抑制ATM/PI3K功能区表达对鼻咽癌细胞CNE1辐射增敏的研究

Radiosensitization of Nasopharyngeal Carcinoma Cell Line CNE1 by Inhibiting the Expression of ATM/PI3K Functional Region

背景与目的:共济失调毛细血管扩张症突变基因(ataxia-telangiectasiamutant,ATM)与细胞辐射敏感性密切相关,已有报道,ATM蛋白(ATM基因编码产物)表达抑制可引起多种恶性肿瘤细胞辐射敏感性的改变。本研究观察ATM/PI3K区反义RNA对鼻咽癌细胞系CNE1ATM蛋白的抑制作用及辐射增敏作用。方法:构建含反义ATM/PI3K区序列逆转录病毒重组体pDOR-atm,经阳离子脂质体转染入CNE1细胞,并命名为CNE1/pDOR-atm。应用半定量RT-PCR法分析反义组和对照组细胞中ATMmRNA的水平,应用流式细胞仪检测表达ATM蛋白的阳性细胞百分率及平均荧光强度,应用集落形成实验及线性二次模型(linear-quadraticmodel,L-Q模型)检测细胞辐射敏感性的变化。结果:在反义组细胞CNE1/pDOR-atm中ATMmRNA指数(mRNAindex,RI)平均为0.23±0.02,在对照组细胞CNE1/pDOR中RI平均为0.51±0.03(P<0.05)。在反义组中ATM蛋白阳性细胞百分率平均为70.8%,ATM蛋白平均荧光强度为1.81±0.12;而对照组中分别为99.3%,4.51±0.18(P<0.01),提示反义组细胞中ATMmRNA水平及蛋白表达抑制。反义组细胞α值为0.40Gy-1,对照组为0.36Gy-1,2Gy辐射增敏比达3.0,提示反义组细胞辐射敏感性增加。结论:抑制CNE1细胞的ATM/PI3K区表达可以达到有效的辐射增敏目的。

BACKGROUND ＆ OBJECTIVE. It is reported that ATM gene is closely correlated to cellular radiosensitivity in several malignant tumors. Suppression of ATM protein expression leads to cellular radiosensitization. This study was to determine whether this effect also exists in nasopharyngeal carcinoma （NPC） cell line CNE1 by inhibiting the expression of ATM protein through antisense RNA of ATM/PI3K region, which is the most important functional fragment of ATM gene. METHODS： The recombinant pDOR-atm expressing antisense RNA of ATM/PI3K segment was constructed with retroviral vector pDOR. CNE1 cells were stably transfected with pDOR-atm by cationic liposome and named CNE1/pDOR-atm. Semi-quantitive RT-PCR was used to detect the level of ATM mRNA. Flow cytometry （FCM） was employed to analyze the percentage of positive cells and mean fluorescence density of protein expressing ATM. Cellular radiosensitivity was evaluated by colony survival assay （CSA） and linear-quadratic model in both CNE1/ pDOR-atm and control cells. RESULTS： The level of ATM mRNA index （RI） was 0.23±0.02 in CNE1/pDOR-atm group, and 0.51±0.03 in control group （P〈0.05）. The percentage of positive cells and mean fluorescence density of proteins expressing ATM were 70.8% and 1.81 ±0.12 in CNE1/pDOR-atm group, while 99.3% and 4.51±0.18 in control group（P〈0.01）. The expression of ATM mRNA and protein was inhibited by antisense RNA. The α value （one function from linear-quadratic model） of CNE1/pDOR-atm group was 0.40 Gy^-1, while it was 0.36 Gy^-1 in control group. The radiosensitizing ratio of surviving fraction at 2 Gy （SF2） was 3.0, indicating that antisense RNA group was more radiosensitive to X-ray than the controls. CONCLUSION, NPC cell line CNE1 could be radiosensitized by the down-regulation of ATM/ PI3K expression.