靶向生存素基因的反义寡核苷酸诱导涎腺黏液表皮样癌细胞凋亡
[Antisense oligonucleotide targeting survivin gene induces cell apoptosis in salivary mucoepidermoid carcinoma].
作者信息
Qi Hong, Guo Jian, Zahng Yin-Cheng, Li Shu-Wei, Yang Li-Lin
机构信息
Department of Oral Pathology, Xi'an Jiaotong University Stomatological Hospital, Xi'an 710004, China.
出版信息
Zhonghua Kou Qiang Yi Xue Za Zhi. 2010 Sep;45(9):525-30.
OBJECTIVE
to determine the effects of survivin antisense oligonucleotide (ASODN) on the expression levels of survivin mRNA and human mucoepidermoid carcinoma cell line (highly metastatic Mc3) apoptosis and to explore the feasibility of survivin gene as the mucoepidermoid carcinoma therapeutic targets.
METHODS
the survivin ASODN was designed and synthesized and then respectively transfected into Mc3 cells. The morphological changes of the Mc3 cells were observed 24, 48 and 72 h after transfection by inverted microscope and the apoptosis rate detected by flow cytometry. Methyl thiazolyl tetrazolium (MTT) assay was used to detect the effect of the transfection on cell poliferation, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method for analysis of apoptotic index, and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to detect the expression of survivin.
RESULTS
in survivin ASODN transfection group, there was less Mc3 cells than in other groups. The suspended cells dropping from the wall increased and showed typical apoptotic changes and time-dependent. Mc3 cell apoptosis rate in survivin ASODN transfection group transfected for 24, 48, 72 h was 12.96%, 14.43%, 22.69%, respectively, which were significantly higher than in other groups (P < 0.01) and time-dependent (P < 0.05). The inhibitory rate of Mc3 cells in survivin ASODN transfection group was 22.35%, 39.04%, 43.46%, which were significantly higher than other groups (P < 0.01) and time-dependent (P < 0.05). The apoptosis index (AI) of Mc3 cells in survivin ASODN transfection group was 11.038%, 12.172%, 18.900%, significantly higher than other groups (P < 0.01) and time-dependent (P < 0.05). The survivin mRNA levels in Mc3 cells were 0.739 ± 0.008, 0.668 ± 0.007, 0.500 ± 0.006, and the relative inhibition rate in these cells was 18.21%, 26.06%, 44.82%, significantly lower than other groups (P < 0.01) and time-dependent manner (P < 0.01).
CONCLUSIONS
survivin ASODN could inhibit the proliferation of Mc3 cells and induce the apoptosis of Mc3 cells. It also can inhibit the expression of survivin mRNA. Survivin can be used as a gene therapy targets for mucoepidermoid carcinoma.
目的
探讨生存素反义寡核苷酸（ASODN）对人黏液表皮样癌细胞系（高转移Mc3细胞）生存素mRNA表达水平及凋亡的影响，探讨生存素基因作为黏液表皮样癌治疗靶点的可行性。
方法
设计并合成生存素ASODN，转染Mc3细胞。转染后24、48及72 h，倒置显微镜下观察Mc3细胞形态变化，流式细胞术检测细胞凋亡率。采用甲基噻唑基四氮唑（MTT）法检测转染对细胞增殖的影响，用末端脱氧核苷酸转移酶介导的dUTP-生物素缺口末端标记（TUNEL）法分析凋亡指数，半定量逆转录聚合酶链反应（RT-PCR）检测生存素表达。
结果
生存素ASODN转染组Mc3细胞数量较其他组少。贴壁细胞脱落、悬浮，呈现典型凋亡变化，且具有时间依赖性。生存素ASODN转染组转染24、48、72 h的Mc3细胞凋亡率分别为12.96%、14.43%、22.69%，显著高于其他组（P<0.01），且具有时间依赖性（P<0.05）。生存素ASODN转染组Mc3细胞抑制率分别为22.35%、39.04%、43.46%，显著高于其他组（P<0.01），且具有时间依赖性（P<0.05）。生存素ASODN转染组Mc3细胞凋亡指数（AI）分别为11.038%、12.172%、18.900%，显著高于其他组（P<0.01），且具有时间依赖性（P<0.05）。Mc3细胞生存素mRNA水平分别为0.739±0.008、0.668±0.007、0.500±0.006，相对抑制率分别为18.21%、26.06%、44.82%，显著低于其他组（P<0.01），且呈时间依赖性（P<0.01）。
结论
生存素ASODN可抑制Mc3细胞增殖，诱导其凋亡，抑制生存素mRNA表达。生存素可作为黏液表皮样癌基因治疗的靶点。
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