E6-AP基因在乳腺癌MDA-MB-231细胞中调节膜联蛋白A2的表达
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摘要
目的检测E6-AP基因及膜联蛋白A2(Annexin A2)在乳腺癌MDA-MB-231细胞中的表达,探讨其对癌细胞增殖、凋亡及浸润转移的影响。方法设计1条无关序列Negative-siRNA作为阴性对照组和针对E6-AP基因的3条特异性E6-AP-siRNAs片段转染至MDA-MB-231细胞内作为实验组,未经处理细胞作为空白对照组,加入脂质体处理的细胞为脂质体组,利用RT-PCR检测干扰E6-AP后在MDA-MB-231细胞中E6-AP和Annexin A2 mRNA相对表达水平。选择出转染效率最高的E6-APsiRNA1组及阴性对照组、空白对照组继续行后续实验。Western blot检测干扰E6-AP后E6-AP和Annexin A2在MDA-MB-231细胞中的蛋白的相对表达水平。利用CCK-8试剂盒法、流式细胞术、Transwell小室侵袭实验分别检测干扰E6-AP后MDA-MB-231细胞的增殖、凋亡、侵袭能力。基因的mRNA及蛋白表达水平、细胞凋亡率及细胞数的组间比较采用方差分析,两两比较采用LSD法,吸光度比较采用重复测量的方差分析。结果转染72 h后,E6-AP基因干扰后各实验组(E6-AP-siRNA1组、E6-AP-siRNA2组、E6-AP-siRNA3组)及空白对照组、阴性对照组及脂质体组中的E6-AP mRNA相对表达水平分别为0.159±0.003、0.325±0.006、0.229±0.007、0.593±0.031、0.594±0.012、0.612±0.016,Annexin A2 mRNA相对表达水平分别为0.929±0.017、1.013±0.082、0.992±0.024、1.341±0.037、1.323±0.010、1.326±0.012,差异均有统计学意义(F=850.792、417.447,P均<0.050)。转染72 h后,E6-AP-siRNA1组、空白对照组和阴性对照组中E6-AP及Annexin A2蛋白相对表达水平为分别为0.271±0.017、0.492±0.018、0.477±0.016及0.447±0.034、0.887±0.022、0.849±0.033,组间差异均有统计学意义(F=256.850、350.149,P均<0.050)。转染24、48、72、96 h后,E6-AP-siRNA1组、阴性对照组和空白对照组间比较,不同时间点之间比较,细胞吸光度差异均有统计学意义(F=524.828,P<0.001;F=904.079,P<0.001);分组与时间点存在交互作用(F=28.116,P<0.001)。转染72 h后,空白对照组、阴性对照组、E6-AP-siRNA1组的凋亡率分别为2.959±0.117、3.097±0.070、10.812±0.199,组间差异有统计学意义(F=3110.005,P<0.050)。Transwell检测E6-AP-siRNA1组、空白对照组、阴性对照组中细胞穿透Matrigel胶到达Transwell下室的细胞数分别为99±5、96±6、62±7,组间差异有统计学意义(F=55.404,P<0.001)。结论干扰E6-AP基因可使Annexin A2表达下调,同时可诱导MDA-MB-231细胞的凋亡,其增殖、侵袭能力也受到抑制。
Objective To study the expressions of E6-AP gene and Annexin A2 in breast cancer MDA-MB-231 cells,and explore their effects on the proliferation,apoptosis and invasion and metastasis of cancer cells. Methods Negative-siRNA was transfected into MDA-MB-231 cells as negative control,3 designed E6-AP-siRNA sequences were transfected into MDA-MB-231 cells as experimental groups,the untreated cells served as blank control and the cells with liposome treatment served as liposome group. The mRNA expressions of E6-AP and Annexin A2 in MDA-MB-231 cells were detected by RT-PCR after E6-AP interference. The E6-AP-siRNA1 group with the highest transfection efficiency,along with negative control group and blank control group,was selected for the following experiments. The protein expressions of E6-AP and Annexin A2 in MDA-MB-231 cells after E6-AP interference were detected by Western blot. The proliferation,apoptosis and invasion of MDA-MB-231 cells were detected by CCK-8 kit,flow cytometry and Transwell assay respectively after E6-AP interference. Comparison between groups was performed using analysis of variance, pairwise comparison using LSD method. The optical density was compared by repeated measurement analysis of variance. Results At 72 h after transfection,E6-AP mRNA expressions in E6-APsiRNA1 group,E6-AP-siRNA2 group,E6-AP-siRNA3 group,blank control group,negative control group and liposome group were 0. 159 ± 0. 003,0. 325 ± 0. 006,0. 229 ± 0. 007,0. 593 ± 0. 031,0. 594 ± 0. 012,0. 612± 0. 016 respectively,Annexin A2 mRNA expressions were 0. 929 ± 0. 017,1. 013 ± 0. 082,0. 992 ± 0. 024,1. 341 ± 0. 037,1. 323 ± 0. 010,1. 326 ± 0. 012 respectively,indicating statistically significant differences( F= 850. 792,417. 447,both P < 0. 050). E6-AP and Annexin A2 protein expressions in E6-AP-siRNA1 group,blank control group and negative control group were 0. 271 ± 0. 017,0. 492 ± 0. 018,0. 477 ± 0. 016 and 0. 447 ± 0. 034,0. 887 ± 0. 022,0. 849 ± 0. 033, respectively, indicating statistically significant differences( F = 256. 850,350. 149,both P < 0. 050). There was a significant difference in optical density among E6-AP-siRNA1 group,negative control group and blank control group at 24,48,72,96 h after the transfection( F = 524. 828,P < 0. 001),and there was also a significant difference in optical density among the different time points( F = 904. 079,P < 0. 001). There was an interaction between the grouping and the time points( F = 28. 116,P < 0. 001). The apoptosis rates in blank control group,negative control group and E6-AP-siRNA1 group were 2. 959 ± 0. 117,3. 097 ± 0. 070 and 10. 812 ± 0. 199 respectively, and the difference was significant among groups( F = 3110. 005,P < 0. 050). The number of cells which went through Matrigel gel into lower Transwell chamber in E6-AP-siRNA1 group,blank control group and negative control group was 99 ± 5,96 ± 6,62 ± 7,respectively,suggesting a significant difference among groups( F = 55. 404,P< 0. 001). Conclusion Interference with E6-AP gene may down-regulate the expression of Annexin A2,induce the apoptosis of MDA-MB-231 cells,inhibit the proliferation and invasion ability of MDA-MB-231 cells.
Objective To study the expressions of E6-AP gene and Annexin A2 in breast cancer MDA-MB-231 cells,and explore their effects on the proliferation,apoptosis and invasion and metastasis of cancer cells. Methods Negative-siRNA was transfected into MDA-MB-231 cells as negative control,3 designed E6-AP-siRNA sequences were transfected into MDA-MB-231 cells as experimental groups,the untreated cells served as blank control and the cells with liposome treatment served as liposome group. The mRNA expressions of E6-AP and Annexin A2 in MDA-MB-231 cells were detected by RT-PCR after E6-AP interference. The E6-AP-siRNA1 group with the highest transfection efficiency,along with negative control group and blank control group,was selected for the following experiments. The protein expressions of E6-AP and Annexin A2 in MDA-MB-231 cells after E6-AP interference were detected by Western blot. The proliferation,apoptosis and invasion of MDA-MB-231 cells were detected by CCK-8 kit,flow cytometry and Transwell assay respectively after E6-AP interference. Comparison between groups was performed using analysis of variance, pairwise comparison using LSD method. The optical density was compared by repeated measurement analysis of variance. Results At 72 h after transfection,E6-AP mRNA expressions in E6-APsiRNA1 group,E6-AP-siRNA2 group,E6-AP-siRNA3 group,blank control group,negative control group and liposome group were 0. 159 ± 0. 003,0. 325 ± 0. 006,0. 229 ± 0. 007,0. 593 ± 0. 031,0. 594 ± 0. 012,0. 612± 0. 016 respectively,Annexin A2 mRNA expressions were 0. 929 ± 0. 017,1. 013 ± 0. 082,0. 992 ± 0. 024,1. 341 ± 0. 037,1. 323 ± 0. 010,1. 326 ± 0. 012 respectively,indicating statistically significant differences( F= 850. 792,417. 447,both P < 0. 050). E6-AP and Annexin A2 protein expressions in E6-AP-siRNA1 group,blank control group and negative control group were 0. 271 ± 0. 017,0. 492 ± 0. 018,0. 477 ± 0. 016 and 0. 447 ± 0. 034,0. 887 ± 0. 022,0. 849 ± 0. 033, respectively, indicating statistically significant differences( F = 256. 850,350. 149,both P < 0. 050). There was a significant difference in optical density among E6-AP-siRNA1 group,negative control group and blank control group at 24,48,72,96 h after the transfection( F = 524. 828,P < 0. 001),and there was also a significant difference in optical density among the different time points( F = 904. 079,P < 0. 001). There was an interaction between the grouping and the time points( F = 28. 116,P < 0. 001). The apoptosis rates in blank control group,negative control group and E6-AP-siRNA1 group were 2. 959 ± 0. 117,3. 097 ± 0. 070 and 10. 812 ± 0. 199 respectively, and the difference was significant among groups( F = 3110. 005,P < 0. 050). The number of cells which went through Matrigel gel into lower Transwell chamber in E6-AP-siRNA1 group,blank control group and negative control group was 99 ± 5,96 ± 6,62 ± 7,respectively,suggesting a significant difference among groups( F = 55. 404,P< 0. 001). Conclusion Interference with E6-AP gene may down-regulate the expression of Annexin A2,induce the apoptosis of MDA-MB-231 cells,inhibit the proliferation and invasion ability of MDA-MB-231 cells.
引文
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[12]Hajjar KA.The biology of annexin A2:from vascular fibrinolysis to innate immunity[J].Trans Am Clin Climatol Assoc,2015,126:144-155.
[13]Lauritzen SP,Boye TL,Nylandsted J.Annexins are instrumental for efficient plasma membrane repair in cancer cells[J].Semin Cell Dev Biol,2015,45:32-38.
[14]Jeon YR,Kim SY,Lee EJ,et al.Identification of annexin II as a novel secretory biomarker for breast cancer[J].Proteomics,2013,13(21):3145-3156.
[15]Deng S,Jing B,Xing T,et al.Overexpression of annexin A2 is associated with abnormal ubiquitination in breast cancer[J].Genomics Proteomics Bioinformatics,2012,10(3):153-157.
[16]Wolyniec K,Shortt J,de Stanchina E,et al.E6AP ubiquitin ligase regulates PML-induced senescence in Myc-driven lymphomagenesis[J].Blood,2012,120(4):822-832.
[17]Lochab S,Pal P,Kanaujiya JK,et al.Proteomic identification of E6AP as a molecular target of tamoxifen in MCF7 cells[J].Proteomics,2012,12(9):1363-1377.
[18]侯令密,谢少利,陈茂山,等.E6相关蛋白调节膜联蛋白A2的表达对三阴性乳腺癌裸鼠移植瘤的影响[J/CD].中华妇幼临床医学杂志:电子版,2016,12(30):274-279.
[2]SalomèM,Campos J,Keeshan K.TRIB2 and the ubiquitin proteasome system in cancer[J].Biochem Soc Trans,2015,43(5):1089-1094.
[3]Ando H,Wen ZM,Kim HY,et al.Intracellular composition of fatty acid affects the processing and function of tyrosinase through the ubiquitin-proteasome pathway[J].Biochem J,2006,394(Pt 1):43-50.
[4]Mattern MR,Wu J,Nicholson B.Ubiquitin-based anticancer therapy:carpet bombing with proteasome inhibitors vs surgical strikes with E1,E2,E3,or DUB inhibitors[J].Biochim Biophys Acta,2012,1823(11):2014-2021.
[5]Hillemanns P,Jentschke M,Evans TG,et al.Detection of E6-AP as a potential therapeutic target in cervical specimen of HPV-infected women[J].Arch Gynecol Obstet,2014,289(6):1281-1286.
[6]Deng S,Jing B,Xing T,et al.Overexpression of annexin A2 is associated with abnormal ubiquitination in breast cancer[J].Genomics Proteomics Bioinformatics,2012,10(3):153-157.
[7]Deng S,Wang J,Hou L,et al.Annexin A1,A2,A4 and A5 play important roles in breast cancer,pancreatic cancer and laryngeal carcinoma,alone and/or synergistically[J].Oncol Lett,2013,5(1):107-112.
[8]Deng S,Huang C.E3 ubiquitin ligases in regulating stress fiber,lamellipodium,and focal adhesion dynamics[J].Cell Adh Migr,2014,8(1):49-54.
[9]Zhang J,Song R,Li Y,et al.Integration of microarray profiles associated with cardiomyopathy and the potential role of E6-AP in apoptosis[J].Mol Med Rep,2014,9(2):621-625.
[10]Lokman NA,Ween MP,Oehler MK,et al.The role of Annexin A2 in tumorigenesis and cancer progression[J].Cancer Microenviron,2011,4(2):199-208.
[11]Li PG,Yang YL,Ge YT.Roles of annexin A2 for the regulation of the growth and cytoskeleton of tumor cells[J].Sheng Li Ke Xue Jin Zhan,2010,41(6):457-460.
[12]Hajjar KA.The biology of annexin A2:from vascular fibrinolysis to innate immunity[J].Trans Am Clin Climatol Assoc,2015,126:144-155.
[13]Lauritzen SP,Boye TL,Nylandsted J.Annexins are instrumental for efficient plasma membrane repair in cancer cells[J].Semin Cell Dev Biol,2015,45:32-38.
[14]Jeon YR,Kim SY,Lee EJ,et al.Identification of annexin II as a novel secretory biomarker for breast cancer[J].Proteomics,2013,13(21):3145-3156.
[15]Deng S,Jing B,Xing T,et al.Overexpression of annexin A2 is associated with abnormal ubiquitination in breast cancer[J].Genomics Proteomics Bioinformatics,2012,10(3):153-157.
[16]Wolyniec K,Shortt J,de Stanchina E,et al.E6AP ubiquitin ligase regulates PML-induced senescence in Myc-driven lymphomagenesis[J].Blood,2012,120(4):822-832.
[17]Lochab S,Pal P,Kanaujiya JK,et al.Proteomic identification of E6AP as a molecular target of tamoxifen in MCF7 cells[J].Proteomics,2012,12(9):1363-1377.
[18]侯令密,谢少利,陈茂山,等.E6相关蛋白调节膜联蛋白A2的表达对三阴性乳腺癌裸鼠移植瘤的影响[J/CD].中华妇幼临床医学杂志:电子版,2016,12(30):274-279.