背景:骨肉瘤(OS)是儿童和青少年人群中的原发性恶性骨肿瘤。长链非编码RNA(LncRNA),如血浆-细胞瘤变异型易位1(PVT1),已成为OS转移的重要调节因子。最近的研究表明,信号转导和转录激活因子3(STAT3)信号的激活,这可能是由PVT1控制,抑制铁凋亡,促进癌症的恶性进展。因此,本研究旨在确定PVT1在OS发病机制中的作用,并探讨PVT1是否通过调节STAT3/GPX4通路介导的铁凋亡影响OS进展.
方法:用sh-PVT1质粒转染人OS细胞系MG63,抑制PVT1的表达,有或没有与STAT3过表达质粒共转染。通过实时定量聚合酶链反应(RT-qPCR)确定PVT1的表达。扩散,迁移,入侵,使用细胞计数试剂盒-8(CCK8)确定MG63细胞的凋亡,Transwell分析,和流式细胞术。丙二醛(MDA)的水平,Fe2+,和谷胱甘肽(GSH)通过ELISA试剂盒测定,而活性氧(ROS)水平是通过免疫荧光测定的。Westernblot(WB)检测STAT3、p-STAT3和谷胱甘肽过氧化物酶4(GPX4)蛋白表达水平。
结果:PVT1在MG63细胞中表达显著增加。当用sh-PVT1质粒敲除PVT1时,扩散,迁移,MG63细胞的侵袭能力明显受到抑制,而细胞凋亡率上调。进一步的研究表明,与PVT1敲低MG63细胞表现出升高的MDA水平,Fe2+,ROS。此外,抑制PVT1的表达导致GSH水平降低并抑制p-STAT3和GPX4的表达。当sh-PVT1与STAT3过表达质粒共转染MG63细胞时,MDA水平的增加,Fe2+,ROS被下调,GSH的表达减少,p-STAT3和GPX4上调。
结论:PVT1通过激活STAT3/GPX4通路抑制铁凋亡促进OS转移。靶向PVT1可能是OS治疗的一种新的治疗策略。
BACKGROUND: Osteosarcoma (OS) is a primary malignant bone tumor in the pediatric and adolescent populations. Long non-coding RNAs (LncRNAs), such as plasma-cytoma variant translocation 1 (PVT1), have emerged as significant regulators of OS metastasis. Recent studies have indicated that activation of signal transducer and activator of transcription 3 (STAT3) signaling, which might be controlled by PVT1, inhibits ferroptosis to promote the malignant progression of cancer. Therefore, the present study aimed to determine the role of PVT1 in OS pathogenesis and investigate whether PVT1 affects OS progression by regulating STAT3/GPX4 pathway-mediated ferroptosis.
METHODS: The human OS cell line MG63 were transfected with sh-PVT1 plasmid to inhibit PVT1 expression, with or without co-transfection with a STAT3 overexpression plasmid. The expression of PVT1 was determined by real-time quantitative polymerase chain reaction (RT-qPCR). The proliferation, migration, invasion, and apoptosis of MG63 cells were determined using the cell counting kit-8 (CCK8), Transwell assay, and flow cytometry. The levels of malondialdehyde (MDA), Fe2+, and glutathione (GSH) were determined by ELISA kits, whereas reactive oxygen species (ROS) level was determined by immunofluorescence. The protein expression levels of STAT3, p-STAT3, and glutathione peroxidase 4 (GPX4) were detected by western blot (WB).
RESULTS: PVT1 expression was significantly increased in MG63 cells. When knocking down PVT1 with sh-PVT1 plasmid, the proliferation, migration, and invasion of MG63 cells were markedly inhibited, while the rate of apoptosis was upregulated. Further investigation revealed that MG63 cells with PVT1 knockdown exhibited elevated levels of MDA, Fe2+, and ROS. In addition, the inhibition of PVT1 expression resulted in decreased levels of GSH and inhibited expression of p-STAT3 and GPX4. When sh-PVT1 was co-transfected with STAT3 overexpression plasmid in MG63 cells, the increased levels of MDA, Fe2+, and ROS were downregulated, and the decreased expressions of GSH, p-STAT3, and GPX4 were upregulated.
CONCLUSIONS: PVT1 promotes OS metastasis by activating the STAT3/GPX4 pathway to inhibit ferroptosis. Targeting PVT1 might be a novel therapeutic strategy for OS treatment.