Background

Long non-coding RNAs (lncRNAs) have been demonstrated to play important roles in various tumors, including chordoma. The purpose of this study was to investigate the role and mechanism of lncRNA X-inactive specific transcript (XIST) in chordoma.

Methods

RNA levels and protein levels were measured by real-time quantitative polymerase chain reaction (RT‑qPCR) and western blot assay, respectively. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2′-deoxyuridine (EdU) assay and colony formation assay. Tanswell assay was used to examine cell migration and invasion. Cellular glycolysis was examined via the measurement of extracellular acidification rate (ECAR) and lactate production. The interaction between microRNA-320d (miR-320d) and XIST or ADP-ribosylation factor 6 (ARF6) was predicted by bioinformatics analysis and verified by a dual-luciferase reporter and RNA-pull down assays. The xenograft tumor model was used to explore the biological function of XIST in vivo.

Results

XIST was overexpressed in chordoma tissues. XIST knockdown suppressed chordoma cell proliferation, migration, invasion, and glycolysis. XIST acted as a sponge of miR-320d. Moreover, miR-320d overexpression inhibited the proliferation, migration, invasion, and glycolysis of chordoma cells. ARF6 was a direct target of miR-320d, and XIST upregulated ARF6 expression via sponging miR-320d. Furthermore, overexpression of ARF6 reversed the inhibitory effects of XIST knockdown on chordoma cell proliferation, migration, invasion, and glycolysis. Importantly, XIST silencing blocked xenograft tumor growth in vivo.

Conclusion

XIST knockdown inhibited chordoma progression via regulating the miR-320d/ARF6 axis, providing a novel insight into chordoma pathogenesis.

中文翻译：

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XIST 海绵 miR-320d 通过调节 ARF6 促进脊索瘤进展

背景

长链非编码 RNA (lncRNA) 已被证明在包括脊索瘤在内的各种肿瘤中发挥重要作用。本研究的目的是研究 lncRNA X 失活特异性转录物 (XIST) 在脊索瘤中的作用和机制。

方法

RNA 水平和蛋白质水平分别通过实时定量聚合酶链反应 (RT-qPCR) 和蛋白质印迹法测定。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑 (MTT) 测定、5-乙炔基-2'-脱氧尿苷 (EdU) 测定和集落形成测定来评估细胞增殖。Tanswell测定用于检查细胞迁移和侵袭。通过测量细胞外酸化率 (ECAR) 和乳酸产生来检查细胞糖酵解。通过生物信息学分析预测 microRNA-320d (miR-320d) 和 XIST 或 ADP-核糖基化因子 6 (ARF6) 之间的相互作用，并通过双荧光素酶报告基因和 RNA 下拉分析进行验证。采用异种移植肿瘤模型探索XIST在体内的生物学功能。

结果

XIST 在脊索瘤组织中过度表达。XIST 敲低抑制了脊索瘤细胞的增殖、迁移、侵袭和糖酵解。XIST 充当 miR-320d 的海绵。此外，miR-320d 过表达抑制脊索瘤细胞的增殖、迁移、侵袭和糖酵解。ARF6 是 miR-320d 的直接靶标，XIST 通过海绵化 miR-320d 上调 ARF6 表达。此外，ARF6 的过表达逆转了 XIST 敲低对脊索瘤细胞增殖、迁移、侵袭和糖酵解的抑制作用。重要的是，XIST 沉默阻止了体内异种移植肿瘤的生长。

结论

XIST 敲低通过调节 miR-320d/ARF6 轴抑制脊索瘤进展，为脊索瘤发病机制提供了新的见解。