modification level in LUAD cells. Interestingly, we found that METTL3 knockdown also reduced VGF expression by increasing H3K36me3 modification at the VGF promoter. Further research revealed that METTL3 knockdown upregulated the expression of histone methylase SETD2, the major H3K36me3 methyltransferase, by methylating the m6A site in the 3'UTR of SETD2 mRNA in LUAD cells.ConclusionsOverall, our results reveal that the expression of VGF in LUAD cells is regulated spatio-temporally by METTL3 through both transcriptional (via histone modifications) and post-transcriptional (via m6A modifications) mechanisms. The synergistic effect of these multiple epigenetic mechanisms provides new opportunities for the diagnosis and precision treatment of tumors.

Curdione induces ferroptosis mediated by m6A methylation via METTL14 and YTHDF2 in colorectal cancer

BackgroundCurdione is a sesquiterpene isolated from Curcumae Rhizoma that possesses high biological activity and extensive pharmacological effects. As a traditional Chinese medicine, Curcumae Rhizoma can inhibit the development of many types of cancer, especially colorectal cancer. However, the anti-colorectal mechanism of its monomer curdione remains unclear.MethodsColorectal cancer (CRC) cells were treated with curdione at doses of 12.5 μM, 25 μM, and 50 μM, and then the cells’ activity was measured with methyl thiazolyl tetrazolium (MTT). Nude mice were administered different doses of curdione subcutaneously and oxaliplatin by tail vein injection, and then hematoxylin–eosin (HE) staining was adopted to examine tumor histology. Moreover, flow cytometry was applied to detect reactive oxygen species in cells and tissues. Kits were employed to detect the levels of iron ions, malondialdehyde, lipid hydroperoxide, and glutathione. Polymerase chain reaction (PCR) and Western blotting were adopted to detect ferroptosis and m6A modification-related factors. A methylation spot hybridization assay was performed to measure changes in overall methylation. SLC7A11 and HOXA13 were measured by MeRIP-qPCR. The shRNA-METTL14 plasmid was constructed to verify the inhibitory effect of curdione on CRC.ResultsA dose-dependent decrease in activity was observed in curdione-treated cells. Curdione increased the accumulation of reactive oxygen species in CRC cells and tumor tissues, greatly enhanced the levels of malondialdehyde, lipid hydroperoxide and Fe2+, and lowered the activity of glutathione. According to the qPCR and Western blot results, curdione promoted the expression of METTL14 and YTHDF2 in CRC cells and tissues, respectively, and decreased the expression of SLC7A11, SLC3A2, HOXA13, and glutathione peroxidase 4. Additionally, in animal experiments, the curdione-treated group showed severe necrosis of tumor cells, as displayed by HE staining. Furthermore, compared with the control group, levels of m6A modifying factors (namely, SLC7A11 and HOXA13) were increased in the tissues after drug intervention. METTL14 knockdown was followed by an increase in CRC cell activity and glutathione levels. However, the levels of reactive oxygen species, malondialdehyde, and iron ions decreased. The expression levels of SLC7A11, SLC3A2, HOXA13, and GPX4 were all increased after METTL14 knockdown.ConclusionThe results suggest that curdione induces ferroptosis in CRC by virtue of m6A methylation.

METTL3 Promotes the Differentiation of Goat Skeletal Muscle Satellite Cells by Regulating MEF2C mRNA Stability in a m6A-Dependent Manner.

The development of mammalian skeletal muscle is a highly complex process involving multiple molecular interactions. As a prevalent RNA modification, N6-methyladenosine (m6A) regulates the expression of target genes to affect mammalian development. Nevertheless, it remains unclear how m6A participates in the development of goat muscle. In this study, methyltransferase 3 (METTL3) was significantly enriched in goat longissimus dorsi (LD) tissue. In addition, the global m6A modification level and differentiation of skeletal muscle satellite cells (MuSCs) were regulated by METTL3. By performing mRNA-seq analysis, 8050 candidate genes exhibited significant changes in expression level after the knockdown of METTL3 in MuSCs. Additionally, methylated RNA immunoprecipitation sequencing (MeRIP-seq) illustrated that myocyte enhancer factor 2c (MEF2C) mRNA contained m6A modification. Further experiments demonstrated that METTL3 enhanced the differentiation of MuSCs by upregulating m6A levels and expression of MEF2C. Moreover, the m6A reader YTH N6-methyladenosine RNA binding protein C1 (YTHDC1) was bound and stabilized to MEF2C mRNA. The present study reveals that METTL3 enhances myogenic differentiation in MuSCs by regulating MEF2C and provides evidence of a post-transcriptional mechanism in the development of goat skeletal muscle.

METTL14 inhibits malignant progression of oral squamous cell carcinoma by targeting the autophagy-related gene RB1CC1 in an m6A-IGF2BP2-dependent manner.

N6-methyladenosine (m6A) plays crucial roles in tumorigenesis and autophagy. However, the underlying mechanisms mediated by m6A and autophagy in the malignant progression of oral squamous cell carcinoma (OSCC) remain unclear. In the present study, we revealed that down-regulated expression of METTL14 was correlated with advanced clinicopathological characteristics and poor prognosis in OSCC. METTL14 knockdown significantly inhibited autophagy and facilitated malignant progression in vitro, and promoted tumor growth and metastasis in vivo. A cell model of rapamycin-induced autophagy was established to identify RB1CC1 as a potential target gene involved in m6A-regulated autophagy in OSCC, through RNA sequencing and methylated RNA immunoprecipitation sequencing (meRIP-seq) analysis. Mechanistically, we confirmed that METTL14 posttranscriptionally enhanced RB1CC1 expression in an m6A-IGF2BP2-dependent manner, thereby affecting autophagy and progression in OSCC, through methylated RNA immunoprecipitation qRT-PCR (meRIP-qPCR), RNA stability assays, mutagenesis assays and dual-luciferase reporter. Collectively, our findings demonstrated that METTL14 serves as an OSCC suppressor by regulating the autophagy-related gene RB1CC1 through m6A modification, which may provide a new insight for the diagnosis and therapy of OSCC.

METTL3-mediated N6-methyladenosine exacerbates ferroptosis via m6A-IGF2BP2-dependent mitochondrial metabolic reprogramming in sepsis-induced acute lung injury.

Neutrophil extracellular traps (NETs), released by polymorphonuclear neutrophils (PMNs), exert a robust antimicrobial function in infectious diseases such as sepsis. NETs also contribute to the pathogenesis and exacerbation of sepsis. Although the lung is highly vulnerable to infections, few studies have explored the role of NETs in sepsis-induced acute lung injury (SI-ALI). We demonstrate that NETs induce SI-ALI via enhanced ferroptosis in alveolar epithelial cells. Our findings reveal that the excessive release of NETs in patients and mice with SI-ALI is accompanied by upregulation of ferroptosis depending on METTL3-induced m6A modification of hypoxia-inducible factor-1α (HIF-1α) and subsequent mitochondrial metabolic reprogramming. In addition to conducting METTL3 overexpression and knockdown experiments in vitro, we also investigated the impact of ferroptosis on SI-ALI caused by NETs in a caecum ligation and puncture (CLP)-induced SI-ALI model using METTL3 condition knockout (CKO) mice and wild-type mice.Our results indicate the crucial role of NETs in the progression of SI-ALI via NET-activated METTL3 m6A-IGF2BP2-dependent m6A modification of HIF-1α, which further contributes to metabolic reprogramming and ferroptosis in alveolar epithelial cells.

M6A-modified circABCC4 promotes stemness and metastasis of prostate cancer by recruiting IGF2BP2 to increase stability of CCAR1.

Prostate cancer (PCa) is a malignant tumor of the urinary system. CircABCC4 has been demonstrated to promote the development of PCa; however, its regulatory mechanisms in PCa progression remain largely unknown. We found that circABCC4 was highly expressed in PCa tissues and cells, and elevated circABCC4 level indicated a poor overall survival of PCa patients. METTL3 overexpression increased circABCC4 expression via m6A modification in PCa cells. Functionally, knockdown of circABCC4 or METTL3 repressed PCa cell stemness, migration, and invasion in vitro and delayed PCa cancer growth and metastasis in vivo. circABCC4 knockdown-mediated inhibition in PCa cell stemness and metastasis could be counteracted by overexpression of wild-type circABCC4 with m6A sites. Mechanistically, circABCC4 recruited IGF2BP2 protein to CCAR1 mRNA, thereby enhancing CCAR1 mRNA stability and subsequent activation of the Wnt/β-catenin pathway. Overexpression of CCAR1 counteracted the inhibitory effect of circABCC4 silencing on PCa cell stemness and metastasis. These results revealed that m6A-modified circABCC4 by METTL3 facilitated PCa cell stemness and metastasis by interacting with IGF2BP2 to increase the stability and expression of CCAR and subsequent expression of Wnt/β-catenin target genes. Our findings suggest circABCC4 as a promising therapeutic target for PCa.

LncRNA THRIL, transcriptionally activated by AP-1 and stabilized by METTL14-mediated m6A modification, accelerates LPS-evoked acute injury in alveolar epithelial cells

Acute lung injury (ALI) and its extreme manifestation, acute respiratory distress syndrome (ARDS), are life-threatening diseases in intensive care units. LncRNA THRIL plays a crucial role in regulating the inflammatory response; however, the potential function of THRIL in ALI/ARDS and the associated mechanism remain unclear. In our study, we found that THRIL was upregulated in the serum of ALI/ARDS patients, and its increased expression was positively correlated with the inflammatory cytokines IL-17. In LPS-induced A549 cells, knockdown of THRIL inhibited the release of the proinflammatory cytokines TNF-α, IL-1β, IL-17, and IL-6, decreased the number of monodansylcadaverine-positive cells and LC3-II with immunofluorescence staining, decreased the expression of autophagy marker ATG7 and Beclin1, and increased expression of p62. Mechanistically, the transcription factor AP-1 bound directly to the THRIL promoter region and activated its transcription by c-Jun upon LPS exposure. Moreover, m6A modification of THRIL was increased in LPS-treated A549 cells, and METTL14 knockdown significantly abolished m6A modification and reduced stabilization of THRIL mRNA. In conclusion, our findings reveal that THRIL, transcriptionally activated by AP-1 and modified by METTL14-mediated m6A modification, induces autophagy in LPS-treated A549 cells, suggesting the potential application of THRIL for ALI/ARDS therapy.

Klf6 aggravates myocardial ischemia/reperfusion injury by activating Acsl4-mediated ferroptosis.

Ferroptosis is closely related to myocardial ischemia/reperfusion (I/R) damage. Kruppel-like factor 6 (Klf6) can aggravate renal I/R injury. We aimed to elucidate the role of Klf6 in myocardial I/R damage as well as its potential mechanism. Myocardial I/R mice model and hypoxia/reoxygenation (H/R)-treated HL-1 cells were established. The levels of Fe2+ , MDA, lipid ROS, and ferroptosis-related proteins were measured for assessing ferroptosis. Infarct area, H&E staining, cardiac function, and cell viability were detected for evaluating myocardial injury. Immunohistochemistry, immunofluorescence, western blot, and RT-qPCR were applied for detecting the levels of related genes. The m6A modification of Klf6, as well as the relationships between Klf6 and Mettl3, Igf2bp2, or Acsl4 promoter, was evaluated using MeRIP, RNA immunoprecipitation, RNA pull-down, chromatin immunoprecipitation, and luciferase reporter assay accordingly.Klf6 protein and mRNA levels, as well as Klf6 m6A modification, were elevated in HL-1 cells subjected to H/R and in the heart tissues from I/R mice. In H/R-challenged HL-1 cells, the binding relationships between Klf6 mRNA and Igf2bp2 or Mettl3 were confirmed; moreover, Igf2bp2 or Mettl3 knockdown decreased the Klf6 level and inhibited Klf6 mRNA stability. Klf6 knockdown restrained H/R-triggered cell viability loss, improved I/R-induced myocardial injury, and inhibited ferroptosis in myocardial I/R damage models. Klf6 directly bound to the Acsl4 promoter and positively regulated its expression. Acsl4 overexpression compromised the Klf6 knockdown-generated protective effect in HL-1 cells.m6A modification-regulated Klf6 aggravated myocardial I/R damage through activating Acsl4-mediated ferroptosis, thereby providing one potential target for the treatment of myocardial I/R.

METTL14 reverses liver fibrosis by inhibiting NOVA2 through an m6A-YTHDF2-dependent mechanism.

N6-methyladenosine (m6A), the most prevalent internal RNA modification in eukaryotic cells, is dynamically regulated in response to a wide range of physiological and pathological states. Nonetheless, the involvement of METTL14-induced m6A in liver fibrosis (LF) has yet to be established. In vitro, HSC cell lines with knock-down and overexpression of METTL14 were constructed, and the effects of METTL14 gene on the phenotypic function of activated HSCs were observed. The proliferation rate was measured by CCK8 and EDU, the cell proliferation cycle was measured by flow detector, the migration rate was measured by Transwell, and the contractility of F-actin was observed after phalloidin staining. The downstream target gene NOVA2 of METTL14 was screened by combined sequencing of MeRIP-seq and RNA-seq, combined with signal analysis. Adeno-associated virus (AAV) was injected into the tail vein in vivo to knock down the expression of METTL14, so as to further observe the role of METTL14 in the progress of LF. our research showed that the methylase METTL14 content was decreased in hepatic tissue from patients with LF, leading to a lowered degree of m6A modification. Functionally, we discovered that knocking down m6A methyltransferase METTL14 led to increased HSC activation and a substantial worsening of LF. Mechanically, as shown in a multiomics study of HSCs, depleting METTL14 levels decreased m6A deposition onNOVA2 mRNA transcripts, which prompted the activation of YTHDF2 to detect and degrade the decrease of NOVA2 mRNA. METTL14 functioned as a profibrotic gene by suppressing NOVA2 activity in a mechanism dependent on m6A-YTHDF2. Moreover, knocking down METTL14 exacerbated LF, while NOVA2 prevented its development and partly reversed the damage.

METTL3-Mediated Maturation of miR-99a-5p Promotes Cell Migration and Invasion in Oral Squamous Cell Carcinoma by Targeting ZBTB7A.

METTL3 is an important methyltransferase in N(6)-methyladenosine (m6A) modification. Recently, METTL3 mediates methylation of pri-microRNA (miRNA) to accelerate miRNA maturation, regulating tumor development. This study explored whether METTL3 mediated miR-99a-5p to influence oral squamous cell carcinoma (OSCC) cell metastasis. MiR-99a-5p, ZBTB7A, and MATTL3 expression was measured using quantitative real-time PCR. Biological behaviors were assessed using cell counting kit-8, flow cytometry, Transwell assay, as well as western blot. Luciferase reporter assay evaluated the interaction between miR-99a-5p and ZBTB7A. METTL3-regulated pri-miR-99a-5p processing was determined by RNA binding protein immunoprecipitation (RIP) and methylated RNA immunoprecipitation (MeRIP) assays. The consequences clarified that miR-99a-5p was upregulated in OSCC cells. Downregulation of miR-99a-5p suppressed cellular viability, migration, invasion, and epithelial-mesenchymal transition (EMT), and induced apoptosis. ZBTB7A acted as a miR-99a-5p target and reversed the effects on cellular behaviors induced by miR-99a-5p inhibitor. m6A content and METTL3 expression were increased in OSCC cells. METTL3 promoted the m6A modification of pri-miR-99a-5p and thereby facilitated miR-99a-5p processing. Moreover, knockdown of METTL3 inhibited OSCC metastasis by downregulating miR-99a-5p. Taken together, METTL3 promoted miR-99a-5p maturation in an m6A-dependent manner, which further targets ZBTB7A to accelerate the progression of OSCC. These findings suggest potential targets for OSCC therapy.