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 osteosarcoma

Methods

Differentially expressed circRNAs in the osteosarcoma cell lines SaOS2 and MG63 and in the normal human osteoblast cell line hFOB1.19 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. qRT-PCR, 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.

中文翻译：

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Hsa_circ_0008934通过靶向miR-145-5p来增强E2F3表达，从而促进骨肉瘤细胞的增殖和迁移。

目的

探讨hsa_circ_0008934在骨肉瘤中的作用及其调控骨肉瘤发生发展的分子机制

方法

通过下一代RNA测序鉴定出骨肉瘤细胞SaOS2和MG63以及正常人成骨细胞细胞hFOB1.19中差异表达的circRNA。通过定量实时聚合酶链反应（qRT-PCR）和RT-PCR分析分别分析了hsa_circ_0008934的表达和圆形形态。分别使用MTS方法，集落形成分析，流式细胞仪和transwell系统评估hsa_circ_0008934沉默或过表达的SaOS2和MG63细胞的增殖，凋亡，细胞周期进程，迁移和侵袭。hsa_circ_0008934的亚细胞分布通过荧光原位杂交揭示。hsa_circ_0008934与microRNA的结合已通过双重萤光素酶报告基因检测得以证实。hsa_circ_0008934在骨肉瘤中的致癌作用是通过裸鼠体内肿瘤发生试验确定的。使用qRT-PCR，western印迹，TUNEL分析和免疫组化（IHC）检测hsa_circ_0008934在骨肉瘤细胞中的致瘤性。

结果

SaOS2和MG63细胞中的许多circRNA与hFOB1.19细胞中的差异表达。在SaOS2和MG63细胞中，Hsa_circ_0008934的表达显着升高。Hsa_circ_0008934沉默显着降低了SaOS2细胞的增殖，增强了细胞凋亡，阻断了细胞周期进程并削弱了其迁移和侵袭能力。相反的细胞改变是通过在MG63细胞中过表达hsa_circ_0008934来实现的。Hsa_circ_0008934主要分布在骨肉瘤细胞质中，并在骨肉瘤细胞中表达E2F3。此外，它直接与miR-145-5p结合以抑制E2F3表达并增强裸鼠MG63细胞的肿瘤发生能力。qRT-PCR显示，细胞内注射hsa_circ_0008934慢病毒会导致hsa_circ_0008934过表达和miR-145-5p下调。蛋白质印迹证实E2F3被上调。此外，TUNEL分析表明hsa_circ_0008934的过表达抑制了肿瘤细胞的凋亡。IHC检测显示hsa_circ_0008934过表达可以促进Ki67和PC​​NA的表达。

结论

hsa_circ_0008934表达升高可通过海绵化miR-145-5p增强E2F3表达来促进骨肉瘤细胞的增殖和迁移。