m6A去甲基化酶FTO下调miR-485-5p促进HER2阳性乳腺癌恶性进展的机制研究

N6-methyladenosine (m6A) internal modification in RNAs has been shown to play critical roles in the development of cancers. The fat mass and obesity-associated protein (FTO) is the first identified genuine demethylase of m6A modification. However, the expressions and the functions of FTO in breast cancer remain unknown. Our previous study demonstrated a high expression of FTO in HER2-positive breast cancer, which is related to poor prognosis of the patients. Down-regulated FTO inhibited HER2-positive breast cancer cells invasion and migration. RNA-seq and MeRIP-seq results prompted that miR-485-5p contains target sequences for m6A methylation and may also target to HER2 3'UTRs. Down-regulated FTO inhibited HER2 expression and increased miR-485-5p expression; Low expression of miR-485-5p was observed in HER2-positive breast cancer, which is negative related to FTO expression. In this proposal, we further extend our research to clarify the expression and clinical significance of FTO, as well as its promoting effects on the malignant phenotype of HER-positive breast cancer. We also explore the transcriptional regulation mechanisms of HER2 overexpression via reducing miR-485 m6A modification and down-regulating miR-485-5p expression by FTO in HER-positive breast cancer. Finally, we explore whether the interventions of FTO can enhance the therapeutic response of trastuzumab in mouse model. Our work aims to provide new strategies for diagnosis and treatment of HER2-positive breast cancer.

RNA甲基化修饰在肿瘤恶性进展中具有重要作用，但m6A去甲基化酶FTO在乳腺癌的表达调控及功能尚未见报道。我们发现FTO高表达仅在HER2阳性乳腺癌提示不良预后；敲降FTO抑制HER2阳性乳腺癌细胞的侵袭和迁移；测序结果显示miR-485-5p同时存在m6A甲基化修饰位点及HER2 3'UTRs结合位点；敲降FTO可抑制HER2表达，上调miR-485-5p表达；miR-485-5p在HER2 阳性乳腺癌中呈低表达，且与FTO表达呈负相关。本项目拟进一步明确FTO和m6A甲基化水平在HER2阳性乳腺癌中的表达情况及临床意义；探讨FTO高表达对癌细胞恶性表型的促进作用；阐明其通过减少miR-485的m6A甲基化修饰下调miR-485-5p，促进HER2表达的转录调控机制；最后在小鼠模型上明确靶向干预FTO能否增强癌细胞对曲妥珠单抗的敏感性，为HER2阳性乳腺癌的临床诊治提供新策略。

N6-甲基腺嘌呤（N6-methyladenosine, m6A）是真核生物mRNA中最常见的一种转录后修饰方式。m6A修饰与乳腺癌等多种恶性肿瘤进展和转移密切相关。现已发现，参与m6A修饰的蛋白质主要包括甲基化酶、去甲基化酶以及甲基化识别蛋白，前两者分别催化目标RNA的N6腺苷甲基化和去甲基化。FTO是一种Fe2+/α-酮戊二酸依赖性双加氧酶，属于AlkB甲基化酶家族成员，在细胞内主要介导m6A的去甲基化修饰。近年来的研究发现，FTO在体内具有重要的功能，其异常表达与肥胖、心肌纤维化、癌症等多种疾病的发生发展有关，但FTO是否参与乳腺癌进展以及相关机制问题尚不明确。.为了解决上述关键问题，项目组首先评估了FTO在乳腺癌组织标本中的表达以及与患者预后之间的相关性，发现乳腺癌组织FTO表达水平显著高于癌旁组织，且FTO高表达特异性提示HER2阳性乳腺癌患者预后不良；后续的细胞功能实验中，项目组利用RNA干扰、质粒过表达等技术，从正反两方面证实FTO可促进HER2阳性乳腺癌细胞的迁移及侵袭能力；为进一步探讨FTO促进HER2阳性乳腺癌恶性表型的潜在机制，项目组进一步利用RNA-seq筛选了FTO调控的下游靶点，发现FTO可负向调控miR-181b-3p的表达，后者通过介导ARL5B降解或翻译抑制过程，从而促进HER2阳性乳腺癌细胞的迁移及侵袭。.本项目首次报道并揭示了m6A去甲基化酶FTO通过miR-181b-3p/ARL5B通路调控HER2阳性乳腺癌进展的新机制，为后续开发针对该类型乳腺癌的特异性干预措施提供实验依据。