Petri Belinda J, Piell Kellianne M, Avila-Valdes Bailey L, Stanley Christian G, Winkler Laura J, Brown Johnny Tyler, Ulett Rye, Sanchez Gabriela, Chariker Julia H, Rouchka Eric C, Klinge Carolyn M
Department of Biochemistry and Molecular Genetics, University of Louisville School of Medicine, Louisville, KY, USA.
Kentucky IDeA Networks of Biomedical Research Excellence Bioinformatics Core, University of Louisville School of Medicine, Louisville, KY, USA.
Funct Integr Genomics. 2025 Jul 9;25(1):149. doi: 10.1007/s10142-025-01658-2.
The role of epitranscriptomic changes in the development of acquired endocrine therapy (ET)- resistance in estrogen receptor α (ER) expressing breast cancer (BC) is unknown. We tested the hypothesis that inhibition of METTL3, the methyltransferase responsible for the mRNA modification N-6 methyladenosine (m6A), alters m6A modifications and differentially regulates the abundance of mRNA transcripts in ET-sensitive MCF-7 versus resistant LCC9 ER + human BC cells. Differential m6A modifications were identified using direct mRNA sequencing (DRS) performed on five replicates for each cell line ± 1 µM STM2457, a selective METTL3 inhibitor, using Nanopore MinION long read RNA-seq. Parallel short read Illumina RNA-seq quantified differential transcript abundance in the same samples. Selected results were validated by RT-qPCR, m6A-RIP-qPCR, reporter assays, and western blot analysis. Statistical analysis combined m6Anet, a machine-learning algorithm designed to call m6A modified bases, with a generalized linear model following a binomial distribution analysis to identify significant differential m6A modification ratios (DMR). Distinct METTL3 dependent m6A modification patterns in LCC9 and MCF-7 cells were observed in differentially expressed genes (DEG) associated with ET-resistance, including EEF1A2, ACTB, FLNA, PDIA6, AMIGO2, TPT1, XBP1, and CITED4. Select results were validated in additional ET-resistant BC cell lines. m6A-RIP-RT-qPCR validated specific m6A sites. We examined the proximity of m6A sites to estrogen receptor α (ER α)-mRNA binding sites reported in MCF-7 cells. ACTB, PDIA6, and XBP1 demonstrated a short-range proximity, with m6A sites located within 100 bp of ERα binding sites, suggesting a role for m6A in influencing ERα-mRNA binding. Our work provides a framework for integrating DRS and DEG omics data. Our results suggest a role for dysregulation of m6A modifications in pathways implicated in ET resistance in BC.
表观转录组变化在雌激素受体α（ER）阳性乳腺癌（BC）获得性内分泌治疗（ET）耐药发展中的作用尚不清楚。我们检验了以下假设：抑制负责mRNA修饰N-6甲基腺苷（m6A）的甲基转移酶METTL3，会改变m6A修饰，并在ET敏感的MCF-7细胞与耐药的LCC9 ER+人BC细胞中差异调节mRNA转录本的丰度。使用直接mRNA测序（DRS）鉴定差异m6A修饰，对每个细胞系进行五次重复实验，±1µM STM2457（一种选择性METTL3抑制剂），采用纳米孔MinION长读长RNA测序。并行的短读长Illumina RNA测序对相同样本中的差异转录本丰度进行定量。选定的结果通过RT-qPCR、m6A-RIP-qPCR、报告基因检测和蛋白质免疫印迹分析进行验证。统计分析将m6Anet（一种用于识别m6A修饰碱基的机器学习算法）与遵循二项分布分析的广义线性模型相结合，以识别显著的差异m6A修饰率（DMR）。在与ET耐药相关的差异表达基因（DEG）中，包括EEF1A2、ACTB、FLNA、PDIA6、AMIGO2、TPT1、XBP1和CITED4，在LCC9和MCF-7细胞中观察到了不同的METTL3依赖性m6A修饰模式。在其他ET耐药BC细胞系中验证了选定的结果。m6A-RIP-RT-qPCR验证了特定的m6A位点。我们研究了m6A位点与MCF-7细胞中报道的雌激素受体α（ERα）-mRNA结合位点的接近程度。ACTB、PDIA6和XBP1表现出短距离接近，m6A位点位于ERα结合位点的100bp范围内，表明m6A在影响ERα-mRNA结合中发挥作用。我们的工作为整合DRS和DEG组学数据提供了一个框架。我们的结果表明，m6A修饰失调在BC中与ET耐药相关的通路中发挥作用。
