Department of Clinical Laboratory, The Second Hospital of Shandong University, No. 247 Beiyuan Street, Jinan, 250033, China.
Department of Clinical Laboratory, Qilu Hospital, Shandong University, Jinan, 250012, Shandong Province, China.
Mol Cancer. 2019 Mar 19;18(1):43. doi: 10.1186/s12943-019-0981-7.
Oxaliplatin resistance is a major challenge for treatment of advanced colorectal cancer (CRC). Both acquisition of epithelial-mesenchymal transition (EMT) and suppressed drug accumulation in cancer cells contributes to development of oxaliplatin resistance. Aberrant expression of small noncoding RNA, miR-128-3p, has been shown to be a key regulator in tumorigenesis and cancer development. However, its roles in the progression of CRC and oxaliplatin-resistance are largely unknown.
Oxaliplatin-resistant CRC and normal intestinal FHC cells were transfected with a miR-128-3p expression lentivirus. After transfection, FHC-derived exosomes were isolated and co-cultured with CRC cells. miR-128-3p expression in resistant CRC cells, FHC cells, and exosomes was quantified by quantitative real-time PCR (RT-qPCR). The mRNA and protein levels of miR-128-3p target genes in resistant CRC cells were quantified by RT-qPCR and western blot, respectively. The effects of miR-128-3p on CRC cell viability, apoptosis, EMT, motility and drug efflux were evaluated by CCK8, flow cytometry, Transwell and wound healing assays, immunofluorescence, and atomic absorption spectrophotometry. Xenograft models were used to determine whether miR-128-3p loaded exosomes can re-sensitize CRC cells to oxaliplatin in vivo.
In our established stable oxaliplatin-resistant CRC cell lines, in vitro and vivo studies revealed miR-128-3p suppressed EMT and increased intracellular oxaliplatin accumulation. Importantly, our results indicated that lower miR-128-3p expression was associated with poor oxaliplatin response in advanced human CRC patients. Moreover, data showed that miR-128-3p-transfected FHC cells effectively packaged miR-128-3p into secreted exosomes and mediated miR-128-3p delivery to oxaliplatin-resistant cells, improving oxaliplatin response in CRC cells both in vitro and in vivo. In addition, miR-128-3p overexpression up-regulated E-cadherin levels and inhibited oxaliplatin-induced EMT by suppressing Bmi1 expression in resistant cells. Meanwhile, it also decreased oxaliplatin efflux through suppressed expression of the drug transporter MRP5.
Our results demonstrate that miR-128-3p delivery via exosomes represents a novel strategy enhancing chemosensitivity in CRC through negative regulation of Bmi1 and MRP5. Moreover, miR-128-3p may be a promising diagnostic and prognostic marker for oxaliplatin-based chemotherapy.
奥沙利铂耐药是治疗晚期结直肠癌（CRC）的主要挑战。上皮-间充质转化（EMT）的获得和癌细胞中药物积累的抑制都有助于奥沙利铂耐药的发展。微小非编码 RNA，miR-128-3p 的异常表达已被证明是肿瘤发生和癌症发展的关键调节剂。然而，其在 CRC 进展和奥沙利铂耐药中的作用在很大程度上仍是未知的。
用 miR-128-3p 表达慢病毒转染奥沙利铂耐药的 CRC 和正常肠 FHC 细胞。转染后，分离 FHC 衍生的外泌体并与 CRC 细胞共培养。通过实时定量 PCR（RT-qPCR）定量检测耐药 CRC 细胞、FHC 细胞和外泌体中的 miR-128-3p 表达。通过 RT-qPCR 和 Western blot 分别定量检测耐药 CRC 细胞中 miR-128-3p 靶基因的 mRNA 和蛋白水平。通过 CCK8、流式细胞术、Transwell 和划痕愈合实验、免疫荧光和原子吸收分光光度法评估 miR-128-3p 对 CRC 细胞活力、凋亡、EMT、迁移和药物外排的影响。使用异种移植模型来确定载有 miR-128-3p 的外泌体是否可以在体内使 CRC 细胞重新对奥沙利铂敏感。
在我们建立的稳定的奥沙利铂耐药 CRC 细胞系中，体外和体内研究表明 miR-128-3p 抑制 EMT 并增加细胞内奥沙利铂积累。重要的是，我们的结果表明，晚期人类 CRC 患者中 miR-128-3p 表达较低与奥沙利铂反应不良相关。此外，数据表明，miR-128-3p 转染的 FHC 细胞可有效地将 miR-128-3p 包装到分泌的外泌体中，并将 miR-128-3p 递送至奥沙利铂耐药细胞，从而在体外和体内均提高 CRC 细胞对奥沙利铂的反应。此外，miR-128-3p 过表达通过抑制耐药细胞中 Bmi1 的表达，上调 E-钙黏蛋白水平并抑制奥沙利铂诱导的 EMT。同时，它还通过抑制药物转运蛋白 MRP5 的表达降低了奥沙利铂的外排。
我们的结果表明，通过外泌体递送 miR-128-3p 通过负调控 Bmi1 和 MRP5 来增强 CRC 的化疗敏感性，是一种新的策略。此外，miR-128-3p 可能是基于奥沙利铂化疗的有前途的诊断和预后标志物。
