Institute of Tumor Biology, Jiangsu Province Academy of Clinical Medicine, Nanjing, Jiangsu 210029, PR China.
Oncol Rep. 2013 May;29(5):1769-76. doi: 10.3892/or.2013.2297. Epub 2013 Feb 21.
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease and is usually resistant to chemotherapy. MicroRNA‑181b (miR-181b) has been reported to be associated with chemoresistance in various types of cancer. In this study, we investigated the effects of miR-181b on the chemosensitivity of PDAC cells to gemcitabine and the underlying molecular events. miR-181b mimics and inhibitors were synthesized for transient gene transfection in vitro. Lentivirus carrying miR-181b mimics were used to infect PDAC cells for nude mouse xenograft assays by implanting infected PDAC cells into recipient mice. Cell viability was determined by MTT assays, while gene expression was assessed using qRT-PCR, western blot analysis and enzyme-linked immunosorbent assay (ELISA). miR-181b targeting BCL-2 expression was assessed by a dual-luciferase activity assay. The data showed that miRNA-181b expression sensitized PDAC cells to gemcitabine treatment. Although gemcitabine-resistant PDAC cell sublines (SW1990/GR and CFPAC-1/GR) expressed higher levels of miRNA-181b, gemcitabine induced higher levels of apoptosis in PDAC cells transfected with miRNA-181b mimics. The nude mouse xenograft assay data showed that miR-181b transfection also sensitized the cells to gemcitabine treatment in vivo. Molecularly, bioinformatics data predicted that miR-181b was able to bind to BCL-2 mRNA 3'UTR. The dual luciferase activity assay revealed that miRNA-181b downregulated BCL-2 expression. The results from western blot analysis showed a reduced BCL-2 expression following miR-181b transfection but an enhanced caspase-3 activity in miRNA-181b mimic-transfected PDAC cells. This study demonstrates that miRNA-181b sensitizes PDAC cells to gemcitabine by targeting BCL-2.
胰腺导管腺癌（PDAC）是一种高度致命的疾病，通常对化疗有抗性。有报道称，microRNA-181b（miR-181b）与各种类型癌症的化疗耐药性有关。在这项研究中，我们研究了 miR-181b 对 PDAC 细胞对吉西他滨化疗敏感性的影响及其潜在的分子事件。合成了 miR-181b 模拟物和抑制剂用于体外瞬时基因转染。携带 miR-181b 模拟物的慢病毒用于感染 PDAC 细胞，通过将感染的 PDAC 细胞植入受体小鼠中进行裸鼠异种移植实验。通过 MTT 测定法测定细胞活力，通过 qRT-PCR、western blot 分析和酶联免疫吸附测定（ELISA）评估基因表达。通过双荧光素酶活性测定评估 miR-181b 对 BCL-2 表达的靶向作用。数据表明，miR-181b 表达使 PDAC 细胞对吉西他滨治疗敏感。尽管吉西他滨耐药 PDAC 细胞亚系（SW1990/GR 和 CFPAC-1/GR）表达更高水平的 miR-181b，但 miR-181b 模拟物转染的 PDAC 细胞中吉西他滨诱导更高水平的细胞凋亡。裸鼠异种移植实验数据表明，miR-181b 转染也使细胞在体内对吉西他滨治疗敏感。分子水平上，生物信息学数据预测 miR-181b 能够与 BCL-2 mRNA 3'UTR 结合。双荧光素酶活性测定显示 miR-181b 下调 BCL-2 表达。western blot 分析结果显示，miR-181b 转染后 BCL-2 表达降低，但 miR-181b 模拟物转染的 PDAC 细胞中 caspase-3 活性增强。本研究表明，miR-181b 通过靶向 BCL-2 使 PDAC 细胞对吉西他滨敏感。
