BACKGROUND Retinoblastoma (RB) is the most frequent pediatric retinal tumor. In the present study, to elucidate chemoresistance mechanisms and identify potential biomarkers in RB, we utilized RNA sequencing (RNAseq) technological platforms to reveal transcriptome profiles and identify any differentially expressed genes (DEGs) between an etoposide drug-resistant subline (Y79/EDR) and parental Y79 cells. METHODS To test whether Y79/EDR cells showed resistance to antineoplastic agents for RB, we treated the cells with etoposide, carboplatin and vincristine and analyzed them with a Cell Counting Kit-8 (CCK-8). Y79/EDR and parental Y79 cells were used for RNAseq and bioinformatics analysis to enable a genome-wide review of DEGs between the two lines using the DESeq R package (1.10.1). Then, DEG enrichment in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was analyzed with KOBAS software. Next, real-time quantitative reverse transcription polymerase chain reaction (real time QRT-PCR) and cytotoxicity assays were performed to experimentally and functionally validate the identified candidate biomarkers. RESULTS Y79/EDR cells showed resistance to etoposide, carboplatin and vincristine at different concentrations. In total, 524 transcripts were differentially expressed in Y79/EDR cells based on analysis of fragments per kilobase of transcript per million fragments mapped (FPKM); among these, 57 genes were downregulated and 467 genes were upregulated in Y79/EDR cells compared to parental Y79 cells. We selected candidate DEGs, including ARHGAP9, HIST1H4H, RELN, DDIT4, HK2, STC1 and PFKFB4, for mRNA expression validation with real time QRT-PCR assays and found that the expression levels determined by real time QRT-PCR were consistent with the RNAseq data. Further studies involving downregulation of ARHGAP9 with a specific siRNA showed that ARHGAP9 altered the cellular sensitivity of Y79 cells to etoposide and carboplatin. CONCLUSION Our initial findings provided a genomic view of the transcription profiles of etoposide-induced acquired resistance in RB. Follow-up studies indicated that ARHGAP9 might be a chemoresistance biomarker in RB, providing insight into potential therapeutic targets for overcoming acquired chemoresistance in RB. These findings can aid in understanding and overcoming chemoresistance during treatment of RB in the clinic.

中文翻译：

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人视网膜母细胞瘤Y79细胞和依托泊苷抗性亚系之间不同的转录组概况揭示了化学耐药机制。

背景技术视网膜母细胞瘤（RB）是最常见的小儿视网膜肿瘤。在本研究中，为阐明化学抗药性机制并确定RB中潜在的生物标记，我们利用RNA测序（RNAseq）技术平台揭示了转录组图谱并鉴定了依托泊苷抗药性亚系（Y79 / EDR）之间的任何差异表达基因（DEG）。和亲代Y79细胞。方法为了测试Y79 / EDR细胞是否对RB的抗肿瘤药具有抗性，我们用依托泊苷，卡铂和长春新碱处理细胞，并用Cell Counting Kit-8（CCK-8）进行了分析。Y79 / EDR和亲代Y79细胞用于RNAseq和生物信息学分析，以使用DESeq R软件包（1.10.1）在两株系之间对DEGs进行全基因组范围的检查。然后，使用KOBAS软件分析了京都基因与基因组百科全书（KEGG）途径中的DEG富集。接下来，进行实时定量逆转录聚合酶链反应（实时QRT-PCR）和细胞毒性测定，以通过实验和功能验证所鉴定的候选生物标记。结果Y79 / EDR细胞在不同浓度下均对依托泊苷，卡铂和长春新碱具有耐药性。根据对每百万片段映射图谱（FPKM）的每千个转录本碱基的片段分析，总共有524个转录物在Y79 / EDR细胞中差异表达。其中，与亲本Y79细胞相比，Y79 / EDR细胞中57个基因被下调，而467个基因被上调。我们选择了候选DEG，包括ARHGAP9，HIST1H4H，RELN，DDIT4，HK2，STC1和PFKFB4，使用实时QRT-PCR分析进行mRNA表达验证，发现实时QRT-PCR确定的表达水平与RNAseq数据一致。进一步涉及用特定siRNA下调ARHGAP9的研究表明，ARHGAP9改变了Y79细胞对依托泊苷和卡铂的细胞敏感性。结论我们的初步发现为依托泊苷诱导的RB获得性耐药的转录概况提供了基因组学观点。后续研究表明，ARHGAP9可能是RB中的化学抗药性生物标志物，从而为克服RB中获得的化学抗性的潜在治疗靶点提供了见识。这些发现有助于在临床上治疗RB期间理解和克服化学耐药性。