摘要
目的近年来,受体型蛋白酪氨酸酶O(protein tyrosine phosphatase receptor type O,PTPRO)被公认为是一种新的参与肿瘤发展和转移的抑癌基因。本研究通过分析PTPRO过表达对食管鳞癌细胞增殖及放射敏感性的影响,初步探讨影响放射敏感性的机制。方法构建pCR-PTPRO-HA真核表达载体;采用脂质体转染法获得PTPRO过表达组(PTPROc1组和PTPROc2组)及空载组(Vector组)细胞株;通过实时逆转录-聚合酶链反应(real-time reverse transcription-polymerase chain reaction,RT-PCR)、蛋白质印迹法验证PTPRO表达;应用四甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)法、平板克隆形成及软琼脂克隆形成实验检测细胞体外增殖能力;使用X射线分别照射PTPROc1组、PTPROc2组和Vector组细胞株,不同细胞组再按照射剂量分为5个梯度,分别为0、1、2、4和6Gy,照射后行平板克隆形成实验评估细胞体外放射敏感性,并采用流式细胞术分析其对细胞周期和X射线照射前后TE1细胞凋亡的影响。结果成功构建pCR-PTPRO-HA真核表达载体;RT-PCR、蛋白质印迹法验证PTPRO mRNA及蛋白过表达成功;克隆形成实验结果表明,PTPROc1组、PTPROc2组、未转染组(TE1组)和Vector组细胞克隆形成数分别为112±6、118±4、314±7和315±4,4组间差异有统计学意义,F=42.125,P<0.001,其中PTPROc1组细胞克隆形成数低于TE1组(P=0.008)和Vector组(P=0.009),PTPROc2组细胞克隆形成数低于TE1组(P=0.012)和Vector组(P=0.013);软琼脂克隆形成实验结果表明,PTPROc1组、PTPROc2组、TE1组和Vector组细胞克隆形成数分别为12±2、11±1、52±3和51±3,4组间差异有统计学意义,F=17.687,P=0.015,其中PTPROc1组和PTPROc2组细胞克隆形成数均低于TE1组和Vector组,均P<0.001;流式细胞术检测结果表明,PTPROc1组、PTPROc2组和Vector组的G2/M期细胞占比分别为7.31±0.38、8.52±0.28和2.33±0.24,差异有统计学意义,F=41.223,P<0.001,其中PTPROc1和PTPROc2组较Vector组细胞所占比例增加,差异有统计学意义,均P<0.001;PTPROc1组、PTPROc2组和Vector组的G0/G1期细胞占比分别为77.18±0.27、77.75±0.21和73.75±0.34,差异有统计学意义,F=19.215,P<0.001,其中PTPROc1和PTPROc2组较Vector组细胞所占比例增加,均P<0.001;PTPROc1组、PTPROc2组和Vector组的S期分别为15.51±0.11、13.73±0.16和23.92±0.44,差异有统计学意义,F=28.221,P<0.001,其中PTPROc1组和PTPROc2组较Vector组细胞所占比例减少,均P<0.001;X线照射后结果显示,PTPROc1组、PTPROc2组和Vector组的ID50值分别为1.42±0.02、1.46±0.02和2.11±0.03,差异有统计学意义,F=32.255,P<0.001,其中PTPROc1组和PTPROc2组较Vector组降低,均P<0.001;PTPROc1组、PTPROc2组和Vector组2Gy射线照射后的存活分数分别为0.38±0.006、0.34±0.009和0.57±0.003,差异有统计学意义,F=25.645,P<0.001,PTPROc1组和PTPROc2组较Vector组降低,均P<0.001;AnnexinⅤ-FITC/PI双染细胞凋亡实验表明,未放射处理前PTPROc1组、PTPROc2组和Vector组的凋亡率分别为(13.91±0.31)%、(14.03±0.22)%和(2.32±0.32)%,差异有统计学意义,F=28.887,P<0.001,其中PTPROc1组和PTPROc2组较Vector组凋亡率增高,均P<0.001;放射治疗后,PTPROc1组凋亡率为(26.33±0.82)%,PTPROc2组凋亡率为(25.43±0.95)%,均较放疗前增高,差异有统计学意义,均P<0.001。结论过表达PTPRO对食管癌细胞具有抑制增殖及放射增敏作用,其放射增敏机制可能与PTPRO的过表达引起细胞周期再分布和细胞早期凋亡有关。
OBJECTIVE In recent years,protein tyrosine phosphatase receptor type O(PTPRO)has been recognized as a novel tumor suppressor gene involved in tumor development and metastasis.This study was to investigate the effects of PTPRO overexpression on proliferation and radiosensitivity of esophageal squamous carcinoma cells,and to explore the mechanisms affecting radiosensitivity.METHODS The pCR-PTPRO-HA eukaryotic expression vector was constructed.The PTPRO overexpression group(PTPROc1 group and PTPROc2 group)and the Vector group were obtained by lipofection.PTPRO expression was verified by real-time reverse transcription-polymerase chain reaction(RT-PCR)and Western blotting.Methyl thiazolyl tetrazolium(MTT)method,plate cloning and soft AGAR cloning were used to test the proliferation ability of cells in vitro.X-rays were used to irradiate the PTPROc1 group,PTPROc2 group and Vector group.The different cell groups were divided into 5 gradients according to the dose,which were 0,1,2,4 and 6 Gy,respectively.After irradiation,the plate cloning assay was used to evaluate the in vitro radiosensitivity of the cells,and the effects of cell cycle and TE1 cell apoptosis before and after X-ray irradiation were analyzed by flow cytometry.RESULTS The pCR-PTPRO-HA eukaryotic expression vector was successfully constructed.RT-PCR and Western blotting experiments confirmed the successful expression of PTPRO mRNA and protein.The results of colony formation showed that the number of cell clones in the PTPROc1 group,PTPROc2 group,untransfected group(TE1 group)and Vector group were 112±6,118±4,314±7 and 315±4,respectively.There were significant differences between the four groups,F=42.125,P<0.001.The number of cell clone formation in PTPROc1 group was lower than TE1 group(P=0.008)and Vector group(P=0.009).The number of cell clone formation in PTPROc2 group was lower than TE1 group(P=0.012)and Vector group(P=0.013).The results of soft agar colony formation showed that the number of cell clone formation in PTPROc1 group,PTPROc2 group,TE1 group and Vector group were 12±2,11±1,52±3 and 51±3,respectively,the difference between the four groups was statistically significant(F=17.687,P=0.015),in which the number of cell clone formation in PTPROc1 group and PTPROc2 group was lower than that in TE1 group and Vector group,all P<0.001.The results of flow cytometry cell cycle experiments showed that the G2/M phase of PTPROc1 group,PTPROc2 group and Vector group were7.31±0.38,8.52±0.28 and 2.33±0.24,respectively,and the difference was statistically significant,F=41.223,P<0.001.The proportion of cells in the PTPROc1 and PTPROc2 groups was higher than that in the Vector group,and the difference was statistically significant,all P<0.001.The G0/G1 phase of PTPROc1 group,PTPROc2 group and Vector group were 77.18±0.27,77.75±0.21 and 73.75±0.34,respectively.The difference was statistically significant,F=19.215,P<0.001.The percentage of cells in PTPROc1 and PTPROc2 group was higher than that in Vector group,all P<0.001.The S phase of PTPROc1 group,PTPROc2 group and Vector group was 15.51±0.11,13.73±0.16 and23.92±0.44,respectively,and the difference was statistically significant,F=28.221,P<0.001.The proportion of cells in PTPROc1 group and PTPROc2 group was lower than that in Vector group,all P<0.001.After X-ray irradiation,the ID50 values of PTPROc1 group,PTPROc2 group and Vector group were 1.42±0.02,1.46±0.02 and 2.11±0.03,respectively,and the difference was statistically significant(F=32.255,P<0.001),among which PTPROc1 group and PTPROc2 group was lower than Vector group,all P<0.001.The survival scores of PTPROc1 group,PTPROc2 group and Vector group after 2 Gy irradiation were 0.38±0.006,0.34±0.009 and 0.57±0.003,respectively.The difference was statistically significant,F=25.645,P<0.001.The survival scores of PTPROc1 group and PTPROc2 group were lower than those of Vector group,P<0.001.AnnexinⅤ-FITC+PI double-staining apoptotic test showed that before irradiation,the apoptotic rates of PTPROc1,PTPROc2 and Vector groups were 13.91±0.31,14.03±0.22 and 2.32±0.32,respectively.The difference was statistically significant,F=28.887,P<0.001.The apoptotic rates of PTPROc1 and PTPROc2 groups were higher than those of Vector group,P<0.001.After radiotherapy,the apoptotic rate of PTPROc1 group(26.33±0.82)and PTPROc2 group(25.43±0.95)were higher than those before radiotherapy,and the difference was statistically significant,P<0.001.CONCLUSIONS Overexpression of PTPRO can inhibit proliferation and radiosensitization of esophageal cancer cells.The mechanism of radiosensitization may be related to cell cycle redistribution and early cell apoptosis induced by overexpression of PTPRO.
作者
叶娟
许镒洧
方玫玫
肖佳
YE Juan;XU Yi-ivei;FANG Mei-mei;XIAO Jia(Department of Head and Neck Oncology Affiliated Hospital of Zunyi Medical College,Zunyi 563000,P.R.China;Cancer Hospital Affiliated to Shantou University Medical College,Shantou 515000,P.R.China;Department of Oncology,First People's Hospital of Yue yang tYueyang 414000,P.R.China)
出处
《中华肿瘤防治杂志》
CAS
北大核心
2019年第23期1762-1770,共9页
Chinese Journal of Cancer Prevention and Treatment
基金
广东省教育厅青年创新人才类项目（2015KQNCX044）
遵义医学院附属医院硕士启动基金﹝院字（2012）07号﹞
