Liu Xiaobing, Luo Xing, Wu Yuqi, Xia Ding, Chen Wei, Fang Zhenqiang, Deng Jianping, Hao Yaxing, Yang Xia, Zhang Teng, Zhou Luqiang, Wu Yingbing, Wang Qingqing, Xu Jie, Hu Xiaoyan, Li Longkun
Department of Urology, Xinqiao Hospital, Third Military Medical University, Chongqing, China.
Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Cell Physiol Biochem. 2018;50(1):261-276. doi: 10.1159/000494004. Epub 2018 Oct 3.
BACKGROUND/AIMS: Treatment options for metastatic castrate-resistant prostate cancer (mCRPC) are limited and typically centered on paclitaxel-based chemotherapy. In this study, we aimed to evaluate whether miR-34a attenuates chemoresistance to paclitaxel by regulating target genes associated with drug resistance.
We used data from The Cancer Genome Atlas to compare miR-34a expression levels in prostate cancer (PC) tissues with normal prostate tissues. The effects of miR-34a inhibition and overexpression on PC proliferation were evaluated in vitro via Cell Counting Kit-8 (CCK-8) proliferation, colony formation, apoptosis, and cell-cycle assays. A luciferase reporter assay was employed to identify the interactions between miR-34a and specific target genes. To determine the effects of up-regulation of miR-34a on tumor growth and chemo-resistance in vivo, we injected PC cells overexpressing miR-34a into nude mice subcutaneously and evaluated the rate of tumor growth during paclitaxel treatment. We examined changes in the expression levels of miR-34a target genes JAG1 and Notch1 and their downstream genes via miR-34a transfection by quantitative reverse transcription PCR (qRT-PCR) and western blot assay.
miR-34a served as an independent predictor of reduced patient survival. MiR-34a was down-regulated in PC-3PR cells compared with PC-3 cells. The CCK-8 assay showed that miR-34a overexpression resulted in increased sensitivity to paclitaxel while miR-34a down-regulation resulted in chemoresistance to paclitaxel in vitro. A study of gain and loss in a series of functional assays revealed that PC cells expressing miR-34a were chemosensitive. Furthermore, the overexpression of miR-34a increased the sensitivity of PC-3PR cells to chemotherapy in vivo. The luciferase reporter assay confirmed that JAG1 and Notch1 were directly targeted by miR-34a. Interestingly, western blot analysis and qRT-PCR confirmed that miR-34a inhibited the Notch1 signaling pathway. We found that miR-34a increased the chemosensitivity of PC-3PR cells by directly repressing the TCF1/ LEF1 axis.
Our results showed that miR-34a is involved in the development of chemosensitivity to paclitaxel. By regulating the JAG1/Notch1 axis, miR-34a or its target genes JAG1 or Notch1 might serve as potential predictive biomarkers of response to paclitaxel-based chemotherapy and/or therapeutic targets that will help to overcome chemoresistance at the mCRPC stage.
背景/目的：转移性去势抵抗性前列腺癌（mCRPC）的治疗选择有限，通常以紫杉醇为基础的化疗为主。在本研究中，我们旨在评估miR-34a是否通过调节与耐药相关的靶基因来减弱对紫杉醇的耐药性。
我们使用来自癌症基因组图谱的数据，比较前列腺癌（PC）组织与正常前列腺组织中miR-34a的表达水平。通过细胞计数试剂盒-8（CCK-8）增殖、集落形成、凋亡和细胞周期检测，在体外评估miR-34a抑制和过表达对PC增殖的影响。采用荧光素酶报告基因检测来确定miR-34a与特定靶基因之间的相互作用。为了确定miR-34a上调对体内肿瘤生长和化疗耐药性的影响，我们将过表达miR-34a的PC细胞皮下注射到裸鼠体内，并评估紫杉醇治疗期间的肿瘤生长速率。我们通过定量逆转录PCR（qRT-PCR）和蛋白质免疫印迹分析，检测miR-34a转染后miR-34a靶基因JAG1和Notch1及其下游基因表达水平的变化。
miR-34a是患者生存降低的独立预测因子。与PC-3细胞相比，PC-3PR细胞中miR-34a表达下调。CCK-8检测显示，miR-34a过表达导致对紫杉醇的敏感性增加，而miR-34a下调导致体外对紫杉醇耐药。一系列功能检测的增减研究表明，表达miR-34a的PC细胞具有化疗敏感性。此外，miR-34a过表达增加了PC-3PR细胞在体内对化疗的敏感性。荧光素酶报告基因检测证实JAG1和Notch1是miR-34a的直接靶点。有趣的是，蛋白质免疫印迹分析和qRT-PCR证实miR-34a抑制Notch1信号通路。我们发现miR-34a通过直接抑制TCF1/LEF1轴增加PC-3PR细胞的化疗敏感性。
我们的结果表明，miR-34a参与了对紫杉醇化疗敏感性的发展。通过调节JAG1/Notch1轴，miR-34a或其靶基因JAG1或Notch1可能作为基于紫杉醇化疗反应的潜在预测生物标志物和/或治疗靶点，有助于克服mCRPC阶段的化疗耐药性。
