Zhang Kai, Liu Zixiang, Wang Zhidong, Zhou Zhangzhe, Shao Xiaofeng, Hua Xi, Mao Haiqing, Yang Huilin, Ren Ke, Chen Kangwu
Department of Orthopedic Surgery, The First Affiliated Hospital of Soochow University, Suzhou, China.
Collaborative Innovation Center of Sichuan for Elderly Care and Health, Chengdu Medical College, Chengdu, China.
Front Oncol. 2021 Sep 21;11:743718. doi: 10.3389/fonc.2021.743718. eCollection 2021.
Chordoma, an extremely rare malignant tumor, remains difficult to be cured because of its strong local invasiveness and high recurrence rate. Long non-coding RNAs (lncRNAs) have been demonstrated to play multiple roles in various cancers. The purpose of this study was to investigate the modulatory function of lncRNA MDFIC-7 in chordoma and to elucidate its underlying mechanisms.
Quantitative real-time polymerase chain reaction was performed to detect the expression of lncRNA MDFIC-7 in tumor tissues and adjacent nontumorous tissues collected from 15 chordoma patients, as well as in chordoma cell lines. Gene silencing and overexpression experiments were carried out by RNA interference and lentiviral transduction. The effect of lncRNA MDFIC-7 on the proliferation of chordoma cells was evaluated by cell counting kit-8 assay, colony formation assay and xenograft tumor experiments. RNA immunoprecipitation and dual luciferase reporter assays were conducted to evaluate the binding between lncRNA MDFIC-7 and miRNA-525-5p and the interaction between miR-525-5p and the 3' untranslated region of ADP-ribosylation factor 6 (ARF6) mRNA. The glycolytic capacity and mitochondrial function of chordoma cells were measured by the Seahorse Bioscience XF96 Extracellular Flux Analyzer.
The expression of lncRNA MDFIC-7 was higher in chordoma tumor tissues than in adjacent non-tumor tissues. Downregulation of lncRNA MDFIC-7 reduced colony formation and cell proliferation in chordoma cells and decreased xenograft tumor growth in a nude mouse model. Moreover, lncRNA MDFIC-7 knockdown attenuated the Warburg effect in chordoma cells and xenograft tumors. LncRNA MDFIC-7 knockdown elevated miR-525-5p levels and decreased ARF6 expressions. Overexpression of ARF6 reversed the inhibitory effect of lncRNA MDFIC-7 knockdown on cell proliferation and the Warburg effect in chordoma cells and xenograft tumors. Mechanistically, lncRNA MDFIC-7, as a molecular sponge of miR-525-5p, negatively regulated miR-525-5p expression and promoted the gene expression of ARF6, a miR-525-5p target.
Our findings demonstrate that lncRNA MDFIC-7 acts as a molecular sponge to competitively bind to miR-525-5p and promote expression of ARF6. The lncRNA MDFIC-7/miR-525-5p/ARF6 axis regulates chordoma progression and the Warburg effect in chordoma, suggesting that lncRNA MDFIC-7 and miR-525-5p could be promising therapeutic targets for the treatment of chordoma.
脊索瘤是一种极其罕见的恶性肿瘤，因其强大的局部侵袭性和高复发率，仍然难以治愈。长链非编码RNA（lncRNAs）已被证明在各种癌症中发挥多种作用。本研究的目的是探讨lncRNA MDFIC-7在脊索瘤中的调节功能，并阐明其潜在机制。
采用定量实时聚合酶链反应检测15例脊索瘤患者肿瘤组织及癌旁非肿瘤组织以及脊索瘤细胞系中lncRNA MDFIC-7的表达。通过RNA干扰和慢病毒转导进行基因沉默和过表达实验。采用细胞计数试剂盒-8法、集落形成试验和异种移植瘤实验评估lncRNA MDFIC-7对脊索瘤细胞增殖的影响。进行RNA免疫沉淀和双荧光素酶报告基因检测，以评估lncRNA MDFIC-7与miRNA-525-5p之间的结合以及miR-525-5p与ADP-核糖基化因子6（ARF6）mRNA的3'非翻译区之间的相互作用。用Seahorse Bioscience XF96细胞外通量分析仪检测脊索瘤细胞的糖酵解能力和线粒体功能。
lncRNA MDFIC-7在脊索瘤肿瘤组织中的表达高于癌旁非肿瘤组织。lncRNA MDFIC-7的下调减少了脊索瘤细胞的集落形成和细胞增殖，并降低了裸鼠模型中异种移植瘤的生长。此外，lncRNA MDFIC-7的敲低减弱了脊索瘤细胞和异种移植瘤中的瓦伯格效应。lncRNA MDFIC-7的敲低提高了miR-525-5p水平并降低了ARF6表达。ARF6的过表达逆转了lncRNA MDFIC-7敲低对脊索瘤细胞和异种移植瘤细胞增殖和瓦伯格效应的抑制作用。机制上，lncRNA MDFIC-7作为miR-525-5p的分子海绵，负向调节miR-525-5p表达并促进miR-525-5p靶标ARF6的基因表达。
我们的研究结果表明，lncRNA MDFIC-7作为分子海绵竞争性结合miR-525-5p并促进ARF6表达。lncRNA MDFIC-7/miR-525-5p/ARF6轴调节脊索瘤进展和脊索瘤中的瓦伯格效应，提示lncRNA MDFIC-7和miR-525-5p可能是治疗脊索瘤的有前景的治疗靶点。
