Department of Neurosurgery, Hangzhou Xiasha Hospital, Sir Run Run Shaw Hospital, Medical College, Zhejiang University, Hangzhou, People’s Republic of China.
Neuro Oncol. 2013 May;15(5):578-88. doi: 10.1093/neuonc/not004. Epub 2013 Feb 14.
Nemo-like kinase (NLK) is an evolutionarily conserved protein kinase involved in Wnt/beta-catenin signaling, which has been reported to be associated with gliomagenesis. In the present study, we aimed to identify a concrete mechanism of Wnt/beta-catenin pathway regulation by microRNAs (miRNAs) in glioma.
Quantitative reverse-transcription polymerase chain reaction and in situ hybridization were conducted to detect the expression of miR-92b. The cell proliferation rate and cell cycle kinetics were detected using 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay and flow cytometry, cell invasion and migration were evaluated using Transwell assay and wound healing assay, and cell apoptosis was detected using annexin V staining. Furthermore, the relevant molecules regulating proliferation and invasion were examined using Western blot analysis, immunohistochemistry, and immunofluorescence staining. Luciferase reporter assay was used to identify the direct regulation of NLK by miR-92b and beta-catenin/TCF4 activity.
We first showed that the expression of miR-92b was elevated in both glioma samples and glioma cells. Furthermore, down-regulation of miR-92b triggered growth inhibition, induced apoptosis, and suppressed invasion of glioma in vitro and in vivo. Luciferase assay and Western blot analysis revealed that NLK is a direct target of miR-92b. Restoring expression of NLK inhibited glioma proliferation and invasion. Mechanistic investigation revealed that miR-92b deletion suppressed beta-catenin/TCF-4 transcription activity by targeting NLK. Moreover, expression of NLK was inversely correlated with miR-92b in glioma samples and was predictive of patient survival in a retrospective analysis.
Our findings identify a role for miR-92b in glioma proliferation and invasion after activation of Wnt/beta-catenin signaling via NLK.
Nemo 样激酶（NLK）是一种进化上保守的蛋白激酶，参与 Wnt/β-连环蛋白信号通路，据报道与神经胶质瘤的发生有关。在本研究中，我们旨在确定 microRNAs（miRNAs）在神经胶质瘤中调节 Wnt/β-连环蛋白通路的具体机制。
采用定量逆转录聚合酶链反应和原位杂交检测 miR-92b 的表达。采用 3-(4,5)-二甲基噻唑(-z-y1)-3,5-二苯基四唑溴盐（MTT）法和流式细胞术检测细胞增殖率和细胞周期动力学，采用 Transwell 法和划痕愈合试验检测细胞侵袭和迁移，采用 Annexin V 染色检测细胞凋亡。此外，采用 Western blot 分析、免疫组织化学和免疫荧光染色检测调节增殖和侵袭的相关分子。采用荧光素酶报告基因检测 NLK 是否受 miR-92b 及β-连环蛋白/TCF4 活性的直接调控。
我们首先表明 miR-92b 在神经胶质瘤样本和神经胶质瘤细胞中均呈高表达。此外，miR-92b 的下调可触发体外和体内神经胶质瘤的生长抑制、诱导凋亡和抑制侵袭。荧光素酶测定和 Western blot 分析表明，NLK 是 miR-92b 的直接靶标。恢复 NLK 的表达可抑制神经胶质瘤的增殖和侵袭。机制研究表明，miR-92b 通过靶向 NLK 抑制β-连环蛋白/TCF-4 转录活性。此外，在神经胶质瘤样本中 NLK 的表达与 miR-92b 呈负相关，在回顾性分析中 NLK 的表达与患者生存相关。
我们的研究结果表明，miR-92b 通过 NLK 激活 Wnt/β-连环蛋白信号通路后在神经胶质瘤的增殖和侵袭中发挥作用。
