High-throughput RNA sequencing for screening the circular RNA expression profiling associated with chemo-resistance inosteosarcoma
10.3760/cma.j.issn.0253-2352.2019.06.003
- VernacularTitle:高通量测序筛选与骨肉瘤化疗耐药相关的环状RNA分子表达谱
- Author:
KunPeng ZHU
1
,
2
;
ChunLin ZHANG
;
XiaoLong MA
Author Information
1. 同济大学附属上海市第十人民医院骨科 200072
2. 同济大学骨肿瘤研究所
- Keywords:
Sarcoma;
RNA;
Drug resistance,neoplasm;
MicroRNAs;
High-throughput screening assays
- From:
Chinese Journal of Orthopaedics
2019;39(6):336-345
- CountryChina
- Language:Chinese
-
Abstract:
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.Moreover,thedifferently expressedcircRNA-hsa_circ_0004674 with the highestfold change was highly expressedin the chemotherapy-resistant osteosarcoma cells and tissues,associated with poor prognosis in osteosarcoma patients.Some potential endogenous competitive RNA (ceRNA) regulatory pathways associated with hsa_circ_0004674,such as hsa_circ_0004674-miR-490-3p-ABCC2 and hsa_circ_0004674-miR-1254-EGFR,were constructed by use of the authoritative databases (target scan and miRanda) and literature searching and the miRNA response element sequences (MREs) between miRNAs that have a potential ceRNA regulatory relationship with hsa_circ_0004674 were alsopredicted.Conclusion CircRNA is closely related to tumor progression and may play a role in ostoosarcoma chemo-resistance.Besides,hsa_circ_0004674 may be a potential candidate for reversing drug resistance of osteosarcoma.
