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Abstract：
BACKGROUND: RNA m5C methylation has been extensively implicated in the occurrence and development of tumors. As the main methyltransferase, NSUN2 plays a crucial regulatory role across diverse tumor types. However, the precise impact of NSUN2-mediated m5C modification on breast cancer (BC) remains unclear. Our study aims to elucidate the molecular mechanism underlying how NSUN2 regulates the target gene HGH1 (also known as FAM203) through m5C modification, thereby promoting BC progression. Additionally, this study targets at preliminarily clarifying the biological roles of NSUN2 and HGH1 in BC.
METHODS: Tumor and adjacent tissues from 5 BC patients were collected, and the m5C modification target HGH1 in BC was screened through RNA sequencing (RNA-seq) and single-base resolution m5C methylation sequencing (RNA-BisSeq). Methylation RNA immunoprecipitation-qPCR (MeRIP-qPCR) and RNA-binding protein immunoprecipitation-qPCR (RIP-qPCR) confirmed that the methylation molecules NSUN2 and YBX1 specifically recognized and bound to HGH1 through m5C modification. In addition, proteomics, co-immunoprecipitation (co-IP), and Ribosome sequencing (Ribo-Seq) were used to explore the biological role of HGH1 in BC.
RESULTS: As the main m5C methylation molecule, NSUN2 is abnormally overexpressed in BC and increases the overall level of RNA m5C. Knocking down NSUN2 can inhibit BC progression in vitro or in vivo. Combined RNA-seq and RNA-BisSeq analysis identified HGH1 as a potential target of abnormal m5C modifications. We clarified the mechanism by which NSUN2 regulates HGH1 expression through m5C modification, a process that involves interactions with the YBX1 protein, which collectively impacts mRNA stability and protein synthesis. Furthermore, this study is the first to reveal the binding interaction between HGH1 and the translation elongation factor EEF2, providing a comprehensive understanding of its ability to regulate transcript translation efficiency and protein synthesis in BC cells.
CONCLUSIONS: This study preliminarily clarifies the regulatory role of the NSUN2-YBX1-m5C-HGH1 axis from post-transcriptional modification to protein translation, revealing the key role of abnormal RNA m5C modification in BC and suggesting that HGH1 may be a new epigenetic biomarker and potential therapeutic target for BC.
METHODS: Tumor and adjacent tissues from 5 BC patients were collected, and the m5C modification target HGH1 in BC was screened through RNA sequencing (RNA-seq) and single-base resolution m5C methylation sequencing (RNA-BisSeq). Methylation RNA immunoprecipitation-qPCR (MeRIP-qPCR) and RNA-binding protein immunoprecipitation-qPCR (RIP-qPCR) confirmed that the methylation molecules NSUN2 and YBX1 specifically recognized and bound to HGH1 through m5C modification. In addition, proteomics, co-immunoprecipitation (co-IP), and Ribosome sequencing (Ribo-Seq) were used to explore the biological role of HGH1 in BC.
RESULTS: As the main m5C methylation molecule, NSUN2 is abnormally overexpressed in BC and increases the overall level of RNA m5C. Knocking down NSUN2 can inhibit BC progression in vitro or in vivo. Combined RNA-seq and RNA-BisSeq analysis identified HGH1 as a potential target of abnormal m5C modifications. We clarified the mechanism by which NSUN2 regulates HGH1 expression through m5C modification, a process that involves interactions with the YBX1 protein, which collectively impacts mRNA stability and protein synthesis. Furthermore, this study is the first to reveal the binding interaction between HGH1 and the translation elongation factor EEF2, providing a comprehensive understanding of its ability to regulate transcript translation efficiency and protein synthesis in BC cells.
CONCLUSIONS: This study preliminarily clarifies the regulatory role of the NSUN2-YBX1-m5C-HGH1 axis from post-transcriptional modification to protein translation, revealing the key role of abnormal RNA m5C modification in BC and suggesting that HGH1 may be a new epigenetic biomarker and potential therapeutic target for BC.
摘要：
背景：RNAm5C甲基化与肿瘤的发生和发展密切相关。作为主要的甲基转移酶，NSUN2在不同类型的肿瘤中起着至关重要的调节作用。然而,NSUN2介导的m5C修饰对乳腺癌(BC)的确切影响尚不清楚.我们的研究旨在阐明NSUN2如何通过m5C修饰调节靶基因HGH1（也称为FAM203）的分子机制，从而促进BC进展。此外,本研究旨在初步阐明NSUN2和HGH1在BC中的生物学作用。
方法：收集5例BC患者的肿瘤及癌旁组织，通过RNA测序(RNA-seq)和单碱基分辨率m5C甲基化测序(RNA-BisSeq)筛选BC中m5C修饰靶标HGH1。甲基化RNA免疫沉淀-qPCR(MeRIP-qPCR)和RNA结合蛋白免疫沉淀-qPCR(RIP-qPCR)证实甲基化分子NSUN2和YBX1通过m5C修饰特异性识别并结合HGH1。此外,蛋白质组学，免疫共沉淀（co-IP），采用核糖体测序(Ribo-Seq)研究HGH1在BC中的生物学作用。
结果：作为主要的m5C甲基化分子，NSUN2在BC中异常过表达，并增加RNAm5C的总体水平。敲除NSUN2可以在体外或体内抑制BC进展。组合的RNA-seq和RNA-BisSeq分析将HGH1鉴定为异常m5C修饰的潜在靶标。我们阐明了NSUN2通过m5C修饰调节HGH1表达的机制，一个涉及与YBX1蛋白相互作用的过程，它们共同影响mRNA的稳定性和蛋白质的合成。此外，本研究首次揭示HGH1与翻译延伸因子EEF2之间的结合相互作用，全面了解其在BC细胞中调节转录本翻译效率和蛋白质合成的能力。
结论：本研究初步阐明了NSUN2-YBX1-m5C-HGH1轴从转录后修饰到蛋白质翻译的调控作用，揭示了异常RNAm5C修饰在BC中的关键作用，并提示HGH1可能是新的表观遗传生物标志物和BC的潜在治疗靶标。
方法：收集5例BC患者的肿瘤及癌旁组织，通过RNA测序(RNA-seq)和单碱基分辨率m5C甲基化测序(RNA-BisSeq)筛选BC中m5C修饰靶标HGH1。甲基化RNA免疫沉淀-qPCR(MeRIP-qPCR)和RNA结合蛋白免疫沉淀-qPCR(RIP-qPCR)证实甲基化分子NSUN2和YBX1通过m5C修饰特异性识别并结合HGH1。此外,蛋白质组学，免疫共沉淀（co-IP），采用核糖体测序(Ribo-Seq)研究HGH1在BC中的生物学作用。
结果：作为主要的m5C甲基化分子，NSUN2在BC中异常过表达，并增加RNAm5C的总体水平。敲除NSUN2可以在体外或体内抑制BC进展。组合的RNA-seq和RNA-BisSeq分析将HGH1鉴定为异常m5C修饰的潜在靶标。我们阐明了NSUN2通过m5C修饰调节HGH1表达的机制，一个涉及与YBX1蛋白相互作用的过程，它们共同影响mRNA的稳定性和蛋白质的合成。此外，本研究首次揭示HGH1与翻译延伸因子EEF2之间的结合相互作用，全面了解其在BC细胞中调节转录本翻译效率和蛋白质合成的能力。
结论：本研究初步阐明了NSUN2-YBX1-m5C-HGH1轴从转录后修饰到蛋白质翻译的调控作用，揭示了异常RNAm5C修饰在BC中的关键作用，并提示HGH1可能是新的表观遗传生物标志物和BC的潜在治疗靶标。
