MiRNA-21在激素非依赖前列腺癌细胞中的作用及其分子机制实验研究
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摘要
目的
研究在激素非依赖性前列腺癌细胞中E2和AR-miRNA-21通路的内在关联性及其重要意义。分析非依赖前列腺癌中miRNA-21调控的下游分子机制。
方法
通过RT-PCR检查有无E2培养下非依赖性细胞系PC3在转染miRNA-21 inhibitor前后miRNA-21及AR的表达情况。通过CCK-8和FCM检测转染前后细胞的增殖能力。通过western blot检测有无E2培养下PC3细胞AR蛋白的表达情况。通过western blot检测在转染miRNA-21 inhibitor前后PTEN、p-Akt和MMP-2的表达情况。
结果
RT-PCR检测显示:E2环境培养下转染前miRNA-21的表达明显高于不含E2的培养基中的表达;在无E2的培养基中转染后miRNA-21的表达明显低于转染前的表达;在含有E2的培养基中转染后的miRNA-21的表达明显低于转染前得细胞组。
Cck-8方法测转染前PC3细胞系在不同环境下和转染miRNA-21 inhibitor前后的增殖能力,显示在含有E2的环境下PC3细胞增殖能力明显增强;无论有没有E2,转染后PC3的增值能力均明显比未转染组弱。
FCM检测转染miRNA-21抑制剂后,对应与转染前PC3细胞的增殖都明显的减慢。
RT-PCR检测AR表达发现,转染前后AR的表达变化不具有显著性(P>0.05),但E2能促进AR的表达增加。
western blot测定PTEN、p-Akt和MMP-2在转染前后的PC3细胞中表达水平。转染后PTEN的表达明显高于转染前;转染后p-Akt和MMP-2的表达明显低于转染前。
结论
1. E2通过诱导AR表达在非依赖性前列腺癌的进展中起重要作用。
2. E2可能通过AR或其他途径诱导miRNA-21的表达。
3. miRNA-21在前列腺癌的非依赖性进程中起重要作用,转染miRNA-21 inhibitor前列腺癌细胞的增值能力明显降低提示miRNA-21 inhibitor有希望成为非依赖性前列腺癌的一个潜在治疗之路。
4. MiR-21负面调节PTEN的表达同时上调p-Akt和MMP-2的表达,提示miRNA-21可能通过PTEN-pAkt/MMP-2通路调控前列腺癌的进展。
Objective
To study the relationship and the significance of E2 with AR-miRNA-21 pathway, Analyze the downstream regulation mechanisms of miRNA-21 in hormone-independence prostate cancer.
Methods
Respectively, in the presence of E2 and E2 in the special medium does not contain cultured PC3 and then were transected with miRNA-21 inhibitor. Detect cell proliferation with CCK-8 and FCM, while the expression of gene and protein with RT-PCR and western blot.
Results
RT-PCR analysis showed that before transfection the expression of miRNA-21 in E2 environment cultured was significantly higher than in the medium without E2; In the presence of E2 the expression of miRNA-21 after transfection was significantly lower than untransfected cell group.
Cck-8:in the presence of E2 the PC3 cell proliferation was significantly enhanced ; With or without E2, PC3 after transfection, the capacity of amplification was significantly weaker than the untransfected group.
FCM: make sure that the miRNA-21 inhibitor is useful to mitotic cycle in PC3.
AR is not different between before transfection and behind transfection (p>0.05), EGF can increase expression of AR by RT-PCR,but E2 can promote increased expression of AR.
Western blot show that the expression of PTEN behind transfection is higher than before,but p-Act and MMP-2 behind transfection is lower than before.
Conclusions
1. E2 through inducting of AR expression in prostate cancer progression plays an important role.
2. E2 may induce the expression of miRNA-21 by AR or other means.
3. miRNA-21 plays an important role in the process of transfection in hormone-independence prostate cancer. Transfected with miRNA-21 inhibitor of prostate cancer cells significantly reduced the amplification capacity and prompted miRNA-21-inhibitor with prostate cancer will be a potential way of biological treatment.
4. miRNA-21 negatively regulate the expression of PTEN also increased p-Akt and the expression of MMP-2, suggesting that miRNA-21 pathway may be through PTEN-pAkt/MMP-2 control the progress of prostate cancer.
研究在激素非依赖性前列腺癌细胞中E2和AR-miRNA-21通路的内在关联性及其重要意义。分析非依赖前列腺癌中miRNA-21调控的下游分子机制。
方法
通过RT-PCR检查有无E2培养下非依赖性细胞系PC3在转染miRNA-21 inhibitor前后miRNA-21及AR的表达情况。通过CCK-8和FCM检测转染前后细胞的增殖能力。通过western blot检测有无E2培养下PC3细胞AR蛋白的表达情况。通过western blot检测在转染miRNA-21 inhibitor前后PTEN、p-Akt和MMP-2的表达情况。
结果
RT-PCR检测显示:E2环境培养下转染前miRNA-21的表达明显高于不含E2的培养基中的表达;在无E2的培养基中转染后miRNA-21的表达明显低于转染前的表达;在含有E2的培养基中转染后的miRNA-21的表达明显低于转染前得细胞组。
Cck-8方法测转染前PC3细胞系在不同环境下和转染miRNA-21 inhibitor前后的增殖能力,显示在含有E2的环境下PC3细胞增殖能力明显增强;无论有没有E2,转染后PC3的增值能力均明显比未转染组弱。
FCM检测转染miRNA-21抑制剂后,对应与转染前PC3细胞的增殖都明显的减慢。
RT-PCR检测AR表达发现,转染前后AR的表达变化不具有显著性(P>0.05),但E2能促进AR的表达增加。
western blot测定PTEN、p-Akt和MMP-2在转染前后的PC3细胞中表达水平。转染后PTEN的表达明显高于转染前;转染后p-Akt和MMP-2的表达明显低于转染前。
结论
1. E2通过诱导AR表达在非依赖性前列腺癌的进展中起重要作用。
2. E2可能通过AR或其他途径诱导miRNA-21的表达。
3. miRNA-21在前列腺癌的非依赖性进程中起重要作用,转染miRNA-21 inhibitor前列腺癌细胞的增值能力明显降低提示miRNA-21 inhibitor有希望成为非依赖性前列腺癌的一个潜在治疗之路。
4. MiR-21负面调节PTEN的表达同时上调p-Akt和MMP-2的表达,提示miRNA-21可能通过PTEN-pAkt/MMP-2通路调控前列腺癌的进展。
Objective
To study the relationship and the significance of E2 with AR-miRNA-21 pathway, Analyze the downstream regulation mechanisms of miRNA-21 in hormone-independence prostate cancer.
Methods
Respectively, in the presence of E2 and E2 in the special medium does not contain cultured PC3 and then were transected with miRNA-21 inhibitor. Detect cell proliferation with CCK-8 and FCM, while the expression of gene and protein with RT-PCR and western blot.
Results
RT-PCR analysis showed that before transfection the expression of miRNA-21 in E2 environment cultured was significantly higher than in the medium without E2; In the presence of E2 the expression of miRNA-21 after transfection was significantly lower than untransfected cell group.
Cck-8:in the presence of E2 the PC3 cell proliferation was significantly enhanced ; With or without E2, PC3 after transfection, the capacity of amplification was significantly weaker than the untransfected group.
FCM: make sure that the miRNA-21 inhibitor is useful to mitotic cycle in PC3.
AR is not different between before transfection and behind transfection (p>0.05), EGF can increase expression of AR by RT-PCR,but E2 can promote increased expression of AR.
Western blot show that the expression of PTEN behind transfection is higher than before,but p-Act and MMP-2 behind transfection is lower than before.
Conclusions
1. E2 through inducting of AR expression in prostate cancer progression plays an important role.
2. E2 may induce the expression of miRNA-21 by AR or other means.
3. miRNA-21 plays an important role in the process of transfection in hormone-independence prostate cancer. Transfected with miRNA-21 inhibitor of prostate cancer cells significantly reduced the amplification capacity and prompted miRNA-21-inhibitor with prostate cancer will be a potential way of biological treatment.
4. miRNA-21 negatively regulate the expression of PTEN also increased p-Akt and the expression of MMP-2, suggesting that miRNA-21 pathway may be through PTEN-pAkt/MMP-2 control the progress of prostate cancer.
引文
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11 Volinia S, Calin GA, Liu CG, et al. A microRNA expression signature of human solid tumors defines cancer gene targets [J]. ProcNatl Acad Sci USA. 2006 Feb 14; 103(7): 2257-61.
12 Zhu S, Wu H, Wu F, et al. MicroRNA-21 targets tumor suppressor genes in invasion and metastasis [J]. Cell Res. 2008 Mar; 18(3):350-9.
13 Kasahara K, Taguchi T, Yamasaki I, et al. Detection of genetic alterations in advancedprostate cancer by comparative genomic hybridization [J]. Cancer Genet Cytogenet. 2002 Aug; 137(1):59-63.
14 Li T, Li D, Sha J, et al. MicroRNA-21 directly targets MARCKS and promotes apoptosis resistance and invasion in prostate cancer cells [J]. Biochem Biophys Res Commun. 2009 Jun 5; 383(3):280-5.
15 Meng F, Henson R, Wehbe-Janek H, et al. MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer [J]. Gastroenterology. 2007 Aug; 133(2): 647-658.
16 Rosivatz E. Inhibiting PTEN [J]. Biochem Soc Trans. 2007 Apr; 35(Pt2): 257-259.
17 Roy S, Khanna S, Hussain SR,et al. MicroRNA expression in response to murine myocardial infarction: miR-21 regulates fibroblast metalloprotease-2 via phosphatase and tensin homologue [J].Cardiovasc Res. 2009 Apr 1; 82(1):21-9.
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20 Han G, Buchanan G, Ittmann M, et al. Mutation of the androgen receptor causes oncogenic transformation of the prostate [J]. PNAS ,2005 Jan 25;,102 (4) :1151-1156
21 Visakorpi T, Hyytinen E, Koivisto P, et al. In vivo amplification of t he androgen receptor gene and progression of human prostate cancer [J]. Nat Genet ,1995 Apr; 9(4) :401-406
22 Mangelsdorf DJ, Thummel C, Beato M, et al. The nuclear receptor superfamily: the second decade [J]. Cell 1995 Dec 15; 83(6): 835-9.
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33 Littlepage LE, Sternlicht MD, Rougier N, et al. Matrix metalloproteinases contribute distinct roles in neuroendocrine prostate carcinogenesis, metastasis, and angiogenesis progression [J]. Cancer Res. 2010 Mar; 70(6): 2224-2234.
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2 Gandellini P, Folini M, Zaffaroni N, et al. Towards the definition of prostate cancer-related microRNAs: where are we now [J]? Trends Mol Med. 2009 Sep;15(9): 381-90.
3 Bartel, DP. MicroRNAs: genomics, biogenesis, mechanism, and function [J]. Cell.2004 Jan 23; 116(2):281-97.
4 Bartel, D.P. MicroRNAs: target recognition and regulatory functions [J]. Cell. 2009 Jan 23; 136(2): 215-33.
5 Winter J, Jung S, Keller S, et al. Many roads to maturity: microRNA biogenesis pathways and their regulation [J]. Nat Cell Biol. 2009 Mar; 11(3): 228-234.
6 Lugli G, Torvik VI, Larson J, et al. Expression of microRNAs and their precursors in synaptic fractions of adult mouse forebrain [J]. Neurochem. 2008 Jul; 106(2); 650-661
7 Kedde M, Strasser MJ, Boldajipour B, et al. RNA-binding protein Dnd1 inhibits microRNA access to target Mrna [J]. Cell.2007 Dec 28; 131(7), 1273-1286
8 Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets [J]. Cell. 2005 Jan 14; 120(1): 15-20
9 Medina PP, Slack FJ. microRNAs and cancer: an overview [J]. Cell Cycle. 2008 Aug 15; 7(16): 2485-2492
10 Chan JA, Krichevsky AM, Kosik KS. MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells [J]. Cancer Res. 2005 Jul 15; 65(14): 6029-33.
11 Volinia S, Calin GA, Liu CG, et al. A microRNA expression signature of human solid tumors defines cancer gene targets [J]. ProcNatl Acad Sci USA. 2006 Feb 14; 103(7): 2257-61.
12 Zhu S, Wu H, Wu F, et al. MicroRNA-21 targets tumor suppressor genes in invasion and metastasis [J]. Cell Res. 2008 Mar; 18(3):350-9.
13 Kasahara K, Taguchi T, Yamasaki I, et al. Detection of genetic alterations in advancedprostate cancer by comparative genomic hybridization [J]. Cancer Genet Cytogenet. 2002 Aug; 137(1):59-63.
14 Li T, Li D, Sha J, et al. MicroRNA-21 directly targets MARCKS and promotes apoptosis resistance and invasion in prostate cancer cells [J]. Biochem Biophys Res Commun. 2009 Jun 5; 383(3):280-5.
15 Meng F, Henson R, Wehbe-Janek H, et al. MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer [J]. Gastroenterology. 2007 Aug; 133(2): 647-658.
16 Rosivatz E. Inhibiting PTEN [J]. Biochem Soc Trans. 2007 Apr; 35(Pt2): 257-259.
17 Roy S, Khanna S, Hussain SR,et al. MicroRNA expression in response to murine myocardial infarction: miR-21 regulates fibroblast metalloprotease-2 via phosphatase and tensin homologue [J].Cardiovasc Res. 2009 Apr 1; 82(1):21-9.
18 Furukawa K, Kumon Y, Harada H, et al. PTEN gene transfer suppresses the invasive potential of human malignant gliomas by regulating cell invasion-related molecules [J]. Int J Oncol 2006 Jul; 29(1):73-81.
19 Suzuki H, Akakura K, Komiya A. et al. CAG polymorphic repeat lengt hs in androgen receptor gene among Japanese prostate cancer patients: potential predictor of prognosis after endocrine t herapy [J]. Prostate. 2002 May 15; 51(3): 219-224
20 Han G, Buchanan G, Ittmann M, et al. Mutation of the androgen receptor causes oncogenic transformation of the prostate [J]. PNAS ,2005 Jan 25;,102 (4) :1151-1156
21 Visakorpi T, Hyytinen E, Koivisto P, et al. In vivo amplification of t he androgen receptor gene and progression of human prostate cancer [J]. Nat Genet ,1995 Apr; 9(4) :401-406
22 Mangelsdorf DJ, Thummel C, Beato M, et al. The nuclear receptor superfamily: the second decade [J]. Cell 1995 Dec 15; 83(6): 835-9.
23 Yuan X, Balk SP.Mechanisms mediating androgen receptor reactivation after castration [J]. Urol Oncol 2009 Jan-Feb; 27(1): 36-41.
24 Koivisto P. Aneuploidy and rapid cell proliferation in recurrent prostate cancers with androgen receptor gene amplification [J]. Prostate Cancer Prostatic Dis ,1997 Sep;1 (1): 21-25
25 Bohl CE, Wenqing G, Duane DM, et al. Structural Basis for Antagonism and Resistance of Bicalutamide in Prostate Cancer [J]. Proc. Natl. Acad. Sci. USA 2005 Apr 26; 102(17), 6201-6206.
26 Isbarn H, Boccon-Gibod L, Carroll PR, et al. Androgen Deprivation Therapy for the Treatment of Prostate Cancer: Consider Both Benefits and Risks [J]. Eur. Urol. 2009 Jan; 55(1): 62-75.
27 Ribas J, Ni X, Haffner M. et al. miR-21: An Androgen Receptor Regulated MicroRNA that Promotes Hormone-Dependent and Hormone-Independent Prostate Cancer Growth [J]. Cancer Res 2009 Sep 15;69(18):7165-9
28 Zhu S, Wu H, Wu F, et al.MicroRNA-21 targets tumor suppressor genes in invasion and metastasis [J]. Cell Res 2008 Mar;18(3):350-9.
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