下调垂体肿瘤转化基因1表达对前列腺癌LNCaP-AI细胞衰老的影响
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摘要
目的:探讨下调垂体肿瘤转化基因1(PTTG1)的表达对人去势抵抗前列腺癌LNCaP-AI细胞衰老的影响。方法:采用体外诱导的人去势抵抗前列腺癌LNCaP-AI细胞模型, LNCaP-AI细胞转染靶向PTTG1基因的siRNA为siRNA-PTTG1组,只添加转染试剂为Mock组,转染阴性序列为NC组。于含雄激素培养液(FBS)/去除雄激素培养液(CSS)中培养各组细胞,细胞计数实验比较各组细胞数量变化。采用细胞衰老-β-半乳糖苷酶(SA-β-Gal)染色试剂盒检测衰老细胞数目;Western印迹检测β-半乳糖苷酶蛋白1(Glb1)、细胞周期相关蛋白(p-21~(CIP1)、p-27~(Kip1))、异染色质蛋白1γ(HP1γ)的表达情况。结果:siRNA有效抑制了LNCaP-AI细胞中PTTG1的表达。siRNA-PTTG1组在FBS中培养,细胞数量增加,而在CSS中培养,细胞数量呈减少趋势;Mock组及NC组在上述不同培养液中细胞数量均呈增长趋势,siRNA-PTTG1组与Mock组和NC组比较差异有统计学意义(P<0.05)。SA-β-Gal染色检测衰老细胞数量,Mock组、NC组、siRNA-PTTG1组细胞染色阳性率分别为(11.3±1.24)%、(12.4±1.15)%、(63.5±2.35)%,siRNA-PTTG1组显著高于Mock组和NC组(P<0.05)。Western印迹检测转染效率显示Mock组、NC组及siRNA-PTTG1组中PTTG1相对表达量分别为0.56±0.02、0.61±0.02、0.21±0.01,p-21~(CIP1)相对表达量分别为0.20±0.02、0.21±0.03、0.32±0.03,p-27~(Kip1)相对表达量分别为0.20±0.03、0.22±0.01、0.38±0.02,GlB1相对表达量为0.13±0.01、0.15±0.01、0.24±0.01,HP1γ相对表达量分别为0.26±0.01、0.27±0.02、0.41±0.01;siRNA-PTTG1组与Mock组和NC组比较差异均有统计学意义(P<0.05)。结论 :下调PTTG1基因表达可促进人去势抵抗前列腺癌细胞株LNCaP-AI细胞衰老。
Objective: To investigate the effect of the down-regulated expression of pituitary tumor-transforming gene 1(PTTG1) on the senescence of human castration-resistant prostate cancer LNCaP-AI cells. Methods: Human castration-resistant prostate cancer LNCaP-AI cells were induced in vitro and transfected with siRNA targeting PTTG1(the siRNA-PTTG1 group), the reagent lip3000 only(the mock group) or siRNA negative control vector(the NC group). All the cells were cultured in fetal bovine serum(FBS) or charcoal-stripped bovine serum(CSS) and counted with the cell counting chamber. The senescence characteristics of the transfected LNCaP-AI cells were examined by senescence-associated β-galactosidase(SA-β-Gal) staining, and the expressions of the senescence-related β-galactosidase-1-like proteins(Glb1), the cyclin-dependent kinase inhibitors p-21~(CIP1) and p-27~(Kip1), and the chromatin-regulating heterochromatin protein 1γ(HP1γ) were detected by Western blot. Results: The expression of PTTG1 in the human prostate cancer LNCaP-AI cells was significantly reduced in the siRNA-PTTG1 group compared with those in the mock and NC groups(0.21 ± 0.01 vs 0.56 ± 0.02 and 0.61 ± 0.02, P < 0.05). Culture with FBS markedly increased while that with CSS decreased the number of LNCaP-AI cells transfected with siRNA, but both FBS and CSS enhanced the proliferation of the LNCaP-AI cells in the mock and NC groups. SA-β-Gal staining revealed that reducing the expression of PTTG1 induced a remarkably higher positive rate of the LNCaP-AI cells in the siRNA-PTTG1 than in the mock and NC groups([63.5 ± 2.35]% vs [11.3 ± 1.24]% and [12.4 ± 1.15]%, P < 0.05). The siRNA-PTTG1 group, in comparison with the mock and NC groups, showed a significantly down-regulated expression of PTTG1(0.21 ± 0.01 vs 0.56 ± 0.02 and 0.61 ± 0.02, P < 0.05), but up-regulated expressions of p-21~(CIP1)(0.32 ± 0.03 vs 0.20 ± 0.02 and 0.21 ± 0.03, P < 0.05), p-27~(Kip1)(0.38 ± 0.02 vs 0.20 ± 0.03 and 0.22 ± 0.01, P < 0.05), Glb1(0.24 ± 0.01 vs 0.13 ± 0.01 and 0.15 ± 0.01, P < 0.05), and HP1γ(0.41 ± 0.01 vs 0.26 ± 0.01 and 0.27 ± 0.02, P < 0.05) in the LNCaP-AI cells. Conclusion: Down-regulated expression of PTTG1 induces senescence of human castration-resistant prostate cancer LNCaP-AI cells.
Objective: To investigate the effect of the down-regulated expression of pituitary tumor-transforming gene 1(PTTG1) on the senescence of human castration-resistant prostate cancer LNCaP-AI cells. Methods: Human castration-resistant prostate cancer LNCaP-AI cells were induced in vitro and transfected with siRNA targeting PTTG1(the siRNA-PTTG1 group), the reagent lip3000 only(the mock group) or siRNA negative control vector(the NC group). All the cells were cultured in fetal bovine serum(FBS) or charcoal-stripped bovine serum(CSS) and counted with the cell counting chamber. The senescence characteristics of the transfected LNCaP-AI cells were examined by senescence-associated β-galactosidase(SA-β-Gal) staining, and the expressions of the senescence-related β-galactosidase-1-like proteins(Glb1), the cyclin-dependent kinase inhibitors p-21~(CIP1) and p-27~(Kip1), and the chromatin-regulating heterochromatin protein 1γ(HP1γ) were detected by Western blot. Results: The expression of PTTG1 in the human prostate cancer LNCaP-AI cells was significantly reduced in the siRNA-PTTG1 group compared with those in the mock and NC groups(0.21 ± 0.01 vs 0.56 ± 0.02 and 0.61 ± 0.02, P < 0.05). Culture with FBS markedly increased while that with CSS decreased the number of LNCaP-AI cells transfected with siRNA, but both FBS and CSS enhanced the proliferation of the LNCaP-AI cells in the mock and NC groups. SA-β-Gal staining revealed that reducing the expression of PTTG1 induced a remarkably higher positive rate of the LNCaP-AI cells in the siRNA-PTTG1 than in the mock and NC groups([63.5 ± 2.35]% vs [11.3 ± 1.24]% and [12.4 ± 1.15]%, P < 0.05). The siRNA-PTTG1 group, in comparison with the mock and NC groups, showed a significantly down-regulated expression of PTTG1(0.21 ± 0.01 vs 0.56 ± 0.02 and 0.61 ± 0.02, P < 0.05), but up-regulated expressions of p-21~(CIP1)(0.32 ± 0.03 vs 0.20 ± 0.02 and 0.21 ± 0.03, P < 0.05), p-27~(Kip1)(0.38 ± 0.02 vs 0.20 ± 0.03 and 0.22 ± 0.01, P < 0.05), Glb1(0.24 ± 0.01 vs 0.13 ± 0.01 and 0.15 ± 0.01, P < 0.05), and HP1γ(0.41 ± 0.01 vs 0.26 ± 0.01 and 0.27 ± 0.02, P < 0.05) in the LNCaP-AI cells. Conclusion: Down-regulated expression of PTTG1 induces senescence of human castration-resistant prostate cancer LNCaP-AI cells.
引文
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[8] Xiang W, Wu X, Huang C, et al. PTTG1 regulated by miR-146a-3p promotes bladder cancer migration, invasion, metastasis and growth. Oncotarget, 2016, 8(1): 664-678.
[9] 曹希亮, 宋晓明, 于文朝, 等. 垂体肿瘤转化基因1在雄激素非依赖性前列腺癌发生过程中的表达变化. 中华男科学杂志, 2016, 22(8): 686-691.
[10] Gomez-Cabello D, Adrados I, Gamarra D, et al. DGCR8-mediated disruption of miRNA biogenesis induces cellular senescence in primary fibroblasts. Aging cell, 2013, 12(5): 923-931.
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[12] Tong Y, Zhao W, Zhou C, et al. PTTG1 attenuates drug-induced cellular senescence. PLoS One, 2011, 6(8): e23754.
[13] 曹希亮, 魏洋洋, 宋晓明, 等. 下调基因PTTG1表达对前列腺癌LNCaP-AI细胞增殖、侵袭和凋亡的影响. 中华男科学杂志, 2017, 23(7): 589-597.
[14] Dimri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA, 1995, 92(20): 9363-9367
[15] Lee BY, Han JA, Im JS, et al. Senescence-associated beta-galactosidase is lysosomal beta-galactosidase. Aging Cell, 2006, 5(2): 187-195.
[16] Wagner J, Damaschke N, Yang B, et al. Overexpression of the novel senescence marker β-galactosidase (GLB1) in prostate cancer predicts reduced PSA recurrence. PLoS One, 2015, 10(4): e0124366.
[17] Bringold F, Serrano M. Tumor suppressors and oncogenes in cellular senescence. Exp Gerontol, 2000, 35(3): 317-329.
[18] Campisi J. Fragile fugue: p53 in aging, cancer and IGF signaling. Nat Med, 2004, 10(3): 231-232.
[19] Lowe SW. Activation of p53 by oncogenes. Endocr Relat Cancer, 1999, 6(1): 45-48.
[20] Zhang R, Adams PD. Heterochromatin and its relationship to cell senescence and cancer therapy. Cell Cycle, 2007, 6(7): 784-789.
[21] Xue W, Zender L, Miething C, et al. Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas. Nature, 2007, 445(7128): 656-660.
[22] Van Deursen JM. The role of senescent cells in aging. Nature, 2014, 509(7501): 439-446.
[23] Salama R, Sadaie M, Hoare M, et al. Cellular senescence and its effector programs. Genes Dev, 2014, 28(2): 99-114.
[24] Takeuchi S, Takahashi A, Motoi N, et al. Intrinsic cooperation between p16INK4a and p21Waf1/Cip1 in the onset of cellular senescence and tumor suppression in vivo. Cancer Res, 2010, 70(22): 9381-9390.
[25] Zweegman S, Engelhardt M, Larocca A, et al. Elderly patients with multiple myeloma: Towards a frailty approach? Curr Opin Oncol, 2017, 29(5): 315-321.
[26] D?rr JR, Yu Y, Milanovic M, et al. Synthetic lethal metabolic targeting of cellular senescence in cancer therapy. Nature, 2013, 501(7467): 421-425.
[27] Mccormick JR, Blute ML, Yang B, et al. MP50-05 synthetic lethal metabolic targeting of cellular senescence in prostate cancer with the repurposed drug metformin. J Urol, 2016, 195(4s): e673-e674.
[2] Noll JE, Vandyke K, Hewett DR, et al. PTTG1 expression is associated with hyperproliferative disease and poor prognosis in multiple myeloma. J Hematol Oncol, 2015, 8(1): 106-122.
[3] Demeure MJ, Coan KE, Grant CS, et al. PTTG1 over-expression in adrenocortical cancer is associated with poor survival and represents a potential therapeutic target. Surgery, 2013, 154(6): 1405-1416.
[4] Zhang Z, Jin B, Jin Y, et al. PTTG1, A novel androgen responsive gene is required for androgen-induced prostate cancer cell growth and invasion. Exp Cell Res, 2017, 350(1):1-8.
[5] Cao XL, Gao JP, Wang W, et al. Expression of pituitary tumor transforming gene 1 is an independent factor of poor prognosis in localized or locally advanced prostate cancer cases receiving hormone therapy. Asian Pac J Cancer Prev, 2012, 13(7): 3083-3088.
[6] Huang SQ, Liao QJ, Wang XW, et al. RNAi-mediated knockdown of pituitary tumor-transforming gene-1 (PTTG1) suppresses the proliferation and invasive potential of PC3 human prostate cancer cells. Braz J Med Biol Res. 2012, 45(11): 995-1001.
[7] Yu YC, Yang PM, Chuah QY, et al. Radiation-induced senescence in securin-deficient cancer cells promotes cell invasion involving the IL-6/STAT3 and PDGF-BB/PDGFR pathways. Sci Rep, 2013, 3(4): 1675-1685.
[8] Xiang W, Wu X, Huang C, et al. PTTG1 regulated by miR-146a-3p promotes bladder cancer migration, invasion, metastasis and growth. Oncotarget, 2016, 8(1): 664-678.
[9] 曹希亮, 宋晓明, 于文朝, 等. 垂体肿瘤转化基因1在雄激素非依赖性前列腺癌发生过程中的表达变化. 中华男科学杂志, 2016, 22(8): 686-691.
[10] Gomez-Cabello D, Adrados I, Gamarra D, et al. DGCR8-mediated disruption of miRNA biogenesis induces cellular senescence in primary fibroblasts. Aging cell, 2013, 12(5): 923-931.
[11] Schraml E, Grillari J. From cellular senescence to age-associated diseases: The miRNA connection. Longev Healthspan, 2012, 1(1): 10.
[12] Tong Y, Zhao W, Zhou C, et al. PTTG1 attenuates drug-induced cellular senescence. PLoS One, 2011, 6(8): e23754.
[13] 曹希亮, 魏洋洋, 宋晓明, 等. 下调基因PTTG1表达对前列腺癌LNCaP-AI细胞增殖、侵袭和凋亡的影响. 中华男科学杂志, 2017, 23(7): 589-597.
[14] Dimri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA, 1995, 92(20): 9363-9367
[15] Lee BY, Han JA, Im JS, et al. Senescence-associated beta-galactosidase is lysosomal beta-galactosidase. Aging Cell, 2006, 5(2): 187-195.
[16] Wagner J, Damaschke N, Yang B, et al. Overexpression of the novel senescence marker β-galactosidase (GLB1) in prostate cancer predicts reduced PSA recurrence. PLoS One, 2015, 10(4): e0124366.
[17] Bringold F, Serrano M. Tumor suppressors and oncogenes in cellular senescence. Exp Gerontol, 2000, 35(3): 317-329.
[18] Campisi J. Fragile fugue: p53 in aging, cancer and IGF signaling. Nat Med, 2004, 10(3): 231-232.
[19] Lowe SW. Activation of p53 by oncogenes. Endocr Relat Cancer, 1999, 6(1): 45-48.
[20] Zhang R, Adams PD. Heterochromatin and its relationship to cell senescence and cancer therapy. Cell Cycle, 2007, 6(7): 784-789.
[21] Xue W, Zender L, Miething C, et al. Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas. Nature, 2007, 445(7128): 656-660.
[22] Van Deursen JM. The role of senescent cells in aging. Nature, 2014, 509(7501): 439-446.
[23] Salama R, Sadaie M, Hoare M, et al. Cellular senescence and its effector programs. Genes Dev, 2014, 28(2): 99-114.
[24] Takeuchi S, Takahashi A, Motoi N, et al. Intrinsic cooperation between p16INK4a and p21Waf1/Cip1 in the onset of cellular senescence and tumor suppression in vivo. Cancer Res, 2010, 70(22): 9381-9390.
[25] Zweegman S, Engelhardt M, Larocca A, et al. Elderly patients with multiple myeloma: Towards a frailty approach? Curr Opin Oncol, 2017, 29(5): 315-321.
[26] D?rr JR, Yu Y, Milanovic M, et al. Synthetic lethal metabolic targeting of cellular senescence in cancer therapy. Nature, 2013, 501(7467): 421-425.
[27] Mccormick JR, Blute ML, Yang B, et al. MP50-05 synthetic lethal metabolic targeting of cellular senescence in prostate cancer with the repurposed drug metformin. J Urol, 2016, 195(4s): e673-e674.