Department of Spine Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China.
Department of Spine Surgery, The Third Affiliated Hospital, Southern Medical University, Guangzhou, China.
J Cell Biochem. 2019 Sep;120(9):15678-15687. doi: 10.1002/jcb.28837. Epub 2019 May 8.
The aim of this study was to explore the regulatory mechanism of circRNA_100876/ microRNA-136 (miR-136) axis in the development and progression of osteosarcoma cancer. Quantitative real-time polymerase chain reaction (RT-qPCR) was used to evaluate the expression levels of circRNA_100876 and miR-136 in osteosarcoma cancer samples and the adjacent nontumor tissues. Then, cell proliferation, cell cycle, apoptosis, and migration of circRNA_100876-knocked down cells were analyzed by in vitro and in vivo experiments, using cell counting kit-8 (CCK-8), flow cytometry, and transwell and tumorigenesis assays. The expression of circRNA_100876 was found to be significantly upregulated in osteosarcoma, and was closely correlated with the tumor size and tumor differentiation degree. In addition, the knockdown of circRNA_100876 could significantly inhibit the tumor growth, both in vitro and in vivo. Flow cytometry and Western blot analysis results showed that the downregulation of circRNA_100876 inhibited osteosarcoma cells proliferation via promoting apoptosis and arresting more cells in the G2/M stage, as suggested by the expression of apoptosis and cell cycle pathway-related proteins, which changed consistently. Furthermore, the level of miR-136 was negatively correlated with the expression of circRNA_100876, and miR-136 inhibitors were able to reverse the suppression of cell proliferation induced by silencing circRNA_100876. Our study demonstrates that the dysregulation of circRNA_100876 could induce apoptosis and arrest the cell cycle at G2/M stage, followed by suppression of cell proliferation in osteosarcoma, while silencing miR-136 could restore the cell growth. Therefore, circRNA_100876 might serve as a promising biomarker and treatment target for osteosarcoma.
本研究旨在探索环状 RNA_100876/微小 RNA-136（miR-136）轴在骨肉瘤发生发展中的调控机制。采用实时定量聚合酶链反应（RT-qPCR）检测骨肉瘤组织及相邻非肿瘤组织中环状 RNA_100876 和 miR-136 的表达水平。然后，通过体外和体内实验分析环状 RNA_100876 敲低细胞的增殖、细胞周期、凋亡和迁移，采用细胞计数试剂盒-8（CCK-8）、流式细胞术和 Transwell 及肿瘤生成实验。结果发现，环状 RNA_100876 在骨肉瘤中表达明显上调，且与肿瘤大小和肿瘤分化程度密切相关。此外，环状 RNA_100876 的敲低可显著抑制体外和体内肿瘤的生长。流式细胞术和 Western blot 分析结果表明，下调环状 RNA_100876 通过促进细胞凋亡和将更多细胞阻滞在 G2/M 期来抑制骨肉瘤细胞的增殖，这与凋亡和细胞周期通路相关蛋白的表达一致，这些蛋白的表达也随之发生变化。此外，miR-136 的水平与环状 RNA_100876 的表达呈负相关，miR-136 抑制剂能够逆转沉默环状 RNA_100876 诱导的细胞增殖抑制。本研究表明，环状 RNA_100876 的失调可诱导骨肉瘤细胞凋亡和阻滞细胞周期于 G2/M 期，随后抑制细胞增殖，而沉默 miR-136 可恢复细胞生长。因此，环状 RNA_100876 可能成为骨肉瘤有前途的生物标志物和治疗靶点。
