circUSP34 accelerates osteosarcoma malignant progression by sponging miR-16-5p.

Osteosarcoma (OS) is a primary and highly malignant mesenchymal tissue tumor. The specific pathological mechanism underlying disease initiation or progression remains unclear. Circular RNAs (circRNAs) are a type of covalently circular RNA with a head-to-tail junction site. In this study, we aimed to investigate the sponging mechanism between circRNAs and microRNAs (miRNAs) in OS. Based on the inhibited effect of miR-16-5p reported on OS, circUSP34 was analyzed as a sponge of miR-16-5p via Starbase. We found that circUSP34 promoted the proliferation, migration, and invasion of OS in vitro and in vivo. circUSP34 increased but miR-16-5p decreased in OS by qRT-PCR. Function assays showed that the malignancy of OS cells, including proliferation, migration, and invasion, was inhibited after knocking out circUSP34. Western blotting results showed that the expression level of vimentin and Ki-67 decreased. Similarly, miR-16-5p mimic compromised the proliferation, migration, and invasion of OS cells. FISH assay results indicated that circUSP34 and miR-16-5p were colocalized in the cytoplasm. The sponging mechanism of circUSP34 and miR-16-5p was verified by dual-luciferase reporter assay, RNA immunoprecipitation (RIP), and RNA pull down assays. Interestingly, the miR-16-5p inhibitor partly reversed the inhibitory effect of sh-circUSP34 on the malignancy of OS cells. Further, mice tumors for IHC indicated that vimentin, N-cadherin, and Ki-67 protein expression decreased, but E-cadherin protein expression increased. Collectively, circUSP34 promoted OS malignancy, including proliferation, migration, and invasion, by sponging miR-16-5p. It can serve as a potential therapeutic target and biomarker.

Lou J ，Zhang H ，Xu J ，Ren T ，Huang Y ，Tang X ，Guo W ... -  《-》

Circular RNA circTADA2A promotes osteosarcoma progression and metastasis by sponging miR-203a-3p and regulating CREB3 expression.

Wu Y ，Xie Z ，Chen J ，Chen J ，Ni W ，Ma Y ，Huang K ，Wang G ，Wang J ，Ma J ，Shen S ，Fan S ... -  《Molecular Cancer》

WTAP-mediated m6A modification of circ_0032463 promotes osteosarcoma progression by sponging miR-145-5p and regulating GFRA1 expression.

Osteosarcoma (OS) is the most frequent bone malignancy in humans. Previous evidence suggest that circ_0032463 is an oncogenic circular RNA (circRNA) in various cancers, including OS. However, the molecular mechanism of circ_0032463 involved in OS is still unclear. Circ_0032463, microRNA-145-5p (miR-145-5p), GDNF receptor alpha 1 (GFRA1), and Wilms tumor 1-associated protein (WTAP) levels were determined using real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation, apoptosis, migration, invasion, and angiogenesis were analyzed using 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell, and tube formation assays. Western blot analysis was performed to measure matrix metalloproteinase 2 (MMP2), MMP9, GFRA1, and WTAP protein levels. Binding between miR-145-5p and circ_0032463 or GFRA1 was confirmed using a dual-luciferase reporter and pull-down assay. The biological role of circ_0032463 on OS cell growth was also analyzed using a xenograft tumor model in vivo. Methylated RNA immunoprecipitation assay validated the interaction between WTAP and circ_0032463. Circ_0032463, GFRA1, and WTAP levels were increased, and miR-145-5p was decreased in OS tissues and cells. Circ_0032463 deficiency might hinder OS cell proliferation, migration, invasion, angiogenesis, and promote apoptosis in vitro. Mechanically, circ_0032463 worked as a miR-145-5p sponge to increase GFRA1 expression. Repression of circ_0032463 knockdown on tumor cell growth was proved in vivo. Besides, N6-methyladenosine (m6A) modification facilitates the biogenesis of circ_0032463. Taken together, m6A-mediated biogenesis of circ_0032463 facilitates OS cell malignant biological behavior partly via regulating the miR-145-5p/GFRA1 axis, suggesting a promising molecular marker for OS treatment.

Huang Z ，Chen P ，Liu Y 《-》

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》

Tanshinone I restrains osteosarcoma progression by regulating circ_0000376/miR-432-5p/BCL2 axis.

Circular RNAs (circRNAs) have been identified as important regulators in cancer progression. Nevertheless, little is known about the biological function of circ_0000376 in the progression of osteosarcoma (OS). Cell viability, colony formation ability, apoptosis, and motility were analyzed by Cell Counting Kit-8 assay, colony formation assay, flow cytometry, and transwell assays. Cellular glycolytic metabolism was analyzed using commercial kits. RT-qPCR and Western blot assay were performed to analyze RNA and protein expression in OS tissues and cells. Starbase software was used to establish circRNA-microRNA (miRNA)-messenger RNA linkage, and intermolecular interaction was verified by dual-luciferase reporter assay. Xenograft tumor assay was conducted to analyze the effects of Tanshinone I (Tan I) and circ_0000376 on xenograft tumor growth in vivo. Tan I treatment suppressed the viability, migration, invasion, and glycolysis and triggered the apoptosis of OS cells. Tan I treatment markedly down-regulated circ_0000376 expression in OS cells. The addition of circ_0000376 plasmid largely rescued the malignant behaviors of OS cells upon Tan I exposure. Circ_0000376 interacted with miR-432-5p in OS cells. Circ_0000376 overexpression-mediated protective effects in Tan I-induced OS cells were partly attenuated by the accumulation of miR-432-5p. miR-432-5p bound to the 3' untranslated region (3'UTR) of B-cell leukemia/lymphoma 2 (BCL2) in OS cells. miR-432-5p interference-induced effects in Tan I-treated OS cells were partly overturned by the silence of BCL2. Circ_0000376 can act as miR-432-5p sponge to up-regulate BCL2 expression in OS cells. Circ_0000376 silencing contributed to the anti-tumor effect of Tan I on the growth of xenograft tumors in vivo. Tan I exerted an anti-tumor role in OS progression by targeting circ_0000376/miR-432-5p/BCL2 axis.

Ye B ，Qiao K ，Zhao Q ，Jiang Z ，Hu N ，Wang F ... -  《-》