METTL3 Expression Research Articles

LncRNA RASAL2-AS1 promotes METTL14-mediated m6A methylation in the proliferation and progression of head and neck squamous cell carcinoma

BackgroundLong non-coding RNAs (lncRNAs) are key regulators of the 6-methyladenosine (m6A) epigenetic modification, playing a role in the initiation and progression of tumors. However, the regulatory mechanisms in head and neck squamous cell carcinoma (HNSCC) remain elusive. In this study, we investigated the molecular regulatory mechanisms of the lncRNA RASAL2-AS1 in the occurrence and development of HNSCC tumors.MethodsA bioinformatics analysis was conducted to analyze the expression level of RASAL2-AS1 in HNSCC and normal tissues. RASAL2-AS1 mRNA and protein levels were detected using RT-PCR and Western blotting. Wound healing, transwell assays, flow cytometry, M6A dot blot, and RNA immunoprecipitation experiments were conducted to explore the regulatory role of the RASAL2-AS1 and downstream targets METTL14/LIS1 signaling pathway in HNSCC. Immunohistochemical examination was conducted to evaluate the expression of METTL14 and LIS1 in HNSCC and normal tissues. A tumor xenograft model of BALB/c nude mice was established to assess the impact of RASAL2-AS1 on cell proliferation and growth.ResultsRASAL2-AS1 high expression in HNSCC and cells deteriorated with survival rates of HNSCC. RASAL2-AS1 overexpression in HNSCC accelerated cell migration, colony formation, cell proliferation, cell cycle in S stage, while RASAL2-AS1 knockdown in HNSC cells inhibited cell cycle in G1 stage. After silencing METTL14, the above effects induced by overexpression of the RASAL2-AS1 were reversed. RASAL2-AS1 overexpression prompted LIS1 expression, whereas RASAL2-AS1 silencing reduced LIS1 levels in HNSCC cells, which was confirmed by immunohistological staining. Results demonstrated elevated expression of METTL14 or LIS1 in tongue cancer tissues. Overexpression of RASAL2-AS1 promoted tumor weight and tumor volume, which was counteracted by pcDNA3.1 RASAL2-AS1 plus silencing METTL14 and METTL14 and LIS1 were significantly decreased.ConclusionOur study highlights the functional importance of the LncRNA RASAL2-AS1 in HNSCC and might assist in the development of a prognostic stratification and therapeutic approach. Which regulates HNSCC with the dependence of m6a manner.

Abstract 3233: EP102: Pharmacological Inhibition of METTL3 causes tumor growth inhibition and prolongs survival in preclinical models of NSCLC, ovarian and squamous cell carcinoma

Abstract METTL3 is the RNA methyltransferase predominantly responsible for the addition of N-6-methyladenosine (m6A), the most abundant epigenetic modification to mRNA. The prevalence of m6A and the activity and expression of METTL3 have been linked to the appearance and progression of numerous hematological and solid tumor cancers. EPICS has discovered and optimized small molecule inhibitors of METTL3 (“M3i”). The aim of the current study is to evaluate the efficacy of M3i in various CDX mouse tumor models. M3i compound potency was evaluated in vitro by measuring enzymatic inhibition of METTL3/14 by scintillation proximity assay (IC50 = 2 nM). The anti-proliferative effects of M3i were measured in cell viability assays following a 72h treatment of A549 (NSCLC, IC50 = 190 nM), SKOV3 (ovarian, IC50 = 9 nM) and FaDu (hypopharyngeal squamous cell carcinoma, IC50 = 24 nM) cells. Complementary data in all three cell lines demonstrates a clear concentration-response for a target engagement biomarker (m6A; IC50 = 2-8 nM, across cell lines) as well as M3i-induced changes in biomarkers of oncogenic drivers. M3i (30 mg/kg, IP, QD) was tested in vivo in mouse models using FaDu (subcutaneous implant) in addition to A549 and SKOV3 (both orthotopic implants); all animal models were performed by LabCorp (MI, USA). Daily M3i treatment significantly prolonged survival in all three disease models consistent with efficacy on tumor growth inhibition as measured by imaging (A549, SKOV3) or direct measurement of tumor volume in the case of subcutaneous implant (FaDu). M3i has previously been shown to be effective in mouse CDX and PDX models of AML. The current results extend the application of METTL3 inhibitors to include the treatment of various types of solid tumors. The efficacy of M3i as a single-agent on tumor growth inhibition and survival advances the validation of METTL3 as a cancer target and provides additional evidence for the applicability of mRNA epigenetic mechanisms to the treatment of solid tumors. Citation Format: Graeme Fraser, Catherine Sorlet, Nicolas Parmentier, Elodie Brunel, Christian Coremans, Sarah Rorive, Anne-France Hartiel, Killian Oukoloff, Guillaume Dutheuil. EP102: Pharmacological Inhibition of METTL3 causes tumor growth inhibition and prolongs survival in preclinical models of NSCLC, ovarian and squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3233.

Abstract 1737: METTL3-mediated m6A modification promotes tumor progression in upper tract urothelial carcinoma

Abstract Upper tract urothelial carcinoma (UTUC), comprising 5% of urothelial neoplasms, is a rare malignancy arising from the transitional epithelium of the renal pelvis and ureter. Its rarity and diverse histology pose diagnostic and therapeutic challenges, often lead to delayed diagnoses. The absence of targeted therapies, coupled with a limited understanding of molecular pathogenesis, underscores the ongoing research in urological oncology for effective UTUC management. N6-methyladenosine (m6A) is the predominant RNA methylation in mammalian cells, with its inscription primarily orchestrated by METTL3, a crucial methyltransferase. METTL3 has been implicated in various cancers, affecting critical processes like proliferation, migration, and tumor progression. However, the specific mechanisms and biological function of how METTL3/m6A regulates the progression of UTUC remain unclear. In our study, we found that m6A modification and METTL3 expression were elevated in aggressive UTUC, both are negatively correlated with unfavorable clinical outcomes and poor patient survival. By overexpressing METTL3 in less aggressive UTUC cell lines BFTC909 and KTCC28, the abilities of cell proliferation, migration, invasion, and colony formation increased. Notably, overexpression of METTL3 also increased tumor growth and metastasis in the in vivo orthotopic mouse model. In contrast, knockdown of METTL3 decreases tumor progression in a highly aggressive UTUC cell line KTCC28M. Furthermore, our comparative analysis of RNA-seq and m6A MeRIP-seq datasets unveiled the regulation of 1660 genes by METTL3. This diverse set includes genes associated with tumor growth and metastasis (such as MYC, JAK2, and STAT3), as well as RNA processing-related genes which their critical roles in modulating mRNA stability and splicing (such as GEMIN5 and PABPC1L). Together, we noticed METTL3/m6A is a crucial protagonist in the oncogenic landscape of UTUC, which suggested that it can be used as a target for novel therapeutic strategy for UTUC treatment. Citation Format: LI-JIE LIN, PEI-HUA PENG, KAI-WEN HSU. METTL3-mediated m6A modification promotes tumor progression in upper tract urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1737.

Abstract 4417: Development of biochemical screening assays to facilitate drug discovery in RNA m6A modification regulators

Abstract Over the recent years, knowledge of RNA modifications and their regulation has expanded extensively, providing novel insights and strategies to explore potential therapeutics for pathogenesis of various diseases, including cancer. Several chemical modifications in RNA have been identified so far, among these N6-methyladenosine (m6A) modification is the most abundant and well-studied epitranscriptomic marker found in mRNA and long noncoding RNA. Abnormal m6A expression is proven to be associated with tumorigenesis, cancer stemness and drug resistance of cancers. Methylation in 6-Adenosin of RNA is a dynamic and reversible process tightly regulated by its writer, m6A methyltransferase complex and erasers, FTO and ALKBH5. m6A methyltransferase complex contains METTL3-METTL14-WTAP as core components. METTL3 is the catalytic component, which is activated by heterodimer formation with METTL14. High METTL3 expression is found in several cancers such as breast, lung, liver, gastric, colorectal, AML and a METTL3 catalytic inhibitor is found to delay AML progression in mouse models. Protein that binds RNA m6A modification to execute it signaling are known as m6A readers. Readers are categorized into three main classes, YTH domain proteins, IGF2 mRNA-binding proteins and heterogeneous nuclear ribonucleoproteins. YTH family readers are found to have oncogenic roles in several cancers including AML, breast, lung, CRC and glioblastoma. Recent studies suggest inhibition of m6A binding of individual YTH family proteins is a promising therapeutic strategy however, potent inhibitors are yet to be identified. This poster summarizes current assays we have developed to facilitate cancer therapeutic discovery in m6A related protein targets. We present development and validation of an assay suitable identify in METTL3 catalytic inhibitors using our proprietary hotspot technology. We also show development of HTRF based biochemical assays for all five YTH family protein YTHDF1-3 and YTHDC1-2, to screen for molecules that disrupt protein-m6A interaction. We further study the selectivity of an FDA approved drug Tegaserod, a reported YTHDF1 inhibitor by a structure based virtual screening, between YTH family proteins. Citation Format: Safnas F. AbdulSalam, Sung Won Oh, Brenna P. Lee, Joseph J. Ferry, John R. Ries, Kurumi Y. Horiuchi. Development of biochemical screening assays to facilitate drug discovery in RNA m6A modification regulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4417.

METTL3 Promotes Osteosarcoma Metastasis via an m6A-dependent Epigenetic Activity of CBX4.

Osteosarcoma cells are prone to metastasis, and the mechanism of N6-methyladenosine (m6A) methylation modification in this process is still unclear. Methylation modification of m6A plays an important role in the development of osteosarcoma, which is mainly due to abnormal expression of enzymes related to methylation modification of m6A, which in turn leads to changes in the methylation level of downstream target genes messenger RNA (mRNA) leading to tumor development. We analyzed the expression levels of m6A methylation modification-related enzyme genes in GSE12865 whole-genome sequencing data. And we used shRNA (short hairpin RNA) lentiviral interference to interfere with METTL3 (Methyltransferase 3) expression in osteosarcoma cells. We studied the cytological function of METTL3 by Cell Counting Kit-8 (CCK8), flow cytometry, migration and other experiments, and the molecular mechanism of METTL3 by RIP (RNA binding protein immunoprecipitation), Western blot and other experiments. We found that METTL3 is abnormally highly expressed in osteosarcoma and interferes with METTL3 expression in osteosarcoma cells to inhibit metastasis, proliferation, and apoptosis of osteosarcoma cells. We subsequently found that METTL3 binds to the mRNA of CBX4 (chromobox homolog 4), a very important regulatory protein in osteosarcoma metastasis, and METTL3 regulates the mRNA and protein expression of CBX4. Further studies revealed that METTL3 inhibited metastasis of osteosarcoma cells by regulating CBX4. METTL3 has been found to be involved in osteosarcoma cells metastasis by CBX4 affecting the protein expression of matrix metalloproteinase 2 (MMP2), MMP9, E-Cadherin and N-Cadherin associated with osteosarcoma cells metastasis. These results suggest that the combined action of METTL3 and CBX4 plays an important role in the regulation of metastasis of osteosarcoma, and therefore, the METTL3-CBX4 axis pathway may be a new potential therapeutic target for osteosarcoma.

20(R)-Panaxatriol enhances METTL3-mediated m6A modification of STUB1 to inhibit autophagy and exert antitumor effects in Triple-Negative Breast Cancer cells

BackgroundAberrant activation of autophagy in triple-negative breast cancer (TNBC) has led researchers to investigate potential therapeutic strategies targeting this process. The regulation of autophagy is significantly influenced by METTL3. Our previous research has shown that the Panax ginseng-derived compound, 20(R)-panaxatriol (PT), has potential as an anti-tumor agent.However, it remains unclear whether PT can modulate autophagy through METTL3 to exert its anti-tumor effects. ObjectiveOur objective is to investigate whether PT can regulate autophagy in TNBC cells and elucidate the molecular mechanisms. Study DesignFor in vitro experiments, we employed SUM-159-PT and MDA-MB-231 cells. While in vivo experiments involved BALB/c nude mice and NOD/SCID mice. MethodsIn vitro, TNBC cells were treated with PT, and cell lines with varying expression levels of METTL3 were established. We assessed the impact on tumor cell activity and autophagy by analyzing autophagic flux, Western Blot (WB), and methylation levels. In vivo, subcutaneous transplantation models were established in BALB/c nude and NOD/SCID mice to observe the effect of PT on TNBC growth. HE staining and immunofluorescence were employed to analyze histopathological changes in tumor tissues. MeRIP-seq and dual-luciferase reporter gene assays were used to identify key downstream targets. Additionally, the silencing of STIP1 Homology And U-Box Containing Protein 1 (STUB1) explored PT's effects. The mechanism of PT's action on STUB1 via METTL3 was elucidated through mRNA stability assays, mRNA alternative splicing analysis, and nuclear-cytoplasmic mRNA separation. ResultsIn both in vivo and in vitro experiments, it was discovered that PT significantly upregulates the expression of METTL3, leading to autophagy inhibition and therapeutic effects in TNBC. Simultaneously, through MeRIP-seq analysis and dual-luciferase reporter gene assays, we have demonstrated that PT modulates STUB1 via METTL3, influencing autophagy in TNBC cells. Furthermore, intriguingly, PT extends the half-life of STUB1 mRNA by enhancing its methylation modification, thereby enhancing its stability. ConclusionIn summary, our research reveals that PT increases STUB1 m6A modification through a METTL3-mediated mechanism in TNBC cells, inhibiting autophagy and further accentuating its anti-tumor properties. Our study provides novel mechanistic insights into TNBC pathogenesis and potential drug targets for TNBC.

Specific deletion of Mettl3 in IECs triggers the development of spontaneous colitis and dysbiosis of T lymphocytes in mice.

The enzymatic core component of m6A writer complex, Mettl3, plays a crucial role in facilitating the development and progress of gastric and colorectal cancer (CRC). However, its underlying mechanism in regulating intestinal inflammation remains unclear and poorly investigated. First, the characteristics of Mettl3 expression in inflammatory bowel diseases (IBD) patients were examined. Afterward, we generated the mice line with intestinal epithelial cells (IECs)-specific deletion of Mettl3 verified by various experiments. We continuously recorded and compared the physiological status including survival rate etc. between the two groups. Subsequently, we took advantage of staining assays to analyze mucosal damage and immune infiltration of Mettl3WT and Mettl3KO primary IECs. Bulk RNA sequencing was used to pursuit the differential expression of genes (DEGs) and associated signaling pathways after losing Mettl3. Pyroptosis-related proteins were to determine whether cell death was caused by pyroptosis. Eventually, CyTOF was performed to probe the difference of CD45+ cells, especially CD3e+ T-cell clusters after losing Mettl3. In IBD patients, Mettl3 was highly expressed in the inner-nucleus of IECs while significantly decreased upon acute intestinal inflammation. IECs-specific deletion of Mettl3 KO mice triggered a wasting phenotype and developed spontaneous colitis. The survival rate, body weight, and intestinal length observed from 2 to 8 weeks of Mettl3KO mice were significantly lower than Mettl3WT mice. The degree of mucosal damage and immune infiltration in Mettl3KO were even more serious than in their WT littermate. Bulk RNA sequencing demonstrated that DEGs were dramatically enriched in NOD-signaling pathways due to the loss of Mettl3. The colonic epithelium was more prone to pyroptosis after losing Mettl3. Subsequently, CyTOF revealed that T cells have altered significantly in Mettl3KO. Furthermore, there was abnormal proliferation of CD4+ T and markedly exhaustion of CD8 + T in Mettl3KO mice. In severe IBD patients, Mettl3 is located in the inner-nucleus of IECs and declined when intestinal inflammation occurs. Subsequently, Mettl3 prevented mice from developing colitis.

METTL3-dependent m6A modification of PSEN1 mRNA regulates craniofacial development through the Wnt/β-catenin signaling pathway

Craniofacial malformations, often associated with syndromes, are prevalent birth defects. Emerging evidence underscores the importance of m6A modifications in various bioprocesses such as stem cell differentiation, tissue development, and tumorigenesis. Here, in vivo, experiments with zebrafish models revealed that mettl3-knockdown embryos at 144 h postfertilization exhibited aberrant craniofacial features, including altered mouth opening, jaw dimensions, ethmoid plate, tooth formation and hypoactive behavior. Similarly, low METTL3 expression inhibited the proliferation and migration of BMSCs, HEPM cells, and DPSCs. Loss of METTL3 led to reduced mRNA m6A methylation and PSEN1 expression, impacting craniofacial phenotypes. Co-injection of mettl3 or psen1 mRNA rescued the level of Sox10 fusion protein, promoted voluntary movement, and mitigated abnormal craniofacial phenotypes induced by mettl3 knockdown in zebrafish. Mechanistically, YTHDF1 enhanced the mRNA stability of m6A-modified PSEN1, while decreased METTL3-mediated m6A methylation hindered β-catenin binding to PSEN1, suppressing Wnt/β-catenin signaling. Pharmacological activation of the Wnt/β-catenin pathway partially alleviated the phenotypes of mettl3 morphant and reversed the decreases in cell proliferation and migration induced by METTL3 silencing. This study elucidates the pivotal role of METTL3 in craniofacial development via the METTL3/YTHDF1/PSEN1/β-catenin signaling axis.

Enhancing m6A modification of lncRNA through METTL3 and RBM15 to promote malignant progression in bladder cancer

Bladder cancer is one of the most prominent malignancies affecting the urinary tract, characterized by a poor prognosis. Our previous research has underscored the pivotal role of m6A methylation in the progression of bladder cancer. Nevertheless, the precise relationship between N6-methyladenosine (m6A) regulation of long non-coding RNA (lncRNA) and bladder cancer remains elusive. This study harnessed sequencing data and clinical records from 408 bladder cancer patients in the TCGA database. Employing R software, we conducted bioinformatics analysis to establish an m6A-lncRNA co-expression network. Analyzing the differences between high and low-risk groups, particularly at the immunological level, and subsequently investigating the primary regulatory factors of these lncRNA, validating the findings through experiments, and exploring their specific cellular functions. We identified 50 m6A-related lncRNA with prognostic significance through univariate Cox regression analysis. In parallel, we employed a LASSO-Cox regression model to pinpoint 11 lncRNA and calculate risk scores for bladder cancer patients. Based on the median risk score, patients were categorized into low-risk and high-risk groups. The high-risk cohort exhibited notably lower survival rates than their low-risk counterparts. Further analysis pointed to RBM15 and METTL3 as potential master regulators of these m6A-lncRNA. Experimental findings also shed light on the upregulated expression of METTlL3 and RBM15 in bladder cancer, where they contributed to the malignant progression of tumors. The experimental findings demonstrated a significant upregulation of METTL3 and RBM15 in bladder cancer specimens, implicating their contributory role in the oncogenic progression. Knockdown of METTL3 and RBM15 resulted in a marked attenuation of tumor cell proliferation, invasion, and migration, which was concomitant with a downregulation in the cellular m6A methylation status. Moreover, these results revealed that RBM15 and METTL3 function in a synergistic capacity, positing their involvement in cancer promotion via the upregulation of m6A modifications in long non-coding RNAs. Additionally, this study successfully developed an N-methyl-N-nitrosourea (MNU)-induced rat model of in situ bladder carcinoma, confirming the elevated expression of RBM15 and METTL3, which paralleled the overexpression of m6A-related- lncRNAs observed in bladder cancer cell lines. This congruence underscores the potential utility of these molecular markers in in vivo models that mirror human malignancies. This study not only offers novel molecular targets,but also enriches the research on m6A modification in bladder cancer, thereby facilitating its clinical translation.

METTL3 as a novel diagnosis and treatment biomarker and its association with glycolysis, cuproptosis and ceRNA in oesophageal carcinoma.

METTL3 has been shown to be involved in regulating a variety of biological processes. However, the relationship between METTL3 expression and glycolysis, cuproptosis-related genes and the ceRNA network in oesophageal carcinoma (ESCA) remains unclear. ESCA expression profiles from databases were obtained, and target genes were identified using differential analysis and visualization. Immunohistochemistry (IHC) staining assessed METTL3 expression differences. Functional enrichment analysis using GO, KEGG and GSEA was conducted on the co-expression profile of METTL3. Cell experiments were performed to assess the effect of METTL3 interference on tumour cells. Correlation and differential analyses were carried out to assess the relationship between METTL3 with glycolysis and cuproptosis. qRT-PCR was used to validate the effects of METTL3 interference on glycolysis-related genes. Online tools were utilized to screen and construct ceRNA networks based on the ceRNA theory. METTL3 expression was significantly higher in ESCA compared to the controls. The IHC results were consistent with the above results. Enrichment analysis revealed that METTL3 is involved in multiple pathways associated with tumour development. Significant correlations were observed between METTL3 and glycolysis-related genes and cuproptosis-related gene. Experiments confirmed that interfered with METTL3 significantly inhibited glucose uptake and lactate production in tumour cells, and affected the expression of glycolytic-related genes. Finally, two potential ceRNA networks were successfully predicted and constructed. Our study establishes the association between METTL3 overexpression and ESCA progression. Additionally, we propose potential links between METTL3 and glycolysis, cuproptosis and ceRNA, presenting a novel targeted therapy strategy for ESCA.

Dietary selenomethionine reduced oxidative stress by resisting METTL3-mediated m6A methylation level of Nrf2 to ameliorate LPS-induced liver necroptosis in laying hens

Selenomethionine (SeMet) as the main form of daily dietary selenium, occupies essential roles in providing antioxidant and anti-inflammatory properties, which alleviates inflammatory liver damage. N6-methyladenosine (m6A) is one of the most prevalent and abundant internal transcriptional modifications that regulate gene expression. To investigate the protective mechanism of SeMet on liver injury and the regulatory effect of m6A methylation modification, we established the model by supplementing dietary SeMet, and LPS as stimulus in laying hens. LMH cells were intervened with SeMet (0.075 μM) and/or LPS (60 μg/mL). Subsequently, histopathology and ultrastructure of liver were observed. Western Blot, qRT-PCR, colorimetry, MeRIP-qPCR, fluorescent probe staining and AO/EB were used to detect total m6A methylation level, m6A methylation level of Nrf2, ROS, inflammatory and necroptosis factors. Studies showed that SeMet suppressed LPS-induced upregulation of total m6A methylation levels and METTL3 expression. Interestingly, SeMet reduced the m6A methylation level of Nrf2, activated antioxidant pathways and alleviated oxidative stress. LMH cells were transfected with 50 μm siMETTL3. SeMet/SiMETTL3 reversed the LPS-induced reduction in Nrf2 mRNA stability, slowed down its degradation rate. Moreover, LPS induced oxidative stress, led to necroptosis and activated NF-κB to promote the expression of inflammatory factors. SeMet/SiMETTL3 alleviated LPS-induced necroptosis and inflammation. Altogether, SeMet enhanced antioxidant and anti-inflammatory capacity by reducing METTL3-mediated m6A methylation levels of Nrf2, ultimately alleviating liver damage. Our findings provided new insights and therapeutic target for the practical application of dietary SeMet in the treatment and prevention of liver inflammation, and supplied a reference for comparative medicine.

Alteration of RNA Demethylases by Cholesterol Crystals

Atherosclerosis is a chronic inflammatory disease of the arteries that can lead to cardiovascular diseases. Cholesterol crystals are one of the elements found in atherosclerotic plaques, and they play a significant role in the development and progression of the disease. In this study, we investigated the effects of cholesterol crystals on mRNA modification in macrophages, which are involved in plaque formation and inflammation. We used RAW 264.7 cells, a murine macrophage cell line, and exposed them to cholesterol crystals. We found that cholesterol crystals stimulated the production of pro-inflammatory cytokines, including IL-1β, IL-6, and TNFα. Furthermore, cholesterol crystals were taken up by the macrophages and were detected within the cells. Interestingly, cholesterol crystals upregulated demethylating enzymes FTO and ALKBH5, which are involved in RNA modification. However, the expression of methyltransferases, including METTL3, METTL14, NSUN2, and WTAP remained unchanged. These results suggest that cholesterol crystals can modulate mRNA modification by affecting demethylating enzymes and pro-inflammatory cytokines, potentially contributing to the development of atherosclerosis. Understanding the molecular mechanisms underlying cholesterol crystalinduced atherosclerosis may provide new insights into preventive and therapeutic strategies for cardiovascular diseases.

M6A modification regulates tumor suppressor DIRAS1 expression in cervical cancer cells

ABSTRACT DIRAS family GTPase 1 (DIRAS1) has been reported as a potential tumor suppressor in other human cancer. However, its expression pattern and role in cervical cancer remain unknown. Knockdown of DIRAS1 significantly promoted the proliferation, growth, migration, and invasion of C33A and SiHa cells cultured in vitro. Overexpression of DIRAS1 significantly inhibited the viability and motility of C33A and SiHa cells. Compared with normal cervical tissues, DIRAS1 mRNA levels were significantly lower in cervical cancer tissues. DIRAS1 protein expression was also significantly reduced in cervical cancer tissues compared with para-cancerous tissues. In addition, DIRAS1 expression level in tumor tissues was significantly negatively correlated with the pathological grades of cervical cancer patients. DNA methylation inhibitor (5-Azacytidine) and histone deacetylation inhibitor (SAHA) resulted in a significant increase in DIRAS1 mRNA levels in C33A and SiHa cells, but did not affect DIRAS1 protein levels. FTO inhibitor (FB23–2) significantly down-regulated intracellular DIRAS1 mRNA levels, but significantly up-regulated DIRAS1 protein levels. Moreover, the down-regulation of METTL3 and METTL14 expression significantly inhibited DIRAS1 protein expression, whereas the down-regulation of FTO and ALKBH5 expression significantly increased DIRAS1 protein expression. In conclusion, DIRAS1 exerts a significant anti-oncogenic function and its expression is significantly downregulated in cervical cancer cells. The m6A modification may be a key mechanism to regulate DIRAS1 mRNA stability and protein translation efficiency in cervical cancer.

Multi-omics sequencing revealed endostar combined with cisplatin treated non small cell lung cancer via anti-angiogenesis

BackgroundEndostar, an anti-angiogenic drug, has been approved for treating non-small cell lung cancer (NSCLC). At present, endostar combined with radiotherapy or chemotherapy has achieved ideal results in the treatment of some tumors, but there is a lack of application and study in NSCLC. This study investigated the therapeutic effect and potential mechanism of endostar combined with cisplatin (EC) in NSCLC.MethodsHE staining, TUNEL staining, immunofluorescence, colony formation ability, and cell migration ability were used to evaluate the anti-tumor activity of EC. The expressions of FMOD, VEGF, FGF-2, and PDGF-B were detected by western blotting and qPCR. The target of combination therapy was analyzed by m6A sequencing and RNA sequencing. METTL3 knockdown and overexpressed A549 cells were constructed and co-cultured with HUVECs to further evaluate the effect of METLL3 on combination therapy.ResultsCombination therapy significantly reduced the colony formation and migration ability of NSCLC cells, induced cell apoptosis, and inhibited the tube formation ability of HUVECs. The results of m6A sequencing and RNA sequencing showed that the EC could down-regulate the expression level of FMOD in tumor tissues, which might be related to the reduction of its m6A methylation modification regulatory enzyme METTL3. Restricting FMOD expression could reduce the expression of FGF2, TGF-β1, VEGF and PDGF-B. Moreover, overexpression of METTLE almost abolished the anti-tumor effect of EC and promoted angiogenesis.ConclusionsEndostar combined with cisplatin might exert anti-tumor effects by down-regulating the expression of METTL3 and FMOD.

METTL14 enhances the m6A modification level of lncRNA MSTRG.292666.16 to promote the progression of non-small cell lung cancer

Backgroundm6A modification has close connection with the occurrence, development, and prognosis of tumors. This study aimed to explore the roles of m6A modification and its related mechanisms in non-small cell lung cancer (NSCLC).MethodsNSCLC tissues and their corresponding para-cancerous tissues were collected to determine the m6A levels of total RNA/lncRNAs and the expression of m6A modification-related genes/lncRNAs. Then, A549 cells were transfected with si-METTL14 or oe-METTL14, and the cell transfection efficiency was assessed. Subsequently, the viability, apoptosis, cell colony formation, migration and invasion of the different cells were determined. Finally, the nude mouse tumorigenicity experiments were performed to observe the effects of METTL14 in vivo.ResultsCompared to the para-NSCLC tissues, the m6A level and METTL14 expression were both significantly increased in the NSCLC tissues (P < 0.05). Based on the expression of METTL14 in the different cell lines, A549 cells were chosen for further experiments. Then, the A549 cells with METTL14 knockdown and overexpression were successfully established, as well as it was found that METTL14 knockdown could inhibit the viability, colony formation, migration, and invasion of A549 cells, while facilitate their apoptosis. In vivo experiments also showed that METTL14 knockdown could inhibit tumor formation and growth. Additionally, the m6A level of MSTRG.292666.16 was higher in the NSCLC tissues; and after METTL14 knockdown, the expression and m6A level of MSTRG.292666.16 were both significantly reduced in A549 cells, and vice versa.ConclusionMETTL14 may promote the progression of NSCLC through up-regulating MSTRG.292666.16 and enhance its m6A modification level.

Linc-smad7 is involved in the regulation of lipid synthesis in mouse mammary epithelial cells

Long intergenic non-coding RNA(lincRNA) is transcribed from the intermediate regions of coding genes and plays a pivotal role in the regulation of lipid synthesis. N6-methyladenosine (m6A) modification is widely prevalent in eukaryotic mRNAs and serves as a regulatory factor in diverse biological processes. This study aims to delineate the mechanism by which Linc-smad7 mediates m6A methylation to regulate milk fat synthesis. Tissue expression analysis in this study revealed a high expression of Linc-smad7 in breast tissue during pregnancy. Cell proliferation assays, including CCK8 and EdU assays, demonstrated that Linc-smad7 had no significant impact on the proliferation of mammary epithelial cells. However, during mammary epithelial cell differentiation, the overexpression of Linc-smad7 led to reduced lipid formation, whereas interference with Linc-smad7 promoted lipogenesis. Mechanistically, Linc-smad7 was found to modulate RNA m6A levels, as evidenced by dot blot assays and methylated RNA immunoprecipitation sequencing (MeRIP-Seq). Subsequent validation through RT-qPCR corroborated these findings, aligning with the m6A sequencing outcomes. Furthermore, co-transfection experiments elucidated that Linc-smad7 regulates lipid synthesis in mammary epithelial cells by influencing the expression of METTL14. In summary, these findings underscore the regulatory role of Linc-smad7 in controlling METTL14 gene expression, thereby mediating m6A modifications to regulate lipid synthesis in mammary epithelial cells.

Electroacupuncture Alleviates Cerebral Ischemia-reperfusion Injury by Regulating the S1PR2/TLR4/NLRP3 Signaling Pathway via m6A Methylation of lncRNA H19.

All rats were randomly divided into five groups: the SHAM group, MCAO group, MCAO+EA (MEA) group, MCAO+METTL3 overexpression+EA (METTL3) group and MCAO+lncRNA H19 overexpression+EA (lncRNA H19) group. The middle cerebral artery occlusion (MCAO) rats were established to mimic CIR injury. The overexpression of lncRNA H19 and METTL3 was induced by stereotactic injection of lentiviruses into the rat lateral ventricles. The rats in the MEA, METTL3, and lncRNA H19 groups were treated with EA therapy on "Renzhong" (DU26) and "Baihui" (DU20) acupoints (3.85/6.25Hz; 1mA). Besides, the neurological deficit scoring, cerebral infarction area, pathological changes in brain tissue, total RNA m6A level, and the expression of METTL3, S1PR2, TLR4, NLRP3 and lncRNA H19 were detected in this experiment. EA improved the neurological deficit scoring, cerebral infarction area, and pathological injury in MCAO rats, while these beneficial effects of EA on CIR injury were attenuated by the overexpression of METTL3 or lncRNA H19. More importantly, EA down-regulated the total RNA m6A level and the expression of METTL3, S1PR2, TLR4, NLRP3 and lncRNA H19 in MCAO rats. Instead, the overexpression of METTL3 or lncRNA H19 was found to reverse the EA-induced down-regulation. The findings indicated that EA might down-regulate the S1PR2/TLR4/NLRP3 signaling pathway via m6A methylation of lncRNA H19 to alleviate CIR injury. Our findings provide a new insight into the molecular mechanism of EA on CIR injury.

Targeting the m6A RNA methyltransferase METTL3 attenuates the development of kidney fibrosis

Kidney fibrosis is a major mechanism underlying chronic kidney disease (CKD). N6-methyladenosine (m6A) RNA methylation is associated with organ fibrosis. We investigated m6A profile alterations and the inhibitory effect of RNA methylation in kidney fibrosis in vitro (TGF-β-treated HK-2 cells) and in vivo (unilateral ureteral obstruction [UUO] mouse model). METTL3-mediated signaling was inhibited using siRNA in vitro or the METTL3-specific inhibitor STM2457 in vivo and in vitro. In HK-2 cells, METTL3 protein levels increased in a dose- and time-dependent manner along with an increase in the cellular m6A levels. In the UUO model, METTL3 expression and m6A levels were significantly increased. Transcriptomic and m6A profiling demonstrated that epithelial-to-mesenchymal transition- and inflammation-related pathways were significantly associated with RNA m6A methylation. Genetic and pharmacologic inhibition of METTL3 in HK-2 cells decreased TGF-β-induced fibrotic marker expression. STM2457-induced inhibition of METTL3 attenuated the degree of kidney fibrosis in vivo. Furthermore, METTL3 protein expression was significantly increased in the tissues of CKD patients with diabetic or IgA nephropathy. Therefore, targeting alterations in RNA methylation could be a potential therapeutic strategy for treating kidney fibrosis.

Dynamics of N6-methyladenosine modification during aging and their potential roles in the degeneration of intervertebral disc.

The N6-methyladenosine (m6A) dynamics in the progression of intervertebral disc (IVD) aging remain largely unknown. This study aimed to explore the distribution and pattern of m6A modification in nucleus pulpous (NP) tissues of rats at different ages. Histological staining and MRI were performed to evaluate the degeneration of IVD. The expression of m6A modifiers was analyzed using qRT-PCR and western blot. Subsequently, methylated RNA immunoprecipitation next generation sequencing and RNA-seq were conducted to identify differences in m6A methylome and transcriptome of NP tissues. Compared to 2-month-old rats, we found significant changes in the global m6A level and the expression of Mettl3 and FTO in NP tissues from 20-month-old rats. During the progression of NP aging, there were 1126 persistently differentially m6A peaks within 931 genes, and 51 persistently differentially expressed genes. GO and KEGG analyses showed that these m6A peaks and m6A modified genes were mainly engaged in the biological processes and pathways of intervertebral disc degermation (IDD), such as extracellular matrix metabolism, angiogenesis, inflammatory response, mTOR and AMPK signaling pathways. Meanwhile, conjoint analyses and Venn diagram revealed a total of 405 aging related genes contained significant methylation and expression levels in 20-month-old rats in contrast to 2-month-old and 10-month-old rats. Moreover, it was found that four aging related genes with hypermethylated modification including BUB1, CA12, Adamts1, and Adamts4 depicted differentially expressed at protein level, of which BUB1 and CA12 were decreased, while Adamts1 and Adamts4 were increased during the progression of NP aging. Collectively, this study elucidated the distribution and pattern of m6A modification during the aging of IVD. Furthermore, the m6A modified genes were involved in the IDD related biological processes and pathways. These findings may provide novel insights into the mechanisms and therapies of IDD from the perspective of aging.

A Characterization of the RNA Modification Response to Starvation under Low Temperatures in Large Yellow Croaker (Larimichthys crocea)

Emerging evidence shows that N6-methyladenosine (m6A) is a post-transcriptional RNA modification that plays a vital role in regulation of gene expression, fundamental biological processes, and physiological functions. To explore the effect of starvation on m6A methylation modification in the liver of Larimichthys crocea (L. crocea) under low temperatures, the livers of L. crocea from cold and cold + fasting groups were subjected to MeRIP-seq and RNA-seq using the NovaSeq 6000 platform. Compared to the cryogenic group, the expression of RNA methyltransferases mettl3 and mettl14 was upregulated, whereas that of demethylase fto and alkbh5 was downregulated in the starved cryogenic group. A Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the differentially m6A-modified genes were mainly enriched in steroid biosynthesis, DNA replication, ribosome biogenesis in eukaryotes, PPAR, ECM-receptor interaction, lysine degradation, phosphatidylinositol, and the MAPK signaling pathway, suggesting that L. crocea responds to starvation under low-temperature stress through m6A methylation modification-mediated cell growth, proliferation, innate immunity, and the maintenance of lipid homeostasis. This study advances understanding of the physiological response mechanism exerted by m6A methylation modification in starved L. crocea at low temperatures.