EZH2-miR-494-FBP1促进骨肉瘤细胞增殖作用及分子机制研究

Osteosarcoma is a primary malignant tumor that occurs in the metaphysis of adolescents. Studies have shown that the epigenetic gene EZH2 and miR-494 are closely related to the development of osteosarcoma, but the specific mechanism is still unknown. Our previous vitro and vivo experiments found that EZH2 inhibitor GSK343 can significantly inhibit osteosarcoma proliferation and promote its apoptosis; high-throughput sequencing showed significant changes in miR-494 and FBP1 expression with GSK343 treatment of osteosarcoma cells; bioinformatics analysis showed there was a binding site between miR-494 and FBP1 3'UTR end, suggesting that malignant proliferation of osteosarcoma cells may be related to the EZH2-miR-494-FBP1 signal axis. In this study, the regulatory axis of EZH2-miR-494-FBP1 was confirmed by luciferase dual fluorescence reporter system and quantitative PCR; correlation between EZH2 and miR-494 and FBP1 expression in osteosarcoma samples was detected and analyzed at molecular and protein levels; vitro and vivo experiments confirmed the inhibitory effect of blocking EZH2-miR-494-FBP1 signal on the malignant proliferation of osteosarcoma cells, and elucidated the molecular mechanism of EZH2 promoting osteosarcoma cell proliferation, providing a theoretical basis for for targeted treatment and drug development of osteosarcoma.

骨肉瘤是好发于青少年长骨干骺端的原发性恶性肿瘤，研究显示表观遗传基因EZH2及miR-494与骨肉瘤发展密切相关，但具体机制尚不明。我们前期体内外实验发现EZH2抑制剂GSK343可显著抑制骨肉瘤增殖，促进其凋亡；高通量测序显示GSK343处理骨肉瘤细胞后miR-494及FBP1基因表达出现显著变化；生物信息学分析显示miR-494与FBP1 3’UTR端存在结合位点，提示骨肉瘤细胞恶性增殖可能与EZH2-miR-494-FBP1信号轴有关。本研究拟利用荧光素酶双荧光报告系统、定量PCR等技术确证EZH2-miR-494-FBP1调控轴；在分子及蛋白水平检测及分析骨肉瘤样本中EZH2与miR-494及FBP1表达的相关性；体内外实验确证阻断EZH2-miR-494-FBP1信号对骨肉瘤细胞恶性增殖的抑制作用，阐明EZH2促进骨肉瘤细胞增殖的分子机制，为骨肉瘤靶向治疗及药物开发提供理论依据。

骨肉瘤是一种分化缺陷病，与成骨分化相关基因和表观遗传改变有关。表观遗传的改变在肿瘤的发生发展中起着重要作用，研究表明表观遗传蛋白EZH2在多种肿瘤中表达异常，但其在骨肉瘤中的作用机制尚不明确。在本项目中，我们提出EZH2-miR-494-FBP1信号轴促进骨肉瘤细胞恶性增殖有关的假说。为了证实上述假说，我们利用小分子抑制剂、慢病毒干扰、定量PCR及免疫印迹等技术，证实EZH2的小分子抑制剂GSK343可显著抑制骨肉瘤增殖及转移，促进细胞凋亡及自噬性死亡；GSK343处理骨肉瘤细胞后miR-494显著上调而FBP1的表达显著下调；生物信息学分析及双荧光报告系统证实miR-494与FBP1 3′UTR端存在结合位点，并进一步确证阻断EZH2-miR-494-FBP1调控轴对骨肉瘤细胞恶性增殖的抑制作用，从而阐明EZH2促进骨肉瘤细胞增殖的分子机制，为骨肉瘤靶向治疗及小分子药物开发提供新的理论依据。我们也通过使用小分子抑制剂GSK126，证实GSK126能够抑制骨肉瘤细胞增殖和诱导细胞程序性死亡，申请专利“GSK126在制备抗骨肉瘤的药物中的应用”，涉及抗骨肉瘤的新型药物的开发。本研究为基础研究向临床转化运用提供了新的理论依据。. 在本项目的资助下，我们还拓展研究了FBP1在卵巢癌和乳腺癌的作用。在卵巢癌中，FBP1在卵巢癌组织中的高表达，促进细胞周期转换及细胞转移；敲低FBP1可能通过下调EZH2/H3K27me3而减弱细胞增殖并增强细胞对顺铂的敏感性。在乳腺癌中，FBP1及其靶标c-Myc在乳腺癌组织中的高表达；敲低FBP1通过下调cyclinA2和上调cyclinB1及p-CDC2的表达而抑制乳腺癌细胞增殖，通过下调MMP2的表达来减少细胞迁移，通过诱导细胞凋亡增强乳腺癌细胞对顺铂的敏感性。这些结果表明FBP1可用于卵巢癌和乳腺癌的诊断和治疗的潜在的新型生物学标记。

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