摘要
目的通过高通量测序筛选与骨肉瘤化疗耐药相关的环状RNA表达谱,并初步分析、鉴定其可能的分子功能。方法采用CCK-8实验检测三对配对的耐药和敏感人骨肉瘤细胞系(MG63/DXR vs MG63、U2OS/DXR vs U2OS、KHOS/DXR vs KHOS)对于三种常用骨肉瘤化疗药物(多柔比星、顺铂、甲氨蝶呤)的敏感性。后采用下一代高通量RNA测序技术,在三对配对的化疗多药耐药和敏感的人骨肉瘤细胞系中进行circRNA表达谱的比较分析。使用实时定量PCR(qRT-PCR)在化疗耐药骨肉瘤细胞系和组织中确认测序数据的可靠性和准确性。此外,还进行包括GO、KEGG通路分析和circRNA-miRNA网络构建在内的多种生物信息学分析,以预测差异表达的circRNA的潜在分子功能,构建相关的可能调控通路或网络。结果三种骨肉瘤耐药细胞(MG63/DXR、U2OS/DXR、KHOS/DXR)对于三种常见的骨肉瘤化疗药物均较对照组细胞(MG63、U2OS、KHOS)具有明显的抵抗性;RNA测序共检测到80个组间差异表达的circRNAs,相对于药物敏感的骨肉瘤细胞,药物抵抗骨肉瘤细胞系中,57个circRNAs表达上调,23个circRNAs表达下调(组间差异倍数≥2或≤0.5)。随机选择其中的10个circRNA行qRT-PCR验证,发现有9个qRT-PCR的检测结果与测序结果一致;此外,KEGG通路分析结果显示56个通路在差异表达的circRNAs中被显著富集,包括糖酵解/糖异生、ABC转运蛋白、VEGF信号通路等等。此外,发现在化疗耐药的骨肉瘤细胞和组织中,上调倍数最高的circRNA-hsa_circ_0004674明显高表达,与骨肉瘤患者的预后不良有关。通过权威数据库(TargetScan和miRanda)和文献搜索,构建了与hsa_circ_0004674相关的一些潜在的内源竞争性RNA(ceRNA)调控通路,例如hsa_circ_0004674-miR-490-3p-ABCC2和hsa_circ_0004674-miR-1254-EGFR等,并预测了与hsa_circ_0004674存在潜在ceRNA调控关系的miRNA之间的miRNA反应元件序列。结论CircRNA与肿瘤的进展密切相关,可能在�
Objective To screen the expression profile of circular RNA associated with chemo-resistance in osteosarcoma, and to analyze and identify its possible molecular functions. Methods Three pairs of matched drug-resistant and sensitive human osteosarcoma cell lines (MG63/DXR vs MG63, U2OS/DXR vs U2OS, KHOS/DXR vs KHOS) were first tested by CCK-8 assay for three commonly used osteosarcoma chemotherapy drugs (doxorubicin, cisplatin, and methotrexate);then, using next-generation high-throughput RNA sequencing technology, comparative analysis of circRNA expression was performed in three pairs of matched multidrug-resistant and sensitive human osteosarcoma cell lines;followed by real-time quantitative PCR (qRT-PCR) to confirm the reliability and accuracy of sequencing data in chemotherapy-resistant osteosarcoma cell lines and tissues. In addition, a variety of bioinformatics analyses, including GO, KEGG pathway analysis and the construction of circRNA-miRNA networks, were performed to predict potential molecular functions of differentially expressed circRNAs and to construct relevant regulatory pathways or networks. Results Three osteosarcoma-resistant cells (MG63/DXR, U2OS/DXR, KHOS/DXR) were significantly resistant to the three common osteosarcoma chemotherapy drugs compared with control cells (MG63, U2OS, KHOS), which laid a solid foundation for the further experiments. RNA sequencing revealed a total of 80 circRNAs differentially expressed between the two groups. Compared with drug-sensitive osteosarcoma cells, 57 circRNAs were upregulated and 23 circRNAs weredownregulated in the drug-resistant osteosarcoma cell lines (fold change≥ 2 or ≤0.5, P<0.05). Tenrandomly selected circRNAs were verified by qRT-PCR and the results showed that 9 of the 10 circRNAswere consistent with the sequencing results. In addition, KEGG pathway analysis showed that 56 pathways were significantly enriched in differentially expressed circRNAs, including Glycolysis/gluconeogenesis, ABC transporter, VEGF signaling pathway, and so on. Moreove
作者
朱昆鹏
张春林
马小龙
Zhu KunPeng;Zhang ChunLin;Ma XiaoLong(Department of Orthopaedic Surgery, Shanghai Tenth People's Hospital, Tongji University, School of Medicine, Shanghai 200072, China;Institute of Bone Tumor, Tongji University, School of Medicine, Shanghai 200072, China)
出处
《中华骨科杂志》
CAS
CSCD
北大核心
2019年第6期336-345,共10页
Chinese Journal of Orthopaedics
基金
国家自然科学基金(81572630
81872174).
关键词
肉瘤
RNA
抗药性
肿瘤
微RNAS
高通量筛选分析
Sarcoma
RNA
Drug resistance, neoplasm
MicroRNAs
High-throughput screening assays
