Table 7_Deciphering the oncogenic network: how C1QTNF1-AS1 modulates osteosarcoma through miR-34a-5p and glycolytic pathways.doc

IntroductionOsteosarcoma (OS), a prevalent metastatic cancer among young individuals, is associated with a grim prognosis. Long non-coding RNAs (lncRNAs), including C1QTNF1-AS1, are pivotal regulators of cancer cell proliferation and motility. As an oncogene, C1QTNF1-AS1 is implicated in various tumor types, such as colorectal, pancreatic, hepatocellular carcinomas, and OS. The aim of this study was to investigate the functions and underlying mechanisms of C1QTNF1-AS1 in the progression of osteosarcoma.
MethodsThis investigation focused on elucidating the functional roles and mechanisms of C1QTNF1-AS1 in OS cells. Bioinformatics tools were utilized to identify the interaction between microRNA miR-34a-5p and C1QTNF1-AS1, as well as the targeting of LDHA and PDK3 by miR-34a-5p. Dual-luciferase reporter assays and RNA immunoprecipitation were employed to validate these interactions. Expression profiles of C1QTNF1-AS1, miR-34a-5p, LDHA, and PDK3 in osteosarcoma cells were analyzed using RT-PCR and western blot analyses, revealing their intricate relationships. The impact of these molecules on OS cell proliferation, invasion, and migration was assessed through CCK-8, Transwell, and Cell scratch assay. Moreover, the effects on aerobic glycolysis in OS cells were examined by quantifying ATP levels, lactate production, glucose uptake capacity, and the extracellular acidification rate.
ResultsThe findings indicated a significant decrease in C1QTNF1-AS1 expression levels in OS cells compared to normal osteoblasts. A parallel downregulation trend of miR-34a-5p was also observed in OS cells. Silencing C1QTNF1-AS1 led to a marked upregulation of LDHA and PDK3 in OS cells, which was partially attenuated by miR-34a-5p mimics. Functional evaluations demonstrated that suppression of C1QTNF1-AS1 accelerated OS cell growth, motility, invasiveness, and the Warburg effect. Conversely, the overexpression of miR-34a-5p mitigated these stimulatory effects, suggesting a regulatory role in modulating OS progression.
DiscussionOur research emphasizes the critical role of C1QTNF1-AS1 in the pathogenesis of osteosarcoma (OS). We discovered that the downregulation of C1QTNF1-AS1 indirectly upregulates the expression of LDHA and PDK3 by suppressing miR-34a-5p, which functions as a regulator of the Warburg effect. This cascade of events promotes OS progression by enhancing glycolytic metabolism and supplying energy for cancer cell growth, migration, and invasion. These findings suggest a potential therapeutic target and highlight the importance of understanding the regulatory network involving lncRNAs in cancer metabolism and progression.

引言 骨肉瘤（Osteosarcoma, OS）是青少年群体中高发的转移性恶性肿瘤，预后极差。长链非编码RNA（long non-coding RNAs, lncRNAs）是调控癌细胞增殖与运动能力的关键分子，C1QTNF1-AS1即为其中一员。作为癌基因，C1QTNF1-AS1已被证实在结直肠癌、胰腺癌、肝细胞癌及骨肉瘤等多种肿瘤中发挥作用。本研究旨在探究C1QTNF1-AS1在骨肉瘤发生发展中的功能及潜在作用机制。
方法 本研究聚焦阐明C1QTNF1-AS1在骨肉瘤细胞中的功能与作用机制。借助生物信息学工具，本研究预测了微小RNA（microRNA, miR-34a-5p）与C1QTNF1-AS1的结合互作，以及miR-34a-5p对乳酸脱氢酶A（LDHA）和丙酮酸脱氢酶激酶3（PDK3）的靶向调控关系。采用双荧光素酶报告基因实验与RNA免疫沉淀实验验证上述互作关系。通过实时荧光定量逆转录PCR（RT-PCR）与蛋白质印迹实验，检测骨肉瘤细胞中C1QTNF1-AS1、miR-34a-5p、LDHA及PDK3的表达水平，以明确其间的复杂调控关联。采用CCK-8实验、Transwell实验与细胞划痕实验，检测上述分子对骨肉瘤细胞增殖、侵袭与迁移能力的影响。此外，通过检测ATP含量、乳酸生成量、葡萄糖摄取能力及细胞外酸化率，分析上述分子对骨肉瘤细胞有氧糖酵解的调控作用。
结果 研究发现，与正常成骨细胞相比，骨肉瘤细胞中C1QTNF1-AS1的表达水平显著下调。同时，骨肉瘤细胞中miR-34a-5p的表达亦呈现下调趋势。敲低C1QTNF1-AS1可显著上调骨肉瘤细胞中LDHA与PDK3的表达，而miR-34a-5p模拟物可部分逆转这一效应。功能实验结果显示，抑制C1QTNF1-AS1可增强骨肉瘤细胞的增殖、运动、侵袭能力以及瓦伯格效应（Warburg effect）。反之，过表达miR-34a-5p可削弱上述促癌效应，提示miR-34a-5p在骨肉瘤进展中发挥负向调控作用。
讨论 本研究证实了C1QTNF1-AS1在骨肉瘤发病机制中的关键调控作用。本研究发现，C1QTNF1-AS1表达下调可通过抑制miR-34a-5p的表达，间接上调LDHA与PDK3的转录水平，而miR-34a-5p本身即为瓦伯格效应的调控因子。这一调控轴通过增强糖酵解代谢、为癌细胞增殖、迁移与侵袭提供能量，进而促进骨肉瘤的进展。本研究结果提示C1QTNF1-AS1可作为骨肉瘤潜在的治疗靶点，同时也凸显了阐明长链非编码RNA参与的肿瘤代谢与进展调控网络的重要意义。