背景:小檗碱(BBR),黄连的一种异喹啉生物碱,已经发现对各种人类恶性肿瘤有强大的活性,包括乳腺癌.然而,BBR在乳腺癌中的潜在抗肿瘤机制仍然知之甚少。
方法:将乳腺癌细胞培养并用不同剂量(0、20、40和60μM)的BBR处理48小时。扩散,凋亡,入侵,和迁移使用3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2H-四唑溴化物(MTT)进行评估,5-乙炔基-2'-脱氧尿苷(EdU),流式细胞术,transwell,和伤口愈合试验。成纤维细胞生长因子7(FGF7),甲基转移酶样3(METTL3),使用实时定量聚合酶链反应(RT-qPCR)和蛋白质印迹测量胰岛素样生长因子2mRNA结合蛋白3(IGF2BP3)的mRNA水平和蛋白质水平。使用甲基化RNA免疫沉淀(MeRIP)-qPCR和RNA免疫沉淀(RIP)测定评估METTL3和FGF7m6A之间的相互作用。使用RIP测定法分析IGF2BP3和FGF7mRNA之间的结合能力。
结果:BBR治疗阻碍了乳腺癌细胞的增殖,入侵,迁移,诱导细胞凋亡。乳腺癌组织中FGF7表达上调,而其水平在BBR处理的肿瘤细胞中降低。FGF7上调减轻了BBR对乳腺癌细胞恶性行为的抑制。在机制上,METTL3通过m6A-IGF2BP3依赖性机制稳定FGF7mRNA,并自然改善FGF7表达。BBR治疗在体内抑制乳腺癌生长。
结论:BBR治疗部分通过调节METTL3介导的FGF7mRNA的m6A修饰来阻断乳腺癌细胞的生长和转移,为乳腺癌的治疗提供了一个有希望的治疗靶点。
BACKGROUND: Berberine (BBR), an isoquinoline alkaloid from Coptidis rhizoma, has been found to have powerful activities against various human malignancies, including breast cancer. However, the underlying antitumor mechanisms of BBR in breast cancer remain poorly understood.
METHODS: Breast cancer cells were cultured and treated with different doses (0, 20, 40, and 60 μM) of BBR for 48 h. Cell viability, proliferation, apoptosis, invasion, and migration were assessed using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT), 5-ethynyl-2\'-deoxyuridine (EdU), flow cytometry, transwell, and wound healing assays. Fibroblast growth factor 7 (FGF7), methyltransferase-like 3 (METTL3), and insulin-like growth factor-2 mRNA-binding protein 3 (
IGF2BP3) mRNA levels and protein levels were measured using real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Interaction between METTL3 and FGF7 m6A was assessed using methylated RNA immunoprecipitation (MeRIP)-qPCR and RNA immunoprecipitation (RIP) assay. Binding ability between
IGF2BP3 and FGF7 mRNA was analyzed using RIP assay.
RESULTS: BBR treatment hindered breast cancer cell proliferation, invasion, migration, and induced apoptosis. FGF7 expression was upregulated in breast cancer tissues, while its level was reduced in BBR-treated tumor cells. FGF7 upregulation relieved the repression of BBR on breast cancer cell malignant behaviors. In mechanism, METTL3 stabilized FGF7 mRNA through the m6A-
IGF2BP3-dependent mechanism and naturally improved FGF7 expression. BBR treatment inhibited breast cancer growth in vivo.
CONCLUSIONS: BBR treatment blocked breast cancer cell growth and metastasis partly by regulating METTL3-mediated m6A modification of FGF7 mRNA, providing a promising therapeutic target for breast cancer treatment.