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Abstract：
Objective: Most patients with osteosarcoma (OS) have an extremely poor prognosis. The primary purpose of this investigation was to explore the biological effect of Lnc-CLSTN2-1:1 on OS and the potential processes involved. Materials and procedures: We selected differentially overexpressed Lnc-CLSTN2-1:1 from our laboratory\'s existing RNA sequence analysis data (fibroblast osteoblast (hFOB 1.19) and three osteosarcoma cell lines (HOS, MG63, and U2OS) as the research object. Next, we detected Lnc-CLSTN2-1:1 in the osteosarcoma HOS cell line and fibroblast cells using qRT-PCR. We evaluated cell proliferation ability using EdU incorporation test, CCK-8 test, and cell clone formation; cell invasion and migration were assessed using the Transwell test, while flow cytometry examined cell cycle, apoptosis, and reactive oxygen species (ROS); Subsequently, the activity changes of selenase (GPx) glutathione peroxidase and (TrxR) thioredoxin reductase were detected. In addition, changes in related proteins were analyzed through Western blotting. Results: The expression of Lnc-CLSTN2-1:1 in osteosarcoma cells was significantly increased. The proliferation, invasion, and migration of osteosarcoma cells were significantly inhibited by knockdown of the expression of Lnc-CLSTN2-1:1, and the cell cycle-related signaling pathway PI3K/AKT/GSK-3β/cycinD1 was also inhibited. However, insulin-like growth factor-1 (igf-1) could reverse this process. In addition, we examined the activity of two selenophenases (TrxR and GPx) and the changes of ROS before and after Lnc-CLSTN2-1:1 knockdown. The results showed that both TrxR and GPx activities were reduced after Lnc-CLSTN2-1:1 knockdown, resulting in the inhibition of antioxidant stress levels, while intracellular ROS levels were high, which eventually caused killing effects on tumor cells due to the imbalance between oxidative stress and antioxidant stress. Conclusion: Our results showed that Lnc-CLSTN2-1:1 enhanced anti-oxidative stress TrxR and GPx selenoprotein activities through the PI3K/AKT signaling pathway while counteracting the loss of reactive oxygen species ROS produced by mitochondria to osteosarcoma cells, which protected osteosarcoma cells and thus promoted the proliferation and metastatic ability of OS.
摘要：
目的：大多数骨肉瘤（OS）患者的预后极差。这项研究的主要目的是探索Lnc-CLSTN2-1：1对OS的生物学效应以及所涉及的潜在过程。材料和程序：我们从实验室现有的RNA序列分析数据（成纤维细胞成骨细胞（hFOB1.19）和三种骨肉瘤细胞系（HOS，以MG63、U2OS)为研讨对象。接下来,我们使用qRT-PCR检测了骨肉瘤HOS细胞系和成纤维细胞中的Lnc-CLSTN2-1：1。我们使用EdU掺入试验评估细胞增殖能力，CCK-8试验,和细胞克隆形成；使用Transwell测试评估细胞侵袭和迁移，而流式细胞术检查细胞周期，凋亡，和活性氧(ROS)；随后，检测了硒酶（GPx）谷胱甘肽过氧化物酶和（TrxR）硫氧还蛋白还原酶的活性变化。此外,通过Western印迹分析相关蛋白的变化.结果:Lnc-CLSTN2-1:1在骨肉瘤细胞中的表达明显增加。扩散,入侵,敲低Lnc-CLSTN2-1:1的表达可显著抑制骨肉瘤细胞的迁移,同时抑制细胞周期相关信号通路PI3K/AKT/GSK-3β/cycinD1。然而,胰岛素样生长因子-1（igf-1）可以逆转这一过程。此外,我们检查了两种硒烯酶（TrxR和GPx）的活性以及Lnc-CLSTN2-1：1敲低前后ROS的变化。结果表明，Lnc-CLSTN2-1:1敲低后，TrxR和GPx活性均降低，导致抗氧化应激水平的抑制，虽然细胞内ROS水平很高，由于氧化应激和抗氧化应激之间的失衡，最终对肿瘤细胞产生杀伤作用。结论：我们的结果表明，Lnc-CLSTN2-1：1通过PI3K/AKT信号通路增强了抗氧化应激TrxR和GPx硒蛋白的活性，同时抵消了线粒体产生的活性氧ROS向骨肉瘤细胞的损失，保护骨肉瘤细胞，从而促进OS的增殖和转移能力。
