C-Met antisense oligodeoxynucleotide inhibits growth of glioma cells
C-Met antisense oligodeoxynucleotide inhibits growth of glioma cells
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DOI：
10.1016/j.surneu.2005.11.024
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发表时间：
2006-06-01
影响因子：
--
通讯作者：
Zhang, Jie
中科院分区：
文献类型：
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作者：
Chu, Shenghua;Yuan, Xianhou;Zhang, Jie
背景：C-Met 是一种受体酪氨酸激酶及其配体肝细胞生长因子，在细胞增殖、运动和侵袭中至关重要，并且已知在神经胶质瘤中过度表达。因此，我们研究的目的是研究转染的羧基荧光素-5-琥珀酰亚胺酯（FAM）标记的c-Met反义寡核苷酸（ASODN）对胶质瘤细胞生长的影响。方法：用LIPOFECTAMINE PLUS Reagent封装缀合的FAM标记的c-Met ASODN，然后添加到人胶质瘤细胞系U251中。将培养的细胞分为5组：对照组、500 nmol/L无义寡核苷酸(NSODN)组、250 nmol/L ASODN组、500 nmol/L ASODN组和750 nmol/L ASODN组。荧光显微镜观察c-Met ASODN在细胞内的分布；通过甲基噻唑四唑测定检测细胞生长。还用流式细胞仪检测了U251细胞的凋亡。采用半定量逆转录酶聚合酶链反应和Western blot检测c-Met信使RNA（mRNA）和蛋白的表达。结果：FAM标记的c-Met ASODN组细胞用FAM标记的c-Met ASODN-LIPOFECTAMINE PLUS试剂复合物处理3小时后，在荧光显微镜下可见到细胞质和细胞核中出现蓝色荧光。反义（AS）寡核苷酸导致细胞活力统计上显着降低（P < .05），而 NSODN 没有这种变化。 ASODN 也显着抑制细胞生长 (P < .05)。转染后，250、500和750 nmol/L ASODN诱导显着的细胞凋亡反应，24小时内约4.67% 2.86%、8.65% 3.18%和12.76% +/- 3.15% (P < .05)和7.79% +/- 1.92%、11.43% +/- 48 小时内分别为 1.54% 和 15.78% +/- 1.86% (P < .01)。然而，500 nmol/L NSODN 直到转染后 48 小时才诱导任何明显的细胞凋亡反应（P > .05）。 ASODN 处理的细胞中 c-Met mRNA 显着丢失，而 NSODN 处理中未见这种情况。 c-Met ASODN转染48小时后蛋白水平显着下降。结论：靶向c-Met的反义寡核苷酸可以被认为是最有效的AS化合物，它可以抑制细胞生长并诱导细胞凋亡。这提供了c-Met通过作为癌基因在神经胶质瘤的肿瘤进展中发挥作用的证据，并表明c-Met ASODN可能为人类神经胶质瘤的治疗提供一种新方法。 (c) 2006 Elsevier Inc. 保留所有权利。
Background: C-Met, a receptor tyrosine kinase, and its ligand, hepatocyte growth factor, are critical in cellular proliferation, motility, and invasion and are known to be overexpressed in gliomas. The aim of our study was therefore to investigate the effect of transfected caroboxyfluorescein-5-succimidyl ester (FAM)-labeled c-Met antisense oligonucleotide (ASODN) on growth of glioma cells.Methods: Conjugated FAM-labeled c-Met ASODN was encapsulated by LIPOFECTAMINE PLUS Reagent and then added into the human glioma cell line U251. Cultured cells were divided into 5 groups: control group, 500 nmol/L nonsense oligonucleotide (NSODN) group, 250 nmol/L ASODN group, 500 nmol/L ASODN group, and 750 nmol/L ASODN group. The intracellular distribution of c-Met ASODN was observed with fluorescence microscopy; cell growth was detected by methyl thiazole tetrazolium assay. The apoptosis of U251 cells was also examined with a flow cytometer. Semiquantitative reverse transcriptase polymerase chain reaction and Western blot examinations were carried for expression of c-Met messenger RNA (mRNA) and protein.Results: The blue fluorescence was seen in the cytoplast and nuclei of cells of FAM-labeled c-Met ASODN groups with fluorescence microscopy after the cells were treated with FAM-labeled c-Met ASODN-LIPOFECTAMINE PLUS Reagent complex for 3 hours. Antisense (AS) oligonucleotide caused a statistically significant reduction of cell viability (P < .05),whereas NSODN had no such changes. The cell growth was also significantly inhibited by ASODN (P < .05). After transfection, 250, 500, and 750 nmol/L ASODN induced significant apoptotic response, about 4.67% 2.86%, 8.65% 3.18%, and 12.76% +/- 3.15% for 24 hours (P < .05) and 7.79% +/- 1.92%, 11.43% +/- 1.54%, and 15.78% +/- 1.86% for 48 hours (P < .01), respectively. However, 500 nmol/L NSODN did not induce any significant apoptotic response until 48 hours after transfection (P > .05). A significant loss of c-Met mRNA was presented in ASODN-treated cells, and this was not seen in treatment with NSODN. Protein level was significantly decreased 48 hours after c-Met ASODN trasfected.Conclusions: Antisense oligonucleotide targeting c-Met can be identified as a most potent AS compound, which can inhibit cell growth and induce cell apoptosis. This provides evidence that c-Met plays a role in tumor progression of glioma by acting as an oncogene and suggests that c-Met ASODN may provide a novel approach to therapy for human glioma. (c) 2006 Elsevier Inc. All rights reserved.