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Abstract：
Retinoblastoma (RB) is a childhood eye tumor, caused by RB1 mutation. Though diagnosing RB is easier, prognosticating RB is limited to examining the patient under anesthesia and imaging technique. The aim of the study is to find exosomal miRNA biomarkers to prognosticate RB. Exosomes were isolated from one control - MIO-M1 and two RB cell lines - WERI-Rb-1 and NCC-RbC-51. Small RNA sequencing was performed on exosomal miRNA isolated from the three cell lines. miRNAs specific to each cell line were shortlisted. A total of 243, 606 and 400 miRNAs were identified in MIO-M1, WERI-Rb-1 and NCC-RbC-51 cell lines respectively. Nine miRNAs were shortlisted based on adjusted p value and literature, MIO-M1 specific (n = 1), WERI-RB-1 specific (n = 2), NCC-RbC-51 specific (n = 2) and miRNAs common to both RB cell lines (n = 4) were chosen. Validation was done using specific Taqman miRNA assays.miRNA validation was carried out on cell lines, cell line derived exosomes, primary RB tissues and exosomes isolated from serum of the RB patients. Validation of the miRNAs in cell lines and exosomes derived from the cell lines, confirmed the sequencing data. However, only 2 miRNAs - hsa-miR-301b-3p and hsa-miR-216b-5p were upregulated in the primary RB tissues. None of the miRNAs had significant expression in the serum exosomes of RB patients. Therefore, serum exosomal miRNA may not be ideal for prognosticating RB.Further research on other body fluids like CSF and vitreous could serve as potential source for biomarkers for prognosticating RB.
摘要：
视网膜母细胞瘤（RB）是一种儿童眼部肿瘤，由RB1突变引起。虽然诊断RB更容易，预测RB仅限于在麻醉和成像技术下检查患者。该研究的目的是寻找外泌体miRNA生物标志物来预测RB。从一个对照-MIO-M1和两个RB细胞系-WERI-Rb-1和NCC-RbC-51分离外泌体。对从三种细胞系分离的外泌体miRNA进行小RNA测序。每个细胞系特异性的miRNA入围。在MIO-M1、WERI-Rb-1和NCC-RbC-51细胞系中分别鉴定了总共243、606和400种miRNA。根据调整后的p值和文献，有9个miRNA入围，MIO-M1特异性（n=1），WERI-RB-1特异性（n=2），选择两种RB细胞系(n=4)共同的NCC-RbC-51特异性(n=2)和miRNA。使用特异性TaqmanmiRNA测定进行验证。在细胞系上进行miRNA验证，细胞系来源的外泌体，从RB患者血清中分离的原代RB组织和外泌体。细胞系中的miRNA和源自细胞系的外泌体的验证，确认了测序数据。然而,只有2个miRNA-hsa-miR-301b-3p和hsa-miR-216b-5p在原代RB组织中上调。这些miRNA在RB患者的血清外泌体中均无显著表达。因此,血清外泌体miRNA可能不是预测RB的理想选择。对CSF和玻璃体等其他体液的进一步研究可以作为预测RB的生物标志物的潜在来源。
