Hsa_circ_0001017 promotes cell proliferation, migration and invasion in osteosarcoma by sponging miR-145-5p.

Circular RNAs (circRNAs) have displayed important roles in the development and progression of various cancers. However, the functions of the majority of circRNAs in osteosarcoma (OS) remain unknown. Circular RNA microarray analysis was performed in three OS cell lines (Saos-2, U2OS and MG63) and normal vascular endothelial cells. The co-differentially expressed circRNAs (CDECs) were identified in OS cell lines with the criterion of FDR (false discovery rate) < 0.05 and |fold change (FC)|> 2. Quantitative real-time PCR was used to validate the expression levels of selected CDECs. A series of functional assays, including MTT assay, flow cytometry and transwell assay were conducted in OS cells. The interaction between circRNA and miRNAs was confirmed by luciferase reporter assay and RNA immunoprecipitation assay. A total of 241 CDECs, including 75 upregulated and 166 downregulated CDECs, were identified in three OS cell lines compared with normal vascular endothelial cells. PCR validation showed that hsa_circ_0000704, hsa_circ_0001017 and hsa_circ_0005035 were all highly expression in the three OS cell lines, compared with osteoblast cell lines (HECC, hFOB1.19 and HFF-1). Functionally, overexpression of circ_0001017 significantly promoted the cell proliferation, migration and invasion and decreased apoptosis in U2OS cells. Knockdown of circ_0001017 obtained the opposite results. Circ_0001017 may downregulate miR-145-5p through direct binding. Furthermore, the expression of miR-145-5p was negatively regulated by circ_0001017 in OS cells. In addition, further functional studies indicated that miR-145-5p inhibitor eliminated the effects caused by si-circ_0001017 in OS cells. In conclusion, our study suggested that circ_0001017 may be a novel oncogenic factor during the progression and development of OS by targeting miR-145-5p.

Yang Q ，Yu H ，Hu K 《Journal of Orthopaedic Surgery and Research》

Hsa_circ_0008934 promotes the proliferation and migration of osteosarcoma cells by targeting miR-145-5p to enhance E2F3 expression.

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. 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. Elevated hsa_circ_0008934 expression promotes the proliferation and migration of osteosarcoma cells by sponging miR-145-5p to enhance E2F3 expression.

Li S ，Zeng M ，Yang L ，Tan J ，Yang J ，Guan H ，Kuang M ，Li J ... -  《-》

Circ_0001174 facilitates osteosarcoma cell proliferation, migration, and invasion by targeting the miR-186-5p/MACC1 axis.

Studies of aberrantly expressed circular RNAs (circRNAs) can provide insights into the molecular mechanisms of osteosarcoma (OS). However, the role of circ_0001174 in OS progression remains unknown. This study is aimed to identify differentially expressed circRNAs and messenger RNAs (mRNAs) in patients with OS and to investigate potential regulatory ways of circ_0001174. High-throughput sequencing was performed to screen aberrantly expressed circRNAs and mRNAs between tumor and paracancerous tissues from patients with OS. Several bioinformatics tools were used to analyze the functions and pathways of the differentially expressed genes between the tissues. Cell counting kit-8, cell migration and invasion assays were performed to evaluate the functions of the critical circRNAs. RNA interference experiments, quantitative real-time polymerase chain reaction (RT-qPCR) and western blotting were used to explore the relationship between miR-186-5p and circ_0001174 or metastasis-associated in colon cancer 1 (MACC1). Compared with the paracancerous tissues, 109 circRNAs and 1264 mRNAs were differentially expressed in the OS tissues, including 88 circRNAs and 707 mRNAs that were upregulated and 21 circRNAs and 557 mRNAs that were downregulated. The expression of four upregulated and four downregulated circRNAs was validated using RT-qPCR; the results were consistent with the sequencing data, and circ_0001174 was found to be significantly upregulated in 16 pairs of OS tissues and OS cell lines (fold change > 2.0, P value < 0.05). Knockdown of circ_0001174 inhibited the proliferation, migration, and invasion of OS cells. Additionally, circ_0001174 directly and negatively modulated the expression of miR-186-5p and positively regulated the expression of MACC1. Abnormally high expression of circ_0001174 may promote the proliferation, migration, and invasion of OS cells through up-regulating MACC1 by sponging miR-186-5p. These results provide insight into therapeutic targets for preventing and treating OS.

Lin F ，Wang X ，Zhao X ，Ren M ，Wang Q ，Wang J ... -  《Journal of Orthopaedic Surgery and Research》

Circular RNA hsa_circ_0069117 suppresses proliferation and migration of osteosarcoma cells lines via miR-875-3p/PF4V1 axis.

Osteosarcoma (OS) is one of the most common malignant bone tumors in children and adolescents. Circular RNAs (circRNAs) are critical regulators involved in multiple physiological and pathological processes. However, the underlying regulatory mechanisms of circRNA in OS are still not fully understood. The circRNA expression profiles were downloaded from the Gene Expression Omnibus (GEO) database and analyzed by GEO2R. Bioinformatics analysis was performed to predict the potential target miRNAs of hsa_circ_0069117 and its downstream mRNAs. The co-expression of hsa_circ_0069117/miR-875-3p/PF4V1 axis was further validated in OS tissue samples via quantitative real-time PCR (qRT-PCR). Luciferase reporter gene plasmids containing the sequence of PF4V1 and hsa_circ_0069117 were constructed to verify the putative sites of miR-875-3p. Gain/loss-of-function assays were performed to verify the effect of hsa_circ_0069117 on miR-875-3p/PF4V1 expression and related pathways via qRT-PCR and Western blot. Cell counting kit-8 (CCK-8) and wound-healing assays were performed to evaluate the effect of hsa_circ_0069117 on cell proliferation and migration of MG63 and U2OS, respectively. We identified hsa_circ_0069117 as the most markedly dysregulated circRNA in OS cell lines. Bioinformatics analysis indicated that hsa_circ_0069117 might inhibit the expression of miR-875-3p, thereby promoting the expression of platelet factor 4 variant 1 (PF4V1). The expression of miR-875-3p was negatively correlated to hsa_circ_0069117 and PF4V1 in clinical samples. Luciferase reporter gene assays confirmed the binding sites of miR-875-3p on hsa_circ_0069117 and PF4V1. Gain/loss-of-function and rescue assays further indicated that hsa_circ_0069117 could significantly promote the expression of PF4V1 by sponging miR-875-3p, thereby inhibiting the proliferation and migration of OS cells by suppressing ERK1 and AKT. Our study revealed that hsa_circ_0069117 is an anti-OS molecule that could substantially attenuate cell proliferation and migration of OS, which may provide a novel and reliable molecular target for the treatment of OS patients.

Zhang Z ，Zhou L ，Zhou S ，Li X ... -  《Journal of Orthopaedic Surgery and Research》

Circular RNA hsa_circ_0000073 contributes to osteosarcoma cell proliferation, migration, invasion and methotrexate resistance by sponging miR-145-5p and miR-151-3p and upregulating NRAS.

Li X ，Liu Y ，Zhang X ，Shen J ，Xu R ，Liu Y ，Yu X ... -  《Aging-US》