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

发布时间：2018-05-14 23:19

本文选题：鼻咽肿瘤 + 环氧合酶-2　；参考：《南方医科大学》2011年硕士论文

【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), P0.05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,P0.001),细胞组别与时间之间有交互效应(F=15.73,P0.001),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,P0.001),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,P0.001),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,P0.001)。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。
[Abstract]:Background and purpose of the study :

Nasopharyngeal carcinoma ( NPC ) is one of the most common malignant tumors in China . Its pathogenesis is characterized by obvious regional distribution , strong metastasis tendency and close relationship with EB virus . Radiotherapy is the most commonly accepted and effective treatment method for nasopharyngeal carcinoma .

COX - 2 is one of the two isozymes of cyclooxygenase , which is inducible in vivo . It is difficult to find in most normal tissues . When stimulated by some stimulating factors such as growth factors , cytokines and cancer promoting agents , the expression of COX - 2 can be increased . The inhibition of COX - 2 expression can significantly increase the killing ability of chemotherapy and radiotherapy for nasopharyngeal carcinoma cells .

shRNAmir ( microRNA - adapted , shRNA ) is a hairpin - shaped small interfering RNA modified on the basis of microRNA structure , and shRNAmir similar to miRNA is transcribed in the cell by using the shRNAmir expression vector , and is more safe and effective than the conventional shRNA , and is an up - to - date technology for artificially inducing RNA interference silencing gene expression .

In the early stage of the project , we have completed the following research : silencing the expression of COX - 2 by using RNA interference technique , so that the cell cycle of nasopharyngeal carcinoma appears G0 / G1 phase arrest , and the proliferation ability is obviously decreased ;
The anti - COX - 2 shRNAmir ( ? ) lentivirus expression vector was successfully constructed by using RNA interference technique based on the structure of miR - 155 . After 48 to 72 hours of lentivirus infection , the positive C666 - 1 cells emitted green fluorescent light . After three months of continuous passage , the expression of COX - 2 mRNA was detected .

Based on previous studies , this study further explores the effects of COX - 2 gene silencing on the growth , metastasis and sensitivity of nasopharyngeal carcinoma cells ( NPC ) cells and provides a theoretical basis for the comprehensive treatment of nasopharyngeal carcinoma patients .

method

5 . Effect of COX - 2 silencing on cell cycle induced by radiation

2 脳 103 exponential growth cells were inoculated into 96 well plates , the cell suspension volume was 100ul , cultured in incubator for 1 , 2 , 3 , 4 , 5 days , and 10 ul of CCK - 8 solution was added to each well . The OD value at 450nm was measured by microplate reader . Six replicate wells were set in each group , and the average value was taken to plot the growth curve in vitro .

2 . Scratch test

2 脳 105 cells were seeded on 6 - well plates . Each group of cells was divided into three complex wells . When the cells grew to about 90 % confluence , the culture medium was washed twice with 200 ul tip . The cells were washed twice to remove the cells . After addition of the culture medium , the cells were washed twice to remove the cells . Then the cells were taken for sampling . The relative widths of the scratches were measured with the image J software . The displacement force curve was drawn and the independent experiment was repeated three times .

3 . Sensitivity changes of nasopharyngeal carcinoma cells to cisplatin after COX - 2 silencing

Two groups of cells were added into 96 well plates according to 5000 cells / well , medium 100ul was added into each well , the culture medium was discarded after 24 h , the final concentration was 0.5 , 1 , 2 , 4 , 8 , 16 , 32 ug / ml , and the final concentration was 0.5 , 1 , 2 , 4 , 8 , 16 , 32 ug / ml . After 24 h , 10 ul of CCK - 8 reagent was added . After incubation for 1h , the inhibitory rate of cell growth was calculated , and half of the inhibition concentration was calculated by SPSS 13.0 software .

4 . Cell cycle experiment

The cells were cultured for 24 h in a medium containing 6 ug / ml of cis - platinum or no cisplatin , or 24 and 48 hours after irradiation with 2 Gy or 0 Gy of sham irradiation . After 2 times of washing with PBS , the cells were added to the staining solution containing 50 ug / ml RNase and 50 ul / ml PI . After 30 min at room temperature , the percentage of each phase of cell cycle was detected by flow cytometry .

5 . Cloning formation experiment

Two groups of cells were inoculated into 6 - well plates according to a predetermined number . Each group was set with 6 dose groups of 0 , 1 , 2 , 4 , 6 and 8 Gy . After irradiation , the cells were cultured in a 5 % CO2 incubator . The results were analyzed by using GraphPad Prism 5.0 software . The parameters of L - Q model , 尾 , 伪 / 尾 , SF2 and multi - target were calculated . Then , the parameters D0 , Dq , and N were calculated .

6 . Radiosensitivity test in vivo

Female BALB / c nude mice , 4 - 6 week old , 18 - 22 g , SPF condition were fed . Two groups of cells were respectively made into single cell suspension , the cell concentration was adjusted to 1 脳 106 / ml with serum - free 1640 medium . The tumor volume ( V ) = ( long diameter 脳 short diameter 2 ) / 2.10 - 14d was measured by vernier caliper in the right hind limb of nude mice .
( 2 ) COX - 2 ( - ) control group without any treatment ;
( 3 ) COX - 2 ( + ) experimental group was given 10 Gy X - ray irradiation ;
( 4 ) COX - 2 ( - ) experimental group was given 10 Gy X - ray irradiation . 6 rats in each group were sacrificed 2 weeks later , the nude mice were sacrificed and the tumor inhibition rate was calculated .

7 . Statistical Analysis

Statistical analysis was carried out with SPSS 13.0 software , and the experimental data was expressed by mean 卤 SD , and the comparison of the two samples was conducted using One - Way ANOVA or Independent - Sample T Test of two independent samples . The results showed that the difference was statistically significant by using SNK method ( Student - Neuman - Keeper ) and P0.05 .

Results :

1 . Effect of COX - 2 gene silencing on proliferation and in vitro migration of nasopharyngeal carcinoma cells

Anti - COX - 2 C666 - 1 and Anti - GL - 2 C666 - 1 cells were inoculated with 96 well plates for 5 days . The growth curve was plotted on the basis of OD value at 450nm . The results showed that there was no interaction between the two groups ( F = 5.927 , P = 0.019 ) , but there was no interaction between cell group and days ( F = 1.264 , P = 0.296 ) .

The effect of COX - 2 stable silencing on the in vitro migration of C666 - 1 in nasopharyngeal carcinoma cells was detected by cell scratch test . The results of variance analysis showed that the difference between the two groups had statistical significance ( F = 157.01 , P0.001 ) , and there was an interaction effect between the two groups ( F = 15.73 , P0.001 ) . The migration distance of the two groups was different with time . Anti - COX - 2 C666 - 1 cells migrated more than Anti - GL - 2 C666 - 1 .

2 . Changes of the sensitivity of C666 - 1 cells to cisplatin after COX - 2 silencing

The IC50 of anti - COX - 2 C666 - 1 and Anti - GL - 2C666 - 1 cells on cisplatin was 3.682 卤 0.980 and 8.819 卤 2.354 , respectively , and the IC50 values of anti - COX - 2 C666 - 1 and Anti - GL - 2C666 - 1 cells were 3.682 卤 0.980 and 8.819 卤 2.354 respectively .

3 . Effect of COX - 2 on Cell Cycle Changes Induced by Cisplatin

The results of flow cytometry showed that the percentage of G0 / G1 phase cells was 70.260 % and 60.353 % ( t = - 2.726 , P = 0.053 ) , and the percentage of S - phase cells was 24.27 % and 32.180 % ( t = - 3.954 , P = 0.112 ) , respectively . The percentage of S - phase and G2 / M phase increased . There was no significant difference between the two groups ( t = - 1.636 , P = 0.177 ) . The amplitude of G2 / M phase increased by 0.500 % and 7.633 % , respectively . There were no obvious apoptotic peaks in the G2 / M phase after COX - 2 silencing , but there were no obvious apoptotic peaks before and after the action of cisplatin , suggesting that the cell G2 / M phase block induced by cisplatin was decreased after COX - 2 silencing , but there was no obvious effect on apoptosis .

4 . Effect of COX - 2 silencing on the radiosensitivity of C666 - 1 cell in nasopharyngeal carcinoma

The values of D0 , Dq , F2 and 伪 / 尾 in the two groups were 6.5356 卤 0.0375 , 0.3961 卤 0.0160 , 6.5356 卤 0.861 , 9.4231 卤 0.9599 , respectively .

1 . Effect of COX - 2 gene silencing on the growth characteristics of nasopharyngeal carcinoma cells

The results of flow cytometry showed that there was no significant difference between G0 / G1 and S phase in the cell cycle of Anti - GL - 2 C666 - 1 and Anti - COX - 2 C666 - 1 , but the percentage of G2 / M cells was 7.500 % and 5.470 % , respectively ( t = - 3.952 , P = 0 . 017 ) . The G2 / M phase of anti - GL - 2 C666 - 1 cells increased by 18.430 % ( t = 9.184 , P = 0.001 ) . The G2 / M phase of anti - GL - 2 C666 - 1 cells increased by 18.430 % ( t = 9.184 , P = 0.001 ) . The G2 / M phase of the two groups were 24.397 % and 12.393 % ( t = 4.646 , P = 0.01 ) after 2Gy irradiation , 17.357 % and 6.923 % respectively before irradiation ( t = 3.320 , P = 0.029 ) . However , there were no sub - G1 phase reflecting apoptosis before and after irradiation , indicating that COX - 2 silencing significantly reduced the G2 / M phase block induced by radiation , but had no obvious effect on apoptosis .

6 . Effect of COX - 2 silencing on radiation sensitivity of nude mice in vivo

There was no significant difference in growth curve between the two groups ( F = 3.61 , P = 0.084 ) , and the tumor inhibition rates of anti - GL - 2 and Anti - COX - 2 groups were 32.72 % and 51.76 % , respectively .

Conclusion :

1 . COX - 2 silencing reduces the proliferation and in vitro migration ability of nasopharyngeal carcinoma cells .

2 . The sensitivity of cisplatin to nasopharyngeal carcinoma ( C666 - 1 ) was enhanced after COX - 2 silencing , and cell G2 / M arrest induced by cisplatin was reduced .

3 . After the COX - 2 silencing , the sensitivity of external radiotherapy in nasopharyngeal carcinoma was enhanced , and the G2 / M phase block of the cells induced by radiation was reduced .

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.63

【参考文献】