Zhang Luyao, He Liang, Zhang Hua, Chen Yan
Cell Physiol Biochem. 2018;47(4):1432-1441. doi: 10.1159/000490834. Epub 2018 Jun 19.
BACKGROUND/AIMS: Platinum-based chemotherapy is one of the most important strategies for treatment of colorectal cancer. To improve the therapeutic efficiency, adjuvant drugs were sought to sensitize colorectal cancer cells to platinum-based agents such as cisplatin. As previous research has shown that miRNAs are associated with chemosensitivity, we aimed to alter miRNA regulation in colorectal cancer cells to increase their chemosensitivity.
MTT assays were performed to determine the viability of HT29, SW480, and LoVo cells. Quantitative real time polymerase chain reaction (qRT-PCR) was performed to examine the expression of miR-20a in these cell lines. Regulation of the miR-20a/ASK1 axis was confirmed by western blotting and luciferase reporter assays. After treatment with miR-20a inhibitor (anti-miR-20a) and cisplatin, production of reactive oxygen species (ROS), mitochondrial membrane potential, and apoptosis were measured by flow cytometry. Activation of ASK1, Bcl-xl, JNK, and caspase-9, -7, and -3 was detected by western blotting.
miR-20a was overexpressed in colorectal cancer cell lines. Furthermore, knockdown of miR-20a increased the sensitivity of colorectal cancer cells to cisplatin treatment in vitro and in vivo. We demonstrated that the ASK1 gene was the target of miR-20a, and knockdown of miR-20a increased the expression of ASK1 in colorectal cancer cells. As cisplatin treatment induced production of ROS, knockdown of miR-20a enhanced ROS signaling through promoting the phosphorylation of ASK1. Phosphorylation of JNK and the subsequent mitochondrial apoptosis were triggered by the combination of cisplatin and anti-miR-20a.
Knockdown of miR-20a enhanced sensitivity of colorectal cancer cells to cisplatin through the ROS/ASK1/JNK pathway.
背景/目的：铂类化疗是治疗结直肠癌最重要的策略之一。为提高治疗效果，人们寻求辅助药物使结直肠癌细胞对顺铂等铂类药物敏感。既往研究表明，微小RNA（miRNA）与化疗敏感性相关，因此我们旨在改变结直肠癌细胞中的miRNA调控，以提高其化疗敏感性。
采用MTT法检测HT29、SW480和LoVo细胞的活力。通过定量实时聚合酶链反应（qRT-PCR）检测这些细胞系中miR-20a的表达。通过蛋白质印迹法和荧光素酶报告基因检测法证实miR-20a/凋亡信号调节激酶1（ASK1）轴的调控。在用miR-20a抑制剂（抗miR-20a）和顺铂处理后，通过流式细胞术检测活性氧（ROS）的产生、线粒体膜电位和细胞凋亡情况。通过蛋白质印迹法检测ASK1、Bcl-xl、c-Jun氨基末端激酶（JNK）以及半胱天冬酶-9、-7和-3的激活情况。
miR-20a在结直肠癌细胞系中过表达。此外，敲低miR-20a可在体外和体内提高结直肠癌细胞对顺铂治疗的敏感性。我们证明ASK1基因是miR-20a的靶标，敲低miR-20a可增加结直肠癌细胞中ASK1的表达。由于顺铂治疗可诱导ROS产生，敲低miR-20a通过促进ASK1的磷酸化增强了ROS信号传导。顺铂与抗miR-20a联合触发了JNK的磷酸化以及随后的线粒体凋亡。
敲低miR-20a通过ROS/ASK1/JNK途径增强了结直肠癌细胞对顺铂的敏感性。
