Department of Medical Oncology, The First Affiliated Hospital of Xi'an Jiaotong University, No. 277 Yanta West Road, Xi'an, Shaanxi, 710061, People's Republic of China.
Department of Respiratory, Third Hospital of Xi'an, Xi'an, Shaanxi, 710018, People's Republic of China.
J Exp Clin Cancer Res. 2018 Mar 12;37(1):52. doi: 10.1186/s13046-018-0677-7.
Multidrug resistance is the main obstacle for hepatocellular carcinoma (HCC) treatment. miR-32-5p is involved in HCC progression but its function in multidrug resistance is still unclear. Here we aim to find out the function of miR-32-5p in inducing multidrug resistance and its underlying mechanisms of transforming sensitive cell to resistant cell.
We detected the expression of miR-32-5p and PTEN in the multidrug-resistant cell line (Bel/5-FU) and the sensitive cell line (Bel7402), HCC and para-carcinoma liver tissues through real-time PCR. Dual-luciferase reporter assay verified PTEN is the target of miR-32-5p. Exosomes from sensitive and multidrug resistant cell line were obtained and confirmed through ultracentrifuge and Nano Analyzer. Gain- and loss-of-function experiments, rescue experiments, a PI3K/Akt pathway inhibitor, an exosome biogenesis inhibitor, and nude mice xenograft models were used to determine the underlying mechanisms of miR-32-5p and PTEN, as well as exosomal miR-32-5p in inducing multidrug resistance in vitro and in vivo.
miR-32-5p was significantly elevated but PTEN was reduced in Bel/5-FU. An inverse correlation between miR-32-5p and PTEN was confirmed in HCC cell lines and patients; moreover, high expression of miR-32-5p and low expression of PTEN were positively associated with poor prognosis. Over-expression of miR-32-5p activated the PI3K/Akt pathway by suppressing PTEN and induced multidrug resistance via exosomes through promoting angiogenesis and epithelial-mesenchymal transition (EMT).
Our study demonstrated that the multidrug-resistant cell, Bel/5-FU delivers miR-32-5p to sensitive cell, Bel7402 by exosomes and activates the PI3K/Akt pathway to further induce multidrug resistance by modulating angiogenesis and EMT.
多药耐药是肝细胞癌（HCC）治疗的主要障碍。miR-32-5p 参与 HCC 进展，但在多药耐药中的作用尚不清楚。本研究旨在探讨 miR-32-5p 诱导多药耐药的功能及其将敏感细胞转化为耐药细胞的潜在机制。
通过实时 PCR 检测多药耐药细胞系（Bel/5-FU）和敏感细胞系（Bel7402）、肝癌及癌旁肝组织中 miR-32-5p 和 PTEN 的表达。双荧光素酶报告基因实验验证 PTEN 是 miR-32-5p 的靶基因。超速离心和 Nano Analyzer 获得并验证敏感和多药耐药细胞系的外泌体。通过 gain- 和 loss-of-function 实验、挽救实验、PI3K/Akt 通路抑制剂、外泌体生物发生抑制剂以及裸鼠异种移植模型，确定 miR-32-5p 和 PTEN 以及外泌体 miR-32-5p 在体外和体内诱导多药耐药的潜在机制。
Bel/5-FU 中 miR-32-5p 显著升高，而 PTEN 降低。在 HCC 细胞系和患者中证实 miR-32-5p 与 PTEN 呈负相关；此外，miR-32-5p 高表达和 PTEN 低表达与不良预后呈正相关。过表达 miR-32-5p 通过抑制 PTEN 激活 PI3K/Akt 通路，并通过促进血管生成和上皮间质转化（EMT），通过外泌体诱导多药耐药。
本研究表明，耐药细胞 Bel/5-FU 通过外泌体将 miR-32-5p 传递给敏感细胞 Bel7402，并通过调节血管生成和 EMT 激活 PI3K/Akt 通路，进一步诱导多药耐药。
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