Multi-drug resistance is a major challenge to hepatocellular carcinoma (HCC) treatment, and the over-expression or deletion of microRNA (miRNA) expression is closely related to the drug-resistant properties of various cell lines. However, the underlying molecular mechanisms remain unclear. CCK-8, EdU, flow cytometry, and transmission electron microscopy were performed to determine cell viability, proliferation, apoptosis, autophagic flow, and nanoparticle characterization, respectively. In this study, the results showed that the expression of miR-26b was downregulated following doxorubicin treatment in human HCC tissues. An miR-26b mimic enhanced HCC cell doxorubicin sensitivity, except in the absence of p53 in Hep3B cells. Delivery of the proteasome inhibitor, MG132, reversed the inhibitory effect of miR-26b on the level of p53 following doxorubicin treatment. Tenovin-1 (an MDM2 inhibitor) protected p53 from ubiquitination-mediated degradation only in HepG2 cells with wild type p53. Tenovin-1 pretreatment enhanced HCC cell resistance to doxorubicin when transfected with an miR-26b mimic. Moreover, the miR-26b mimic inhibited doxorubicin-induced autophagy and the autophagy inducer, rapamycin, eliminated the differences in the drug sensitivity effect of miR-26b. In vivo, treatment with sp94dr/miR-26b mimic nanoparticles plus doxorubicin inhibited tumor growth. Our current data indicate that miR-26b enhances HCC cell sensitivity to doxorubicin through diminishing USP9X-mediated p53 de-ubiquitination caused by DNA damaging drugs and autophagy regulation. This miRNA-mediated pathway that modulates HCC will help develop novel therapeutic strategies.

中文翻译：

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miR-26b 通过 USP9X 依赖性 p53 降解和自噬调节增强肝细胞癌对阿霉素的敏感性

多药耐药是肝细胞癌（HCC）治疗的一大挑战，microRNA（miRNA）表达的过度表达或缺失与各种细胞株的耐药特性密切相关。然而，潜在的分子机制仍不清楚。CCK-8、EdU、流式细胞术和透射电子显微镜分别测定细胞活力、增殖、凋亡、自噬流和纳米颗粒表征。在这项研究中，结果表明，在人 HCC 组织中，阿霉素治疗后 miR-26b 的表达下调。miR-26b 模拟增强 HCC 细胞多柔比星敏感性，除了在 Hep3B 细胞中不存在 p53。蛋白酶体抑制剂MG132的递送，逆转了阿霉素治疗后 miR-26b 对 p53 水平的抑制作用。Tenovin-1（一种 MDM2 抑制剂）仅在具有野生型 p53 的 HepG2 细胞中保护 p53 免受泛素化介导的降解。当用 miR-26b 模拟物转染时，Tenovin-1 预处理增强了 HCC 细胞对阿霉素的抗性。此外，miR-26b 模拟物抑制多柔比星诱导的自噬，而自噬诱导剂雷帕霉素消除了 miR-26b 药物敏感性效应的差异。在体内，用 sp94dr/miR-26b 模拟纳米颗粒加多柔比星治疗可抑制肿瘤生长。我们目前的数据表明，miR-26b 通过减少由 DNA 损伤药物和自噬调节引起的 USP9X 介导的 p53 去泛素化来增强 HCC 细胞对阿霉素的敏感性。这种调节 HCC 的 miRNA 介导的途径将有助于开发新的治疗策略。