shRNAmir慢病毒载体沉默COX-2对鼻咽癌细胞生长转移及放化疗敏感性的影响
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摘要
研究背景及目的：
鼻咽癌(nasopharyngeal carcinoma, NPC)是我国常见的恶性肿瘤之一,其发病以明显的区域分布、强烈的转移倾向、与EB病毒的密切关系为显著特征。放射治疗是鼻咽癌目前公认和有效的治疗手段,但因其严重的并发症及后遗症,治疗效果并不稳定,5年总生存率始终徘徊在50%左右。因此,探究鼻咽癌发病的分子机制,寻找新的治疗靶点,提高现有治疗手段的疗效,是当代肿瘤学研究的一项迫切任务。
COX-2是环氧合酶两种同工酶的其中一种,在体内呈诱导性表达,在大多数正常组织中难以发现。当受到一些刺激因子如生长因子、细胞因子及促癌剂的刺激时,其表达量会成倍增加。抑制COX-2的表达可明显增加化疗及放疗对鼻咽癌细胞的杀伤能力。
shRNAmir(microRNA-adapted,shRNA)是在microRNA结构基础上改造出的发夹状小干扰RNA,利用shRNAmir表达载体,在细胞内转录出与miRNA相似的shRNAmir,比传统的shRNA更加安全有效,是人工诱导RNA干扰沉默基因表达的最新技术。
本课题前期已完成如下研究工作：应用RNA干扰技术沉默COX-2的表达,使得鼻咽癌细胞周期出现G0/G1期阻滞,且增殖能力明显下降；应用基于miR-155结构的RNA干扰技术,成功构建了Anti-COX-2 shRNAmir(?)慢病毒表达载体,经慢病毒感染48-72h后,阳性C666-1细胞发出绿色萤光,感染效率最高达90.5%,经流式细胞仪分选出带荧光的细胞经连续传代3个月后,带光率仍超99%,RT-PCR几乎检测不到COX-2基因mRNA的表达,获得了稳定抑制COX-2表达的鼻咽癌细胞亚系,证明shRNAmir慢病毒载体具备获得稳定持久的RNA干扰作用,在转录后水平沉默COX-2基因,达到了“基因敲除”的效果,远优于Anti-COX-2 siRNA的干扰效果,应用于鼻咽癌分子机制研究,为下一步的分子靶向治疗和基因-放射联合治疗奠定基础。
在前期研究工作的基础上,本研究进一步探讨COX-2基因沉默后对鼻咽癌细胞生长、转移及对放化疗敏感性的影响,为鼻咽癌患者的综合治疗提供理论依据。
方法
1.检测COX-2沉默后对鼻咽癌细胞生长特性的影响
2×103个指数生长期细胞接种于96孔板中,每孔细胞悬液量为100ul,置于培养箱中分别培养1、2、3、4、5天,向每孔加入10ul的CCK-8溶液,将培养板置于培养箱中孵育1h,用酶标仪测定在450nm处的OD值,以相对应OD值表示细胞增殖能力大小。每组设6个重复孔,取平均值,绘制体外生长曲线。
2.划痕实验
2×105个细胞接种于6孔板,每组细胞作3个复孔,待细胞长至约90%汇合时,吸干培养基,用200ul tip头垂直在培养孔中部轻轻划过,力度以刮落细胞而不在培养板上留痕为准。PBS洗2遍以冲去脱落细胞,加入培养基后继续培养。每个孔随机选取9个有划痕穿过的视野,分别观察0h,6h,12h,24h时细胞细胞划痕处细胞运动变化,并拍照取样,应用Image J软件测划痕相对宽度,绘制迁移力量值曲线,独立实验重复3次。
3.检测COX-2沉默后鼻咽癌细胞对顺铂的敏感性变化
两组细胞分别按5000个/孔加入96孔板中,每孔加入培养基100ul,培养24h后弃去培养基,加入含有不同浓度顺铂的培养基,使其终浓度分别为0.5、1、2、4、8、16、32ug/ml,每组3个复孔,并设不接种细胞的空白培养基调零组和加入等体积溶剂的对照组,作用24h后加入10ul CCK-8试剂,置于培养箱中孵育1h后,利用酶标仪测各孔450nm处的吸光度A450,计算细胞生长的抑制率,并应用SPSS13.0软件计算半数抑制浓度。
4.细胞周期实验
指数生长期细胞在含有6ug/ml的顺铂或不含顺铂的培养基中培养24h,或经2Gy照射或0Gy假照射24及48h后,以不含EDTA的胰酶消化后收集细胞,加入70%冰乙醇中4℃过夜,PBS洗涤2遍后,避光加入含有50ug/ml的RNA酶和50ul/ml PI的染色液中,室温染色30min后,流式细胞仪检测细胞周期各时相所占百分比。
5.克隆形成实验
两组细胞按预定数量接种于6孔板中,每组均设0、1、2、4、6、8Gy六个剂量组,每一剂量组3个复孔。照射后的细胞置于37℃、5%CO2培养箱内培养。当出现肉眼可见克隆时,终止培养,甲醇固定,1%结晶紫乙醇溶液染色。显微镜下计数含50个细胞以上的克隆数,计算克隆形成率和存活分数。以3次照射的存活分数均值进行分析,运用GraphPad Prism 5.0软件进行L-Q模型和多靶单击模型曲线拟合,绘制剂量存活曲线,并计算L-Q模型参数α、β、α/β、SF2和多靶单击模型参数D0、Dq、和N,其中logN=Dq/D0,计算放射增敏比。
6.体内放疗敏感性实验
雌性BALB/c裸鼠,4-6周龄,18～22g, SPF条件下饲养。将两组细胞分别制成单细胞悬液,用无血清1640培养基调节细胞浓度至1×106/ml。在裸鼠右后肢皮下接种细胞悬液0.1ml,实验中每2天用游标卡尺测量肿瘤的最大径和与之垂直的横径,肿瘤体积(V)=(长径×短径2)/2。10-14d后,当肿瘤体积达到约200mm3时,将荷瘤裸鼠分为4组：①COX-2(+)对照组,无任何处理；②COX-2(-)对照组,无任何处理；③COX-2(+)实验组,给予10GyX线照射；④COX-2(-)实验组,给予10Gy X线照射。每组6只,2周后断颈处死裸鼠,计算抑瘤率。
7.统计学分析
采用SPSS13.0软件进行统计分析,实验数据以均数±标准差表示,两样本均数的比较才采用完全随机设计资料的方差分析(One-Way ANOVA)或两独立样本的t检验(Independent-Sample T Test)。增殖实验和划痕实验采用析因设计的方差分析,多重比较采用SNK法(Student-Neuman-Keuls), P<0.05表示差异有统计学意义。
结果：
1.COX-2基因沉默后对鼻咽癌细胞增殖和体外迁移能力的影响
Anti-COX-2 C666-1和Anti-GL-2 C666-1细胞,接种96孔板连续观察5天,依据450nm处OD值绘制生长曲线,析因分析结果表明,两种细胞增殖差异有统计学意义(F=5.927,P=0.019),但细胞组别与天数之间无交互效应(F=1.264,P=0.296),说明COX-2基因沉默后细胞增殖能力有所下降,但两组细胞增殖速度随时间变化趋势相同。
细胞划痕实验检测COX-2稳定沉默后对鼻咽癌细胞C666-1体外迁移能力的影响,析因设计的方差分析结果显示,随着培养时间的延长,两组间划痕宽度差异有统计学意义(F=157.01,P<0.001),细胞组别与时间之间有交互效应(F=15.73,P<0.001),说明随着时间的变化,两组细胞体外迁移距离的变化趋势不同,Anti-COX-2 C666-1细胞迁移能力较Anti-GL-2 C666-1有所减弱。
2.COX-2沉默后鼻咽癌C666-1细胞对顺铂敏感性的变化
析因分析结果显示,不同浓度顺铂作用24h后,两组细胞增殖抑制率差异有统计学意义(F=22.219,P<0.001),经计算得出Anti-COX-2 C666-1和Anti-GL-2C666-1细胞对顺铂的IC50分别为3.682±0.980和8.819±2.354,两组间IC50差异有统计学意义(t=-3.489,P=0.025)。
3.COX-2沉默后对顺铂引起细胞周期变化的影响
流式细胞仪分析结果表明,加顺铂前Anti-COX-2 C666-1和Anti-GL-2C666-1细胞周期中,G0/G1期细胞比例分别为70.260%和60.353%(t=-2.726,P=0.053),S期细胞比例分别为24.270%和32.180%(t=-2.033,P=0.112),差异均无统计学意义,G2/M期细胞比例差别有统计学意义,分别为5.470%和7.500%(t=-3.954, P=0.017)。6ug/ml的顺铂作用24h后,两组细胞S期与G2/M期比例均升高,两组均出现S期和G2/M期阻滞,S期比例升高幅度分别为24.967%和32.200%,两组间差异无统计学意义(t=--1.636,P=0.177),G2/M期比例升高幅度分别为0.500%和7.633%,两组间差异有统计学意义(t=-7.501,P=0.002)。COX-2沉默后G2/M期升高比例减少。但顺铂作用前后均未出现明显的凋亡峰,说明COX-2沉默后降低了顺铂引起的细胞G2/M期阻滞,但对细胞凋亡无明显影响。
4.COX-2沉默后对鼻咽癌C666-1细胞体外放疗敏感性的影响
COX-2沉默前后Do值分别为2.0157±0.0515、1.6333±0.0751,Dq值分别为0.8037±0.0487、0.5643±0.0399,SF2分别为0.5551±0.0375、0.3961±0.0160,α/β分别为6.5356±0.8461、9.4231±0.9599,两组间D0、Dq、F2、α/β值差异均有统计学意义,放射增敏比SER=1.4014。
5.COX-2沉默后对辐射引起细胞周期的影响
流式细胞仪分析结果表明,照射前Anti-GL-2 C666-1和Anti-COX-2 C666-1细胞周期中,G0/G1以及S期细胞比例差异均无统计学意义,但G2/M期细胞比例差别有统计学意义,分别为7.500%和5.470%(t=-3.952,P=0.017)。照射2Gy后24h,G2期细胞比例均增高,分别为45.833%和23.900%(t=11.999,P<0.001),两组细胞均出现G2/M期阻滞,但Anti-GL-2 C666-1细胞G2/M期升高比例更大,为38.333%,而Anti-COX-2 C666-2细胞G2/M期比例升高18.430%(t=9.184,P=0.001)。照射2Gy后48h,两组细胞G2/M期的比例分别为24.397%和12.393%(t=4.646,P=0.01),较照射前分别增加了17.357%和6.923%(t=3.320,P=0.029)。但照射前后均未出现反映细胞凋亡的亚G1期(sub-G1 phase),说明COX-2沉默后显著降低了辐射引起的细胞G2/M期阻滞,但对细胞凋亡无明显影响。
6.COX-2沉默后对体内荷瘤裸鼠辐射敏感性的影响
未放疗组,Anti-GL-2和Anti-COX-2两组裸鼠移植瘤生长曲线几乎重合,COX-2沉默前后对皮下移植瘤的生长情况差异无统计学意义(F=3.061,P=0.084),加入10Gy单纯放疗后,Anti-GL-2和Anti-COX-2两组的抑瘤率分别为32.72%和51.76%,两组间皮下移植瘤的生长情况差异有统计学意义(F=63.468,P<0.001)。
结论：
1.COX-2沉默后降低了鼻咽癌细胞的增殖及体外迁移能力；
2.COX-2沉默后增强顺铂对鼻咽癌C666-1细胞的敏感性,降低了顺铂引起的细胞G2/M期阻滞；
3.COX-2沉默后增强鼻咽癌体内外放疗的敏感性,降低了辐射引起的细胞G2/M期阻滞。
BACKGROUND AND OBJECTIVE
Nasopharyngeal carcinoma(NPC) is a common malignant epithelial tumor in China, with significant features of distinct endemic distribution, marked tendency to metastasis and close relationship with the EB virus. Radiotherapy is the mainstay treatment for this tumor. But due to its serious complications and sequela, treatment results are unsatisfactory and 5-year overall survival rate has remained at about 50%. Therefore, exploring the molecular mechanism of pathogenesis of NPC and searching for new therapeutic targets to enhance the efficacy of existing strageties is the urgent task of contemporary oncology investigation.
COX-2 is one of the two cyclooxygenase isoforms, which is usually absent from most normal cells and tissues, but is induced by growth factors, tumor promoters and cytokines. Inhibiting the expression of COX-2 can enhance tumor response to radio-or chemotherapy.
ShRNAmir is a hairpin small interfering RNA, which is reconstructed on the base of the microRNA structure, representing the latest technology of artificial induction of RNA interference and silencing gene expression by utilizing the shRNAmir vector in order to transcribe into shRNAmir which is similar to miRNA,more safely and effectively than the traditional one.
In our previous study, we have silenced the COX-2 expression in NPC cells with the application of RNA interference technology, causing the cell cycle arrested in G0/G1 phase and proliferation decreased. With the application of RNA interference technology based on the structure of miR-155, we successfully constructed Anti-COX-2 shRNAmir lentiviral vector. After 48-72h infection of lentivirus to NPC cells, the positive cells emit green fluorescence and the infection efficiency can up to 90.5%.Then selected by flow cytometry, the proportion of fluorescent cells still over 99% after 3 months of serial subculture. COX-2 mRNA gene expression can nearly undetectable by RT-PCR and the NPC subline which has a stable inhibition of COX-2 expression is obtained. It proved that shRNAmir can produce an increased and more consistent knockdown compared with anti-COX-2 siRNA. Application of this technology to the study of the molecular mechanism of NPC is laying foundations for further molecular targeted therapy and gene-radiotherapy.
On the basis of the previous study, we aim to explore the possible role of COX-2 gene in the proliferation, metastasis and sensitivity to chemo- or radiotherapy for nasopharyngeal carcinoma and provide a theoretical foundation for comprehensive treatment of nasopharyngeal carcinoma patients.
Methods:
1. The influence of COX-2 gene silencing on the growth characteristics of nasopharyngeal carcinoma cells
Exponential phase cells (2×103cells/well) were planted in the 96-well plate with 100ul cell suspension per well. At 1,2,3,4 and 5d after seeding, cells were treated with 10ul CCK-8 solution for 1h at 37℃. Spectrometric absorbance at wavelength of 450nm was measured on a microplate reader.
2. Wound migration assay
Equal numbers of cells (2×105) were seeded into 6-well culture plate, each group has three duplicate wells. The injury line was made with a sterile plastic pipette tip on cells cultured in plates at 90% confluency. After being rinsed with phosphate-buffered saline (PBS), cells were cultured in complete medium and migration of cells into the wound was observed at different time points, and then photographs were taken under a 40×objective.
3. Sensitivity detection of COX-2 silencing NPC cells to cisplatin
CCK-8 assay was performed to assess the sensitivity of cell to cisplatin. Cell were plated in 96-well culture plates at a density of 5×103 cells/well, with 100ul cell suspension per well.24h after seeding, the medium was replaced with fresh complete medium containing various concentrations of cisplatin. After 24h culture in the presence of cisplatin, lOul of CCK-8 was added to each well for 1h incubation. The OD value was read with microplate reader at a wavelength of 450nm. Growth inhibition rates were calculated and IC50, which was the concentration inducing 50% inhibiton of cells, were analyzed with SPSS 13.0 sofeware.
4. Cell cycle assays
After 24h incubation in the presence of cisplatin at the concentration of 6ug/ml, or after 24h and 48h irradiation at the dose of 2Gy, cells were harvested, washed in PBS, and then fixed with ice-cold 70% ethanol at 4℃overnight. When ready to stain with, cells were rinsed twice in PBS. Then cells were stained with PI at room temperature for 30min. Cell cycle phase distribution was analyzed by flow cytometry.
5. Clonogenic survival assay
Predetermined number of cells were seeded in 6-well culture plates, and exposed to 0,1,2,4,6 and 8 Gy of irradiation. Then cells were incubated at 37℃、5%CO2 for growth. After two weeks of incubation, when colonies were visible, they were fixed with methanol and stained with 1% crystal violet. Colonies which contained more than 50 cells were manually counted. Then planting efficiency (PE) and surviving fraction(SF) were calculated as follows:PE=colony number/inoculating cell number×100%. SF=PE (tested group)/PE (OGy group)×100%. Then survival curves were plotted and radiobiological parameters were calculated.
6. In vivo studies
Female BABL/c nude mice, aged 4-6 weeks, weighting 18-22g were housed in a specific pathogen-free animal facility. Mice were injected subcutaneously in the right hind limb with 1×105 cells. During experiment, two orthogonal dimensional diameters of each tumor were measured using vernier caliper every other day. The tumor volume was calculated by the formula L×W2/2. When tumors reached an average volume of about 200mm3, the mice were divided into four groups:COX-2 (+) control group, COX-2(-) control group, each of these two groups received no treatment; COX-2(+) radiation group, COX-2(-) radiation group, each tumor of these two groups were exposed to lOGy X-ray alone. Two weeks later the tumor-bearing nude mice were killed. Tumor inhibition rate was calculated.
7. Statistical analysis
SPSS 13.0 software package was used to carry out statistical analysis. Results are presented as mean±SD. Comparison of mean was analyzed by One-Way ANOVA or Independent-Sample T Test. Factorial analysis was utilized for analyzing the results of CCK-8 and wound migration assay. Student-Neuman-Keuls method was used for multiple comparing. A P value less than 0.05 was considered statistically significant.
Results:
1. The influence of COX-2 gene silencing on the abilities of proliferation and migration for nasopharyngeal carcinoma cells
Anti-COX-2 C666-1 and Anti-GL-2 C666-1 cells were inoculated in 96-well plates with continuous observation for 5 days. Growth curves were plotted according to the OD value at 450nm. Factorial analysis showed that the ability of proliferation was significantly decreased in the COX-2 negative group(F=5.927, P=0.019).
In vitro wound migration assay was carried out to detect the ability of cell migration in the presence or absence of COX-2 gene expression. Factorial analysis showed that, width of injury line between these two groups was significantly different (F=157.01, P<0.001), indicating that knockdown of COX-2 expression decreased the ability of in vitro migration.
2. Sensitivity alteration of COX-2 silencing NPC cells to cisplatin
Factorial analysis showed that, after 24h treated of various concentrations of cisplatin, inhibition rate of cell proliferation between two groups were significantly different (F=22.219, P<0.001). IC50 calculated by Anti-COX-2 C666-1 cells and Anti-GL-2 C666-1 cells to cisplatin has statistically differences (t=-3.489, P=0.025), the value were 3.682±0.980 and 8.819±2.354, respectively.
3. Alteration of cisplatin-induced cell cycle by silencing COX-2 gene expression
Flow cytometry analysis showed that,before the treatment of cisplatin, no significant difference can be seen in the proportion of both G0/G1 and S phase cells between Anti-COX-2 C666-1 and Anti-GL-2 C666-1 cells. However, a significant difference can be seen at G2/M phase between these two groups. After 24h treatment of cisplatin, S and G2/M peak were increased in both of these two groups. No statistically difference can be seen in the S-phase change between these two groups, but G2/M arrest decreased dramatically in Anti-COX-2 C666-1 24h after treatment of cisplatin, indicating that silencing of COX-2 gene expression significantly decreased the cisplatin-induced G2/M phase arrest.
4. The influence of Sensitivity of COX-2 silencing NPC cells to radiation in vitro
The values of D0, Dq, SF2 andα/βwith C666-1 cells before and after silencing of COX-2 gene expression were all statistically significant. The ratio of enhanced radiotherapy sensitivity was 1.4014.
5. The influence of radiation-induced cell cycle by silencing COX-2 gene expression
Flow cytometry analysis showed that, G2/M peak were increased in both of these two groups 24 and 48h after radiaton, but G2/M arrest decreased dramatically in Anti-COX-2 C666-1 after radiation, indicating that silencing of COX-2 gene expression significantly decreased the radiation-induced G2/M phase arrest.
6. The influence of Sensitivity of COX-2 silencing NPC tumor-bearing nude mice to radiation
In the non-radiotherapy groups, the xenograft tumor growth curves of COX-2(+) and COX-2(-) groups were almost overlaped. After radiotherapy of lOGy alone to both of these two radiation groups, the inhibition rates were significantly different, the value were 32.72% and 51.76%,respectively.
Conclusion:
1.Silencing of COX-2 gene expression decreased the abilities of proliferation and in vitro migration in nasopharyngeal carcinoma cells.
2.Silencing of COX-2 gene expression enhanced the sensitivity of NPC cells to cisplatin and decreased the cisplatin-induced G2/M phase arrest.
3.Silencing of COX-2 gene expression enhanced the sensitivity of NPC cells to radiation both in vitro and in vivo, and decreased the radiation-induced G2/M phase arrest.
鼻咽癌(nasopharyngeal carcinoma, NPC)是我国常见的恶性肿瘤之一,其发病以明显的区域分布、强烈的转移倾向、与EB病毒的密切关系为显著特征。放射治疗是鼻咽癌目前公认和有效的治疗手段,但因其严重的并发症及后遗症,治疗效果并不稳定,5年总生存率始终徘徊在50%左右。因此,探究鼻咽癌发病的分子机制,寻找新的治疗靶点,提高现有治疗手段的疗效,是当代肿瘤学研究的一项迫切任务。
COX-2是环氧合酶两种同工酶的其中一种,在体内呈诱导性表达,在大多数正常组织中难以发现。当受到一些刺激因子如生长因子、细胞因子及促癌剂的刺激时,其表达量会成倍增加。抑制COX-2的表达可明显增加化疗及放疗对鼻咽癌细胞的杀伤能力。
shRNAmir(microRNA-adapted,shRNA)是在microRNA结构基础上改造出的发夹状小干扰RNA,利用shRNAmir表达载体,在细胞内转录出与miRNA相似的shRNAmir,比传统的shRNA更加安全有效,是人工诱导RNA干扰沉默基因表达的最新技术。
本课题前期已完成如下研究工作：应用RNA干扰技术沉默COX-2的表达,使得鼻咽癌细胞周期出现G0/G1期阻滞,且增殖能力明显下降；应用基于miR-155结构的RNA干扰技术,成功构建了Anti-COX-2 shRNAmir(?)慢病毒表达载体,经慢病毒感染48-72h后,阳性C666-1细胞发出绿色萤光,感染效率最高达90.5%,经流式细胞仪分选出带荧光的细胞经连续传代3个月后,带光率仍超99%,RT-PCR几乎检测不到COX-2基因mRNA的表达,获得了稳定抑制COX-2表达的鼻咽癌细胞亚系,证明shRNAmir慢病毒载体具备获得稳定持久的RNA干扰作用,在转录后水平沉默COX-2基因,达到了“基因敲除”的效果,远优于Anti-COX-2 siRNA的干扰效果,应用于鼻咽癌分子机制研究,为下一步的分子靶向治疗和基因-放射联合治疗奠定基础。
在前期研究工作的基础上,本研究进一步探讨COX-2基因沉默后对鼻咽癌细胞生长、转移及对放化疗敏感性的影响,为鼻咽癌患者的综合治疗提供理论依据。
方法
1.检测COX-2沉默后对鼻咽癌细胞生长特性的影响
2×103个指数生长期细胞接种于96孔板中,每孔细胞悬液量为100ul,置于培养箱中分别培养1、2、3、4、5天,向每孔加入10ul的CCK-8溶液,将培养板置于培养箱中孵育1h,用酶标仪测定在450nm处的OD值,以相对应OD值表示细胞增殖能力大小。每组设6个重复孔,取平均值,绘制体外生长曲线。
2.划痕实验
2×105个细胞接种于6孔板,每组细胞作3个复孔,待细胞长至约90%汇合时,吸干培养基,用200ul tip头垂直在培养孔中部轻轻划过,力度以刮落细胞而不在培养板上留痕为准。PBS洗2遍以冲去脱落细胞,加入培养基后继续培养。每个孔随机选取9个有划痕穿过的视野,分别观察0h,6h,12h,24h时细胞细胞划痕处细胞运动变化,并拍照取样,应用Image J软件测划痕相对宽度,绘制迁移力量值曲线,独立实验重复3次。
3.检测COX-2沉默后鼻咽癌细胞对顺铂的敏感性变化
两组细胞分别按5000个/孔加入96孔板中,每孔加入培养基100ul,培养24h后弃去培养基,加入含有不同浓度顺铂的培养基,使其终浓度分别为0.5、1、2、4、8、16、32ug/ml,每组3个复孔,并设不接种细胞的空白培养基调零组和加入等体积溶剂的对照组,作用24h后加入10ul CCK-8试剂,置于培养箱中孵育1h后,利用酶标仪测各孔450nm处的吸光度A450,计算细胞生长的抑制率,并应用SPSS13.0软件计算半数抑制浓度。
4.细胞周期实验
指数生长期细胞在含有6ug/ml的顺铂或不含顺铂的培养基中培养24h,或经2Gy照射或0Gy假照射24及48h后,以不含EDTA的胰酶消化后收集细胞,加入70%冰乙醇中4℃过夜,PBS洗涤2遍后,避光加入含有50ug/ml的RNA酶和50ul/ml PI的染色液中,室温染色30min后,流式细胞仪检测细胞周期各时相所占百分比。
5.克隆形成实验
两组细胞按预定数量接种于6孔板中,每组均设0、1、2、4、6、8Gy六个剂量组,每一剂量组3个复孔。照射后的细胞置于37℃、5%CO2培养箱内培养。当出现肉眼可见克隆时,终止培养,甲醇固定,1%结晶紫乙醇溶液染色。显微镜下计数含50个细胞以上的克隆数,计算克隆形成率和存活分数。以3次照射的存活分数均值进行分析,运用GraphPad Prism 5.0软件进行L-Q模型和多靶单击模型曲线拟合,绘制剂量存活曲线,并计算L-Q模型参数α、β、α/β、SF2和多靶单击模型参数D0、Dq、和N,其中logN=Dq/D0,计算放射增敏比。
6.体内放疗敏感性实验
雌性BALB/c裸鼠,4-6周龄,18～22g, SPF条件下饲养。将两组细胞分别制成单细胞悬液,用无血清1640培养基调节细胞浓度至1×106/ml。在裸鼠右后肢皮下接种细胞悬液0.1ml,实验中每2天用游标卡尺测量肿瘤的最大径和与之垂直的横径,肿瘤体积(V)=(长径×短径2)/2。10-14d后,当肿瘤体积达到约200mm3时,将荷瘤裸鼠分为4组：①COX-2(+)对照组,无任何处理；②COX-2(-)对照组,无任何处理；③COX-2(+)实验组,给予10GyX线照射；④COX-2(-)实验组,给予10Gy X线照射。每组6只,2周后断颈处死裸鼠,计算抑瘤率。
7.统计学分析
采用SPSS13.0软件进行统计分析,实验数据以均数±标准差表示,两样本均数的比较才采用完全随机设计资料的方差分析(One-Way ANOVA)或两独立样本的t检验(Independent-Sample T Test)。增殖实验和划痕实验采用析因设计的方差分析,多重比较采用SNK法(Student-Neuman-Keuls), P<0.05表示差异有统计学意义。
结果：
1.COX-2基因沉默后对鼻咽癌细胞增殖和体外迁移能力的影响
Anti-COX-2 C666-1和Anti-GL-2 C666-1细胞,接种96孔板连续观察5天,依据450nm处OD值绘制生长曲线,析因分析结果表明,两种细胞增殖差异有统计学意义(F=5.927,P=0.019),但细胞组别与天数之间无交互效应(F=1.264,P=0.296),说明COX-2基因沉默后细胞增殖能力有所下降,但两组细胞增殖速度随时间变化趋势相同。
细胞划痕实验检测COX-2稳定沉默后对鼻咽癌细胞C666-1体外迁移能力的影响,析因设计的方差分析结果显示,随着培养时间的延长,两组间划痕宽度差异有统计学意义(F=157.01,P<0.001),细胞组别与时间之间有交互效应(F=15.73,P<0.001),说明随着时间的变化,两组细胞体外迁移距离的变化趋势不同,Anti-COX-2 C666-1细胞迁移能力较Anti-GL-2 C666-1有所减弱。
2.COX-2沉默后鼻咽癌C666-1细胞对顺铂敏感性的变化
析因分析结果显示,不同浓度顺铂作用24h后,两组细胞增殖抑制率差异有统计学意义(F=22.219,P<0.001),经计算得出Anti-COX-2 C666-1和Anti-GL-2C666-1细胞对顺铂的IC50分别为3.682±0.980和8.819±2.354,两组间IC50差异有统计学意义(t=-3.489,P=0.025)。
3.COX-2沉默后对顺铂引起细胞周期变化的影响
流式细胞仪分析结果表明,加顺铂前Anti-COX-2 C666-1和Anti-GL-2C666-1细胞周期中,G0/G1期细胞比例分别为70.260%和60.353%(t=-2.726,P=0.053),S期细胞比例分别为24.270%和32.180%(t=-2.033,P=0.112),差异均无统计学意义,G2/M期细胞比例差别有统计学意义,分别为5.470%和7.500%(t=-3.954, P=0.017)。6ug/ml的顺铂作用24h后,两组细胞S期与G2/M期比例均升高,两组均出现S期和G2/M期阻滞,S期比例升高幅度分别为24.967%和32.200%,两组间差异无统计学意义(t=--1.636,P=0.177),G2/M期比例升高幅度分别为0.500%和7.633%,两组间差异有统计学意义(t=-7.501,P=0.002)。COX-2沉默后G2/M期升高比例减少。但顺铂作用前后均未出现明显的凋亡峰,说明COX-2沉默后降低了顺铂引起的细胞G2/M期阻滞,但对细胞凋亡无明显影响。
4.COX-2沉默后对鼻咽癌C666-1细胞体外放疗敏感性的影响
COX-2沉默前后Do值分别为2.0157±0.0515、1.6333±0.0751,Dq值分别为0.8037±0.0487、0.5643±0.0399,SF2分别为0.5551±0.0375、0.3961±0.0160,α/β分别为6.5356±0.8461、9.4231±0.9599,两组间D0、Dq、F2、α/β值差异均有统计学意义,放射增敏比SER=1.4014。
5.COX-2沉默后对辐射引起细胞周期的影响
流式细胞仪分析结果表明,照射前Anti-GL-2 C666-1和Anti-COX-2 C666-1细胞周期中,G0/G1以及S期细胞比例差异均无统计学意义,但G2/M期细胞比例差别有统计学意义,分别为7.500%和5.470%(t=-3.952,P=0.017)。照射2Gy后24h,G2期细胞比例均增高,分别为45.833%和23.900%(t=11.999,P<0.001),两组细胞均出现G2/M期阻滞,但Anti-GL-2 C666-1细胞G2/M期升高比例更大,为38.333%,而Anti-COX-2 C666-2细胞G2/M期比例升高18.430%(t=9.184,P=0.001)。照射2Gy后48h,两组细胞G2/M期的比例分别为24.397%和12.393%(t=4.646,P=0.01),较照射前分别增加了17.357%和6.923%(t=3.320,P=0.029)。但照射前后均未出现反映细胞凋亡的亚G1期(sub-G1 phase),说明COX-2沉默后显著降低了辐射引起的细胞G2/M期阻滞,但对细胞凋亡无明显影响。
6.COX-2沉默后对体内荷瘤裸鼠辐射敏感性的影响
未放疗组,Anti-GL-2和Anti-COX-2两组裸鼠移植瘤生长曲线几乎重合,COX-2沉默前后对皮下移植瘤的生长情况差异无统计学意义(F=3.061,P=0.084),加入10Gy单纯放疗后,Anti-GL-2和Anti-COX-2两组的抑瘤率分别为32.72%和51.76%,两组间皮下移植瘤的生长情况差异有统计学意义(F=63.468,P<0.001)。
结论：
1.COX-2沉默后降低了鼻咽癌细胞的增殖及体外迁移能力；
2.COX-2沉默后增强顺铂对鼻咽癌C666-1细胞的敏感性,降低了顺铂引起的细胞G2/M期阻滞；
3.COX-2沉默后增强鼻咽癌体内外放疗的敏感性,降低了辐射引起的细胞G2/M期阻滞。
BACKGROUND AND OBJECTIVE
Nasopharyngeal carcinoma(NPC) is a common malignant epithelial tumor in China, with significant features of distinct endemic distribution, marked tendency to metastasis and close relationship with the EB virus. Radiotherapy is the mainstay treatment for this tumor. But due to its serious complications and sequela, treatment results are unsatisfactory and 5-year overall survival rate has remained at about 50%. Therefore, exploring the molecular mechanism of pathogenesis of NPC and searching for new therapeutic targets to enhance the efficacy of existing strageties is the urgent task of contemporary oncology investigation.
COX-2 is one of the two cyclooxygenase isoforms, which is usually absent from most normal cells and tissues, but is induced by growth factors, tumor promoters and cytokines. Inhibiting the expression of COX-2 can enhance tumor response to radio-or chemotherapy.
ShRNAmir is a hairpin small interfering RNA, which is reconstructed on the base of the microRNA structure, representing the latest technology of artificial induction of RNA interference and silencing gene expression by utilizing the shRNAmir vector in order to transcribe into shRNAmir which is similar to miRNA,more safely and effectively than the traditional one.
In our previous study, we have silenced the COX-2 expression in NPC cells with the application of RNA interference technology, causing the cell cycle arrested in G0/G1 phase and proliferation decreased. With the application of RNA interference technology based on the structure of miR-155, we successfully constructed Anti-COX-2 shRNAmir lentiviral vector. After 48-72h infection of lentivirus to NPC cells, the positive cells emit green fluorescence and the infection efficiency can up to 90.5%.Then selected by flow cytometry, the proportion of fluorescent cells still over 99% after 3 months of serial subculture. COX-2 mRNA gene expression can nearly undetectable by RT-PCR and the NPC subline which has a stable inhibition of COX-2 expression is obtained. It proved that shRNAmir can produce an increased and more consistent knockdown compared with anti-COX-2 siRNA. Application of this technology to the study of the molecular mechanism of NPC is laying foundations for further molecular targeted therapy and gene-radiotherapy.
On the basis of the previous study, we aim to explore the possible role of COX-2 gene in the proliferation, metastasis and sensitivity to chemo- or radiotherapy for nasopharyngeal carcinoma and provide a theoretical foundation for comprehensive treatment of nasopharyngeal carcinoma patients.
Methods:
1. The influence of COX-2 gene silencing on the growth characteristics of nasopharyngeal carcinoma cells
Exponential phase cells (2×103cells/well) were planted in the 96-well plate with 100ul cell suspension per well. At 1,2,3,4 and 5d after seeding, cells were treated with 10ul CCK-8 solution for 1h at 37℃. Spectrometric absorbance at wavelength of 450nm was measured on a microplate reader.
2. Wound migration assay
Equal numbers of cells (2×105) were seeded into 6-well culture plate, each group has three duplicate wells. The injury line was made with a sterile plastic pipette tip on cells cultured in plates at 90% confluency. After being rinsed with phosphate-buffered saline (PBS), cells were cultured in complete medium and migration of cells into the wound was observed at different time points, and then photographs were taken under a 40×objective.
3. Sensitivity detection of COX-2 silencing NPC cells to cisplatin
CCK-8 assay was performed to assess the sensitivity of cell to cisplatin. Cell were plated in 96-well culture plates at a density of 5×103 cells/well, with 100ul cell suspension per well.24h after seeding, the medium was replaced with fresh complete medium containing various concentrations of cisplatin. After 24h culture in the presence of cisplatin, lOul of CCK-8 was added to each well for 1h incubation. The OD value was read with microplate reader at a wavelength of 450nm. Growth inhibition rates were calculated and IC50, which was the concentration inducing 50% inhibiton of cells, were analyzed with SPSS 13.0 sofeware.
4. Cell cycle assays
After 24h incubation in the presence of cisplatin at the concentration of 6ug/ml, or after 24h and 48h irradiation at the dose of 2Gy, cells were harvested, washed in PBS, and then fixed with ice-cold 70% ethanol at 4℃overnight. When ready to stain with, cells were rinsed twice in PBS. Then cells were stained with PI at room temperature for 30min. Cell cycle phase distribution was analyzed by flow cytometry.
5. Clonogenic survival assay
Predetermined number of cells were seeded in 6-well culture plates, and exposed to 0,1,2,4,6 and 8 Gy of irradiation. Then cells were incubated at 37℃、5%CO2 for growth. After two weeks of incubation, when colonies were visible, they were fixed with methanol and stained with 1% crystal violet. Colonies which contained more than 50 cells were manually counted. Then planting efficiency (PE) and surviving fraction(SF) were calculated as follows:PE=colony number/inoculating cell number×100%. SF=PE (tested group)/PE (OGy group)×100%. Then survival curves were plotted and radiobiological parameters were calculated.
6. In vivo studies
Female BABL/c nude mice, aged 4-6 weeks, weighting 18-22g were housed in a specific pathogen-free animal facility. Mice were injected subcutaneously in the right hind limb with 1×105 cells. During experiment, two orthogonal dimensional diameters of each tumor were measured using vernier caliper every other day. The tumor volume was calculated by the formula L×W2/2. When tumors reached an average volume of about 200mm3, the mice were divided into four groups:COX-2 (+) control group, COX-2(-) control group, each of these two groups received no treatment; COX-2(+) radiation group, COX-2(-) radiation group, each tumor of these two groups were exposed to lOGy X-ray alone. Two weeks later the tumor-bearing nude mice were killed. Tumor inhibition rate was calculated.
7. Statistical analysis
SPSS 13.0 software package was used to carry out statistical analysis. Results are presented as mean±SD. Comparison of mean was analyzed by One-Way ANOVA or Independent-Sample T Test. Factorial analysis was utilized for analyzing the results of CCK-8 and wound migration assay. Student-Neuman-Keuls method was used for multiple comparing. A P value less than 0.05 was considered statistically significant.
Results:
1. The influence of COX-2 gene silencing on the abilities of proliferation and migration for nasopharyngeal carcinoma cells
Anti-COX-2 C666-1 and Anti-GL-2 C666-1 cells were inoculated in 96-well plates with continuous observation for 5 days. Growth curves were plotted according to the OD value at 450nm. Factorial analysis showed that the ability of proliferation was significantly decreased in the COX-2 negative group(F=5.927, P=0.019).
In vitro wound migration assay was carried out to detect the ability of cell migration in the presence or absence of COX-2 gene expression. Factorial analysis showed that, width of injury line between these two groups was significantly different (F=157.01, P<0.001), indicating that knockdown of COX-2 expression decreased the ability of in vitro migration.
2. Sensitivity alteration of COX-2 silencing NPC cells to cisplatin
Factorial analysis showed that, after 24h treated of various concentrations of cisplatin, inhibition rate of cell proliferation between two groups were significantly different (F=22.219, P<0.001). IC50 calculated by Anti-COX-2 C666-1 cells and Anti-GL-2 C666-1 cells to cisplatin has statistically differences (t=-3.489, P=0.025), the value were 3.682±0.980 and 8.819±2.354, respectively.
3. Alteration of cisplatin-induced cell cycle by silencing COX-2 gene expression
Flow cytometry analysis showed that,before the treatment of cisplatin, no significant difference can be seen in the proportion of both G0/G1 and S phase cells between Anti-COX-2 C666-1 and Anti-GL-2 C666-1 cells. However, a significant difference can be seen at G2/M phase between these two groups. After 24h treatment of cisplatin, S and G2/M peak were increased in both of these two groups. No statistically difference can be seen in the S-phase change between these two groups, but G2/M arrest decreased dramatically in Anti-COX-2 C666-1 24h after treatment of cisplatin, indicating that silencing of COX-2 gene expression significantly decreased the cisplatin-induced G2/M phase arrest.
4. The influence of Sensitivity of COX-2 silencing NPC cells to radiation in vitro
The values of D0, Dq, SF2 andα/βwith C666-1 cells before and after silencing of COX-2 gene expression were all statistically significant. The ratio of enhanced radiotherapy sensitivity was 1.4014.
5. The influence of radiation-induced cell cycle by silencing COX-2 gene expression
Flow cytometry analysis showed that, G2/M peak were increased in both of these two groups 24 and 48h after radiaton, but G2/M arrest decreased dramatically in Anti-COX-2 C666-1 after radiation, indicating that silencing of COX-2 gene expression significantly decreased the radiation-induced G2/M phase arrest.
6. The influence of Sensitivity of COX-2 silencing NPC tumor-bearing nude mice to radiation
In the non-radiotherapy groups, the xenograft tumor growth curves of COX-2(+) and COX-2(-) groups were almost overlaped. After radiotherapy of lOGy alone to both of these two radiation groups, the inhibition rates were significantly different, the value were 32.72% and 51.76%,respectively.
Conclusion:
1.Silencing of COX-2 gene expression decreased the abilities of proliferation and in vitro migration in nasopharyngeal carcinoma cells.
2.Silencing of COX-2 gene expression enhanced the sensitivity of NPC cells to cisplatin and decreased the cisplatin-induced G2/M phase arrest.
3.Silencing of COX-2 gene expression enhanced the sensitivity of NPC cells to radiation both in vitro and in vivo, and decreased the radiation-induced G2/M phase arrest.
引文
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[8]Soo R, Putti T, Tao Q, et al. Overexpression of cyclooxygenase-2 in nasopharyngeal carcinoma and association with epidermal growth factor receptor expression[J]. Arch Otolaryngol Head Neck Surg,2005,131(2):147-152.
[9]Harizi H, Juzan M, Pitard V, et al. Cyclooxygenase-2-issued prostaglandin e(2) enhances the production of endogenous IL-10, which down-regulates dendritic cell functions[J]. J Immunol,2002,168(5):2255-2263.
[10]Tan KB, Putti TC. Cyclooxygenase 2 expression in nasopharyngeal carcinoma: immunohistochemical findings and potential implications[J]. J Clin Pathol,2005, 58(5):535-538.
[11]Watson AJ. Chemopreventive effects of NSAIDs against colorectal cancer:regulation of apoptosis and mitosis by COX-1 and COX-2[J]. Histol Histopathol,1998,13(2):591-597.
[12]Petersen C, Petersen S, Milas L, et al. Enhancement of intrinsic tumor cell radiosensitivity induced by a selective cyclooxygenase-2 inhibitor[J]. Clin Cancer Res,2000,6(6):2513-2520.
[13]Raju U, Nakata E, Yang P, et al. In vitro enhancement of tumor cell radiosensitivity by a selective inhibitor of cyclooxygenase-2 enzyme:mechanistic considerations[J]. Int J Radiat Oncol Biol Phys,2002,54(3):886-894.
[14]Mirzaie-Joniani H, Eriksson D, Sheikholvaezin A, et al. Apoptosis induced by low-dose and low-dose-rate radiation[J]. Cancer,2002,94(4 Suppl):1210-1214.
[15]Nix P, Lind M, Greenman J, et al. Expression of Cox-2 protein in radioresistant laryngeal cancer[J]. Ann Oncol,2004,15(5):797-801.
[16]Farooq M, Haq I, Qureshi AS. Cardiovascular risks of COX inhibition:current perspectives[J]. Expert Opin Pharmacother,2008,9(8):1311-1319.
[17]Li XP, Li G, Peng Y, et al. Suppression of Epstein-Barr virus-encoded latent membrane protein-1 by RNA interference inhibits the metastatic potential of nasopharyngeal carcinoma cells[J]. Biochem Biophys Res Commun,2004,315(1):212-218.
[18]Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21-and 22-nucleotide RNAs[J]. Genes Dev,2001,15(2):188-200.
[19]Hutvagner G, Zamore PD. RNAi:nature abhors a double-strand[J]. Curr Opin Genet Dev,2002,12(2):225-232.
[20]Sijen T, Fleenor J, Simmer F, et al. On the role of RNA amplification in dsRNA-triggered gene silencing[J]. Cell,2001,107(4):465-476.
[21]李刚,李湘平,陈顺金,等.RNA干扰沉默COX-2对鼻咽癌细胞增殖、凋亡及细胞周期的影响[J].广东医学,2009(01).
[22]Grimm D, Streetz KL, Jopling CL, et al. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways[J]. Nature,2006,441 (7092):537-541.
[23]Ambros V. The functions of animal microRNAs[J]. Nature,2004,431(7006):350-355.
[24]Fewell GD, Schmitt K. Vector-based RNAi approaches for stable, inducible and genome-wide screens[J]. Drug Discov Today,2006,11(21-22):975-982.
[25]李刚,李湘平,姜立,等.shRNAmir慢病毒载体沉默鼻咽癌细胞中环氧合酶-2的表达[J].南方医科大学学报,2009,29(06)：1111-4.
[1]李刚,李湘平,姜立,等shRNAmir慢病毒载体沉默鼻咽癌细胞中环氧合酶-2的表达[J].南方医科大学学报,2009,29(06)：1111-4.
[2]Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature,1998,391(6669):806-811.
[3]Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs[J]. Genes Dev,2001,15(2):188-200.
[4]Hutvagner G, Zamore PD. RNAi:nature abhors a double-strand[J]. Curr Opin Genet Dev,2002,12(2):225-232.
[5]Sijen T, Fleenor J, Simmer F, et al. On the role of RNA amplification in dsRNA-triggered gene silencing[J]. Cell,2001,107(4):465-476.
[6]Ambros V. The functions of animal microRNAs[J]. Nature,2004,431(7006):350-355.
[7]Fewell GD, Schmitt K. Vector-based RNAi approaches for stable, inducible and genome-wide screens[J]. Drug Discov Today,2006,11(21-22):975-982.
[8]Herschman HR. Primary response genes induced by growth factors and tumor promoters[J]. Annu Rev Biochem,1991,60:281-319.
[9]Williams CS, Mann M, Dubois RN. The role of cyclooxygenases in inflammation, cancer, and development[J]. Oncogene,1999,18(55):7908-7916.
[10]Turini ME, Dubois RN. Cyclooxygenase-2:a therapeutic target[J]. Annu Rev Med,2002,53:35-57.
[11]Abrahao AC, Castilho RM, Squarize CH, et al. A role for COX2-derived PGE2 and PGE2-receptor subtypes in head and neck squamous carcinoma cell proliferation[J]. Oral Oncol,2010,46(12):880-887.
[12]Chan MW, Wong CY, Cheng AS, et al. Targeted inhibition of COX-2 expression by RNA interference suppresses tumor growth and potentiates chemosensitivity to cisplatin in human gastric cancer cells[J]. Oncol Rep,2007,18(6):1557-1562.
[13]Williams CS, Tsujii M, Reese J, et al. Host cyclooxygenase-2 modulates carcinoma growth[J]. J Clin Invest,2000,105(11):1589-1594.
[14]Uefuji K, Ichikura T, Mochizuki H. Cyclooxygenase-2 expression is related to prostaglandin biosynthesis and angiogenesis in human gastric cancer[J]. Clin Cancer Res,2000,6(1):135-138.
[15]Muraki C, Ohga N, Hida Y, et al. Cyclooxygenase-2 inhibition causes antiangiogenic effects on tumor endothelial and vascular progenitor cells[J]. Int J Cancer,2011.
[16]Tan KB, Putti TC. Cyclooxygenase 2 expression in nasopharyngeal carcinoma: immunohistochemical findings and potential implications[J]. J Clin Pathol,2005,58(5):535-538.
[1]李刚,李湘平,姜立,等shRNAmir慢病毒载体沉默鼻咽癌细胞中环氧合酶-2的表达[J].南方医科大学学报,2009,29(06)：1111-4.
[2]Seve P, Dumontet C. Chemoresistance in non-small cell lung cancer[J]. Curr Med Chem Anticancer Agents,2005,5(1):73-88.
[3]Elder DJ, Halton DE, Playle LC, et al. The MEK/ERK pathway mediates COX-2-selective NSAID-induced apoptosis and induced COX-2 protein expression in colorectal carcinoma cells[J]. Int J Cancer,2002,99(3):323-327.
[4]Watson A J. Chemopreventive effects of NSAIDs against colorectal cancer:regulation of apoptosis and mitosis by COX-1 and COX-2[J]. Histol Histopathol,1998,13(2):591-597.
[5]Petersen C, Petersen S, Milas L, et al. Enhancement of intrinsic tumor cell radiosensitivity induced by a selective cyclooxygenase-2 inhibitor[J]. Clin Cancer Res,2000,6(6):2513-2520.
[6]Raju U, Nakata E, Yang P, et al. In vitro enhancement of tumor cell radiosensitivity by a selective inhibitor of cyclooxygenase-2 enzyme:mechanistic considerations[J]. Int J Radiat Oncol Biol Phys,2002,54(3):886-894.
[7]Amir M, Agarwal HK. Role of COX-2 selective inhibitors for prevention and treatment of cancer[J].Pharmazie,2005,60(8):563-570.
[1]李刚,李湘平,姜立,等shRNAmir漫病毒载体沉默鼻咽癌细胞中环氧合酶-2的表达[J].南方医科大学学报,2009(06).
[2]Herschman H R. Primary response genes induced by growth factors and tumor promoters[J]. Annu Rev Biochem,1991,60:281-319.
[3]Williams C S, Mann M, Dubois R N. The role of cyclooxygenases in inflammation, cancer, and development[J]. Oncogene,1999,18(55):7908-7916.
[4]Turini M E, Dubois R N. Cyclooxygenase-2:a therapeutic target[J]. Annu Rev Med,2002,53:35-57.
[5]Ohno S, Ohno Y, Suzuki N, et al. Multiple roles of cyclooxygenase-2 in endometrial cancer[J]. Anticancer Res,2005,25(6A):3679-3687.
[6]Chen W C, Mcbride W H, Chen S M, et al. Prediction of poor survival by cyclooxygenase-2 in patients with T4 nasopharyngeal cancer treated by radiation therapy:clinical and in vitro studies[J]. Head Neck,2005,27(6):503-512.
[7]Fewell G D, Schmitt K. Vector-based RNAi approaches for stable, inducible and genome-wide screens[J]. Drug Discov Today,2006,11(21-22):975-982.
[8]Tan K B, Putti T C. Cyclooxygenase 2 expression in nasopharyngeal carcinoma: immunohistochemical findings and potential implications[J]. J Clin Pathol,2005,58(5):535-538.
[9]Petersen C, Petersen S, Milas L, et al. Enhancement of intrinsic tumor cell radiosensitivity induced by a selective cyclooxygenase-2 inhibitor[J]. Clin Cancer Res,2000,6(6):2513-2520.
[10]Raju U, Nakata E, Yang P, et al. In vitro enhancement of tumor cell radiosensitivity by a selective inhibitor of cyclooxygenase-2 enzyme:mechanistic considerations[J]. Int J Radiat Oncol Biol Phys,2002,54(3):886-894.
[11]Amir M, Agarwal H K. Role of COX-2 selective inhibitors for prevention and treatment of cancer[J]. Pharmazie,2005,60(8):563-570.
[12]Nix P, Lind M, Greenman J, et al. Expression of Cox-2 protein in radioresistant laryngeal cancer[J]. Ann Oncol,2004,15(5):797-801.
[13]沈瑜糜福顺.肿瘤放射生物学[M][Z].北京：中国医药科技出版社,200164-68.
[14]Hartwell L H, Kastan M B. Cell cycle control and cancer[J]. Science,1994,266(5192):1821-1828.
[15]Shin Y K, Park J S, Kim H S, et al. Radiosensitivity enhancement by celecoxib, a cyclooxygenase (COX)-2 selective inhibitor, via COX-2-dependent cell cycle regulation on human cancer cells expressing differential COX-2 levels[J]. Cancer Res,2005,65(20):9501-9509.
[2]Chua DT, Ma J, Sham JS, et al. Improvement of survival after addition of induction chemotherapy to radiotherapy in patients with early-stage nasopharyngeal carcinoma: Subgroup analysis of two Phase Ⅲ trials[J]. Int J Radiat Oncol Biol Phys,2006, 65(5):1300-1306.
[3]Cho WC. Nasopharyngeal carcinoma:molecular biomarker discovery and progress[J]. Mol Cancer,2007,6:1.
[4]Herschman HR. Primary response genes induced by growth factors and tumor promoters[J]. Annu Rev Biochem,1991,60:281-319.
[5]Williams CS, Mann M, Dubois RN. The role of cyclooxygenases in inflammation, cancer, and development[J]. Oncogene,1999,18(55):7908-7916.
[6]Turini ME, Dubois RN. Cyclooxygenase-2:a therapeutic target[J]. Annu Rev Med,2002, 53:35-57.
[7]Chen WC, Mcbride WH, Chen SM, et al. Prediction of poor survival by cyclooxygenase-2 in patients with T4 nasopharyngeal cancer treated by radiation therapy:clinical and in vitro studies[J]. Head Neck,2005,27(6):503-512.
[8]Soo R, Putti T, Tao Q, et al. Overexpression of cyclooxygenase-2 in nasopharyngeal carcinoma and association with epidermal growth factor receptor expression[J]. Arch Otolaryngol Head Neck Surg,2005,131(2):147-152.
[9]Harizi H, Juzan M, Pitard V, et al. Cyclooxygenase-2-issued prostaglandin e(2) enhances the production of endogenous IL-10, which down-regulates dendritic cell functions[J]. J Immunol,2002,168(5):2255-2263.
[10]Tan KB, Putti TC. Cyclooxygenase 2 expression in nasopharyngeal carcinoma: immunohistochemical findings and potential implications[J]. J Clin Pathol,2005, 58(5):535-538.
[11]Watson AJ. Chemopreventive effects of NSAIDs against colorectal cancer:regulation of apoptosis and mitosis by COX-1 and COX-2[J]. Histol Histopathol,1998,13(2):591-597.
[12]Petersen C, Petersen S, Milas L, et al. Enhancement of intrinsic tumor cell radiosensitivity induced by a selective cyclooxygenase-2 inhibitor[J]. Clin Cancer Res,2000,6(6):2513-2520.
[13]Raju U, Nakata E, Yang P, et al. In vitro enhancement of tumor cell radiosensitivity by a selective inhibitor of cyclooxygenase-2 enzyme:mechanistic considerations[J]. Int J Radiat Oncol Biol Phys,2002,54(3):886-894.
[14]Mirzaie-Joniani H, Eriksson D, Sheikholvaezin A, et al. Apoptosis induced by low-dose and low-dose-rate radiation[J]. Cancer,2002,94(4 Suppl):1210-1214.
[15]Nix P, Lind M, Greenman J, et al. Expression of Cox-2 protein in radioresistant laryngeal cancer[J]. Ann Oncol,2004,15(5):797-801.
[16]Farooq M, Haq I, Qureshi AS. Cardiovascular risks of COX inhibition:current perspectives[J]. Expert Opin Pharmacother,2008,9(8):1311-1319.
[17]Li XP, Li G, Peng Y, et al. Suppression of Epstein-Barr virus-encoded latent membrane protein-1 by RNA interference inhibits the metastatic potential of nasopharyngeal carcinoma cells[J]. Biochem Biophys Res Commun,2004,315(1):212-218.
[18]Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21-and 22-nucleotide RNAs[J]. Genes Dev,2001,15(2):188-200.
[19]Hutvagner G, Zamore PD. RNAi:nature abhors a double-strand[J]. Curr Opin Genet Dev,2002,12(2):225-232.
[20]Sijen T, Fleenor J, Simmer F, et al. On the role of RNA amplification in dsRNA-triggered gene silencing[J]. Cell,2001,107(4):465-476.
[21]李刚,李湘平,陈顺金,等.RNA干扰沉默COX-2对鼻咽癌细胞增殖、凋亡及细胞周期的影响[J].广东医学,2009(01).
[22]Grimm D, Streetz KL, Jopling CL, et al. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways[J]. Nature,2006,441 (7092):537-541.
[23]Ambros V. The functions of animal microRNAs[J]. Nature,2004,431(7006):350-355.
[24]Fewell GD, Schmitt K. Vector-based RNAi approaches for stable, inducible and genome-wide screens[J]. Drug Discov Today,2006,11(21-22):975-982.
[25]李刚,李湘平,姜立,等.shRNAmir慢病毒载体沉默鼻咽癌细胞中环氧合酶-2的表达[J].南方医科大学学报,2009,29(06)：1111-4.
[1]李刚,李湘平,姜立,等shRNAmir慢病毒载体沉默鼻咽癌细胞中环氧合酶-2的表达[J].南方医科大学学报,2009,29(06)：1111-4.
[2]Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature,1998,391(6669):806-811.
[3]Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs[J]. Genes Dev,2001,15(2):188-200.
[4]Hutvagner G, Zamore PD. RNAi:nature abhors a double-strand[J]. Curr Opin Genet Dev,2002,12(2):225-232.
[5]Sijen T, Fleenor J, Simmer F, et al. On the role of RNA amplification in dsRNA-triggered gene silencing[J]. Cell,2001,107(4):465-476.
[6]Ambros V. The functions of animal microRNAs[J]. Nature,2004,431(7006):350-355.
[7]Fewell GD, Schmitt K. Vector-based RNAi approaches for stable, inducible and genome-wide screens[J]. Drug Discov Today,2006,11(21-22):975-982.
[8]Herschman HR. Primary response genes induced by growth factors and tumor promoters[J]. Annu Rev Biochem,1991,60:281-319.
[9]Williams CS, Mann M, Dubois RN. The role of cyclooxygenases in inflammation, cancer, and development[J]. Oncogene,1999,18(55):7908-7916.
[10]Turini ME, Dubois RN. Cyclooxygenase-2:a therapeutic target[J]. Annu Rev Med,2002,53:35-57.
[11]Abrahao AC, Castilho RM, Squarize CH, et al. A role for COX2-derived PGE2 and PGE2-receptor subtypes in head and neck squamous carcinoma cell proliferation[J]. Oral Oncol,2010,46(12):880-887.
[12]Chan MW, Wong CY, Cheng AS, et al. Targeted inhibition of COX-2 expression by RNA interference suppresses tumor growth and potentiates chemosensitivity to cisplatin in human gastric cancer cells[J]. Oncol Rep,2007,18(6):1557-1562.
[13]Williams CS, Tsujii M, Reese J, et al. Host cyclooxygenase-2 modulates carcinoma growth[J]. J Clin Invest,2000,105(11):1589-1594.
[14]Uefuji K, Ichikura T, Mochizuki H. Cyclooxygenase-2 expression is related to prostaglandin biosynthesis and angiogenesis in human gastric cancer[J]. Clin Cancer Res,2000,6(1):135-138.
[15]Muraki C, Ohga N, Hida Y, et al. Cyclooxygenase-2 inhibition causes antiangiogenic effects on tumor endothelial and vascular progenitor cells[J]. Int J Cancer,2011.
[16]Tan KB, Putti TC. Cyclooxygenase 2 expression in nasopharyngeal carcinoma: immunohistochemical findings and potential implications[J]. J Clin Pathol,2005,58(5):535-538.
[1]李刚,李湘平,姜立,等shRNAmir慢病毒载体沉默鼻咽癌细胞中环氧合酶-2的表达[J].南方医科大学学报,2009,29(06)：1111-4.
[2]Seve P, Dumontet C. Chemoresistance in non-small cell lung cancer[J]. Curr Med Chem Anticancer Agents,2005,5(1):73-88.
[3]Elder DJ, Halton DE, Playle LC, et al. The MEK/ERK pathway mediates COX-2-selective NSAID-induced apoptosis and induced COX-2 protein expression in colorectal carcinoma cells[J]. Int J Cancer,2002,99(3):323-327.
[4]Watson A J. Chemopreventive effects of NSAIDs against colorectal cancer:regulation of apoptosis and mitosis by COX-1 and COX-2[J]. Histol Histopathol,1998,13(2):591-597.
[5]Petersen C, Petersen S, Milas L, et al. Enhancement of intrinsic tumor cell radiosensitivity induced by a selective cyclooxygenase-2 inhibitor[J]. Clin Cancer Res,2000,6(6):2513-2520.
[6]Raju U, Nakata E, Yang P, et al. In vitro enhancement of tumor cell radiosensitivity by a selective inhibitor of cyclooxygenase-2 enzyme:mechanistic considerations[J]. Int J Radiat Oncol Biol Phys,2002,54(3):886-894.
[7]Amir M, Agarwal HK. Role of COX-2 selective inhibitors for prevention and treatment of cancer[J].Pharmazie,2005,60(8):563-570.
[1]李刚,李湘平,姜立,等shRNAmir漫病毒载体沉默鼻咽癌细胞中环氧合酶-2的表达[J].南方医科大学学报,2009(06).
[2]Herschman H R. Primary response genes induced by growth factors and tumor promoters[J]. Annu Rev Biochem,1991,60:281-319.
[3]Williams C S, Mann M, Dubois R N. The role of cyclooxygenases in inflammation, cancer, and development[J]. Oncogene,1999,18(55):7908-7916.
[4]Turini M E, Dubois R N. Cyclooxygenase-2:a therapeutic target[J]. Annu Rev Med,2002,53:35-57.
[5]Ohno S, Ohno Y, Suzuki N, et al. Multiple roles of cyclooxygenase-2 in endometrial cancer[J]. Anticancer Res,2005,25(6A):3679-3687.
[6]Chen W C, Mcbride W H, Chen S M, et al. Prediction of poor survival by cyclooxygenase-2 in patients with T4 nasopharyngeal cancer treated by radiation therapy:clinical and in vitro studies[J]. Head Neck,2005,27(6):503-512.
[7]Fewell G D, Schmitt K. Vector-based RNAi approaches for stable, inducible and genome-wide screens[J]. Drug Discov Today,2006,11(21-22):975-982.
[8]Tan K B, Putti T C. Cyclooxygenase 2 expression in nasopharyngeal carcinoma: immunohistochemical findings and potential implications[J]. J Clin Pathol,2005,58(5):535-538.
[9]Petersen C, Petersen S, Milas L, et al. Enhancement of intrinsic tumor cell radiosensitivity induced by a selective cyclooxygenase-2 inhibitor[J]. Clin Cancer Res,2000,6(6):2513-2520.
[10]Raju U, Nakata E, Yang P, et al. In vitro enhancement of tumor cell radiosensitivity by a selective inhibitor of cyclooxygenase-2 enzyme:mechanistic considerations[J]. Int J Radiat Oncol Biol Phys,2002,54(3):886-894.
[11]Amir M, Agarwal H K. Role of COX-2 selective inhibitors for prevention and treatment of cancer[J]. Pharmazie,2005,60(8):563-570.
[12]Nix P, Lind M, Greenman J, et al. Expression of Cox-2 protein in radioresistant laryngeal cancer[J]. Ann Oncol,2004,15(5):797-801.
[13]沈瑜糜福顺.肿瘤放射生物学[M][Z].北京：中国医药科技出版社,200164-68.
[14]Hartwell L H, Kastan M B. Cell cycle control and cancer[J]. Science,1994,266(5192):1821-1828.
[15]Shin Y K, Park J S, Kim H S, et al. Radiosensitivity enhancement by celecoxib, a cyclooxygenase (COX)-2 selective inhibitor, via COX-2-dependent cell cycle regulation on human cancer cells expressing differential COX-2 levels[J]. Cancer Res,2005,65(20):9501-9509.