Yan Hongliang, Wu Weiguang, Ge Hongyu, Li Pengfei, Wang Zheng
*Hebei United University, Tangshan, PR China; †Department of Gynaecology, the Affiliated Hospital of the Chinese People's Armed Forces Logistic College, Tianjin, PR China; ‡Healthy Care Center of Women and Children of Tianjin Beichen, Tianjin, PR China; §Biomechanics Laboratory of Orthopaedic Research Institute of Tian Jin Hospital, Tianjin, China; and ∥Hebei United University, Tangshan, PR China.
Int J Gynecol Cancer. 2015 Jul;25(6):944-52. doi: 10.1097/IGC.0000000000000456.
Genomic loci encoding miR-204, which was predicted to target brain-derived neurotrophic factor (BDNF), were frequently lost in multiple cancer, including epithelial ovarian cancer (EOC). In this study, we aimed to find out the influence of miR-204 expression level on EOC cell anoikis sensitivity and to explore possible mechanisms of this process.
First, we screened EOC cells, which maintain anoikis resistance forming an anoikis pattern. miR-204 expression level and apoptosis were measured, respectively, by quantitative reverse transcriptase polymerase chain reaction and Annexin-V-R-PE/7-amino-actinomycin assay. Then we restored the expression level of miR-204 by transfection with pre-miR-204. miR-204 expression level and apoptosis were measured as before; cell invasion and migration ability were detected by transwell invasion assay and wound-healing assay. The messenger RNA level of BDNF was also detected by quantitative reverse transcriptase polymerase chain reaction; Western blot analysis was performed to assess pAKT expression.
Expression of miR-204 is significantly down-regulated in an anoikis pattern. Restored expression level of miR-204 enables cells to acquire more sensitivity to anoikis and decrease invasive and metastatic behavior, and also results in BDNF down-expression and inhibits activation of mitochondria-dependent pathway through the PI3K/AKT signaling pathway leading to cancer cell anoikis in EOC cells.
miR-204 up-regulation may be linked directly to the sensitivity of EOC cell anoikis by contributing to BDNF down-regulation. Our findings provide a novel mechanism for manipulating miR-204 levels therapeutically to restore anoikis sensitivity.
编码miR - 204的基因组位点在包括上皮性卵巢癌（EOC）在内的多种癌症中经常缺失，miR - 204被预测靶向脑源性神经营养因子（BDNF）。在本研究中，我们旨在探讨miR - 204表达水平对EOC细胞失巢凋亡敏感性的影响，并探索这一过程的可能机制。
首先，我们筛选出具有失巢凋亡抗性并形成失巢凋亡模式的EOC细胞。分别通过定量逆转录聚合酶链反应和膜联蛋白V - R - PE/7 - 氨基放线菌素检测法测量miR - 204表达水平和细胞凋亡情况。然后，我们通过转染pre - miR - 204来恢复miR - 204的表达水平。如前所述测量miR - 204表达水平和细胞凋亡情况；通过Transwell侵袭实验和伤口愈合实验检测细胞侵袭和迁移能力。还通过定量逆转录聚合酶链反应检测BDNF的信使RNA水平；进行蛋白质免疫印迹分析以评估pAKT表达。
在失巢凋亡模式下，miR - 204的表达显著下调。恢复miR - 204的表达水平可使细胞对失巢凋亡获得更高的敏感性，并降低侵袭和转移行为，还导致BDNF表达下调，并通过PI3K/AKT信号通路抑制线粒体依赖性途径的激活，从而导致EOC细胞发生失巢凋亡。
miR - 204的上调可能通过导致BDNF下调而直接与EOC细胞失巢凋亡的敏感性相关。我们的研究结果为通过治疗性调节miR - 204水平来恢复失巢凋亡敏感性提供了一种新机制。
