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METTL3 modulates cell viability and motility in HCC1143 and MDA-MB-231 triple-negative breast cancer cell lines

ABSTRACT: The m6A methyltransferase METTL3 functions as a critical oncogenic driver in triple negative breast cancer (TNBC). However, its specific downstream targets and mechanistic functions in less metastatic TNBC subtypes remain poorly characterized. To address this, we evaluated METTL3 expression and the phenotypic effects of its siRNA-mediated knockdown in healthy mammary epithelial (MCF10A), low-metastatic TNBC (HCC1143) and high-metastatic TNBC (MDA-MB-231) cell lines. We assessed global m6A levels, cell proliferation, cell cycle progression and migration. To uncover specific downstream pathways, transcriptomic profiling was performed on HCC1143 cells, followed by RT-qPCR validation and m6A site prediction. METTL3 depletion reduced global m6A levels and cell viability across all cell lines. Notably, in low-metastatic HCC1143 cells, METTL3 knockdown induced a pronounced G2/M cell cycle arrest and dramatically impaired migration capacity. Transcriptomic analysis of HCC1143 revealed altered expression of genes associated with these observed phenotypic changes. Specifically, critical transcripts harboring predicted m6A motifs, including LIMK1, CCNB2 and CDH1, were significantly dysregulated, pointing to potential alterations in pathways governing cytoskeletal remodeling, actin organization, and cell-cell adhesion. Taken together, we propose that METTL3 promotes cellular proliferation and motility in low-metastatic TNBC by regulating key transcripts involved in cell cycle progression and actin dynamics.

ORGANISM(S): Homo sapiens

PROVIDER: GSE325029 | GEO | 2026/04/17

REPOSITORIES: GEO

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Project description:This study investigates how partial loss of the m6A RNA methyltransferase METTL3 alters the transcriptomic landscape and translational regulation in Triple Negative Breast Cancer cells. m6A is the most abundant internal RNA modification in eukaryotic mRNAs and long non-coding RNAs and plays important roles in RNA metabolism, translation, and gene expression regulation. Dysregulation of the m6A pathway has been implicated in multiple cancers, including triple negative breast cancer (TNBC), where reduced METTL3 expression has been associated with poor prognosis and increased metastatic potential. To investigate how reduced METTL3 dosage affects RNA methylation and downstream cellular processes, we generated CRISPR/Cas9-engineered METTL3 heterozygous knockout (HKO) cell lines in the human TNBC cell line MDA-MB-468. These cells exhibit reduced METTL3 expression and modest global decreases in m6A levels, allowing investigation of the effects of partial m6A depletion, which may better reflect physiologically relevant perturbations observed in cancer. Using Oxford Nanopore Direct RNA Sequencing, we performed transcriptome-wide profiling of m6A modifications in wild-type and METTL3 heterozygous knockout cells. Computational approaches were applied to identify differential m6A sites and estimate modification stoichiometry across transcripts. These data were integrated with functional analyses including proliferation assays, quantitative proteomics, phosphoproteomics, and global protein synthesis measurements to assess the biological consequences of altered m6A levels. The study aims to determine how partial reductions in METTL3 activity reshape the m6A landscape and influence gene expression programs, particularly those related to translational control, growth signalling, and cancer cell proliferation. Our findings demonstrate that modest decreases in global m6A preferentially affect transcripts involved in translational regulation and are associated with increased translational output and proliferative capacity, whereas stronger pharmacological inhibition of METTL3 suppresses translation and cell growth. Overall, this work provides insight into the dose-dependent effects of METTL3 and m6A RNA methylation on gene regulation, highlighting how partial disruption of the m6A machinery can reprogram translational pathways and contribute to tumour progression.

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METTL3 modulates cell viability and motility in HCC1143 and MDA-MB-231 triple-negative breast cancer cell lines

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