Department of Orthopedic Oncology Surgery, Shandong Cancer Hospital and Institute Affiliated to Shandong University, Jinan 250117, China; Shandong Cancer Hospital and Institute Affiliated to Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan 250117, China.
Department of Orthopedics, Jinan City People's Hospital, Jinan 271100, China.
Biomed Pharmacother. 2020 May;125:109964. doi: 10.1016/j.biopha.2020.109964. Epub 2020 Feb 7.
Osteosarcoma is the most common primary malignant bone tumor in children and young adults. RNA N-methyladenosine (mA) is the most abundant internal modification in mammalian mRNA, which is involved in tumorigenesis and tumor progression. It has been reported that methyltransferase-like 3 (METTL3), the first reported m6A "writer", plays critical roles in cancer progression. However, its role and molecular mechanism in osteosarcoma is poor studied. In this study, we aimed to investigate the functional role and underlying mechanism of METTL3 in the progression of osteosarcoma.
We detected the mRNA expression of METTL3 in osteosarcoma cell lines, and immunofluorescence assay was performed to observe the location of METTL3. Cell lines with METTL3 gene overexpression or knockdown were established by pcDNA3.1-METTL3 or siRNA interferences in order to determine the function of METTL3 in osteosarcoma in vitro. Transcriptomic RNA sequencing (RNA-seq) were used to screen the target genes of METTL3 in osteosarcoma.
We found that METTL3 localized in cytoplasm and nucleus of osteosarcoma cells. Silencing METTL3 in SAOS-2 and MG63 cells significantly inhibited the mA methylation level, proliferation, migration, and invasion abilities, as well as promoted cell apoptosis. However, up-regulation of METTL3 had no significant effect on the biological behaviors of U2OS cells. Further mechanism analysis suggested that METTL3 knockdown inhibited the expression of ATPase family AAA domain containing 2 (ATAD2). Moreover, ATAD2 knockdown inhibited the proliferation and invasion of SAOS-2 and MG63 cells, while its overexpression showed a significant increase in cell proliferation and invasion. Furthermore, METTL3 knockdown abrogated the promoting effects of ATAD2 overexpression on osteosarcoma cells proliferation and invasion.
Overall, our study revealed that METTL3 functions as an oncogene in the growth and invasion of osteosarcoma by regulating ATAD2, suggesting a potential therapeutic target for osteosarcoma treatment.
骨肉瘤是儿童和青少年中最常见的原发性恶性骨肿瘤。RNA N6-甲基腺苷（m6A）是哺乳动物 mRNA 中最丰富的内部修饰物，它参与肿瘤发生和肿瘤进展。已经报道称，甲基转移酶样 3（METTL3），第一个被报道的 m6A“写入器”，在癌症进展中发挥关键作用。然而，其在骨肉瘤中的作用和分子机制研究甚少。在本研究中，我们旨在研究 METTL3 在骨肉瘤进展中的功能作用和潜在机制。
我们检测了骨肉瘤细胞系中 METTL3 的 mRNA 表达，并通过免疫荧光分析观察 METTL3 的位置。通过 pcDNA3.1-METTL3 或 siRNA 干扰建立 METTL3 基因过表达或敲低的细胞系，以确定 METTL3 在骨肉瘤中的体外功能。采用转录组 RNA 测序（RNA-seq）筛选骨肉瘤中 METTL3 的靶基因。
我们发现 METTL3 定位于骨肉瘤细胞的细胞质和细胞核中。在 SAOS-2 和 MG63 细胞中沉默 METTL3 显著抑制 m6A 甲基化水平、增殖、迁移和侵袭能力，并促进细胞凋亡。然而，上调 METTL3 对 U2OS 细胞的生物学行为没有显著影响。进一步的机制分析表明，METTL3 敲低抑制了 ATP 酶家族 AAA 结构域包含蛋白 2（ATAD2）的表达。此外，ATAD2 敲低抑制了 SAOS-2 和 MG63 细胞的增殖和侵袭，而过表达则显著增加了细胞的增殖和侵袭。此外，METTL3 敲低消除了 ATAD2 过表达对骨肉瘤细胞增殖和侵袭的促进作用。
总体而言，我们的研究表明，METTL3 通过调节 ATAD2 作为骨肉瘤生长和侵袭的癌基因发挥作用，提示 METTL3 可能成为骨肉瘤治疗的潜在靶点。
