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Abstract：
BACKGROUND: Circular RNAs (circRNAs) have been confirmed to be involved in cancer pathogenesis. However, the underlying mechanism of circRNA endothelin converting enzyme 1 (circECE1) in osteosarcoma (OS) development is still not understood.
METHODS: The expression levels of circECE1, microRNA-588 (miR-588) and RAB3D, member RAS oncogene family (RAB3D) were gauged by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. OS cell proliferation was assessed by cell counting kit-8 (CCK-8) assay and 5-ethynyl-2\'-deoxyuridine (EdU) assay. OS cell apoptosis rate and metastasis were identified by flow cytometry and transwell assay. Dual-luciferase reporter analysis and RNA immunoprecipitation (RIP) assay were performed to confirm the interactions among circECE1, miR-588 and RAB3D. Xenograft tumor models were established to explore circECE1 function in vivo. Immunohistochemistry (IHC) assay was applied to analyze RAB3D level after circECE1 knockdown.
RESULTS: In OS, circECE1 expression was higher than that in normal chondroma tissues. High levels of circECE1 were positively linked to OS cell viability, proliferation, migration and invasion, and negatively linked to OS cell apoptosis rate. It was found that circECE1 was a miR-588 sponge, and miR-588 inhibitor abrogated the influence of si-circECE1 on OS cells. MiR-588 targeted RAB3D to further regulate the pathological process of OS. Moreover, silencing circECE1 blocked OS tumor growth in vivo.
CONCLUSIONS: We elucidated the function of a novel circECE1/miR-588/RAB3D axis in OS progression.
METHODS: The expression levels of circECE1, microRNA-588 (miR-588) and RAB3D, member RAS oncogene family (RAB3D) were gauged by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. OS cell proliferation was assessed by cell counting kit-8 (CCK-8) assay and 5-ethynyl-2\'-deoxyuridine (EdU) assay. OS cell apoptosis rate and metastasis were identified by flow cytometry and transwell assay. Dual-luciferase reporter analysis and RNA immunoprecipitation (RIP) assay were performed to confirm the interactions among circECE1, miR-588 and RAB3D. Xenograft tumor models were established to explore circECE1 function in vivo. Immunohistochemistry (IHC) assay was applied to analyze RAB3D level after circECE1 knockdown.
RESULTS: In OS, circECE1 expression was higher than that in normal chondroma tissues. High levels of circECE1 were positively linked to OS cell viability, proliferation, migration and invasion, and negatively linked to OS cell apoptosis rate. It was found that circECE1 was a miR-588 sponge, and miR-588 inhibitor abrogated the influence of si-circECE1 on OS cells. MiR-588 targeted RAB3D to further regulate the pathological process of OS. Moreover, silencing circECE1 blocked OS tumor growth in vivo.
CONCLUSIONS: We elucidated the function of a novel circECE1/miR-588/RAB3D axis in OS progression.
摘要：
背景：环状RNA（circularRNAs，circRNAs）已被证实参与癌症的发病机制。然而,circRNA内皮素转换酶1（circECE1）在骨肉瘤（OS）发展中的潜在机制尚不清楚。
方法：circECE1，microRNA-588（miR-588）和RAB3D的表达水平，通过定量实时聚合酶链反应（qRT-PCR）和蛋白质印迹测定RAS癌基因家族成员（RAB3D）。通过细胞计数试剂盒-8(CCK-8)测定和5-乙炔基-2'-脱氧尿苷(EdU)测定来评估OS细胞增殖。通过流式细胞术和transwell法鉴定OS细胞的凋亡率和转移。进行双荧光素酶报告基因分析和RNA免疫沉淀(RIP)测定以确认circECE1、miR-588和RAB3D之间的相互作用。建立异种移植肿瘤模型以探索体内circECE1功能。免疫组织化学(IHC)测定用于分析circECE1敲低后的RAB3D水平。
结果：在操作系统中，circECE1表达高于正常软骨瘤组织。高水平的circECE1与OS细胞活力呈正相关，扩散,移民和入侵，与OS细胞凋亡率呈负相关。发现circECE1是miR-588海绵，miR-588抑制剂消除了si-circECE1对OS细胞的影响。MiR-588靶向RAB3D进一步调控OS的病理进程。此外，沉默circECE1阻断了体内OS肿瘤的生长。
结论：我们阐明了新的circECE1/miR-588/RAB3D轴在OS进展中的功能。
方法：circECE1，microRNA-588（miR-588）和RAB3D的表达水平，通过定量实时聚合酶链反应（qRT-PCR）和蛋白质印迹测定RAS癌基因家族成员（RAB3D）。通过细胞计数试剂盒-8(CCK-8)测定和5-乙炔基-2'-脱氧尿苷(EdU)测定来评估OS细胞增殖。通过流式细胞术和transwell法鉴定OS细胞的凋亡率和转移。进行双荧光素酶报告基因分析和RNA免疫沉淀(RIP)测定以确认circECE1、miR-588和RAB3D之间的相互作用。建立异种移植肿瘤模型以探索体内circECE1功能。免疫组织化学(IHC)测定用于分析circECE1敲低后的RAB3D水平。
结果：在操作系统中，circECE1表达高于正常软骨瘤组织。高水平的circECE1与OS细胞活力呈正相关，扩散,移民和入侵，与OS细胞凋亡率呈负相关。发现circECE1是miR-588海绵，miR-588抑制剂消除了si-circECE1对OS细胞的影响。MiR-588靶向RAB3D进一步调控OS的病理进程。此外，沉默circECE1阻断了体内OS肿瘤的生长。
结论：我们阐明了新的circECE1/miR-588/RAB3D轴在OS进展中的功能。
