HERG基因调控NF-κB通道抑制骨肉瘤恶性表型的研究

doi:10.3760/cma.j.issn.1673422X.2016.07.007

摘要/Abstract

摘要： 目的检测HERG(human etheràgogorelated gene)钾离子通道在骨肉瘤中的表达，并探索小干扰RNA(siRNA)沉默HERG表达后对骨肉瘤细胞增殖和凋亡的影响及可能的调控机制。方法采用反转录定量聚合酶链反应(RTPCR)、Western blotting和免疫组织化学法检测骨肉瘤MG63细胞和组织中HERG的表达。实验分组为：HERGsiRNA处理为实验组，controlsiRNA处理为对照组，未行任何处理为空白组。分别采用CCK8法、克隆形成、流式细胞仪和Tunel法检测MG63细胞增殖、生长和凋亡的变化。最后采用Western blotting检测骨肉瘤细胞中核转录因子κB(NFκB)信号通路相关蛋白的表达水平。结果HERG在骨肉瘤细胞和组织中异常高表达。CCK8法结果示，实验组［(75.34±4.45)%］与对照组［(100.60±5.31)%］和空白组［(100.00±5.66)%］的细胞增殖水平相比，差异有统计学意义（t=3.64，P=0.007；t=3.43，P=0.009）。克隆形成结果示，实验组(134.30±11.82)与对照组(225.30±11.56) 和空白组(232.80±12.21)的克隆形成数相比，差异有统计学意义（t=5.51，P=0.002；t=5.80，P=0.001）。流式细胞凋亡结果示，实验组［(28.10±2.21)%］与对照组［(9.36±2.42)%］和空白组［(10.92±2.51)%］的早期凋亡所占比例相比，差异有统计学意义（t=5.72，P=0.005；t=5.14，P=0.007）。Tunel法结果示，实验组［(31.57±2.08)%］与对照组［(10.35±1.82)%］和空白组［(7.96±0.88)%］的凋亡指数相比，差异有统计学意义（t=7.69，P=0.002；t=10.48，P=0.001）。抑制HERG表达可明显下调NFκB信号通路中细胞凋亡抑制蛋白1（cIAP1）、X染色体连锁凋亡抑制蛋白（XIAP）、Bcl2和Survivin的活性，同时还下调磷酸化NFκB抑制蛋白（IκB）α和NFκB p65的表达水平。结论HERG在骨肉瘤中高表达，其通过调控NFκB信号通路参与骨肉瘤细胞增殖和凋亡过程，有望成为骨肉瘤治疗和诊断的新靶点。

关键词: 钾通道, 骨肉瘤, , 细胞增殖, , 细胞凋亡, , NF-κB

Abstract: ObjectiveTo detect the expression of HERG (human etheràgogorelated gene) potassium channel in human osteosarcoma, and explore the effects of silencing HERG by small interfering RNA (siRNA) on the proliferation and apoptosis of osteosarcoma cells and the mechanisms responsible for HERG regulation. MethodsThe expressions of HERG in osteosarcoma MG63 cells and tissues were detected by reverse transcription polymerase chain reaction (RTPCR), Western blotting and immunohistochemistry. Next, osteosarcoma cells were divided into three groups: HERGsiRNA group, controlsiRNA group and blank group. CCK8, colony formation, flow cytometry and Tunel assay were used to measure the proliferation and apoptosis of the osteosarcoma cells. Finally, Western blotting analysis was performed to detect the expression of nuclear factorκB (NFκB) pathway in osteosarcoma cells treated with HERG siRNA. ResultsOsteosarcoma cells and tissues were found to highly express HERG. Inhibition of HERG in the osteosarcoma cells significantly inhibited the cell proliferation and induced cell apoptosis. Compared to controlsiRNA group or blank group, HERGsiRNA could inhibit the proliferation of MG63 cells significantly ［HERGsiRNA group: (75.34±4.45)%; compared to controlsiRNA group: (100.60±5.31)%; t=3.64, P=0.007; compared to blank group: (100.00±5.66)%; t=3.43, P=0.009］. The similar results were obtained from colony formation assay (HERGsiRNA group: 134.30±11.82; compared to controlsiRNA group: 225.30±11.56; t=5.51, P=0.002; compared to blank group: 232.80±12.21; t=5.80, P=0.001). HERGsiRNA transfected MG63 cells demonstrated a significant increase of apoptotic rate compared to controlsiRNA transfected cells or untreated cells ［HERGsiRNA group: (28.10±2.21)%; compared to controlsiRNA group: (9.36±2.42)%; t=5.72, P=0.005; compared to blank group: (10.92±2.51)%; t=5.14, P=0.007］. This result was further confirmed by Tunel assay. The cells transfected with HERGsiRNA (31.57±2.08)% demonstrated extensive apoptosis, compared with the controlsiRNA group ［(10.35±1.82)%; t=7.69, P=0.002)］ or blank group ［(7.96±0.88)%; t=10.48, P=0.001］. Silencing HERG gene downregulated the cIAP1, XIAP, Bcl2, Survivin, PIκBα and NFκB p65 expression, compared to the control groups. ConclusionHERG is highly expressed in osteosarcoma. HERG silencing can suppress osteosarcoma progression through NFκB pathway and suggest that HERG may be a novel molecular target for osteosarcoma therapy and diagnosis.

Key words: Potassium channels, Osteosarcoma, Cell proliferation, Apoptosis, , NF-kappa B

吴进, 陈志达, 曾文容, 林斌, 吴欣宇, 刘庆军. HERG基因调控NF-κB通道抑制骨肉瘤恶性表型的研究[J]. 国际肿瘤学杂志, 2016, 43(7): 508-514.

WU Jin, CHEN Zhi-Da, ZENG Wen-Rong, LIN Bin, WU Xin-Yu, LIU Qing-Jun. HERG suppresses the malignant phenotypes of osteosarcoma via modulating NF-κB pathway[J]. Journal of International Oncology, 2016, 43(7): 508-514.

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