鸡TIGAR基因真核表达质粒的构建及其抗凋亡功能评价
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摘要
【背景】TP53诱导的糖酵解和凋亡调节因子(TP53-induced glycolysis and apoptosis regulator,TIGAR)是p53下游的靶基因,具有调节糖酵解水平和去除活性氧(Reactive oxygen species,ROS)并降低由活性氧诱发的细胞凋亡水平。【目的】构建鸡源TIGAR基因的真核表达质粒,并检测TIGAR基因在DF1细胞中的抗凋亡作用,为建立稳定表达鸡TIGAR基因的细胞系做准备。【方法】根据GenBank(登录号:XM_417232.6)中预测基因设计引物,利用RT-PCR的方法从SPF鸡脾脏中扩增鸡TIGAR基因,将扩增产物克隆至载体(Flag-CMV14)后送公司测序验证;随后构建进化树对鸡TIGAR基因与其他哺乳动物及水生动物的TIGAR基因进行同源性分析。将重组质粒(Flag-TIGAR)转染入DF1细胞24 h后,使用新城疫病毒诱导细胞凋亡,利用Western Blot检测重组质粒表达情况以及Poly ADP-Ribose Polymerase(PARP)裂解情况。此外还将重组质粒(Flag-TIGAR)转染入DF1细胞,并于收样前2 h使用Staurosporine刺激细胞发生凋亡,分别在转染后24、48 h收集样品,并用流式细胞仪检测细胞凋亡情况。【结果】RT-PCR扩增TIGAR基因,在843 bp处出现目的条带与预测相符,构建的TIGAR真核表达质粒(Flag-TIGAR)经测序,结果显示其序列与GenBank上预测的基本一致。Western Blot结果显示在30 h、36 h收集的样品中PARP均被裂解且转染重组质粒(Flag-TIGAR)的实验组与未转染质粒(MOCK)组或转染空载体(Flag-CMV14)组的样品相比,裂解的PARP表达量明显降低,且差异极显著(P<0.01)。流式结果显示:24 h检测转染Flag-CMV14后细胞总凋亡率为11%(早期凋亡7.8%,晚期凋亡3.2%),而转染Flag-TIGAR后细胞总凋亡率仅为4%(早期凋亡3.7%,晚期凋亡0.3%),转染Flag-CMV14的早期凋亡率和晚期凋亡率均高于转染Flag-TIGAR组,且差异显著(P<0.05)。48 h转染Flag-CMV14后细胞总凋亡率为20.3%(早期凋亡14.3%,晚期凋亡6.0%),而转染Flag-TIGAR后细胞总凋亡率为6.4%(早期凋亡4.8%,晚期凋亡1.6%),转染Flag-CMV14组的细胞早期凋亡和晚期凋亡率均高于转染Flag-TIGAR的组,且差异极显著(P<0.01)。【结论】成功扩增出鸡TIGAR基因,并构建了其真核表达质粒,通过Western Blot和流式实验均证实过表达TIGAR后可降低细胞的凋亡程度并有利于细胞存活。
【Background】TP53-induced glycolysis and apoptosis regulator(TIGAR) is a target gene downstream of p53, which can regulate glycolysis level, remove reactive oxygen species(ROS) and reduce apoptosis induced by reactive oxygen species(ROS).【Objective】The objective of this study was to construct the eukaryotic expression plasmid contain of chicken TIGAR gene, and to evaluate its resistance apoptosis effect in DF1 cells, in order to establish the cell line with stable expression of the chicken TIGAR gene. 【Method】According to design primers based on the predicted TIGAR gene published in Genbank(accession number:XM_417232.6), the TIGAR gene from SPF chicken spleen was amplified by RT-PCR, and then amplified products were cloned into expression vector(Flag-CMV14), finally the positive clone was verified by DNA sequencing. Then the phylogenetic tree of chicken TIGAR genes with other mammals and aquatic animals TIGAR gene was constructed to do homology analysis. The recombinant plasmid was transfected into DF1 cells, and infected with Newcastle disease virus to induce apoptosis. The expression of the TIGAR gene and the cleavage level of PARP protein were detected by Western Blot. At the same time the recombinant plasmid(Flag-TIGAR)was transfected into DF1 cells and incubated with apoptosis inducer staurosporine for 2 hours before sample collection, then cells were collected at 24 hours and 48 hours after transfection to evaluate apoptosis level by flow cytometry. 【Result】The TIGAR gene was amplified by RT-PCR, and a band appeared at 843 bp, which was consistent with the prediction. The constructed TIGAR eukaryotic expression plasmid(Flag-TIGAR) was sequenced, and the result showed that the sequence of amplified TIGAR gene were almost consistent with the predicted sequence published on GenBank. Western Blot results showed: cleavaged PARP bands were existed, and the expression level in transfected recombinant plasmid(Flag-TIGAR) group was significantly different(P<0.05)from that in non-transfected plasmid(MOCK) group and transfected empty vector(Flag-CMV14) group. Flow cytometry results showed that the total apoptotic rate was 11%(early apoptosis 7.8%, late apoptosis 3.2%) after transfection with Flag-CMV14 for 24 h, while the total apoptotic rate was decreased to 4%(early apoptosis 3.7%, late apoptosis 0.3%) in Flag-TIGAR transfected group.The early apoptotic rate of Flag-CMV14 transfected group was much higher than that of Flag-TIGAR transfection group, and the difference was very significant(P<0.01), while the late apoptotic rate was also higher than that of Flag-TIGAR transfected group and the difference was significant(P<0.05). The total apoptotic rate was 20.3%(early apoptosis 14.3%, late apoptosis 6.0%) after transfection with Flag-CMV14 for 48 h, while the total apoptotic rate was only 6.4%(early apoptosis 4.8%, late apoptosis 1.6%)after transfection with Flag-TIGAR for 48 h. Both the early apoptotic rate and late apoptotic rate of Flag-CMV14 transfected group were much higher than that of Flag-TIGAR transfected group and the difference was very significant(P<0.01). 【Conclusion】The chicken TIGAR gene was amplified and the eukaryotic expression plasmid was constructed successfully. It was also confirmed that overexpression of chicken TIGAR could obviously reduce the degree of apoptosis and increase cell survival rate.
【Background】TP53-induced glycolysis and apoptosis regulator(TIGAR) is a target gene downstream of p53, which can regulate glycolysis level, remove reactive oxygen species(ROS) and reduce apoptosis induced by reactive oxygen species(ROS).【Objective】The objective of this study was to construct the eukaryotic expression plasmid contain of chicken TIGAR gene, and to evaluate its resistance apoptosis effect in DF1 cells, in order to establish the cell line with stable expression of the chicken TIGAR gene. 【Method】According to design primers based on the predicted TIGAR gene published in Genbank(accession number:XM_417232.6), the TIGAR gene from SPF chicken spleen was amplified by RT-PCR, and then amplified products were cloned into expression vector(Flag-CMV14), finally the positive clone was verified by DNA sequencing. Then the phylogenetic tree of chicken TIGAR genes with other mammals and aquatic animals TIGAR gene was constructed to do homology analysis. The recombinant plasmid was transfected into DF1 cells, and infected with Newcastle disease virus to induce apoptosis. The expression of the TIGAR gene and the cleavage level of PARP protein were detected by Western Blot. At the same time the recombinant plasmid(Flag-TIGAR)was transfected into DF1 cells and incubated with apoptosis inducer staurosporine for 2 hours before sample collection, then cells were collected at 24 hours and 48 hours after transfection to evaluate apoptosis level by flow cytometry. 【Result】The TIGAR gene was amplified by RT-PCR, and a band appeared at 843 bp, which was consistent with the prediction. The constructed TIGAR eukaryotic expression plasmid(Flag-TIGAR) was sequenced, and the result showed that the sequence of amplified TIGAR gene were almost consistent with the predicted sequence published on GenBank. Western Blot results showed: cleavaged PARP bands were existed, and the expression level in transfected recombinant plasmid(Flag-TIGAR) group was significantly different(P<0.05)from that in non-transfected plasmid(MOCK) group and transfected empty vector(Flag-CMV14) group. Flow cytometry results showed that the total apoptotic rate was 11%(early apoptosis 7.8%, late apoptosis 3.2%) after transfection with Flag-CMV14 for 24 h, while the total apoptotic rate was decreased to 4%(early apoptosis 3.7%, late apoptosis 0.3%) in Flag-TIGAR transfected group.The early apoptotic rate of Flag-CMV14 transfected group was much higher than that of Flag-TIGAR transfection group, and the difference was very significant(P<0.01), while the late apoptotic rate was also higher than that of Flag-TIGAR transfected group and the difference was significant(P<0.05). The total apoptotic rate was 20.3%(early apoptosis 14.3%, late apoptosis 6.0%) after transfection with Flag-CMV14 for 48 h, while the total apoptotic rate was only 6.4%(early apoptosis 4.8%, late apoptosis 1.6%)after transfection with Flag-TIGAR for 48 h. Both the early apoptotic rate and late apoptotic rate of Flag-CMV14 transfected group were much higher than that of Flag-TIGAR transfected group and the difference was very significant(P<0.01). 【Conclusion】The chicken TIGAR gene was amplified and the eukaryotic expression plasmid was constructed successfully. It was also confirmed that overexpression of chicken TIGAR could obviously reduce the degree of apoptosis and increase cell survival rate.
引文
[1]RAVINDRA P V,TIWARI A K,RATTA B,BAIS M V,CHATURVEDI U,PALIA S K,SHARMA B,CHAUHAN R S.Time course of Newcastle disease virus-induced apoptotic pathways.Virus Research,2009,144(1-2):350-354.
[2]RAVINDRA P V,TIWARI A K,RATTA B,CHATURVEDI U,PALIAS K,CHAUHAN R S.Newcastle disease virus-induced cytopathic effect in infected cells is caused by apoptosis.Virus Research,2009,141(1):13-20.
[3]KERR J F,WYLLIE A H,CURRIE A R.Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics.British Journal of Cancer,1972,26(4):239-257.
[4]赵明,冯婧,刘勇,杨玲麟.TIGAR调节肺癌细胞的增殖和侵袭能力研究.国际检验医学杂志,2016(06):754-755.ZHAO M,FENG J,LIU Y,YANG L L.TIGAR promotes proliferation and invasiveness of lung cancer cells.International Journal of Laboratory Medicine,2016(06):754-755.(in Chinese)
[5]GREEN D R,CHIPUK J E.p53 and metabolism:Inside the TIGAR.Cell,2006,126(1):30-32.
[6]VOGELSTEIN B,LANE D,LEVINE A J.Surfing the p53 network.Nature,2000,408(6810):307-310.
[7]SHEN M,ZHAO X,ZHAO L,SHI L,AN S,HUANG G,LIU J.Met is involved in TIGAR-regulated metastasis of non-small-cell lung cancer.Molecular Cancer,2018,17(1):88.
[8]ROMEO M,HUTCHISON T,MALU A,WHITE A,KIM J,GARDNER R,SMITH K,NELSON K,BERGESON R,MCKEE R,HARROD C,RATNER L,LUSCHER B,MARTINEZ E,HARRODR.The human T-cell leukemia virus type-1 p30(II)protein activates p53 and induces the TIGAR and suppresses oncogene-induced oxidative stress during viral carcinogenesis.Virology,2018,518(10):3-15.
[9]BENSAAD K,TSURUTA A,SELAK M A,VIDAL M N,NAKANOK,BARTRONS R,GOTTLIEB E,VOUSDEN K H.TIGAR,a p53-inducible regulator of glycolysis and apoptosis.Cell,2006,126(1):107-120.
[10]YU H P,XIE J M,LI B,SUN Y H,GAO Q G,DING Z H,WU H R,QIN Z H.TIGAR regulates DNA damage and repair through pentosephosphate pathway and Cdk5-ATM pathway.Scientific Reports,2015.DOI:10.1038/srep09853
[11]ZHOU J H,ZHANG T T,SONG D D,XIA Y F,QIN Z H,SHENG R.TIGAR contributes to ischemic tolerance induced by cerebral preconditioning through scavenging of reactive oxygen species and inhibition of apoptosis.Scientific Reports,2016.DOI:10.1038/srep27096.
[12]JIANG L B,CAO L,MA Y Q,CHEN Q,LIANG Y,YUAN F L,LI XL,DONG J,CHEN N.TIGAR mediates the inhibitory role of hypoxia on ROS production and apoptosis in rat nucleus pulposus cells.Osteoarthritis and Cartilage,2018,26(1):138-148.
[13]KIM J,DEVALARAJA-NARASHIMHA K,PADANILAM B J.TIGAR regulates glycolysis in ischemic kidney proximal tubules.American Journal of Physiology Renal Physiology,2015,308(4).DOI:10.1152/ajprenal.00459.2014F298-308.
[14]付托.过表达人TIGAR的CHO细胞株构建及其抗凋亡机制研究[D].苏州:苏州大学,2013.FU T.Establishment of CHO cell line overexpressing h TIGAR and mechanism of anti-apoptosis effect[D].Suzhou:Suzhou University,2013.(in Chinese)
[15]HWANG S O,LEE G M.Nutrient deprivation induces autophagy as well as apoptosis in Chinese hamster ovary cell culture.Biotechnology and Bioengineering,2008,99(3):678-685.
[16]WANG H,CHENG Q,LI X,HU F,HAN L,ZHANG H,LI L,GE J,YING X,GUO X,WANG Q.Loss of TIGAR induces oxidative stress and meiotic defects in oocytes from obese mice.Molecular&Cellular Proteomics,2018.DOI:10.1074/mcp.RA118.000620.
[17]LI B,WANG Z,XIE J M,WANG G,QIAN L Q,GUAN X M,SHENX P,QIN Z H,SHEN G H,LI X Q,GAO Q G.TIGAR knockdown enhanced the anticancer effect of aescin via regulating autophagy and apoptosis in colorectal cancer cells.Acta Pharmacologica Sinica,2018.DOI:10.1038/s41401-018-0001-2.
[18]KUMAR B,IQBAL M A,SINGH R K,BAMEZAI R N.Resveratrol inhibits TIGAR to promote ROS induced apoptosis and autophagy.Biochimie,2015,118:26-35.
[19]YE L,ZHAO X,LU J,QIAN G,ZHENG J C,GE S.Knockdown of TIGAR by RNA interference induces apoptosis and autophagy in Hep G2 hepatocellular carcinoma cells.Biochemical and Biophysical Research Communications,2013,437(2):300-306.
[20]CHEUNG E C,LEE P,CETECI F,NIXON C,BLYTH K,SANSOMO J,VOUSDEN K H.Opposing effects of TIGAR-and RAC1-derived ROS on Wnt-driven proliferation in the mouse intestine.Genes&Development,2016,30(1):52-63.
[21]XIE J M,LI B,YU H P,GAO Q G,LI W,WU H R,QIN Z H.TIGARhas a dual role in cancer cell survival through regulating apoptosis and autophagy.Cancer Research,2014,74(18):5127-5138.
[22]CHEN J,ZHANG D M,FENG X,WANG J,QIN Y Y,ZHANG T,HUANG Q,SHENG R,CHEN Z,LI M,QIN Z H.TIGAR inhibits ischemia/reperfusion-induced inflammatory response of astrocytes.Neuropharmacology,2018,131:377-388.
[23]CHEUNG E C,LUDWIG R L,VOUSDEN K H.Mitochondrial localization of TIGAR under hypoxia stimulates HK2 and lowers ROS and cell death.Proceedings of the National Academy of Sciences of the United States of America,2012,109(50):20491-20496.
[24]FENG J,LUO L,LIU Y,FU S,CHEN J,DUAN X,XIANG L,ZHANG Y,WU J,FAN J,WEN Q,ZHANG Y,YANG J,PENG J,ZHAO M,YANG L.TP53-induced glycolysis and apoptosis regulator is indispensable for mitochondria quality control and degradation following damage.Oncology Letters,2018,15(1):155-160.
[25]KO Y H,DOMINGO-VIDAL M,ROCHE M,LIN Z,WHITAKER-MENEZES D,SEIFERT E,CAPPARELLI C,TULUC M,BIRBE RC,TASSONE P,CURRY J M,NAVARRO-SABATE A,MANZANOA,BARTRONS R,CARO J,MARTINEZ-OUTSCHOORN U.TP53-inducible glycolysis and apoptosis regulator(TIGAR)metabolically reprograms carcinoma and stromal cells in breast cancer.The Journal of Biological Chemistry,2016,291(51):26291-26303.
[26]张腾,袁梅,俞同福.肿瘤中TP53诱导的糖酵解和凋亡调节因子及其靶向治疗研究进展.肿瘤,2016(12):1383-1388.ZHANG T,YUAN M,YU T F.Advances in TP53-induced glycolysis and apoptosis regulator in tumor and its targeted therapy.TUMOR,2016(12):1383-1388.(in Chinese)
[27]顾志冬,倪培华,吴蓓颖,吴华成,樊绮诗,肖家诚.TIGAR基因真核表达载体的构建及在HepG_2细胞中的作用初探.诊断学理论与实践,2009(6):617-622.GU Z D,NI P H,WU B Y,WU H C,FAN Y S,XIAO J C.Construction of TIGAR gene eukaryotic expression vector and effect of TIGAR expression in HepG2 cells.Journal of Diagnostics Concepts&Practice,2009(6):617-622.(in Chinese)
[28]MA T,ZHANG Y,ZHANG C,LUO J G,KONG L Y.Downregulation of TIGAR sensitizes the antitumor effect of physapubenolide through increasing intracellular ROS levels to trigger apoptosis and autophagosome formation in human breast carcinoma cells.Biochemical Pharmacology,2017,143:90-106.
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[2]RAVINDRA P V,TIWARI A K,RATTA B,CHATURVEDI U,PALIAS K,CHAUHAN R S.Newcastle disease virus-induced cytopathic effect in infected cells is caused by apoptosis.Virus Research,2009,141(1):13-20.
[3]KERR J F,WYLLIE A H,CURRIE A R.Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics.British Journal of Cancer,1972,26(4):239-257.
[4]赵明,冯婧,刘勇,杨玲麟.TIGAR调节肺癌细胞的增殖和侵袭能力研究.国际检验医学杂志,2016(06):754-755.ZHAO M,FENG J,LIU Y,YANG L L.TIGAR promotes proliferation and invasiveness of lung cancer cells.International Journal of Laboratory Medicine,2016(06):754-755.(in Chinese)
[5]GREEN D R,CHIPUK J E.p53 and metabolism:Inside the TIGAR.Cell,2006,126(1):30-32.
[6]VOGELSTEIN B,LANE D,LEVINE A J.Surfing the p53 network.Nature,2000,408(6810):307-310.
[7]SHEN M,ZHAO X,ZHAO L,SHI L,AN S,HUANG G,LIU J.Met is involved in TIGAR-regulated metastasis of non-small-cell lung cancer.Molecular Cancer,2018,17(1):88.
[8]ROMEO M,HUTCHISON T,MALU A,WHITE A,KIM J,GARDNER R,SMITH K,NELSON K,BERGESON R,MCKEE R,HARROD C,RATNER L,LUSCHER B,MARTINEZ E,HARRODR.The human T-cell leukemia virus type-1 p30(II)protein activates p53 and induces the TIGAR and suppresses oncogene-induced oxidative stress during viral carcinogenesis.Virology,2018,518(10):3-15.
[9]BENSAAD K,TSURUTA A,SELAK M A,VIDAL M N,NAKANOK,BARTRONS R,GOTTLIEB E,VOUSDEN K H.TIGAR,a p53-inducible regulator of glycolysis and apoptosis.Cell,2006,126(1):107-120.
[10]YU H P,XIE J M,LI B,SUN Y H,GAO Q G,DING Z H,WU H R,QIN Z H.TIGAR regulates DNA damage and repair through pentosephosphate pathway and Cdk5-ATM pathway.Scientific Reports,2015.DOI:10.1038/srep09853
[11]ZHOU J H,ZHANG T T,SONG D D,XIA Y F,QIN Z H,SHENG R.TIGAR contributes to ischemic tolerance induced by cerebral preconditioning through scavenging of reactive oxygen species and inhibition of apoptosis.Scientific Reports,2016.DOI:10.1038/srep27096.
[12]JIANG L B,CAO L,MA Y Q,CHEN Q,LIANG Y,YUAN F L,LI XL,DONG J,CHEN N.TIGAR mediates the inhibitory role of hypoxia on ROS production and apoptosis in rat nucleus pulposus cells.Osteoarthritis and Cartilage,2018,26(1):138-148.
[13]KIM J,DEVALARAJA-NARASHIMHA K,PADANILAM B J.TIGAR regulates glycolysis in ischemic kidney proximal tubules.American Journal of Physiology Renal Physiology,2015,308(4).DOI:10.1152/ajprenal.00459.2014F298-308.
[14]付托.过表达人TIGAR的CHO细胞株构建及其抗凋亡机制研究[D].苏州:苏州大学,2013.FU T.Establishment of CHO cell line overexpressing h TIGAR and mechanism of anti-apoptosis effect[D].Suzhou:Suzhou University,2013.(in Chinese)
[15]HWANG S O,LEE G M.Nutrient deprivation induces autophagy as well as apoptosis in Chinese hamster ovary cell culture.Biotechnology and Bioengineering,2008,99(3):678-685.
[16]WANG H,CHENG Q,LI X,HU F,HAN L,ZHANG H,LI L,GE J,YING X,GUO X,WANG Q.Loss of TIGAR induces oxidative stress and meiotic defects in oocytes from obese mice.Molecular&Cellular Proteomics,2018.DOI:10.1074/mcp.RA118.000620.
[17]LI B,WANG Z,XIE J M,WANG G,QIAN L Q,GUAN X M,SHENX P,QIN Z H,SHEN G H,LI X Q,GAO Q G.TIGAR knockdown enhanced the anticancer effect of aescin via regulating autophagy and apoptosis in colorectal cancer cells.Acta Pharmacologica Sinica,2018.DOI:10.1038/s41401-018-0001-2.
[18]KUMAR B,IQBAL M A,SINGH R K,BAMEZAI R N.Resveratrol inhibits TIGAR to promote ROS induced apoptosis and autophagy.Biochimie,2015,118:26-35.
[19]YE L,ZHAO X,LU J,QIAN G,ZHENG J C,GE S.Knockdown of TIGAR by RNA interference induces apoptosis and autophagy in Hep G2 hepatocellular carcinoma cells.Biochemical and Biophysical Research Communications,2013,437(2):300-306.
[20]CHEUNG E C,LEE P,CETECI F,NIXON C,BLYTH K,SANSOMO J,VOUSDEN K H.Opposing effects of TIGAR-and RAC1-derived ROS on Wnt-driven proliferation in the mouse intestine.Genes&Development,2016,30(1):52-63.
[21]XIE J M,LI B,YU H P,GAO Q G,LI W,WU H R,QIN Z H.TIGARhas a dual role in cancer cell survival through regulating apoptosis and autophagy.Cancer Research,2014,74(18):5127-5138.
[22]CHEN J,ZHANG D M,FENG X,WANG J,QIN Y Y,ZHANG T,HUANG Q,SHENG R,CHEN Z,LI M,QIN Z H.TIGAR inhibits ischemia/reperfusion-induced inflammatory response of astrocytes.Neuropharmacology,2018,131:377-388.
[23]CHEUNG E C,LUDWIG R L,VOUSDEN K H.Mitochondrial localization of TIGAR under hypoxia stimulates HK2 and lowers ROS and cell death.Proceedings of the National Academy of Sciences of the United States of America,2012,109(50):20491-20496.
[24]FENG J,LUO L,LIU Y,FU S,CHEN J,DUAN X,XIANG L,ZHANG Y,WU J,FAN J,WEN Q,ZHANG Y,YANG J,PENG J,ZHAO M,YANG L.TP53-induced glycolysis and apoptosis regulator is indispensable for mitochondria quality control and degradation following damage.Oncology Letters,2018,15(1):155-160.
[25]KO Y H,DOMINGO-VIDAL M,ROCHE M,LIN Z,WHITAKER-MENEZES D,SEIFERT E,CAPPARELLI C,TULUC M,BIRBE RC,TASSONE P,CURRY J M,NAVARRO-SABATE A,MANZANOA,BARTRONS R,CARO J,MARTINEZ-OUTSCHOORN U.TP53-inducible glycolysis and apoptosis regulator(TIGAR)metabolically reprograms carcinoma and stromal cells in breast cancer.The Journal of Biological Chemistry,2016,291(51):26291-26303.
[26]张腾,袁梅,俞同福.肿瘤中TP53诱导的糖酵解和凋亡调节因子及其靶向治疗研究进展.肿瘤,2016(12):1383-1388.ZHANG T,YUAN M,YU T F.Advances in TP53-induced glycolysis and apoptosis regulator in tumor and its targeted therapy.TUMOR,2016(12):1383-1388.(in Chinese)
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