Hsa_circ_0008934 promotes the proliferation and migration of osteosarcoma cells by targeting miR-145-5p to enhance E2F3 expression
Hsa_circ_0008934 promotes the proliferation and migration of osteosarcoma cells by targeting miR-145-5p to enhance E2F3 expression
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DOI：
10.1016/j.biocel.2020.105826
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发表时间：
2020-10-01
影响因子：
4
通讯作者：
Li, Jiaying
中科院分区：
文献类型：
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作者：
Li, Shiyuan;Zeng, Ming;Li, Jiaying
目的：探讨hSA_CIRC_0008934在骨肉瘤中的作用及其参与骨肉瘤发生发展调控的分子机制。方法：利用下一代核糖核酸测序技术对骨肉瘤细胞系SaOS2、MG63和正常成骨细胞系hF0B1.19中差异表达的CircRNA进行鉴定。采用实时定量聚合酶链式反应和逆转录聚合酶链式反应分别检测hSA_CIRC_0008934的表达和环状形态。采用四甲基偶氮唑蓝比色法、集落形成实验、流式细胞仪和Transwell系统分别检测hsa_CIRC_0008934沉默或过表达对SaOS_2和MG63细胞增殖、凋亡、细胞周期进程、迁移和侵袭的影响。荧光原位杂交揭示了hSA_CIRC_0008934的亚细胞分布。用双荧光素酶报告实验证实hSA_CIRC_0008934与microRNA的结合。采用裸鼠体内成瘤实验研究hSA_CIRC_0008934在骨肉瘤中的致癌作用。结果：hSA_CIRC_0008934在SaOS2和MG63细胞中的表达明显高于hFOB1.19细胞。HSA_CIRC_0008934在SaOS_2和MG63细胞中的表达显著升高。HSA_CIRC_0008934沉默可显著抑制细胞增殖，促进细胞凋亡，阻断细胞周期进程，降低细胞的迁移和侵袭能力。在MG63细胞中过表达hsA_CIRC_0008934可引起相反的细胞变化。HSA_CIRC_0008934主要分布在胞浆中，对骨肉瘤细胞E2F3的表达有正向调节作用。此外，它还直接与miR-145-5p结合，抑制E2F3的表达，增强MG63细胞在裸鼠体内的成瘤作用。定量逆转录聚合酶链式反应显示，细胞内注射hSA_CIRC_0008934慢病毒后，hSA_CIRC_0008934过表达，miR-145-5p表达下调。Western blotting证实E2F3表达上调。TUNEL法检测结果显示，hSA_CIRC_0008934过表达可抑制肿瘤细胞的凋亡。免疫组化检测显示hSA_CIRC_0008934过表达可促进Ki67和PCNA的表达。结论：hSA_CIRC_0008934的高表达可通过海绵化miR-145-5p促进E2F3的表达，从而促进骨肉瘤细胞的增殖和迁移。
Objective: To investigate the role of hsa_circ_0008934 in osteosarcoma and the molecular mechanism involved in the regulation of the occurrence and development of osteosarcomaMethods: Differentially expressed circRNAs in the osteosarcoma cell lines SaOS2 and MG63 and in the normal human osteoblast cell line hF0B1.19 were identified via next-generation RNA sequencing. The expression and circular morphology of hsa_circ_0008934 were analyzed via quantitative real-time polymerase chain reaction (qRT-PCR) and RT-PCR analysis, respectively. Proliferation, apoptosis, cell cycle progression, migration, and invasion of SaOS2 and MG63 cells with hsa_circ_0008934 silencing or overexpression were assessed using the MTS method, colony formation assay, flow cytometry, and the transwell system, respectively. The subcellular distribution of hsa_circ_0008934 was revealed via fluorescence in situ hybridization. The binding of hsa_circ_0008934 with microRNAs was confirmed using the dual-luciferase reporter assay. The oncogenic roles of hsa_circ_0008934 in osteosarcoma were determined using an in vivo tumorigenesis assay with nude mice. qRTPCR, western blotting, TUNEL assay, and immunohistochemistry (IHC) were used to detect the tumorigenicity of hsa_circ_0008934 in osteosarcoma cells.Results: Many circRNAs were differentially expressed in SaOS2 and MG63 cells than in hFOB1.19 cells. Hsa_circ_0008934 expression was significantly elevated in SaOS2 and MG63 cells. Hsa_circ_0008934 silencing significantly reduced proliferation, enhanced apoptosis, blocked cell cycle progression, and impaired migration and invasion capacities of SaOS2 cells. Opposite cellular alterations were achieved by overexpressing hsa_circ_0008934 in MG63 cells. Hsa_circ_0008934 was mainly distributed in the cytosol and positively regulated E2F3 expression in osteosarcoma cells. In addition, it directly bound with miR-145-5p to repress E2F3 expression and enhanced the tumorigenesis of MG63 cells in nude mice. qRT-PCR revealed that the intracellular injection of hsa_circ_0008934 lentivirus resulted in hsa_circ_0008934 overexpression and miR-145-5p downregulation. Western blotting confirmed that E2F3 was upregulated. Moreover, the TUNEL assay showed that hsa_circ_0008934 overexpression inhibited the apoptosis of tumor cells. IHC detection revealed that the hsa_circ_0008934 overexpression could promote the expression of Ki67 and PCNA.Conclusion: Elevated hsa_circ_0008934 expression promotes the proliferation and migration of osteosarcoma cells by sponging miR-145-5p to enhance E2F3 expression.