Pim-2通过NF-κB通路激活API-5抑制肝癌细胞凋亡
摘要
目的:探讨肝癌细胞中癌基因Pim-2及其可能的下游因子NF-κB与API-5三者之间的关系,初步明确肝癌细胞中Pim-2抗凋亡的信号通路,证实Pim-2可通过NF-κB通路激活API-5,从而抑制肝癌细胞凋亡、促进肝癌的发生发展。
方法:(1)收集临床手术切除的肝细胞癌组织及相应癌旁组织各27例,以同期行非肿瘤性肝叶切除术的正常肝组织11例为对照,设置临床标本实验组共3组:肝细胞癌组织组、癌旁组织组和正常肝组织组(。2)通过Real-time PCR检测上述各组标本中Pim-2、NF-κB和API-5的mRNA水平;通过免疫组织化学和Western blotting分别检测上述各组标本中Pim-2、tAPI-5和pAPI-5的蛋白水平;通过EMSA检测上述各组标本中NF-κB的活性度。(3)构建携带人Pim-2基因的真核表达质粒,并将其转染入非肿瘤性人肝细胞L02细胞株,以未转染细胞及转染空载体的细胞为对照;设计针对Pim-2的特异性SiRNA,导入真核表达载体pGenesil-1,并将其转染入人肝癌细胞HepG2细胞株,以未转染细胞和转染空载体的细胞为对照(前期完成)。(4)设置细胞实验组共8组:非肿瘤性人肝细胞L02细胞组、转染Pim-2的L02细胞组、转染空载体的L02细胞组、人肝癌细胞HepG2细胞组、转染Pim-2 SiRNA的HepG2细胞组、转染空载体的HepG2细胞组、L02/Pim-2+银胶菊内酯(NF-κB信号通路特异性抑制剂)组、HepG2+银胶菊内酯组(。5)通过Real-time PCR检测上述各组细胞中Pim-2,NF-κB和API-5的mRNA水平;通过Western blotting检测上述各组细胞中Pim-2,tAPI-5和pAPI-5的蛋白水平;通过EMSA检测上述各组细胞核蛋白中NF-κB的活性度;通过AnnexinV-FITC/PI双染法检测上述各组细胞的凋亡率。
结果:(1)临床标本实验发现:肝癌组织中Pim-2、NF-κB和API-5的mRNA水平较癌旁组织和正常肝组织中显著增高(p<0.05);肝癌组织中Pim-2、tAPI-5和pAPI-5蛋白呈阳性表达,而癌旁组织和正常肝组织中呈阴性表达;肝癌组织中Pim-2、tAPI-5和pAPI-5的蛋白水平较癌旁组织和正常肝组织中显著增高(p<0.05);肝癌组织中NF-κB活性度较癌旁组织和正常肝组织中显著增高(p<0.05)。(2)细胞实验发现:转染Pim-2的L02细胞中Pim-2的mRNA和蛋白水平较对照组显著增高(p<0.05);转染Pim-2 SiRNA的HepG2细胞中Pim-2的mRNA和蛋白水平较对照组显著降低(p<0.05)。在各实验组细胞中,NF-κB和API-5的mRNA水平随着Pim-2 mRNA水平的变化而平行变化;tAPI-5和pAPI-5的蛋白水平及NF-κB活性度也随着Pim-2蛋白水平的变化而平行变化。在高表达Pim-2的L02/Pim-2细胞和HepG2细胞中加入NF-κB特异性抑制剂银胶菊内酯后,Pim-2 mRNA及蛋白表达无显著变化,但其中NF-κB、API-5的mRNA水平,pAPI-5和tAPI-5的蛋白水平及NF-κB活性度均显著降低(p<0.05)。各实验组细胞凋亡率与Pim-2的表达水平无直接关系,而与pAPI-5的水平呈反向平行变化趋势。
结论:(1)临床标本实验提示:Pim-2、NF-κB和API-5可能是与肝癌发生发展相关的某信号通路上的相关因子。(2)细胞实验进一步证实:Pim-2可通过磷酸化作用激活API-5从而抑制肝癌细胞凋亡,而NF-κB可能是该信号通路的关键调节因子。
Objective: To explore the relationship among oncogene Pim-2 and its possible downstream factors NF-κB and API-5, so as to illuminate the detailed anti-apoptosis mechanism of Pim-2 in hepatocarcinoma cells, and finally confirm that Pim-2 could activate apoptotic protein API-5 to inhibit the apoptosis of hepatocarcinama cells through NF-κB pathway.
Methods: (1) 27 hepatocellular carcinoma tissues (HCC) and 27 corresponding paired noncancerous liver tissues (PNL) were obtained from the surgery specimen in the second affiliated hospital of Chongqing medical university (HCC were diagnosed by pathology. PNL were defined as liver tissues 2cm away from the edge of carcinoma). 11 normal liver tissues (NL) were obtained from non liver disease volunteer patients who receive upper abdominal surgery in the same period in the hospital. Then three experimental groups were set up in the clinical specimen experiment: HCC, PNL and NL. (2) The mRNA levels of Pim-2, NF-κB and API-5 in the tissues were detected by real-time PCR; the protein levels of Pim-2, total API-5 (tAPI-5) and phosphorylated API-5 (pAPI-5) were detected by immunohistochemistry and western blotting; and the NF-κB activity were detected by EMSA. (3) Eukaryotic expression plasmid carrying human Pim-2 gene was transfected into the nontumorous human liver cell line L02. L02 cells transfected with empty vector and the ones without transfection were set up as controls. Pim-2 specific SiRNA were designed and transfected into human liver cancer cell line HepG2. Also, HepG2 cells transfected with empty vector and the ones without transfection were set up as controls (completed in our prior work). (4) Eight experimental groups were set up in the cell experiment: nontumorous human liver cell line (L02), L02 cells transfected with Pim-2 gene (L02/Pim-2), L02 cells transfected with empty vector (L02/Vector), human liver cancer cell line (HepG2), HepG2 cells transfected with Pim-2 SiRNA (HepG2/Pim-2 SiRNA), HepG2 cells transfected with empty vector (HepG2/Vector), L02/Pim-2 cells cultured with NF-κB specific inhibitor parthenolide (L02/Pim-2+parthenolide), and HepG2 cells cultured with parthenolide (HepG2+parthenolide). (5) The mRNA levels of Pim-2, NF-κB and API-5 in the cells were detected by real-time PCR; the protein levels of Pim-2, tAPI-5 and pAPI-5 were detected by western blotting; the NF-κB activity were detected by EMSA; and the cell apoptosis rate were detected by AnnexinV-FITC/PI double staining method.
Results: (1) In the clinical specimen experiment, the mRNA levels of Pim-2, NF-κB and API-5 in HCC group were significantly higher than those in PNL and NL groups (p<0.05); the protein expression of Pim-2, tAPI-5 and pAPI-5 were positive in HCC group but negative in PNL and NL groups; the protein levels of Pim-2, tAPI-5 and pAPI-5 in HCC group were significantly higher than those in PNL and NL groups (p<0.05); the activity of NF-κB in HCC group was significantly higher than those in PNL and NL groups (p<0.05). (2) In the cell experiment, after the transfection of Pim-2 gene, the mRNA and protein levels of Pim-2 were significantly higher in L02/Pim-2 cells than those in L02 cells and L02/vector cells (p<0.05). After the transfection of Pim-2 SiRNA, the mRNA and protein levels of Pim-2 were significantly lower in HepG2/Pim-2 SiRNA cells than those in HepG2 cells and HepG2/vector cells (p<0.05). In these cell groups, the mRNA levels of NF-κB and API-5 were parallel to the mRNA level of Pim-2. The protein levels of tAPI-5 and pAPI-5, as well as the activity of NF-κB, were parallel to the protein level of Pim-2. When parthenolide were added in L02/Pim-2 cells and HepG2 cells which highly expressed Pim-2, the mRNA and protein levels of Pim-2 had no significant changes, while the mRNA levels of NF-κB and API-5, the protein levels of tAPI-5 and pAPI-5, as well as the activity of NF-κB, were all significantly decreased (p<0.05). It was not the Pim-2 but pAPI-5 levels that affected the cell apoptosis rates directly. The cell apoptosis rates were antiparallel to the level of pAPI-5 in all the cell groups.
Conclusions: (1) From the clinical specimen experiments, we could infer that Pim-2, NF-κB and API-5 may be related factors in some signal transduction pathway which is associated with the genesis and deveiopment of liver cancer. (2) The cell experiments convince that Pim-2 could activate API-5 through phosphorylation mechanism so as to inhibit the apoptosis of hepatocarcinoma cells, and NF-κB may be the key regulating factor on this signal transduction pathway.
方法:(1)收集临床手术切除的肝细胞癌组织及相应癌旁组织各27例,以同期行非肿瘤性肝叶切除术的正常肝组织11例为对照,设置临床标本实验组共3组:肝细胞癌组织组、癌旁组织组和正常肝组织组(。2)通过Real-time PCR检测上述各组标本中Pim-2、NF-κB和API-5的mRNA水平;通过免疫组织化学和Western blotting分别检测上述各组标本中Pim-2、tAPI-5和pAPI-5的蛋白水平;通过EMSA检测上述各组标本中NF-κB的活性度。(3)构建携带人Pim-2基因的真核表达质粒,并将其转染入非肿瘤性人肝细胞L02细胞株,以未转染细胞及转染空载体的细胞为对照;设计针对Pim-2的特异性SiRNA,导入真核表达载体pGenesil-1,并将其转染入人肝癌细胞HepG2细胞株,以未转染细胞和转染空载体的细胞为对照(前期完成)。(4)设置细胞实验组共8组:非肿瘤性人肝细胞L02细胞组、转染Pim-2的L02细胞组、转染空载体的L02细胞组、人肝癌细胞HepG2细胞组、转染Pim-2 SiRNA的HepG2细胞组、转染空载体的HepG2细胞组、L02/Pim-2+银胶菊内酯(NF-κB信号通路特异性抑制剂)组、HepG2+银胶菊内酯组(。5)通过Real-time PCR检测上述各组细胞中Pim-2,NF-κB和API-5的mRNA水平;通过Western blotting检测上述各组细胞中Pim-2,tAPI-5和pAPI-5的蛋白水平;通过EMSA检测上述各组细胞核蛋白中NF-κB的活性度;通过AnnexinV-FITC/PI双染法检测上述各组细胞的凋亡率。
结果:(1)临床标本实验发现:肝癌组织中Pim-2、NF-κB和API-5的mRNA水平较癌旁组织和正常肝组织中显著增高(p<0.05);肝癌组织中Pim-2、tAPI-5和pAPI-5蛋白呈阳性表达,而癌旁组织和正常肝组织中呈阴性表达;肝癌组织中Pim-2、tAPI-5和pAPI-5的蛋白水平较癌旁组织和正常肝组织中显著增高(p<0.05);肝癌组织中NF-κB活性度较癌旁组织和正常肝组织中显著增高(p<0.05)。(2)细胞实验发现:转染Pim-2的L02细胞中Pim-2的mRNA和蛋白水平较对照组显著增高(p<0.05);转染Pim-2 SiRNA的HepG2细胞中Pim-2的mRNA和蛋白水平较对照组显著降低(p<0.05)。在各实验组细胞中,NF-κB和API-5的mRNA水平随着Pim-2 mRNA水平的变化而平行变化;tAPI-5和pAPI-5的蛋白水平及NF-κB活性度也随着Pim-2蛋白水平的变化而平行变化。在高表达Pim-2的L02/Pim-2细胞和HepG2细胞中加入NF-κB特异性抑制剂银胶菊内酯后,Pim-2 mRNA及蛋白表达无显著变化,但其中NF-κB、API-5的mRNA水平,pAPI-5和tAPI-5的蛋白水平及NF-κB活性度均显著降低(p<0.05)。各实验组细胞凋亡率与Pim-2的表达水平无直接关系,而与pAPI-5的水平呈反向平行变化趋势。
结论:(1)临床标本实验提示:Pim-2、NF-κB和API-5可能是与肝癌发生发展相关的某信号通路上的相关因子。(2)细胞实验进一步证实:Pim-2可通过磷酸化作用激活API-5从而抑制肝癌细胞凋亡,而NF-κB可能是该信号通路的关键调节因子。
Objective: To explore the relationship among oncogene Pim-2 and its possible downstream factors NF-κB and API-5, so as to illuminate the detailed anti-apoptosis mechanism of Pim-2 in hepatocarcinoma cells, and finally confirm that Pim-2 could activate apoptotic protein API-5 to inhibit the apoptosis of hepatocarcinama cells through NF-κB pathway.
Methods: (1) 27 hepatocellular carcinoma tissues (HCC) and 27 corresponding paired noncancerous liver tissues (PNL) were obtained from the surgery specimen in the second affiliated hospital of Chongqing medical university (HCC were diagnosed by pathology. PNL were defined as liver tissues 2cm away from the edge of carcinoma). 11 normal liver tissues (NL) were obtained from non liver disease volunteer patients who receive upper abdominal surgery in the same period in the hospital. Then three experimental groups were set up in the clinical specimen experiment: HCC, PNL and NL. (2) The mRNA levels of Pim-2, NF-κB and API-5 in the tissues were detected by real-time PCR; the protein levels of Pim-2, total API-5 (tAPI-5) and phosphorylated API-5 (pAPI-5) were detected by immunohistochemistry and western blotting; and the NF-κB activity were detected by EMSA. (3) Eukaryotic expression plasmid carrying human Pim-2 gene was transfected into the nontumorous human liver cell line L02. L02 cells transfected with empty vector and the ones without transfection were set up as controls. Pim-2 specific SiRNA were designed and transfected into human liver cancer cell line HepG2. Also, HepG2 cells transfected with empty vector and the ones without transfection were set up as controls (completed in our prior work). (4) Eight experimental groups were set up in the cell experiment: nontumorous human liver cell line (L02), L02 cells transfected with Pim-2 gene (L02/Pim-2), L02 cells transfected with empty vector (L02/Vector), human liver cancer cell line (HepG2), HepG2 cells transfected with Pim-2 SiRNA (HepG2/Pim-2 SiRNA), HepG2 cells transfected with empty vector (HepG2/Vector), L02/Pim-2 cells cultured with NF-κB specific inhibitor parthenolide (L02/Pim-2+parthenolide), and HepG2 cells cultured with parthenolide (HepG2+parthenolide). (5) The mRNA levels of Pim-2, NF-κB and API-5 in the cells were detected by real-time PCR; the protein levels of Pim-2, tAPI-5 and pAPI-5 were detected by western blotting; the NF-κB activity were detected by EMSA; and the cell apoptosis rate were detected by AnnexinV-FITC/PI double staining method.
Results: (1) In the clinical specimen experiment, the mRNA levels of Pim-2, NF-κB and API-5 in HCC group were significantly higher than those in PNL and NL groups (p<0.05); the protein expression of Pim-2, tAPI-5 and pAPI-5 were positive in HCC group but negative in PNL and NL groups; the protein levels of Pim-2, tAPI-5 and pAPI-5 in HCC group were significantly higher than those in PNL and NL groups (p<0.05); the activity of NF-κB in HCC group was significantly higher than those in PNL and NL groups (p<0.05). (2) In the cell experiment, after the transfection of Pim-2 gene, the mRNA and protein levels of Pim-2 were significantly higher in L02/Pim-2 cells than those in L02 cells and L02/vector cells (p<0.05). After the transfection of Pim-2 SiRNA, the mRNA and protein levels of Pim-2 were significantly lower in HepG2/Pim-2 SiRNA cells than those in HepG2 cells and HepG2/vector cells (p<0.05). In these cell groups, the mRNA levels of NF-κB and API-5 were parallel to the mRNA level of Pim-2. The protein levels of tAPI-5 and pAPI-5, as well as the activity of NF-κB, were parallel to the protein level of Pim-2. When parthenolide were added in L02/Pim-2 cells and HepG2 cells which highly expressed Pim-2, the mRNA and protein levels of Pim-2 had no significant changes, while the mRNA levels of NF-κB and API-5, the protein levels of tAPI-5 and pAPI-5, as well as the activity of NF-κB, were all significantly decreased (p<0.05). It was not the Pim-2 but pAPI-5 levels that affected the cell apoptosis rates directly. The cell apoptosis rates were antiparallel to the level of pAPI-5 in all the cell groups.
Conclusions: (1) From the clinical specimen experiments, we could infer that Pim-2, NF-κB and API-5 may be related factors in some signal transduction pathway which is associated with the genesis and deveiopment of liver cancer. (2) The cell experiments convince that Pim-2 could activate API-5 through phosphorylation mechanism so as to inhibit the apoptosis of hepatocarcinoma cells, and NF-κB may be the key regulating factor on this signal transduction pathway.
引文
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[1] Fox CJ, Hammerman PS, Cinalli RM, et al. The serine/threonine kinase Pim-2 is a transcriptionally regulated apopototic inhibitor [J]. Genes & Dev. 2003, 17: 1841-54.
[2] Bachmann M, Moroy T. The serine/threonine kinase Pim-1 [J]. Int J Biochem Cell Biol. 2005, 37: 726-30.
[3] Manning G, White DB, Martinez R, et al. The protein kinase complement of the human genome [J]. Science. 2002, 298: 1912-34.
[4] Deneen B, Welford SM, Ho T, et al. PIM3 proto-oncogene kinase is a common transcriptional target of divergent EWS/ETS oncoproteins [J]. Mol Cell Biol. 2003, 23: 3897-908.
[5] Fujii C, Nakamoto Y, Lu P, et al. Aberrant expression of serine/threonine kinase Pim-3 in hepatocellular carcinoma development and its role in the proliferation of human hepatoma cell lines [J]. Int J Cancer. 2005, 114(2): 209-18.
[6] Peter S. Hammerman, Casey J. Fox, Morris J. Birnbaum, et al. Pim and Akt oncogenes are independent regulators of hematopoietic cell growth and surviva [J]. BLOOD. 2005, 105: 4477-83.
[7] Eileen White. The pims and outs of survival signaling: role for the Pim-2 protein kinase in the suppression of apoptosis by cytokines [J]. Genes & Dev. 2003, 17: 1813-6.
[8] Andrew Macdonald, David G Campbell, Rachel Toth, et al. Pim kinases phosphorylate multiple sites on Bad and promote14-3-3 binding and dissociation from Bcl-XL [J]. BMC Cell Biology. 2006, 7: 1.
[9] Lizcano JM, Morrice N, Cohen P. Regulation of BAD by cAMPdependent protein kinase is mediated via phosphorylation of a novel site, Ser155 [J]. Biochem J. 2000, 349: 547-57.
[10] Pelengaris S, Khan M, Evan G. c-MYC: more than just a matter of life and death [J]. Nat. Rev Cancer. 2002, 2: 764-76.
[11] Peter S. Hammerman, Casey J. Fox, Ryan M. Cinalli, et al. Lymphocyte Transformation by Pim-2 Is Dependent on Nuclear Factor-κB Activation [J]. Cancer Research. 2004, 64: 8341-8.
[12] Lin A, Karin M. NF-κB in cancer: a marked target [J]. Semin Cancer Biol. 2003,13: 107-14.
[13] Karin M, Lin A.NF-κB at the crossroads of life and death [J]. Nat Immunol. 2002, 3: 221-7.
[14] Aghajanian C, Soignet S, Dizon DS, et al. A phase I trial of the novel proteasome inhibitor PS341 in advanced solid tumor malignancies [J]. Clin Cancer Res. 2002, 8: 2505-11.
[15] Wang Y, Lee AT, Ma JZ, et al. Profiling microRNA expression in hepatocellular carcinoma reveals microRNA-224 up-regulation and apoptosis inhibitor-5 as a microRNA-224-specific target [J]. J Biol Chem. 2008, 283(19): 13205-15.
[16] Peng C, Knebel A, Morrice NA, et al. Pim kinase substrate identification and specificity [J]. J Biochem. 2007, 141(3): 353-62.
[17] Shahbazian D, Parsyan A, Petroulakis E, et al. Control of cell survival and proliferation by mammalian eukaryotic initiation factor 4B [J]. Mol Cell Biol. 2010 Jan 19. [Epub ahead of print]
[18] Qian K, Wang L, Cywin CL, et al. Hit to lead account of the discovery of a new class of inhibitors of Pim kinases and crystallographic studies revealing an unusual kinase binding mode [J]. J Med Chem. 2009, 52(7): 1814-27.
[19] Tao ZF, Hasvold LA, Leverson JD, et al. Discovery of 3H- benzo [4,5]thieno [3,2-d]pyrimidin-4-ones as potent, highly selective, and orally bioavailable inhibitors of the human protooncogene proviral insertion site in moloney murine leukemia virus (PIM) kinases [J]. J Med Chem. 2009, 52(21): 6621-36.
[20] Akué-Gédu R, Rossignol E, Azzaro S, et al. Synthesis, kinase inhibitory potencies, and in vitro antiproliferative evaluation of new Pim kinase inhibitors [J]. J Med Chem. 2009, 52(20): 6369-81.
[21] Chen LS, Redkar S, Bearss D, et al. Pim kinase inhibitor, SGI-1776, induces apoptosis in chronic lymphocytic leukemia cells [J]. Blood. 2009, 114(19): 4150-7.
[22] Xia Z, Knaak C, Ma J, et al. Synthesis and evaluation of novel inhibitors of Pim-1 and Pim-2 protein kinases [J]. J Med Chem. 2009, 52(1): 74-86.
[23] Tong Y, Stewart KD, Thomas S, et al. Isoxazolo[3,4-b]quinoline- 3,4(1H,9H)- diones as unique, potent and selective inhibitors for Pim-1 and Pim-2 kinases: chemistry, biological activities, and molecular modeling [J]. Bioorg Med Chem Lett. 2008, 18(19): 5206-8.
[24] Shu-Qun Zhang, Qing-You Du, Yang Ying, et al. Polymerase synthesis and potential interference of a small-interfering RNA targeting hPim-2. World J Gastroenterol. 2004;10(18):2657-2660.