CD47基因对食道癌细胞增殖的影响
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摘要
本研究将CD47-si RNA转染至食道癌细胞,采用蛋白免疫印迹检测食道癌细胞中CD47蛋白的表达,MTT法检测CD47-siRNA转染组和空质粒转染组(对照组)食道癌细胞增殖状态,蛋白免疫印迹检测CD47-siRNA转染组和空质粒转染组(对照组)食道癌细胞中PCNA蛋白表达,DCFDA染色流式细胞仪检测食道癌细胞CD47-siRNA转染组和空质粒转染组(对照组)中ROS水平,以探究CD47基因对食道癌发生发展的影响。研究结果表明,CD47-si RNA转染组食道癌细胞中CD47蛋白明显低于对照组;CD47-siRNA转染组食道癌细胞增殖率显著低于对照组(p<0.05);CD47-siRNA转染组细胞增殖相关蛋白PCNA低于对照组(p<0.01);CD47-siRNA转染组食道癌细胞中ROS水平明显高于对照组(p<0.05)。本研究初步认为:CD47-siRNA可降低食道癌细胞中CD47蛋白表达,抑制食道癌细胞的增殖并增加食道癌细胞中ROS水平。
In this study, CD47-siRNA was transfected into esophageal cancer cells in order to explore the effect of CD47 gene on the occurrence and development of esophageal cancer. The expression of CD47 protein in esophageal cancer cells was detected by western blotting. MTT assay was used to detect the proliferation status of esophageal cancer cells in CD47-si RNA transfection group and empty plasmid transfection group(control group).PCNA protein expression in esophageal cancer cells of CD47-siRNA transfection group and empty plasmid transfection group(control group) was detected by western blotting. The ROS levels in esophageal cancer cells of CD47-siRNA transfection group and the empty plasmid transfection group(control group) were measured by DCFDA staining flow cytometry. The results indicated that CD47-siRNA transfection group had significantly lower CD47 protein in esophageal cancer cells than the control group. The proliferation rate of esophageal cancer cells in CD47-siRNA transfection group was significantly lower than that in the control group(p<0.05). The PCNA of cell proliferation-related proteins in CD47-siRNA transfection group was lower than that of the control group(p<0.01). The ROS levels in esophageal cancer cells in CD47-siRNA transfection group were significantly higher than the control group(p<0.05). The study preliminarily concluded that CD47-siRNA could reduce the expression of CD47 protein in esophageal cancer cells, which resulted in inhibiting the proliferation of esophageal cancer cells as well as increasing the ROS levels in esophageal cancer cells.
In this study, CD47-siRNA was transfected into esophageal cancer cells in order to explore the effect of CD47 gene on the occurrence and development of esophageal cancer. The expression of CD47 protein in esophageal cancer cells was detected by western blotting. MTT assay was used to detect the proliferation status of esophageal cancer cells in CD47-si RNA transfection group and empty plasmid transfection group(control group).PCNA protein expression in esophageal cancer cells of CD47-siRNA transfection group and empty plasmid transfection group(control group) was detected by western blotting. The ROS levels in esophageal cancer cells of CD47-siRNA transfection group and the empty plasmid transfection group(control group) were measured by DCFDA staining flow cytometry. The results indicated that CD47-siRNA transfection group had significantly lower CD47 protein in esophageal cancer cells than the control group. The proliferation rate of esophageal cancer cells in CD47-siRNA transfection group was significantly lower than that in the control group(p<0.05). The PCNA of cell proliferation-related proteins in CD47-siRNA transfection group was lower than that of the control group(p<0.01). The ROS levels in esophageal cancer cells in CD47-siRNA transfection group were significantly higher than the control group(p<0.05). The study preliminarily concluded that CD47-siRNA could reduce the expression of CD47 protein in esophageal cancer cells, which resulted in inhibiting the proliferation of esophageal cancer cells as well as increasing the ROS levels in esophageal cancer cells.
引文
Chao M.P.,Alizadeh A.A.,Tang C.,Myklebust J.H.,Varghese B.,Gill S.,Jan M.,Cha A.C.,Chan C.K.,Tan B.T.,Park C.Y.,Zhao F.,Kohrt H.E.,Malumbres R.,Briones J.,Gascoyne R.D.,Lossos I.S.,Levy R.,Weissman I.L.,and Majeti R.,2010,Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma,Cell,142(5):699-713
Fujimoto T.T.,Katsutani S.,Shimomura T.,and Fujimura K.,2003,Thrombospondin-bound integrin-associated protein(CD47)physically and functionally modifies integrin alphaI-Ibbeta3 by its extracellular domain,J.Biol.Chem.,278(29):26655-26665
Gervai J.Z.,Galicza J.,Szeltner Z.,Zámborszky J.,and Szüts D.,2017,A genetic study based on PCNA-ubiquitin fusions reveals no requirement for PCNA polyubiquitylation in DNAdamage tolerance,DNA Repair(Amst),54:46-54
Guo E.L.,Zhang J.F.,Yang Y.N.,Miao S.S.,Zhang L.Q.,and Ma J.Q.,2018,Advances in tumor markers of esophageal cancer,Xiandai Zhongliu Yixue(Journal of Modern Oncology),(11):1783-1786(郭二亮,张金峰,杨英男,苗素生,张鲁权,马建群,2018,食管癌肿瘤标志物的研究进展,现代肿瘤医学,(11):1783-1786)
He Y.T.,Hou J.,Chen Z.F.,Qiao C.Y.,Song G.H.,Meng F.S.,Jin H.X.,and Chen C.,2008,Trends in incidence of esophageal and gastric cardia cancer in high-risk areas in China,Eur.J.Cancer Prev.,17(2):71-76
Jaiswal S.,Jamieson C.H.,Pang W.W.,Park C.Y.,Chao M.P.,Majeti R.,Traver D.,van Rooijen N.,and Weissman I.L.,2009,CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis,Cell,138(2):271-285
Kang Y.,and Yin S.M.,2003,Research on physiological function of CD47,Zhongguo Shiyan Xueyexue Zazhi(Journal of Experimental Hematology),11(4):437-440(康颖,尹松梅,2003,CD47生理功能的研究,中国实验血液学杂志,11(4):437-440)
Leclair P.,Liu C.C.,Monajemi M.,Reid G.S.,Sly L.M.,and Lim C.J.,2018,CD47-ligation induced cell death in T-acute lymphoblastic leukemia,Cell Death Dis.,9(5):544
Liang Z.,Sun Z.Y.,Yuan Y.H.,Han Y.,Wang Y.,Gu J.,and Zhang L.J.,2005,The expression of 11 cancer/testis(CT)antigen genes in esophageal carcinoma,Chinese Journal of Oncology,27(9):534-537
Majeti R.,Chao M.P.,Alizadeh A.A.,Pang W.W.,Jaiswal S.,Gibbs K.D.,van Rooijen N.,and Weissman I.L.,2009,CD47 is an adverse prognostic factor and therapeutic anti body target on human acute myeloid leukemia stem cells,Cell,138(2):286-299
Olsson M.,Bruhns P.,Frazier W.A.,Ravetch J.V.,and Oldenborg P.A.,2005,Platelet homeostasis is regulated by platelet expression of CD47 under normal conditions and in passive immune thrombocytopenia,Blood,105(9):3577-3582
Pettersen R.D.,Hestdal K.,Olafsen M.K.,Lie S.O.,and Lindberg F.P.,1999,CD47 signals T cell death,J.Immunol.,162(12):7031-7040
Saini V.,Manral A.,Arora R.,Meena P.,Gusain S.,Saluja D.,and Tiwari M.,2017,Novel synthetic analogs of diallyl disulfide triggers cell cycle arrest and apoptosis via ROSgeneration in MIA PaCa-2 cells,Pharmacol.Rep.,69(4):813-821
Willingham S.B.,Volkmer J.P.,Gentles A.J.,Sahoo D.,Dalerba P.,Mitra S.S.,Wang J.,Contreras-Trujillo H.,Martin R.,Cohen J.D.,Lovelace P.,Scheeren F.A.,Chao M.P.,Weiskopf K.,Tang C.,Volkmer A.K.,Naik T.J.,Storm T.A.,Mosley A.R.,Edris B.,Schmid S.M.,Sun C.K.,Chua M.S.,Murillo O.,Rajendran P.,Cha A.C.,Chin R.K.,Kim D.,Adorno M.,Raveh T.,Tseng D.,Jaiswal S.,Enger P.諂.,Steinberg G.K.,Li G.,So S.K.,Majeti R.,Harsh G.R.,van de Rijn M.,Teng N.N.,Sunwoo J.B.,Alizadeh A.A.,Clarke M.F.,and Weissman I.L.,2012,The CD47-signal regulatory protein alpha(SIRPa)interaction is a therapeutic target for human solid tumors,Proc.Natl.Acad.Sci.USA,109(17):6662-6667
Wu X.J.,Kassie F.,and Mersch-Sundermann V.,2005,The role of reactive oxygen species(ROS)production on diallyl disulfide(DADS)induced apoptosis and cell cycle arrest in human A549 lung carcinoma cells,Mutat.Res.,579(1-2):115-124
Xu L.J.,Yu X.J.,Wei B.,Hui H.X.,Sun Y.,Dai J.,and Chen X.F.,2018,LncRNA SNHG7 promotes the proliferation of esophageal cancer cells and inhibits its apoptosis,Eur.Rev.Med.Pharmacol.Sci.,22(9):2653-2661
Zhang S.W.,Zhang M.,Li G.L.,Wei W.Q.,Meng F.S.,Liu Z.C.,Zheng G.S.,Li N.,and Chen W.Q.,2012,An analysis of incidence and mortality of esophageal cancer in China,2003~2007,Zhongguo Zhongliu(China Cancer),21(4):241-247(张思维,张敏,李光琳,魏文强,孟凡书,刘志才,郑荣寿,李霓,陈万青,2012,2003~2007年中国食管癌发病与死亡分析,中国肿瘤,21(4):241-247)
Zhang Y.,Peng X.,and Zheng Q.,2018,Ropivacaine inhibits the migration of esophageal cancer cells via sodium-channel-independent but prenylation-dependent inhibition of Rac1/JNK/paxillin/FAK,Biochem.Biophys.Res.Commun.,501(4):1074-1079
Fujimoto T.T.,Katsutani S.,Shimomura T.,and Fujimura K.,2003,Thrombospondin-bound integrin-associated protein(CD47)physically and functionally modifies integrin alphaI-Ibbeta3 by its extracellular domain,J.Biol.Chem.,278(29):26655-26665
Gervai J.Z.,Galicza J.,Szeltner Z.,Zámborszky J.,and Szüts D.,2017,A genetic study based on PCNA-ubiquitin fusions reveals no requirement for PCNA polyubiquitylation in DNAdamage tolerance,DNA Repair(Amst),54:46-54
Guo E.L.,Zhang J.F.,Yang Y.N.,Miao S.S.,Zhang L.Q.,and Ma J.Q.,2018,Advances in tumor markers of esophageal cancer,Xiandai Zhongliu Yixue(Journal of Modern Oncology),(11):1783-1786(郭二亮,张金峰,杨英男,苗素生,张鲁权,马建群,2018,食管癌肿瘤标志物的研究进展,现代肿瘤医学,(11):1783-1786)
He Y.T.,Hou J.,Chen Z.F.,Qiao C.Y.,Song G.H.,Meng F.S.,Jin H.X.,and Chen C.,2008,Trends in incidence of esophageal and gastric cardia cancer in high-risk areas in China,Eur.J.Cancer Prev.,17(2):71-76
Jaiswal S.,Jamieson C.H.,Pang W.W.,Park C.Y.,Chao M.P.,Majeti R.,Traver D.,van Rooijen N.,and Weissman I.L.,2009,CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis,Cell,138(2):271-285
Kang Y.,and Yin S.M.,2003,Research on physiological function of CD47,Zhongguo Shiyan Xueyexue Zazhi(Journal of Experimental Hematology),11(4):437-440(康颖,尹松梅,2003,CD47生理功能的研究,中国实验血液学杂志,11(4):437-440)
Leclair P.,Liu C.C.,Monajemi M.,Reid G.S.,Sly L.M.,and Lim C.J.,2018,CD47-ligation induced cell death in T-acute lymphoblastic leukemia,Cell Death Dis.,9(5):544
Liang Z.,Sun Z.Y.,Yuan Y.H.,Han Y.,Wang Y.,Gu J.,and Zhang L.J.,2005,The expression of 11 cancer/testis(CT)antigen genes in esophageal carcinoma,Chinese Journal of Oncology,27(9):534-537
Majeti R.,Chao M.P.,Alizadeh A.A.,Pang W.W.,Jaiswal S.,Gibbs K.D.,van Rooijen N.,and Weissman I.L.,2009,CD47 is an adverse prognostic factor and therapeutic anti body target on human acute myeloid leukemia stem cells,Cell,138(2):286-299
Olsson M.,Bruhns P.,Frazier W.A.,Ravetch J.V.,and Oldenborg P.A.,2005,Platelet homeostasis is regulated by platelet expression of CD47 under normal conditions and in passive immune thrombocytopenia,Blood,105(9):3577-3582
Pettersen R.D.,Hestdal K.,Olafsen M.K.,Lie S.O.,and Lindberg F.P.,1999,CD47 signals T cell death,J.Immunol.,162(12):7031-7040
Saini V.,Manral A.,Arora R.,Meena P.,Gusain S.,Saluja D.,and Tiwari M.,2017,Novel synthetic analogs of diallyl disulfide triggers cell cycle arrest and apoptosis via ROSgeneration in MIA PaCa-2 cells,Pharmacol.Rep.,69(4):813-821
Willingham S.B.,Volkmer J.P.,Gentles A.J.,Sahoo D.,Dalerba P.,Mitra S.S.,Wang J.,Contreras-Trujillo H.,Martin R.,Cohen J.D.,Lovelace P.,Scheeren F.A.,Chao M.P.,Weiskopf K.,Tang C.,Volkmer A.K.,Naik T.J.,Storm T.A.,Mosley A.R.,Edris B.,Schmid S.M.,Sun C.K.,Chua M.S.,Murillo O.,Rajendran P.,Cha A.C.,Chin R.K.,Kim D.,Adorno M.,Raveh T.,Tseng D.,Jaiswal S.,Enger P.諂.,Steinberg G.K.,Li G.,So S.K.,Majeti R.,Harsh G.R.,van de Rijn M.,Teng N.N.,Sunwoo J.B.,Alizadeh A.A.,Clarke M.F.,and Weissman I.L.,2012,The CD47-signal regulatory protein alpha(SIRPa)interaction is a therapeutic target for human solid tumors,Proc.Natl.Acad.Sci.USA,109(17):6662-6667
Wu X.J.,Kassie F.,and Mersch-Sundermann V.,2005,The role of reactive oxygen species(ROS)production on diallyl disulfide(DADS)induced apoptosis and cell cycle arrest in human A549 lung carcinoma cells,Mutat.Res.,579(1-2):115-124
Xu L.J.,Yu X.J.,Wei B.,Hui H.X.,Sun Y.,Dai J.,and Chen X.F.,2018,LncRNA SNHG7 promotes the proliferation of esophageal cancer cells and inhibits its apoptosis,Eur.Rev.Med.Pharmacol.Sci.,22(9):2653-2661
Zhang S.W.,Zhang M.,Li G.L.,Wei W.Q.,Meng F.S.,Liu Z.C.,Zheng G.S.,Li N.,and Chen W.Q.,2012,An analysis of incidence and mortality of esophageal cancer in China,2003~2007,Zhongguo Zhongliu(China Cancer),21(4):241-247(张思维,张敏,李光琳,魏文强,孟凡书,刘志才,郑荣寿,李霓,陈万青,2012,2003~2007年中国食管癌发病与死亡分析,中国肿瘤,21(4):241-247)
Zhang Y.,Peng X.,and Zheng Q.,2018,Ropivacaine inhibits the migration of esophageal cancer cells via sodium-channel-independent but prenylation-dependent inhibition of Rac1/JNK/paxillin/FAK,Biochem.Biophys.Res.Commun.,501(4):1074-1079