Department of Biomedicine, Neuroscience and Advanced Diagnostics (Bi.N.D.), Section of Biology and Genetics, University of Palermo, 90133, Palermo, Italy.
Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, 90128, Palermo, Italy.
BMC Cancer. 2022 May 20;22(1):567. doi: 10.1186/s12885-022-09666-2.
The uncontrolled proliferation of cancer cells determines hypoxic conditions within the neoplastic mass with consequent activation of specific molecular pathways that allow cells to survive despite oxygen deprivation. The same molecular pathways are often the cause of chemoresistance. This study aims to investigate the role of the hypoxia-induced miR-675-5p in 5-Fluorouracil (5-FU) resistance on colorectal cancer (CRC) cells.
CRC cell lines were treated with 5-Fu and incubated in normoxic or hypoxic conditions; cell viability has been evaluated by MTT assay. MiR-675-5p levels were analysed by RT-PCR and loss and gain expression of the miRNA has been obtained by the transfection of miRNA antagomir or miRNA mimic. Total protein expression of different apoptotic markers was analysed through western blot assay. MirWalk 2.0 database search engine was used to investigate the putative targets of the miR-675-5p involved in the apoptotic process. Finally, the luciferase assay was done to confirm Caspase-3 as a direct target of the miR-675-5p.
Our data demonstrated that hypoxia-induced miR-675-5p counteracts the apoptotic signal induced by 5-FU, thus taking part in the drug resistance response. We showed that the apoptotic markers, cleaved PARP and cleaved caspase-3, increased combining miR-675-5p inhibition with 5-FU treatment. Moreover, we identified pro-caspase-3 among the targets of the miR-675-5p.
Our data demonstrate that the inhibition of hypoxia-induced miR-675-5p combined with 5-FU treatment can enhances drug efficacy in both prolonged hypoxia and normoxia, indicating a possible strategy to partially overcome chemoresistance.
癌细胞的不受控制的增殖决定了肿瘤组织中的缺氧条件，随后激活了特定的分子途径，使细胞能够在缺氧的情况下存活。同样的分子途径往往是化疗耐药的原因。本研究旨在探讨缺氧诱导的 miR-675-5p 在 5-氟尿嘧啶（5-FU）对结直肠癌（CRC）细胞耐药中的作用。
用 5-Fu 处理 CRC 细胞系，并在常氧或低氧条件下孵育；通过 MTT 测定法评估细胞活力。通过 RT-PCR 分析 miR-675-5p 水平，并通过转染 miRNA 拮抗剂或 miRNA 模拟物获得 miRNA 的缺失和表达。通过 Western blot 分析不同凋亡标记物的总蛋白表达。使用 MirWalk 2.0 数据库搜索引擎来研究参与凋亡过程的 miR-675-5p 的假定靶标。最后，进行荧光素酶测定以确认 Caspase-3 是 miR-675-5p 的直接靶标。
我们的数据表明，缺氧诱导的 miR-675-5p 抵消了 5-FU 诱导的凋亡信号，从而参与了耐药反应。我们表明，凋亡标记物，PARP 切割和 Caspase-3 切割，在 miR-675-5p 抑制与 5-FU 处理联合使用时增加。此外，我们确定了 pro-caspase-3 是 miR-675-5p 的靶标之一。
我们的数据表明，抑制缺氧诱导的 miR-675-5p 联合 5-FU 治疗可以增强两种延长缺氧和常氧条件下的药物疗效，表明一种部分克服化疗耐药的可能策略。
