PIAS3敲低对前列腺癌DU145细胞增殖、细胞周期和凋亡的影响
|
摘要
背景及研究目的:由于生活和饮食习惯改变等原因,前列腺癌在我国的发病率呈上升趋势。晚期前列腺癌主要的治疗是内分泌治疗,但多数病人在经过12~18个月的内分泌治疗后转变成为激素非依赖性的前列腺癌,失去对去势治疗的敏感性。寻找前列腺癌新的基因治疗靶点是晚期前列腺癌治疗研究的一个重要方向。JAKs/STATs信号转导通路与细胞的增殖、分化和凋亡关系密切,STAT3是该通路的关键转录因子。活化的信号转导子和转录激活子3(STAT3)的蛋白抑制剂(Protein inhibitor of activated signal transducer and activator of transcription 3,PIAS3)是STAT3的特异蛋白抑制剂,通过与活化的STAT3结合而阻止后者与DNA结合,从而抑制STAT3的转录激活活性。近年的研究表明,PIAS3蛋白的异常表达与前列腺癌有关。我们通过构建PIAS3特异性发夹状RNA(shRNA)表达质粒,敲低人前列腺癌细胞系DU145细胞过表达的PIAS3蛋白,研究PIAS3敲低对前列腺癌细胞增殖、细胞周期和凋亡的影响。
方法:构建PIAS3 shRNA表达质粒pSilencer4.1/PIAS3,应用脂质体
LipofectamineTM2000作为转染介质,转染DU145细胞。细胞分为无关序列组和RNA干扰组,每组设3个复孔,分别转染阴性对照质粒和pSilencer4.1/PIAS3质粒。阴性对照质粒表达与已知人、鼠基因序列低同源性(即无关序列)shRNA。逆转录-聚合酶链反应(RT-PCR)检测转染后PIAS3 mRNA表达水平的变化,蛋白免疫印迹(Western)检测转染后PIAS3蛋白表达水平的变化。噻唑蓝(MTT)法检测细胞增殖状态,碘化丙锭(PI)细胞周期检测试剂盒流式细胞术分析细胞周期,磷脂结合蛋白V /碘化丙锭(Annexin V/PI)双标凋亡检测试剂盒流式细胞术检测细胞凋亡。
结果:双向测序证实PIAS3 shRNA表达质粒构建成功。RNA干扰组细胞PIAS3 mRNA和蛋白质表达水平与无关序列组相比明显下降。MTT分析显示PIAS3敲低促进细胞增殖,并存在剂量-效应关系。流式细胞术分析显示:与无关序列组相比,RNA干扰组S期细胞比例增加,G0/G1期细胞比例减少,凋亡细胞比例减少。
结论:PIAS3敲低促进前列腺癌细胞增殖,抑制其凋亡,PIAS3有可能可以成为前列腺癌的一个治疗靶点。
Background and Objective Protein inhibitor of activated signal transducer and activator of transcription 3(PIAS3) is recently shown to play an important role in prostate carcinoma.We studied the effects of PIAS3 knocking down on proliferation, cell cycle and apoptosis of human prostate cancer cell line DU145.
Methods PIAS3 specific short hairpin RNA(shRNA) expressing plasmid was constructed and named pSilencer4.1/PIAS3.DU145 cell was transfected with pSilencer4.1/PIAS3. The proliferation of DU145 cells was analyzed by MTT assay.Cell cycle and apoptosis of DU145 cells were analyzed by flowcytometry.
Results PIAS3 shRNA expressing plasmid was constructed succefully,confirmed by sequencing.Expression of PIAS3 in DU145 was reduced significantly after pSilencer4.1/PIAS3 transfection. MTT assay shown accelerated proliferation after PIAS3 knocking down,and shown dose-effect curve. Flowcytometry shown cells in S phase increased,cells in G0/G1 decreased and percentage of apoptotic cells decreased after PIAS3 knocking down.
Conclusion The knocking down of PIAS3 expression could accelerate DU145 cell proliferation and inhibit cell apoptosis.
方法:构建PIAS3 shRNA表达质粒pSilencer4.1/PIAS3,应用脂质体
LipofectamineTM2000作为转染介质,转染DU145细胞。细胞分为无关序列组和RNA干扰组,每组设3个复孔,分别转染阴性对照质粒和pSilencer4.1/PIAS3质粒。阴性对照质粒表达与已知人、鼠基因序列低同源性(即无关序列)shRNA。逆转录-聚合酶链反应(RT-PCR)检测转染后PIAS3 mRNA表达水平的变化,蛋白免疫印迹(Western)检测转染后PIAS3蛋白表达水平的变化。噻唑蓝(MTT)法检测细胞增殖状态,碘化丙锭(PI)细胞周期检测试剂盒流式细胞术分析细胞周期,磷脂结合蛋白V /碘化丙锭(Annexin V/PI)双标凋亡检测试剂盒流式细胞术检测细胞凋亡。
结果:双向测序证实PIAS3 shRNA表达质粒构建成功。RNA干扰组细胞PIAS3 mRNA和蛋白质表达水平与无关序列组相比明显下降。MTT分析显示PIAS3敲低促进细胞增殖,并存在剂量-效应关系。流式细胞术分析显示:与无关序列组相比,RNA干扰组S期细胞比例增加,G0/G1期细胞比例减少,凋亡细胞比例减少。
结论:PIAS3敲低促进前列腺癌细胞增殖,抑制其凋亡,PIAS3有可能可以成为前列腺癌的一个治疗靶点。
Background and Objective Protein inhibitor of activated signal transducer and activator of transcription 3(PIAS3) is recently shown to play an important role in prostate carcinoma.We studied the effects of PIAS3 knocking down on proliferation, cell cycle and apoptosis of human prostate cancer cell line DU145.
Methods PIAS3 specific short hairpin RNA(shRNA) expressing plasmid was constructed and named pSilencer4.1/PIAS3.DU145 cell was transfected with pSilencer4.1/PIAS3. The proliferation of DU145 cells was analyzed by MTT assay.Cell cycle and apoptosis of DU145 cells were analyzed by flowcytometry.
Results PIAS3 shRNA expressing plasmid was constructed succefully,confirmed by sequencing.Expression of PIAS3 in DU145 was reduced significantly after pSilencer4.1/PIAS3 transfection. MTT assay shown accelerated proliferation after PIAS3 knocking down,and shown dose-effect curve. Flowcytometry shown cells in S phase increased,cells in G0/G1 decreased and percentage of apoptotic cells decreased after PIAS3 knocking down.
Conclusion The knocking down of PIAS3 expression could accelerate DU145 cell proliferation and inhibit cell apoptosis.
引文
1 Liu B,Liao JY,Rao XP,et al Inhibition of STAT1-mediated gene activation by PIAS1.Proc Natl Acad Sci USA,1998,95:10626-10631
2 Chung CD,Liao J,Liu B, et al Specific Inhibition of STAT3 Signal transduction by PIAS3.Science,1997,278:1803-1805
3 Wible BA,Yang Q,Kuryshev YA,et al Cloning and expression of a novel K+ channel regulatory protein,KChAP.J Biol Chem,1998,273(19):11745-11751
4 Wible BA,Wang L,Kuryshey YA,et al Increased K+ efflux and apoptosis induced by the potassium channel modulatory protein KChAP/PIAS3β in prostate cancer cells. J Biol Chem. 2002,277(20):17852-17862
5 Wang L, Banerjee S.Differential PIAS3 expression in human malignancy. Oncol Rep,2004,11(6):1319-1324
6 Long J,Wang G,Matsuura I,et al Activation of SMAD transcriptional activity by protein inhibitor of activated STAT3 (PIAS3).Proc Natl Acad Sci USA,2004, 101:99-104
7 Levy C,Nechushtan H,Razin E.A new role for the STAT3 inhibitor, PIAS3:a repressor of microphthalmia transcription factor.J Biol Chem,2002,277:1962-1966
8 Nakagawa K,Yokosawa H.PIAS3 induces SUMO-1 modification and transcriptional repression of IRF-1.FEBS Lett,2002,530:204-208
9 Deng J, Hua K, Caveney EJ,et al Protein inhibitor of activated STAT3 inhibits adipogenicgene expression. Biochem Biophys Res Commun,2006, 339(3):923-931
10 Starkel P,De Saeger C,Leclercq I,et al Deficient STAT3 DNA-binding is associated with high PIAS3 expression and a positive anti-apoptotic balance in human end-stage alcoholic and hepatitis C cirrhosis. J Hepatol,2005,43(4):687-695
11 Ogata Y, Osaki T, Naka T,et al Overexpression of PIAS3 suppresses cell growth and restores drug sensitivity of human lung cancer cells in association with PI3-K/Akt inactivation.Neoplasia,2006,8(10):817-825
12 Bromberg JF,Wrzeszczynska MH,Devgan G,et al STAT3 as an Oncogene.Cell,1999,98:295-303
13 Cheng J,Zhang D,Zhou C,et al Down-regulation of SHP1 and up-regulation of negative regulators of JAK/STAT signaling in HTLV-1 transformed cell lines and freshly transformed human peripheral blood CD4+ T-Cells.Leuk Res,2004,28(1):71-82
14 苏桂英,李慧艳,潘欣,等.应用酵母双杂交技术筛选与 p53 相互作用的蛋白质.科学技术与工程,2006,6(6):675-677
15 Junicho A, Matsuda T, Yamamoto T,et al Protein inhibitor of activated STAT3 regulates androgen receptor signaling in prostate carcinoma cells.Biochem Biophys Res Commun,2000,278(1):9-13
16 Gross M,Liu B,Tan J,et al Distinct effects of PIAS proteins on androgen-mediated gene activation in prostate cancer cells. Oncogene,2001,20(29):3880-3887
1. D. Max Parkin, Freddie Bray, J. Ferlay.et al. Global Cancer Statistics, 2002, CA Cancer J Clin 2005;55;74-108
2. Ahmedin Jemal, Rebecca Siegel, Elizabeth Ward,et al. Cancer Statistics, 2008, CA Cancer J Clin 2008;58;71-96
3. Brawley OW. Prostate carcinoma incidence and patient mortality. The effects of screening and early detection. Cancer 1997;80:1857–1863
4. 李鸣.PSA 与前列腺癌诊断.老年医学与保健,2007,13(3):137-140
5.刘振伟,项永兵,张薇,等.上海市区 1973~1999 年前列腺癌发病趋势分析,中国卫生统计,2003,6:335-337
6. Gu F. Epidemiological survey of benign prostatic hyperplasia and prostatic cancer in China. ChinMed J ( Engl) , 2000, 113: 299-302.
7. 那彦群,孙则禹,叶章群等.中国泌尿外科疾病诊断治疗指南(2007 版 ),人民卫生出版社,30-32
1. Henrik Gr?nberg. Prostate cancer epidemiology. The Lancet, 2003, 361: 859 -864
2. D. Max Parkin, Freddie Bray, J. Ferlay.et al. Global Cancer Statistics, 2002, CA Cancer J Clin 2005;55;74-108
3. Hayes RB, Liff JM, Pottern LM, et al. Prostate cancer risk in US blacks and whites with a family history of cancer. Int J Cancer 1995;60: 361–64.
4. Tulinius H, Egilsson V, Olafsdottir GH, et al: Risk of prostate, ovarian, and endometrial cancer among relatives of women with breast cancer. BMJ 1992;305:855-857
5. Chan JM, Stampfer MJ, Giovannucci E, et al. Plasma insulin-like growth factor-I and prostate cancer risk: a prospective study. Science 1998; 279: 563–66.
6. Harman SM, Metter EJ, Blackman MR, Landis PK, Carter HB.Serum levels of insulin-like growth factor I (IGF-I), IGF-II, IGFbinding protein-3, and prostate-specific antigen as predictors of clinical prostate cancer. J Clin Endocrinol Metab 2000; 85: 4258–65.
7. Stattin P, Bylund A, Rinaldi S, et al. Plasma insulin-like growth factor-I, insulin-like growth factor-binding proteins, and prostate cancer risk:a prospective study. J Natl Cancer Inst 2000; 92: 1910–17.
8. Augustsson K, Skog K, Jagerstad M, Dickman PW, Steineck G.Dietary heterocyclic amines and cancer of the colon, rectum, bladder,and kidney: a population-based study. Lancet 1999; 353: 703–07.
9. Miller BA, Kolonel LN, Bernstein L, et al., eds. Racial/Ethnic Patterns of Cancer in the United States 1988–1992. NIH Publication No. 96–4104. Bethesda, MD: National Cancer Institute;1996.
10. Cook LS,Goldoft M,Schwartz SM,et al.Incidence of adenocarcinoma of prostate in Asican immigrants to the United States and their descendants.J Urol,1999,161:152-155
11. 李鸣,前列腺癌发病率和影响因素.中国肿瘤.2003,12(12):688-691
12. Glutathione S-transferase pi is upregulated in the stromal compartment of hormone independent prostate cancer.The prostate.56:98-105
13. 管同郁,李鸣,那彦群.生物转化酶基因多态性与前列腺癌易感性的研究.中华外科,2005,43(22):1467-1470
14. Hickey K, Do KA, Green A. Smoking and prostate cancer.Epidemiol Rev 2001; 23: 115–25.
15. Dennis LK, Hayes RB. Alcohol and prostate cancer. Epidemiol Rev 2001; 23: 110–14.
16. Peterson HB, Howards SS. Vasectomy and prostate cancer: the evidence to date. Fertil Steril 1998; 70: 201–03.
17. Lee IM, Sesso HD, Chen JJ, Paffenbarger RS Jr. Does physical activity play a role in the prevention of prostate cancer? Epidemiol Rev 2001; 23: 132–37.
1. Chung Chan D, Liao Jiayu, Liu Bin, et al. Specific inhibition of Stat3 signal transduction by PIAS3[J].Science,1997,278(5344):1803-1805.
2.Wible BA, Wang Liming, Kuryshey YA, et al. Increased K+ efflux and apoptosis induced by the potassium channel modulatory protein KChAP/PIAS3β in prostate cancer cells[J].J Biol Chem, 2002,277(20):17852-17862.
3. 王永俊,施小六,汤建光,等. RNAi 下调 survivin 表达诱导成人神经细胞瘤细胞凋亡[J].基础医学与临床杂志,2006,26(7):745-750.
4. 翁迈,周利群,王建伟,等.PIM-1 蛋白激酶在前列腺癌组织中的表达[J].基础医学与临床杂志,2005,25(6):503-506.
5. Wang Liming, Banerjee S.Differential PIAS3 expression in human malignancy[J]. Oncol Rep,2004,11(6):1319-1324.
6. Junicho A, Matsuda T, Yamamoto T,et al. Protein inhibitor of activated STAT3 regulates androgen receptor signaling in prostate carcinoma cells[J].Biochem Biophys Res Commun,2000,278(1):9-13.
7. Deng Jianbei, Hua Kunjie, Caveney EJ,et al Protein inhibitor of activated STAT3 inhibits adipogenic gene expression[J]. Biochem Biophys Res Commun,2006, 339(3):923-931
8. St?rkel P,Saeger CD, Leclercq I,et al Role of signal transducer and activator of transcription 3 in liver fibrosis progression in chronic hepatitis C-infected patients[J]. Lab Invest, 2007,87 (2):173-181.
9. Ogata Y, Osaki T, Naka T,et al Overexpression of PIAS3 suppresses cell growth and restores drug sensitivity of human lung cancer cells in association with PI3-K/Akt inactivation[J].Neoplasia,2006,8(10):817-825
10. 唐梅,张钲,伏静媛,等.JAK2/STAT3 信号通路介导大鼠血管平滑肌细胞的增殖与凋亡[J].基础医学与临床杂志,2006,26(12):1354-1358
11. Drachenberg DE, Elgamal AA,Rowbotham R, et al. Circulating levels of IL-6 in patients with hormone-refractory prostate cancer[J].Prostate,1999,41:127-133.
2 Chung CD,Liao J,Liu B, et al Specific Inhibition of STAT3 Signal transduction by PIAS3.Science,1997,278:1803-1805
3 Wible BA,Yang Q,Kuryshev YA,et al Cloning and expression of a novel K+ channel regulatory protein,KChAP.J Biol Chem,1998,273(19):11745-11751
4 Wible BA,Wang L,Kuryshey YA,et al Increased K+ efflux and apoptosis induced by the potassium channel modulatory protein KChAP/PIAS3β in prostate cancer cells. J Biol Chem. 2002,277(20):17852-17862
5 Wang L, Banerjee S.Differential PIAS3 expression in human malignancy. Oncol Rep,2004,11(6):1319-1324
6 Long J,Wang G,Matsuura I,et al Activation of SMAD transcriptional activity by protein inhibitor of activated STAT3 (PIAS3).Proc Natl Acad Sci USA,2004, 101:99-104
7 Levy C,Nechushtan H,Razin E.A new role for the STAT3 inhibitor, PIAS3:a repressor of microphthalmia transcription factor.J Biol Chem,2002,277:1962-1966
8 Nakagawa K,Yokosawa H.PIAS3 induces SUMO-1 modification and transcriptional repression of IRF-1.FEBS Lett,2002,530:204-208
9 Deng J, Hua K, Caveney EJ,et al Protein inhibitor of activated STAT3 inhibits adipogenicgene expression. Biochem Biophys Res Commun,2006, 339(3):923-931
10 Starkel P,De Saeger C,Leclercq I,et al Deficient STAT3 DNA-binding is associated with high PIAS3 expression and a positive anti-apoptotic balance in human end-stage alcoholic and hepatitis C cirrhosis. J Hepatol,2005,43(4):687-695
11 Ogata Y, Osaki T, Naka T,et al Overexpression of PIAS3 suppresses cell growth and restores drug sensitivity of human lung cancer cells in association with PI3-K/Akt inactivation.Neoplasia,2006,8(10):817-825
12 Bromberg JF,Wrzeszczynska MH,Devgan G,et al STAT3 as an Oncogene.Cell,1999,98:295-303
13 Cheng J,Zhang D,Zhou C,et al Down-regulation of SHP1 and up-regulation of negative regulators of JAK/STAT signaling in HTLV-1 transformed cell lines and freshly transformed human peripheral blood CD4+ T-Cells.Leuk Res,2004,28(1):71-82
14 苏桂英,李慧艳,潘欣,等.应用酵母双杂交技术筛选与 p53 相互作用的蛋白质.科学技术与工程,2006,6(6):675-677
15 Junicho A, Matsuda T, Yamamoto T,et al Protein inhibitor of activated STAT3 regulates androgen receptor signaling in prostate carcinoma cells.Biochem Biophys Res Commun,2000,278(1):9-13
16 Gross M,Liu B,Tan J,et al Distinct effects of PIAS proteins on androgen-mediated gene activation in prostate cancer cells. Oncogene,2001,20(29):3880-3887
1. D. Max Parkin, Freddie Bray, J. Ferlay.et al. Global Cancer Statistics, 2002, CA Cancer J Clin 2005;55;74-108
2. Ahmedin Jemal, Rebecca Siegel, Elizabeth Ward,et al. Cancer Statistics, 2008, CA Cancer J Clin 2008;58;71-96
3. Brawley OW. Prostate carcinoma incidence and patient mortality. The effects of screening and early detection. Cancer 1997;80:1857–1863
4. 李鸣.PSA 与前列腺癌诊断.老年医学与保健,2007,13(3):137-140
5.刘振伟,项永兵,张薇,等.上海市区 1973~1999 年前列腺癌发病趋势分析,中国卫生统计,2003,6:335-337
6. Gu F. Epidemiological survey of benign prostatic hyperplasia and prostatic cancer in China. ChinMed J ( Engl) , 2000, 113: 299-302.
7. 那彦群,孙则禹,叶章群等.中国泌尿外科疾病诊断治疗指南(2007 版 ),人民卫生出版社,30-32
1. Henrik Gr?nberg. Prostate cancer epidemiology. The Lancet, 2003, 361: 859 -864
2. D. Max Parkin, Freddie Bray, J. Ferlay.et al. Global Cancer Statistics, 2002, CA Cancer J Clin 2005;55;74-108
3. Hayes RB, Liff JM, Pottern LM, et al. Prostate cancer risk in US blacks and whites with a family history of cancer. Int J Cancer 1995;60: 361–64.
4. Tulinius H, Egilsson V, Olafsdottir GH, et al: Risk of prostate, ovarian, and endometrial cancer among relatives of women with breast cancer. BMJ 1992;305:855-857
5. Chan JM, Stampfer MJ, Giovannucci E, et al. Plasma insulin-like growth factor-I and prostate cancer risk: a prospective study. Science 1998; 279: 563–66.
6. Harman SM, Metter EJ, Blackman MR, Landis PK, Carter HB.Serum levels of insulin-like growth factor I (IGF-I), IGF-II, IGFbinding protein-3, and prostate-specific antigen as predictors of clinical prostate cancer. J Clin Endocrinol Metab 2000; 85: 4258–65.
7. Stattin P, Bylund A, Rinaldi S, et al. Plasma insulin-like growth factor-I, insulin-like growth factor-binding proteins, and prostate cancer risk:a prospective study. J Natl Cancer Inst 2000; 92: 1910–17.
8. Augustsson K, Skog K, Jagerstad M, Dickman PW, Steineck G.Dietary heterocyclic amines and cancer of the colon, rectum, bladder,and kidney: a population-based study. Lancet 1999; 353: 703–07.
9. Miller BA, Kolonel LN, Bernstein L, et al., eds. Racial/Ethnic Patterns of Cancer in the United States 1988–1992. NIH Publication No. 96–4104. Bethesda, MD: National Cancer Institute;1996.
10. Cook LS,Goldoft M,Schwartz SM,et al.Incidence of adenocarcinoma of prostate in Asican immigrants to the United States and their descendants.J Urol,1999,161:152-155
11. 李鸣,前列腺癌发病率和影响因素.中国肿瘤.2003,12(12):688-691
12. Glutathione S-transferase pi is upregulated in the stromal compartment of hormone independent prostate cancer.The prostate.56:98-105
13. 管同郁,李鸣,那彦群.生物转化酶基因多态性与前列腺癌易感性的研究.中华外科,2005,43(22):1467-1470
14. Hickey K, Do KA, Green A. Smoking and prostate cancer.Epidemiol Rev 2001; 23: 115–25.
15. Dennis LK, Hayes RB. Alcohol and prostate cancer. Epidemiol Rev 2001; 23: 110–14.
16. Peterson HB, Howards SS. Vasectomy and prostate cancer: the evidence to date. Fertil Steril 1998; 70: 201–03.
17. Lee IM, Sesso HD, Chen JJ, Paffenbarger RS Jr. Does physical activity play a role in the prevention of prostate cancer? Epidemiol Rev 2001; 23: 132–37.
1. Chung Chan D, Liao Jiayu, Liu Bin, et al. Specific inhibition of Stat3 signal transduction by PIAS3[J].Science,1997,278(5344):1803-1805.
2.Wible BA, Wang Liming, Kuryshey YA, et al. Increased K+ efflux and apoptosis induced by the potassium channel modulatory protein KChAP/PIAS3β in prostate cancer cells[J].J Biol Chem, 2002,277(20):17852-17862.
3. 王永俊,施小六,汤建光,等. RNAi 下调 survivin 表达诱导成人神经细胞瘤细胞凋亡[J].基础医学与临床杂志,2006,26(7):745-750.
4. 翁迈,周利群,王建伟,等.PIM-1 蛋白激酶在前列腺癌组织中的表达[J].基础医学与临床杂志,2005,25(6):503-506.
5. Wang Liming, Banerjee S.Differential PIAS3 expression in human malignancy[J]. Oncol Rep,2004,11(6):1319-1324.
6. Junicho A, Matsuda T, Yamamoto T,et al. Protein inhibitor of activated STAT3 regulates androgen receptor signaling in prostate carcinoma cells[J].Biochem Biophys Res Commun,2000,278(1):9-13.
7. Deng Jianbei, Hua Kunjie, Caveney EJ,et al Protein inhibitor of activated STAT3 inhibits adipogenic gene expression[J]. Biochem Biophys Res Commun,2006, 339(3):923-931
8. St?rkel P,Saeger CD, Leclercq I,et al Role of signal transducer and activator of transcription 3 in liver fibrosis progression in chronic hepatitis C-infected patients[J]. Lab Invest, 2007,87 (2):173-181.
9. Ogata Y, Osaki T, Naka T,et al Overexpression of PIAS3 suppresses cell growth and restores drug sensitivity of human lung cancer cells in association with PI3-K/Akt inactivation[J].Neoplasia,2006,8(10):817-825
10. 唐梅,张钲,伏静媛,等.JAK2/STAT3 信号通路介导大鼠血管平滑肌细胞的增殖与凋亡[J].基础医学与临床杂志,2006,26(12):1354-1358
11. Drachenberg DE, Elgamal AA,Rowbotham R, et al. Circulating levels of IL-6 in patients with hormone-refractory prostate cancer[J].Prostate,1999,41:127-133.