吉林大学学报(医学版)  2016, Vol. 42 Issue (03): 419-423

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胡志宏, 赵大力, 谢忠伟, 王梦莹, 胡景坤, 龚守良, 刘扬, 齐亚莉
HU Zhihong, ZHAO Dali, XIE Zhongwei, WANG Mengying, HU Jingkun, GONG Shouliang, LIU Yang, QI Yali
pEgr1-TRAIL重组质粒联合电离辐射对乳腺癌MCF-7细胞死亡受体通路相关基因和蛋白表达的影响
Effects of pEgr1-TRAIL recombinant plasmid combined with ionizing radiation on related gene and protein expressions of death receptor pathway in breast cancer MCF-7 cells
吉林大学学报(医学版), 2016, 42(03): 419-423
Journal of Jilin University (Medicine Edition), 2016, 42(03): 419-423
10.13481/j.1671-587x.20160301

文章历史

收稿日期: 2016-01-23
网络出版时间: 2016-05-17 14:04:05
pEgr1-TRAIL重组质粒联合电离辐射对乳腺癌MCF-7细胞死亡受体通路相关基因和蛋白表达的影响
胡志宏1, 赵大力2, 谢忠伟2, 王梦莹1, 胡景坤1, 龚守良3, 刘扬3, 齐亚莉1     
1. 北华大学公共卫生学院流行病学教研室, 吉林 吉林 132011;
2. 吉林省吉林(市)出入境检验检疫局, 吉林 吉林 132012;
3. 吉林大学公共卫生学院卫生部放射生物学重点实验室, 吉林 长春 130021
摘要: 目的: 将Egr1-TRAIL重组质粒转染人乳腺癌MCF-7细胞且加X线照射,探讨MCF-7细胞中死亡受体通路相关基因和蛋白表达的变化。方法: 将MCF-7细胞分为对照组、空质粒组、pEgr1-TRAIL质粒组、4.0GyX射线组、空质粒+4.0GyX射线组和pEgr1-TRAIL+4.0GyX射线组。Real-timePCR法检测各组MCF-7细胞中DR4、caspase-9和caspase-6mRNA表达水平;Western blotting法检测各组MCF-7细胞中DR4、caspase-9和caspase-6蛋白相对表达水平。结果: 经4.0GyX射线照射后4h,与对照组比较,各组MCF-7细胞中DR4、caspase-9和caspase-6mRNA表达水平升高(P<0.01),8h达最高值,之后开始降低,到24h恢复到照射前水平;各组MCF-7细胞中DR4、caspase-9和caspase-6mRNA表达由高到低的顺序为pEgr1-TRAIL+4.0GyX射线组> 空质粒+4.0GyX射线组> pEgr1-TRAIL质粒组> 空质粒组> 4.0GyX射线组> 对照组,其中pEgr1-TRAIL+4.0GyX射线组MCF-7细胞中DR4、caspase-9和caspase-6mRNA表达水平明显高于其他各组(P<0.01)。MCF-7细胞中DR4、caspase-9和caspase-6蛋白在照射后6h开始表达,12h达高峰,48h后细胞中DR4、caspase-9和caspase-6蛋白表达水平仍高于6h时蛋白表达水平。与对照组比较,其他各组MCF-7细胞中蛋白相对表达水平由高到低的顺序为pEgr1-TRAIL+4.0GyX射线组> 空质粒+4.0GyX射线组> 4.0GyX射线组> pEgr1-TRAIL质粒组> 空质粒组> 对照组,其中pEgr1-TRAIL+4.0GyX射线组MCF-7细胞中蛋白相对表达水平明显高于其他各组。结论: pEgr1-TRAIL重组质粒联合放疗对MCF-7细胞具有杀伤和诱导凋亡的作用,其效果优于单纯质粒或单纯照射。
关键词: pEgr1-TRAIL重组质粒    电离辐射    MCF-7细胞    死亡受体通路    
Effects of pEgr1-TRAIL recombinant plasmid combined with ionizing radiation on related gene and protein expressions of death receptor pathway in breast cancer MCF-7 cells
HU Zhihong1, ZHAO Dali2, XIE Zhongwei2, WANG Mengying1, HU Jingkun1, GONG Shouliang3, LIU Yang3, QI Yali1     
1. Department of Epidemiology, School of Public Health, Beihua University, Jilin 132001, China;
2. Jilin Province (City) Entry and Exit Inspection and Quarantine Bureau, Jilin Province, Jilin 132012, China;
3. Key Laboratory of Radiobiology, Ministry of Health, School of Public Health, Jilin University, Changchun 130021, China
Abstract: Objective: To transfer the pEgr1-TRAIL recombinant plasmid into human breast cancer MCF-7 cells and to combine with ionizing radiation,and to discuss the changes of related gene and protein expressions of death receptor pathway in breast cancer MCF-7 cells. Methods: The MCF-7 cells were divided into empty plasmid,pEgr1-TRAIL plasmid,4.0 Gy X-ray,empty plasmid+4.0 Gy X-ray, and pEgr1 TRAIL+4.0 Gy X-ray groups.Real-time PCR method was used to detect the expression levels of DR4,caspase-9,and caspase-6 mRNA in the MCF-7 cells in various groups;Western blotting method was used to detect the relative expression levels of DR4,caspase-9,and caspase-6 protein in the MCF-7 cells in various groups. Results: Compared with control group, the mRNA expression levels of DR4,caspase-9 and caspase-6 in MCF-7 cells in the other groups began to rise 4 h after 4.0 Gy X-ray irradiation(P<0.01),and they reached to the highest levels 8 h later,then reduced to the level of pre-irradiation 24 h later.The order of these mRNA expression levels from high to low in various groups was pEgr1-TRAIL+4.0 Gy X-ray group>empty plasmid+4.0 Gy X-ray group >pEgr1-TRAIL plasmid group >empty plasmid group >4.0 Gy X-ray group>control group;the expression levels of RD4,caspase-9,and caspase-6 mRNA in the MCF-7 cells in pEgr1-TRAIL+4.0 Gy X-ray group were higher than those in other groups (P<0.01).The relative expression levels of DR4,caspase-9 and caspase-6 proteins began to rise 6 h after irradiation, and reached the peak 12 h later; the relative expression levels of those protein 48 h later were still higher than those 6 h after irrdiation.The order of these protein expression levels from high to low in various group was pEgr1 TRAIL+4.0 Gy X-ray group >empty plasmid+4.0 Gy X-ray group >4.0 Gy X-ray group >pEgr1-TRAIL plasmid group >empty plasmid group >control group.The relative expression levels of DR4,caspase-9,and caspase-6 proteins in the MCF-7 cells in pEgr1-TRAIL+4.0 Gy X-ray group were higher than those in the other groups. Conclusion: pEgr1-TRAIL recombinant plasmid combined with radiation can kill and induce the apoptosis of MCF-7 cells,and its effect is better than recombinant plasmid or ionizing radiation used alone.
Key words: pEgr1-TRAIL recombinant plasmid    ionizing radiation    MCF-7 cell    death receptor pathway    

近年来,我国居民恶性肿瘤的发病率和死亡率呈升高态势,其中肺癌、乳腺癌、肠癌和胃癌等发病率和死亡率上升尤为明显。传统的肿瘤治疗方法为手术切除后给予放、化疗,但其副反应较严重,治疗效果不够理想[1, 2]。肿瘤放射基因治疗既能够减少正常组织的放射损伤,又能促进肿瘤细胞凋亡,目前已成为肿瘤治疗的热点[3]。肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor related apoptosisinducing ligand,TRAIL)是TNF超家族成员,其在诱导肿瘤细胞凋亡的同时不损伤正常细胞[4, 5, 6]。另外,TRAIL还能结合放疗诱导肿瘤细胞凋亡,发挥杀死肿瘤细胞的协同效应[7, 8]。由于TRAIL基因位于Egr-1启动子的下游,电离辐射可以诱导和激活Egr-1基因,调节下游TRAIL基因的表达,进而激活死亡受体通路,诱导肿瘤细胞凋亡[9, 10]。本研究采用pEgr1-TRAIL重组质粒转染人乳腺癌MCF-7细胞,并联合电离辐射,观察人乳腺癌MCF-7细胞中死亡受体通路基因和蛋白表达水平,为乳腺癌的治疗提供实验依据。

1 材料与方法 1.1 细胞、质粒、主要试剂和仪器

人乳腺癌MCF-7细胞系由吉林大学卫生部放射生物学重点实验室保存。pEgr1-TRAIL重组质粒和脂质体由刘扬博士制备。使用含10%胎牛血清的高糖DMEM(Sigma公司,美国)培养,培养条件为37℃、5% CO2、100%饱和湿度的培养箱,人caspase-9、caspase-6和DR4抗体购自美国Santa Cruz公司,Real-time检测试剂盒购自美国Sigma公司,其他试剂为国产分析纯。X射线深部治疗机购自日本Philips公司。

1.2 实验分组和照射方法

将细胞分为对照组、空质粒组、pEgr1-TRAIL质粒组、4.0 GyX射线组、空质粒+4.0 GyX射线组和pEgr1-TRAIL+4.0 GyX射线组。采用X射线深部治疗机进行照射,照射条件为电压180 kV,电流12 mA,滤板厚度为铜0.5 mm、铝1.0 mm,剂量率为0.387 Gy·min-1,靶皮距为60 cm。

1.3 细胞转染

将各组细胞按每孔2.0×105个接种于24孔板,使用不含抗生素的DMEM培养液培养24 h,当细胞密度达到约80%时进行转染。转染体系中脂质体与质粒的比例为3:1。将重组质粒和脂质体采用不含抗生素和胎牛血清的DMEM培养液稀释,至终浓度分别为1和3 g/50 L,5 min内将2种液体混合,共计400 μL,常温反应30 min后,接种于培养板中,置于37℃、5% CO2的培养箱中培养8 h,更换含抗生素和胎牛血清的DMEM培养液继续培养48 h。

1.4 Real-time PCR法检测各组细胞中DR4、caspase-9和caspase-6 mRNA表达水平

将各组细胞接种于6孔培养板,密度为每孔4×105个细胞,设平行6复孔。培养24 h后,给予4.0 Gy X射线照射,照射后4、8、12和24 h收集细胞,提取总RNA,按照Real-time检测试剂盒说明书操作,检测细胞中DR4、caspase-9和caspase-6mRNA表达水平。采用相对定量2-ΔΔCt法计算目的基因相对于内参基因表达的倍数,比较基因的表达差异。每一个样本的ΔCt=Ct(target gene)-Ct(GAPDH),而ΔΔCt=ΔCt(target gene)-ΔCt(calibrator)。

1.5 Western blotting法检测各组细胞中DR4、caspase-9和caspase-6蛋白相对表达水平

将浓度为1×106mL-1各组细胞分别接种于直径为90 mm培养皿中,待细胞达到80%融合后,给予4.0 Gy X射线照射,再培养12 h,收集各处理组细胞,提取总蛋白。Western blotting主要步骤包括灌胶、电泳、转膜、剪膜、封闭、抗体孵育、曝光、显影和定影。采用Bio-Rad凝胶成像系统采集图像,其结果采用QuantityOne4.6.2软件进行分析。蛋白相对表达水平=(处理组基因灰度值/处理组内参灰度值)/(对照组基因灰度值/对照组内参灰度值)。

1.6 统计学分析

采用SPSS19.0统计软件进行统计学分析。各组细胞中DR4、caspase-9和caspase-6 mRNA及蛋白相对表达水平以x± s 表示,组间样本均数比较采用单因素方差分析。以α=0.01为检验水准。

2 结 果 2.1 各组细胞中DR4、caspase-9和caspase-6 mRNA的表达水平

与对照组比较,经4.0 Gy X射线照射后4 h,空质粒组、pEgr1-TRAIL组、 4.0 Gy组X射线、空质粒+4.0 GyX射线组和pEgr1-TRAIL+4.0 Gy X射线组细胞中DR4、caspase-9和caspase-6 mRNA表达水平均升高(P <0.01);8 h达最高值;其中不同时间pEgr1-TRAIL+4.0 Gy X射线组细胞中DR4、caspase-9和caspase-6 mRNA表达水平明显高于其他各组(P<0.01)。见表 13

表 1 照射后不同时间各组人乳腺癌MCF-7细胞中DR4 mRNA的表达水平 Tab. 1 Expression levels of DR4 mRNA in MCF-7 cells in various groups after irradiated for different time
(n=6,x± s )
GroupExpression level of DR4 mRNA
(t/h)481224
Control1111
Empty plasmid5.46±0.34*9.52±0.29*7.54± 0.37*5.28±0.23*
pEgr1-TRAIL6.48±0.31*14.24±0.29*11.87±0.68*3.88±0.59*
4.0 Gy X-ray4.96±0.54*7.49±0.37*5.87±0.47*2.94±0.38*
Empty plasmid+4.0 Gy X-ray13.51± 0.63*26.17±2.41*18.76±0.67*4.78±0.33*
pEgr1-TRAIL+4.0 GyX-ray22.31±0.59*△#○▲109.49±3.67*△#○▲45.16±0.69*△#○▲8.78±0.28*△#○▲
*P<0.01 vs control group;P< 0.01 vs empty plasmid group;#P<0.01 vs pEgr1-TRAIL group;P<0.01 vs4.0 Gy X-ray group;P<0.01 vsempty plasmid+4.0 Gy X-ray group.
表 2 照射后不同时间各组人乳腺癌MCF-7 细胞中 caspase-9 mRNA的表达水平 Tab. 2 Expression levels of caspase-9 mRNA in MCF-7 cells in various groups after irradiated for different time
(n=6,x± s )
GroupExpression level of caspase-9 mRNA
(t/h)481224
Control1111
Empty plasmid5.57±0.35*9.26±0.37*8.56±0.76*2.45±0.29*
pEgr1-TRAIL8.63±0.48*14.56±0.43*11.52±0.49*5.78±0.24*
4.0 GyX-ray4.72±0.61*8.53±0.42*6.17±0.29*2.35±0.18*
Empty plasmid+4.0 Gy X-ray8.79±0.26*24.57±0.61*12.45±0.79*6.73±0.28*
pEgr1-TRAIL+4.0 Gy X-ray16.44±0.39*△#○▲158.47±4.36*△#○▲46.22±0.45*△#○▲7.93 ±0.57*△#○▲
*P<0.01 vs control group;P<0.01 vs empty plasmid group;#P<0.01 vs pEgr1-TRAIL group; P<0.01 vs 4.0 Gy X-ray group;P<0.01 vs empty plasmid+4.0 Gy X-ray group
表 3 照射后不同时间各组人乳腺癌MCF-7细胞中caspase-6 mRNA的表达水平 Tab. 3 Expression levels of caspase-6 mRNA in MCF-7 cells in various groups after irradiated for different time
(n=6,x± s )
GroupExpression level of caspase-6 mRNA
(t/h)481224
Control1111
Empty plasmid6.59±0.44* 12.78±0.24* 13.59±0.45*7.48±0.57*
pEgr1-TRAIL10.44± 0.34* 20.18±0.34* 15.78±0.57*12.76±0.65*
4.0 GyX-ray5.38±0.16* 7.52±0.11* 6.68±0.21*5.49±0.23*
Empty plasmid+4.0 Gy X-ray12.59±0.78* 23.51±0.79* 19.68±0.96*16.47±0.81*
pEgr1-TRAIL+4.0 Gy X-ray27.69±0.82*△#○▲ 167.43±11.78*△#○▲59.53±3.74*△#○▲39.47±6.58*△#○▲
*P<0.01 vs control group;P<0.01 vs empty plasmid group;#P< 0.01 vs pEgr1-TRAIL group; P<0.01 vs 4.0 Gy X-ray group;P<0.01 vs empty plasmid+4.0 Gy X-ray group
2.2 各组MCF-7细胞中DR4、caspase-9和caspase-6蛋白相对表达水平

4.0 Gy X射线照射后6 h,与对照组比较,其他各组MCF-7细胞中DR4、caspase-9和caspase-6蛋白相对表达水平开始升高,12 h后达峰值,随着时间的延长蛋白相对表达水平开始下降,但48 h仍高于正常值;与对照组比较,其他各组MCF-7细胞中蛋白相对表达水平由高到低的顺序为pEgr1-TRAIL+4.0 GyX射线组>空质粒+4.0 Gy X射线组>4.0 GyX射线组>pEgr1-TRAIL质粒组>空质粒组>对照组,其中pEgr1-TRAIL+4.0 GyX射线组细胞中DR4、caspase-9和caspase-6蛋白相对表达水平上升最明显。见图 1

Lane 1:Control group; Lane 2:Empty plasmid group; Lane 3:pEgr1-TRAIL group; Lane 4:4.0 Gy X-raygroup;Lane 5:Empty plasmid+4.0 Gy X-raygroup; Lane 6:pEgr1-TRAIL+4.0 Gy X-raygroup.A:DR4;C:Caspase-9;E:Caspase-6;B,D,F:GAPDH. 图 1 照射不同时间人乳腺癌MCF-7 细胞中DR4、caspase-9和caspase-6蛋白表达电泳图 Fig. 1 Electrophoregram of expressions of DR4,caspase-9, and caspase-6 proteins in MCF-7 cells in various groups after irradiated for different time
3 讨 论

本实验采用pEgr1-TRAIL重组质粒转染MCF-7细胞并联合电离辐射,观察MCF-7细胞死亡受体通路相关DR4、caspase-9和caspase-6mRNA和蛋白表达水平。本研究结果表明:4.0 Gy X射线照射后4 h,各组MCF-7细胞中DR4、caspase-9和caspase-6 mRNA和蛋白表达水平均开始升高,分别于8和12 h达高峰,其中pEgr1-TRAIL+4.0 Gy组细胞中mRNA和蛋白表达水平升高最明显,照射后48 h其mRNA和蛋白表达仍高于正常水平,提示pEgr1-TRAIL重组质粒联合电离辐射具有杀伤和诱导MCF-7细胞凋亡的作用。

细胞凋亡的通路主要包括两方面:一是启动死亡受体通路,二是启动线粒体凋亡通路[11]。本实验通过转染pEgr1-TRAIL重组质粒联合电离辐射启动了MCF-7细胞死亡受体通路,TRAIL可与细胞膜上的DR4结合,形成三聚体,激活caspase-9和caspase-6,使DR4、caspase-9和caspase-6基因和蛋白表达增强,促进了MCF-7细胞凋亡。

虽然TRAIL应用前景良好,并且具有杀伤肿瘤细胞而不杀伤正常细胞的特异性,但其治疗谱并不宽,一些肿瘤对TRAIL也具有抗拒性。电离辐射可杀伤肿瘤细胞,与TRAIL基因的联合应用已取得了明显的效果。研究[12]表明:电离辐射可使某些恶性肿瘤细胞对TRAIL表现出较高的敏感性,这是由于辐射诱导的死亡受体DR表达量升高,从而提高肿瘤细胞对TRAIL的敏感性。体内实验[13]表明:TRAIL和辐射的联合应用可使已建立起来的异种移植乳腺癌发生快速消退。

TRAIL基因可选择性地诱导肿瘤细胞凋亡,而对正常细胞不产生毒性,联合放疗可发挥杀灭肿瘤细胞的协同作用[14, 15]。目前,以TRAIL为基础的抗肿瘤药物研发取得成效,主要用于治疗乳腺癌、甲状腺癌、胰腺癌和结肠癌等实体瘤,其安全性和有效性得到肯定[16, 17]。本研究初步阐明了pEgr1-TRAIL重组质粒联合放疗对MCF-7细胞杀伤及诱导凋亡的作用,为乳腺癌治疗提供了新思路。

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