Table_3_The Identification of Plasma Exosomal miR-423-3p as a Potential Predictive Biomarker for Prostate Cancer Castration-Resistance Development by Plasma Exosomal miRNA Sequencing.docx

Castration-resistant prostate cancer (CRPC) is the major cause of death from prostate cancer. Biomarkers to improve early detection and prediction of CRPC especially using non-invasive liquid biopsies could improve outcomes. Therefore, we investigated the plasma exosomal miRNAs associated with CRPC and their potential for development into non-invasive early detection biomarkers for resistance to treatment. RNA-sequencing, which generated approximately five million reads per patient, was performed to identify differentially expressed plasma exosomal miRNAs in 24 treatment-naive prostate cancer and 24 CRPC patients. RT-qPCR was used to confirm the differential expressions of six exosomal miRNAs, miR-423-3p, miR-320a, miR-99a-5p, miR-320d, miR-320b, and miR-150-5p (p = 7.3 × 10−8, 0.0020, 0.018, 0.0028, 0.0013, and 0.0058, respectively) firstly in a validation cohort of 108 treatment-naive prostate cancer and 42 CRPC patients. The most significant differentially expressed miRNA, miR-423-3p, was shown to be associated with CRPC with area under the ROC curve (AUC) = 0.784. Combining miR-423-3p with prostate-specific antigen (PSA) enhanced the prediction of CRPC (AUC = 0.908). A separate research center validation with 30 treatment-naive and 30 CRPC patients also confirmed the differential expression of miR-423-3p (p = 0.016). Finally, plasma exosomal miR-423-3p expression in CRPC patients was compared to 36 non-CRPC patients under androgen depletion therapy, which showed significantly higher expression in CRPC than treated non-CRPC patients (p < 0.0001) with AUC = 0.879 to predict CRPC with no difference between treatment-naive and treated non-CRPC patients. Therefore, our findings demonstrate that a number of plasma exosomal miRNAs are associated with CRPC and miR-423-3p may serve as a biomarker for early detection/prediction of castration-resistance.

去势抵抗性前列腺癌（CRPC）是前列腺癌死亡的主要原因。开发能够改善早期检测和预测CRPC的生物标志物，特别是利用非侵入性液体活检技术，有望提升治疗结果。因此，本研究探讨了与CRPC相关的血浆外泌体miRNA及其作为治疗抵抗性早期检测生物标志物的潜力。通过对24例未接受治疗的前列腺癌患者和24例CRPC患者的血浆外泌体miRNA进行RNA测序（每例患者产生约五百万个读数），我们鉴定了差异表达的外泌体miRNA。随后，利用实时定量PCR（RT-qPCR）验证了六种外泌体miRNA的差异表达，包括miR-423-3p、miR-320a、miR-99a-5p、miR-320d、miR-320b和miR-150-5p（分别对应的p值为7.3×10−8、0.0020、0.018、0.0028、0.0013和0.0058）。在108例未接受治疗的前列腺癌患者和42例CRPC患者的验证队列中，首先确认了这六种miRNA的差异表达。其中，差异表达最显著的miR-423-3p与CRPC相关，其曲线下面积（AUC）为0.784。将miR-423-3p与前列腺特异性抗原（PSA）结合，可增强CRPC的预测能力（AUC = 0.908）。另一独立研究中心对30例未接受治疗和30例CRPC患者的验证也证实了miR-423-3p的差异表达（p = 0.016）。最终，比较了CRPC患者与36例非CRPC患者在睾酮耗竭治疗下的血浆外泌体miR-423-3p表达，结果显示CRPC患者表达显著升高（p < 0.0001），AUC = 0.879，用于预测CRPC，且治疗初治与非治疗初治的非CRPC患者之间无显著差异。因此，我们的研究结果表明，多种血浆外泌体miRNA与CRPC相关，miR-423-3p可能作为早期检测/预测去势抵抗性的生物标志物。