Aug 2026· Technology in Cancer Research and Treatment· Vol 25· 0 citations· 41 references
Medicine
TL;DR
In vivo, METTL3 overexpression promoted tumor growth, reduced 4-HNE levels, and regulated ferroptosis and FAK/MAPK/ERK pathway proteins, all of which were reversed by COL5A2 knockdown.
Abstract
Introduction METTL3, the core enzyme of m6A methylation, influences gastric cancer (GC) progression, but its role in ferroptosis remains unclear. This study investigated the regulatory role of METTL3 in GC ferroptosis and its underlying mechanism via the downstream target gene collagen type V alpha 2 chain(COL5A2). Method GC cell lines (AGS, MKN-45) and normal GES-1 cells were used. Ferroptosis was induced by erastin and RSL-3. METTL3 overexpression/knockdown models were constructed to assess ferroptosis indicators, including lipid reactive oxygen species(ROS), malondialdehyde(MDA), Fe2+, glutathione(GSH) and malignant phenotypes. m6A sites were predicted and validated by MeRIP-qPCR. m6A readers were knocked down to identify effectors, and mRNA stability was evaluated via RIP and actinomycin D assays. The focal adhesion kinase(FAK)/mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase(ERK)pathway and ferroptosis-related proteins were detected by Western blot, with pathway involvement confirmed using the FAK inhibitor PF-573228. To validate the in vitro findings, a nude mouse xenograft tumor model was established. Results METTL3 was highly expressed in GC cells and suppressed by ferroptosis inducers. METTL3 overexpression inhibited ferroptosis (reducing ROS, MDA, Fe2+, increasing GSH) and promoted proliferation and migration. COL5A2 was positively correlated with METTL3. MeRIP-qPCR confirmed that METTL3 mediates m6A modification of COL5A2’s 3′-UTR. RIP and ActD assays demonstrated that the m6A reader insulin-like growth factor 2 mRNA-binding protein 3(IGF2BP3) maintains COL5A2 mRNA stability. IGF2BP3 knockdown promoted ferroptosis and apoptosis, which were reversed by COL5A2 overexpression. Furthermore, COL5A2 overexpression activated the FAK/MAPK/ERK pathway, upregulated GPX4, and inhibited ferroptosis; these effects were counteracted by the FAK inhibitor. In vivo, METTL3 overexpression promoted tumor growth, reduced 4-HNE levels, and regulated ferroptosis and FAK/MAPK/ERK pathway proteins, all of which were reversed by COL5A2 knockdown. Conclusion METTL3 upregulates COL5A2 expression through m6A modification, with IGF2BP3 maintaining COL5A2 mRNA stability. This subsequently activates the FAK/MAPK/ERK signaling pathway, inhibits ferroptosis, and promotes GC progression. The METTL3/IGF2BP3/COL5A2 axis represents a promising therapeutic target for GC treatment.
BACKGROUND
Gastric cancer (GC) remains one of the leading causes of cancer-related mortality globally and is associated with poor patient prognosis, which highlights an urgent need to identify effective biomarkers and therapeutic targets. N6-methyladenosine (m6A) modification and its reader proteins play a crucial role in tumorigenesis. Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) participates in carcinogenesis and serves as a reader of m6A. However, its exact function and the underlying molecular mechanisms in the progression of GC remain unknown.
AIM
To investigate the role of IGF2BP1 in the progression of GC.
METHODS
The IGF2BP1 expression in GC tissues was assessed via immunohistochemistry and correlated with clinicopathological characteristics. Gain-of-function and loss-of-function experiments were performed to explore the functional roles of IGF2BP1 and its potential downstream target in GC cells and a nude mouse xenograft model. Cell proliferation, migration, and invasion were assessed using cell counting kit-8, wound-healing, and Transwell assays. RNA sequencing, methylated RNA immunoprecipitation quantitative polymerase chain reaction, and dual-luciferase reporter assays were conducted to elucidate the molecular mechanisms involved.
RESULTS
The IGF2BP1 was significantly upregulated in GC tissues and was positively associated with lymph node metastasis and poor overall survival. Functionally, IGF2BP1 knockdown inhibited GC cell proliferation, migration, and invasion in vitro and attenuated tumor growth in vivo . Integrated multi-omics analysis identified heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) as a key downstream target of IGF2BP1. IGF2BP1 recognized and bound to m6A-modified sites within HS6ST2 mRNA, thereby enhancing its stability in an m6A-dependent manner. Rescue experiments confirmed that HS6ST2 mediated the oncogenic effects of IGF2BP1. Additionally, HS6ST2 knockdown inhibited the malignant characteristics of GC cells and triggered apoptosis, which was associated with inactivation of the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway.
CONCLUSION
These findings demonstrated that IGF2BP1 drives GC progression by stabilizing HS6ST2 mRNA via m6A modification. The IGF2BP1/HS6ST2 axis may serve as a potential prognostic biomarker and therapeutic intervention for GC.
Lu Liu, Yi-jing Zhou, Yue Xu et al.· World Journal of Gastrointes...· 0 citations
INTRODUCTION
Methyl-N'-nitro-N-nitrosoguanidine (MNNG) is an environmental carcinogen that induces Gastric Cancer (GC). N7-methylguanosine (m7G) is a prevalent RNA modification closely linked to cancer onset and progression. However, the role of m7G in regulating gene expression during MNNG-induced gastric carcinogenesis remains unclear. This study aims to investigate the role of m7G modification in MNNG-induced GC and to identify potential downstream regulatory genes.
METHODS
Cell proliferation and migration were evaluated using CCK-8 and scratch assays in Malignant transformed cells (MC) and GC cells with different METTL1 expression levels. The m7G MeRIP-seq and whole-transcriptome sequencing were integrated to screen potential genes regulated by m7G modification in MC-30 cells. GO and KEGG analyses were performed for gene function. Candidate gene expression was screened and validated in MC and GC cells by RT-qPCR. Finally, we validated SLC2A3 by analyzing gene expression using the TCGA STAD cohort and 24 pairs of clinical GC samples.
RESULTS
METTL1 knockdown significantly inhibited proliferation and migration by about 25% (p < 0.001). Sequencing analysis identified SLC2A3 as a key METTL1 downstream target, with significantly altered m7G modification levels (fold change > 2, p < 0.05) and enrichment in cancer-related pathways. Clinically, SLC2A3 expression was significantly up-regulated in GC tissues versus normal controls (FC = 2.52, p < 0.001) and was significantly associated with tumor stage and prognosis in GC patients (p < 0.05).
CONCLUSION
Our study revealed that m7G methyltransferase METTL1 plays an oncogenic role in MNNG-induced gastric carcinogenesis. SLC2A3 is a key downstream target of METTL1, which is associated with clinical progression in GC patients. These findings may provide evidence for developing prognostic biomarkers for GC.
Jia-Xian Li, Jiabei Jian, Wen-Zheng Yuan et al.· Current Medicinal Chemistry· 0 citations
BACKGROUND
Osteosarcoma (OS) represents a common primary malignant bone tumor associated with unfavorable clinical outcomes. Growing evidence underscores the crucial involvement of N6-methyladenosine (m6A) modifications in tumor development, but the specific mechanisms underlying the m6A regulatory network in OS remain to be elucidated.
METHODS
Potential key target genes in OS were identified through bioinformatic analyses, followed by the characterization of m6A-related regulatory proteins, specifically, writer and reader proteins, which showed significant associations with these targets. To elucidate the mechanistic role of m6A methylation in regulating UHRF1 expression, a series of in vitro assays were conducted. These included RNA pull-down, MeRIP-PCR, dot blot, dual-luciferase reporter assays, and RNA stability assays, which collectively confirmed the interaction between m6A regulatory proteins and UHRF1 mRNA. For functional investigations, OS cell lines (U2OS, Saos2, and 143B) with gene silencing or overexpression were established, and the role of UHRF1 in cellular proliferation, migration, and invasion was assessed using CCK-8 assays, Transwell migration and invasion assays, flow cytometry, and wound healing assays. In addition, GSH/GSSG ratio, Fe2+ concentration, and ROS levels were measured using commercial assay kits to explore ferroptosis-related functional mechanisms. To validate the in vivo relevance of our findings, a xenograft mouse model was established. Finally, functional rescue experiments were performed to mechanistically confirm the critical role of the ZCCHC4-UHRF1-CDO1 regulatory axis in OS progression.
RESULTS
ZCCHC4, functioning as an m6A methyltransferase, enhances the stability of UHRF1 mRNA by catalyzing its m6A modification, thereby promoting increased expression of UHRF1. In parallel, IGF2BP3, an established m6A reader protein, specifically recognizes and binds to the m6A-modified sites on UHRF1 mRNA, further stabilizing the transcript and modulating its downstream biological functions. METTL3/METTL14 knockdown experiments ruled out the contribution of classical m6A methyltransferases, confirming that ZCCHC4 is the primary methyltransferase for UHRF1. Dual-luciferase assays and bisulfite sequencing revealed that UHRF1 suppresses CDO1 transcription by inducing high methylation of its promoter, thereby reducing ROS/Fe2+ levels and increasing GSH, which in turn blocks ferroptosis. In U2OS, Saos2, and 143B cells, silencing UHRF1 or ZCCHC4 inhibited proliferation, migration, and invasion while activating ferroptosis. Overexpression of UHRF1 had the opposite effect. In vivo models confirmed that UHRF1 silencing inhibited tumor growth. Furthermore, UHRF1 overexpression partially reversed the phenotypes induced by ZCCHC4 knockdown.
CONCLUSION
In summary, this study reveals for the first time the complete molecular mechanism by which ZCCHC4-mediated UHRF1 m6A methylation promotes OS progression through epigenetic suppression of CDO1 transcription and inhibition of ferroptosis. This regulatory axis (ZCCHC4-IGF2BP3-UHRF1-CDO1-ferroptosis) provides multiple therapeutic targets for OS and lays a solid foundation for the future development of anticancer strategies based on the regulation of ferroptosis, while also offering a promising pathway for clinical translation.
Kai Song, Ju Liu, Bowen Han et al.· Bone· 0 citations
BACKGROUND
Bladder cancer (BCa) is a common urological malignancy with high recurrence rates and limited treatment options for advanced disease. Circular RNAs (circRNAs) are emerging as key regulators of cancer progression, but the roles of most circRNAs in BCa remain poorly understood.
OBJECTIVE
To investigate the expression, function and mechanism of circPCMTD1 (hsa_circ_0001801) in BCa, focusing on its m6A methylation and its role as a ceRNA regulating the miR-3681-5p/HPSE/PI3K/AKT axis.
METHODS
circPCMTD1 expression was analyzed in GEO datasets, 30 paired BCa tissues and cell lines by qRT-PCR. Circular structure and stability were confirmed by RNase R and actinomycin D assays. Subcellular localisation was determined by fractionation and FISH. Gain- and loss-of-function experiments assessed circPCMTD1 effects on BCa cell malignancy. A subcutaneous xenograft model was used for in vivo validation. m6A sites were predicted by SRAMP, and circPCMTD1-EIF4A3 interaction validated by RIP. Protein-protein docking predicted EIF4A3-METTL3 binding. miRNA targets were screened by ENCORI and confirmed by qRT-PCR and RNA-FISH. miR-3681-5p targets were identified by transcriptome sequencing and database analysis. Western blotting assessed HPSE, PI3K, AKT and p-AKT. Rescue experiments verified the circPCMTD1/miR-3681-5p/HPSE axis.
RESULTS
circPCMTD1 was upregulated in BCa tissues and cells, stable and cytoplasmic. Knockdown suppressed BCa proliferation, migration, invasion and Tumor growth in vivo, while overexpression promoted these phenotypes. circPCMTD1 contained nine predicted m6A sites, interacted with EIF4A3, and EIF4A3 docked with METTL3. circPCMTD1 overexpression increased METTL3, METTL14, FTO and YTHDF2. circPCMTD1 directly sponged miR-3681-5p. miR-3681-5p acted as a tumor-suppressive miRNA. HPSE was a direct target of miR-3681-5p and overexpressed in BCa. circPCMTD1 positively regulated HPSE, PI3K and p-AKT, whereas miR-3681-5p had opposite effects. Rescue experiments confirmed that circPCMTD1 promoted BCa through sponging miR-3681-5p and activating HPSE/PI3K/AKT.
CONCLUSIONS
EIF4A3 facilitates METTL3-mediated m6A methylation of circPCMTD1. Methylated circPCMTD1 acts as a ceRNA sponging miR-3681-5p, derepressing HPSE and activating PI3K/AKT to drive BCa progression. This study identifies a novel EIF4A3/m6A-circPCMTD1/miR-3681-5p/HPSE/PI3K/AKT axis and suggests potential therapeutic targets for BCa.
Lingbao Xiang, Yutong Li, Xuan Dong et al.· Cellular Signalling· 0 citations
BACKGROUND
N6-methyladenosine (m6A) is the most common RNA modification and plays a key role in the initiation, progression, and relapse of multiple cancers, including hematologic malignancies. However, the role of m6A and m6A regulatory genes in myelodysplastic syndromes (MDS) remains unclear. This study aims to elucidate the function and molecular mechanism of methyltransferase METTL14 in MDS.
METHODS
RT-qPCR was used to assess the expression of multiple m6A regulators, focusing on METTL14 in MDS patients and cell lines. METTL14 overexpressing and knockdown cell lines were established, and CCK-8, EdU, and flow cytometry assays were performed to explore the biological functions of METTL14.Dot blot, MeRIP-Seq, MeRIP-qPCR, RT-qPCR, and Western blot were employed to investigate the underlying molecular mechanism.
RESULTS
Dysregulation of multiple m6A regulators was observed in MDS, among which METTL14 was upregulated. Elevated METTL14 expression increases MDS risk and adverse prognosis, emerging as a biomarker for poor prognosis. METTL14 promoted proliferation and cell-cycle progression of MDS cells while inhibiting apoptosis; corresponding changes were observed in cell cycle and apoptosis markers. METTL14 regulated cellular m6A levels. Downstream target of METTL14 enriched in cell cycle-related pathways, with CCNE1 identified as a critical target. Knockdown of METTL14, actinomycin D, or S-adenosylhomocysteine treatment reduced CCNE1 mRNA and protein levels. Furthermore, METTL14 activated MAPK-ERK and PI3K-AKT signaling via CCNE1 in an m6A-dependent manner, thereby promoting proliferative MDS cells' capacity.
CONCLUSIONS
This study delineates a METTL14/m6A/CCNE1 signaling axis in MDS progression and suggests that METTL14-mediated m6A modification may be a potential therapeutic target for MDS.
