PRMT2下调NF-κB对甲状腺癌细胞增殖及凋亡的影响
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摘要
目的:检测并观察PRMT2在甲状腺癌SW579细胞株中的表达情况;构建稳定表达PRMT2和空载体细胞系SW579-PRMT2-V5、SW579-V5,研究PRMT2对人甲状腺癌SW579细胞生长的影响,并探讨其作用机制。
方法:应用Lipofectamine 2000将真核表达载体和pcDNA3.2 /V5-PRMT2空载体pcDNA3.2 /V5转染入SW579细胞中,经G418抗性筛选得到稳定的克隆并扩大培养成细胞系,观察转染后细胞形态学的变化,采用RT-PCR和Western blot鉴定稳定表达PRMT2的SW579细胞。MTT法检测稳定表达PRMT2对SW579细胞生长的影响,流式细胞仪分析细胞凋亡的变化,Western blot检测转录因子NF-κB及相关基因蛋白表达的变化,PRMT2对信号NF-κB转导通路的影响。
结果:PRMT2在甲状腺癌SW579细胞中有低表达。成功建立了稳定表达PRMT2和空载体的细胞系SW579-PRMT2-V5、SW579-V5,转染后细胞形态学发生了变化,PRMT2显著抑制SW579细胞体外增殖能力,而且在诱导SW579细胞发生凋亡的同时,PRMT2可下调NF-κB活性及其下游靶基因cyclin D1的表达。
结论:1.稳定表达PRMT2和空载体细胞系SW579-PRMT2-V5、SW579-V5构建成功。
2. PRMT2能下调NF-κB及其下游靶基因cyclin D1的表达,促进甲状腺癌细胞凋亡。
Objectives: The Study aims to detect the expression of protein arginine methyltransferase 2 (PRMT2) in the human Thyroid Cancer SW579 cell, to construct the vector of SW579-PRMT2-V5, and to investigate the effect of PRMT2 on the growth of SW579 cells.
Methods: the eukaryotic expression plasmid pcDNA3.2 /V5-PRMT2, and empty vector pcDNA3.2 /V5 were transfect into SW579 cells. The cells are selected by G418. After being transfected, proliferation of SW579 cells was observed by MTT assay. Apoptosis of SW579 cells are tested by using Hoechst 33342 staining and flow cytometry. NF-κB activation and its related genes are detected by Western blot. The expression of PRMT2 mRNA and protein are then detected by reverse transcription-polymerase chain reaction ( RT-PCR) and Western blot.
Results: A low expression level was detected in human Thyroid Cancer SW579 cells. Cells with SW579-PRMT2-V5 expression were successfully established. After infection of PRMT2, the growth of Thyroid Cancer cells was inhibited significantly. Morever, PRMT2 is induced apoptosis in Thyroid Cancer cells. Also, down -regulation of cyclin D1, companied by decrease in NF–κB activation, are obseved in SW579-PRMT2-V5 cells.
Conclusion: 1.Cells with SW579-PRMT2-V5 expressing were successfully established. After transfection of PRMT2, the growth of Thyroid Cancer cells is inhibited significantly.
2.PRMT2 induces apoptosis in Thyroid Cancer cells. Down-regulation of cyclin D1, companied by decrease in NF–κB activation, was also obseved in SW579-PRMT2-V5 cells.
方法:应用Lipofectamine 2000将真核表达载体和pcDNA3.2 /V5-PRMT2空载体pcDNA3.2 /V5转染入SW579细胞中,经G418抗性筛选得到稳定的克隆并扩大培养成细胞系,观察转染后细胞形态学的变化,采用RT-PCR和Western blot鉴定稳定表达PRMT2的SW579细胞。MTT法检测稳定表达PRMT2对SW579细胞生长的影响,流式细胞仪分析细胞凋亡的变化,Western blot检测转录因子NF-κB及相关基因蛋白表达的变化,PRMT2对信号NF-κB转导通路的影响。
结果:PRMT2在甲状腺癌SW579细胞中有低表达。成功建立了稳定表达PRMT2和空载体的细胞系SW579-PRMT2-V5、SW579-V5,转染后细胞形态学发生了变化,PRMT2显著抑制SW579细胞体外增殖能力,而且在诱导SW579细胞发生凋亡的同时,PRMT2可下调NF-κB活性及其下游靶基因cyclin D1的表达。
结论:1.稳定表达PRMT2和空载体细胞系SW579-PRMT2-V5、SW579-V5构建成功。
2. PRMT2能下调NF-κB及其下游靶基因cyclin D1的表达,促进甲状腺癌细胞凋亡。
Objectives: The Study aims to detect the expression of protein arginine methyltransferase 2 (PRMT2) in the human Thyroid Cancer SW579 cell, to construct the vector of SW579-PRMT2-V5, and to investigate the effect of PRMT2 on the growth of SW579 cells.
Methods: the eukaryotic expression plasmid pcDNA3.2 /V5-PRMT2, and empty vector pcDNA3.2 /V5 were transfect into SW579 cells. The cells are selected by G418. After being transfected, proliferation of SW579 cells was observed by MTT assay. Apoptosis of SW579 cells are tested by using Hoechst 33342 staining and flow cytometry. NF-κB activation and its related genes are detected by Western blot. The expression of PRMT2 mRNA and protein are then detected by reverse transcription-polymerase chain reaction ( RT-PCR) and Western blot.
Results: A low expression level was detected in human Thyroid Cancer SW579 cells. Cells with SW579-PRMT2-V5 expression were successfully established. After infection of PRMT2, the growth of Thyroid Cancer cells was inhibited significantly. Morever, PRMT2 is induced apoptosis in Thyroid Cancer cells. Also, down -regulation of cyclin D1, companied by decrease in NF–κB activation, are obseved in SW579-PRMT2-V5 cells.
Conclusion: 1.Cells with SW579-PRMT2-V5 expressing were successfully established. After transfection of PRMT2, the growth of Thyroid Cancer cells is inhibited significantly.
2.PRMT2 induces apoptosis in Thyroid Cancer cells. Down-regulation of cyclin D1, companied by decrease in NF–κB activation, was also obseved in SW579-PRMT2-V5 cells.
引文
[1]史业辉,钱碧云.原发性甲状腺鳞状细胞癌27例分析.中国肿瘤临床, 2004, 31: 456.
[2] Katz JE , Dlakic M , and Clarke S.Automated identification of putative methyltransferases from genomic open reading frames. [J].Mol. Cell. Proteomics. 2003,2(8):525–540.
[3] Meyer R, Wolf SS, Obendorf M. PRMT2, a member of the protein arginine methyltransferase family, is a coactivator of the androgen receptor[J]. Steroid Biochem Mol Biol, 2007 ,107(122): 1214.
[4] Yoshimoto T, Boehm M, Olive M, et al.The arginine methyltransferase PRMT2 binds RB and regulates E2F function[J]. Exp Cell Res. 2006, 312(11): 2040-53.
[5] Ganesh L, Yoshimoto T, Moorthy NC ,et al. Protein methyltransferase 2 inhibits NF-kappaB function and promotes apoptosis[J]. Mol Cell Biol.2006,26(10):3864-74.
[6]张端莲,余红梅,蔡俊伟,等.NF-kBp65在甲状腺肿瘤组织中的表达及临床意义.数理医药学杂志,2005, 18(6): 20-21
[7] Katz JE,Dlakic M,and Clarke S.Automated identification of putative methyltransferases from genomic open reading frames. [J].Mol. Cell. Proteomics. 2003 ,2(8):525–540.
[8] Katsanis N, Yaspo ML, Fisher EM. Identification and mapping of a novel human gene, HRMT1L1, homologous to the rat protein arginine N-methyltransferase 1 (PRMT1) gene[J]. Mamm Genome. 1997 ,8(7):526-529.
[9] Ganesh L, Yoshimoto T, Moorthy NC ,et al. Protein methyltransferase 2 inhibits NF-kappaB function and promotes apoptosis[J].Mol Cell Biol,2006,26(10):3864-3874.
[10] Chen F ,Castranova V ,Shi X. New insights into the role of nuclear factor2kappaB in cell growth regulation [ J ] . Am J Pathol ,2001 ,159 (2) :387 - 397.
[11] Ghosh S , Karin M. Missing pieces in the NF-κB puzzle [J ] . Cell , 2002 , 109 :81 - 96.
[12] Masayuki I. Prognostic significance of cyclin D1 and E cadherin in patients with esophageal squamous cell carcinoma :multinstitution2 al retrospectiveanalysis[J ] . J Am Coll Surg ,2004 , Am Coll Surg ,2004 ,192 (6) :708 -718.
[13] Hinz M ,Krappmann D ,Eichten A , et al . NF-kappa B function in growth control :regulation of cyclin D1 expression and G0/ G12to2 S2Phase transition[J] . Mol Cell Biol ,1999 ,19 (4) :2690– 2698
[14] Chao Qi, Jeffrey Chang, Yiwei Zhu et al. Identification of Protein Arginine Methyltransferase 2 as a Coactivator for Estrogen Receptor[J]. Biol.chem.2002Aug; 277(32):28624-28630.
[15] Scott HS,Antonarakis SE, Lalioti MD et al.Identification and characterization of two putative human arginine methyltransferases (HEMT1L1 and HRMT1L2).[J]. Genomics.2008, 48(3):330-340.
[16] PandoMP, et al, Signal-dependent and -independent degradation of free and NF-kappa B-bound IkappaBalpha [J] biol chem, 2000,275(28):21278-86.
[17] Choi BH , Choi M , J eon HY, et al . Hepatitis B viral X protei2novercomes inhibition of E2F21 activity by pRb on t he human Rb genepromoter [ J ] .DNA Cell Biol , 2001 , 20 ( 2 ) : 75280.
[18] Krek W, Xu G, Livingston DM. Cyclin A2kinase regulation of E2 F21 DNA binding function underlies suppression of a S phase checkpoint [J ] . Cell , 1995 , 29,83 (7) :1149-58
[19] Puga A , Barnes SJ , Dalton TP , et al . Aromatic hydrocarbonreceptorint- eraction wit h t he retinoblastoma protein potentiatesrepression of E2 F2dependent t ranscription and cell cycle arrest[J ] . J Biol Chem , 2000 ,275 (4) :2943-2950.
[20] Pail F, Bibud hendra S. Metal replacement in Zinc .Finger and its Effect on DNA Binding.[J].Environmental health perspectives,1994.102 ( 3):195-198.
[1] Chao Qi, Jeffrey Chang, Yiwei Zhu et al. Identification of Protein Arginine Methyltransferase 2 as a Coactivator for Estrogen Receptor[J]. Biol.chem.2002,277(32):28624-28630.
[2] Meyer R, Wolf SS , Obendorf M. PRMT2, a member of the protein arginine methyltransferasefamily, is a coactivator of the androgen receptor[J]Steroid Biochem Mol Biol.2007, (1-2):1–14.
[3] Yoshimoto T, Boehm M, OliveM,etal.The arginine methyltransferase PRMT- bindsRBandregulatesE2Ffunction.[J].ExpCellRes .Res .2006,312(11):2040-2053.
[4] Ganesh L, Yoshimoto T, Moorthy NC et al. Protein Methyltransferase 2 Inhibits NF-B Functionand Promotes Apoptosis. [J]. Mol. Cell. Res. 2006 ,312 (11): 2040-2053.
[5] Scott HS,Antonarakis SE, Lalioti MD et al.Identification and characterization of two putative human arginine methyltransferases (HEMT1L1 and HRMT1L2).[J]. Genomics.1998, 48(3):330-340.
[6] Katsanis N,Yaspo ML,Fisher EM. Identification and mapping of a novel human gene, HRMTL1,homologous to the rat protein argininr N-methyltransferase. [J]. Mamm Genome.1997 ,8(7):526-529.
[7] Ted M. LAKOWSKI and Adam FRANKEL1. Kinetic Analysis of Human Protein Arginine N-Methyltransferase 2: Formation ofMonomethyl- and Asymmetric Dimethylarginine Residues on Histone H4.[J] Biochemical Journal Immediate Publication. 2009,(2)68-52
[8] PandoMP, et al, Signal-dependent and -independent degradation of free and NF-kappa B-bound IkappaBalpha [J] biol chem, 2000,275(28):21278-86.
[9] Kraus WL,Mcinerney EM,Katzeneuenenbogen BS. Ligand-dependent transcriptionally productive association of the amino-and carboxy-terminal regions of a steroid hormone nuclear receptor[J]:Natl Acad sci USA, 1995,92:12314-12318.
[10] Pail F, Bibud hendra S. Metal replacement in Zinc .Finger and its Effect on DNA Binding.[J].Environmental health perspectives,1994.102 (3):195-198
[11] Evans RM. The steroid and thyroid hormone surperfamily [J]science 1988, 240:889-895.
[12] Palvimo J J , Kallio P J , J konen T , et al . Dominant negative regu2lation oftrans2activation by the rat androgen receptor : roles of theN2terminal domain and heterodimer formation. Mol Endocrinol ,1993 , 7 (11) : 1399～1407.
[13] Puga A , Barnes SJ , Dalton TP , et al . Aromatic hydrocarbonreceptorint- eraction wit h t he retinoblastoma protein potentiatesrepression of E2 F2dependent t ranscription and cell cycle arrest[J ] . J Biol Chem , 2000 ,275 (4):2943-2950.
[14] Choi BH , Choi M , J eon HY, et al . Hepatitis B viral X protei2novercomes inhibition of E2F21 activity by pRb on t he human Rb genepromoter [ J ] . DNA Cell Biol , 2001 , 20 ( 2 ) : 75280.
[15] Krek W, Xu G, Livingston DM. Cyclin A2kinase regulation of E2 F21 DNA binding function underlies suppression of a S phase checkpoint [J ] . Cell , 1995 , 83 (7) :1149-1158.
[2] Katz JE , Dlakic M , and Clarke S.Automated identification of putative methyltransferases from genomic open reading frames. [J].Mol. Cell. Proteomics. 2003,2(8):525–540.
[3] Meyer R, Wolf SS, Obendorf M. PRMT2, a member of the protein arginine methyltransferase family, is a coactivator of the androgen receptor[J]. Steroid Biochem Mol Biol, 2007 ,107(122): 1214.
[4] Yoshimoto T, Boehm M, Olive M, et al.The arginine methyltransferase PRMT2 binds RB and regulates E2F function[J]. Exp Cell Res. 2006, 312(11): 2040-53.
[5] Ganesh L, Yoshimoto T, Moorthy NC ,et al. Protein methyltransferase 2 inhibits NF-kappaB function and promotes apoptosis[J]. Mol Cell Biol.2006,26(10):3864-74.
[6]张端莲,余红梅,蔡俊伟,等.NF-kBp65在甲状腺肿瘤组织中的表达及临床意义.数理医药学杂志,2005, 18(6): 20-21
[7] Katz JE,Dlakic M,and Clarke S.Automated identification of putative methyltransferases from genomic open reading frames. [J].Mol. Cell. Proteomics. 2003 ,2(8):525–540.
[8] Katsanis N, Yaspo ML, Fisher EM. Identification and mapping of a novel human gene, HRMT1L1, homologous to the rat protein arginine N-methyltransferase 1 (PRMT1) gene[J]. Mamm Genome. 1997 ,8(7):526-529.
[9] Ganesh L, Yoshimoto T, Moorthy NC ,et al. Protein methyltransferase 2 inhibits NF-kappaB function and promotes apoptosis[J].Mol Cell Biol,2006,26(10):3864-3874.
[10] Chen F ,Castranova V ,Shi X. New insights into the role of nuclear factor2kappaB in cell growth regulation [ J ] . Am J Pathol ,2001 ,159 (2) :387 - 397.
[11] Ghosh S , Karin M. Missing pieces in the NF-κB puzzle [J ] . Cell , 2002 , 109 :81 - 96.
[12] Masayuki I. Prognostic significance of cyclin D1 and E cadherin in patients with esophageal squamous cell carcinoma :multinstitution2 al retrospectiveanalysis[J ] . J Am Coll Surg ,2004 , Am Coll Surg ,2004 ,192 (6) :708 -718.
[13] Hinz M ,Krappmann D ,Eichten A , et al . NF-kappa B function in growth control :regulation of cyclin D1 expression and G0/ G12to2 S2Phase transition[J] . Mol Cell Biol ,1999 ,19 (4) :2690– 2698
[14] Chao Qi, Jeffrey Chang, Yiwei Zhu et al. Identification of Protein Arginine Methyltransferase 2 as a Coactivator for Estrogen Receptor[J]. Biol.chem.2002Aug; 277(32):28624-28630.
[15] Scott HS,Antonarakis SE, Lalioti MD et al.Identification and characterization of two putative human arginine methyltransferases (HEMT1L1 and HRMT1L2).[J]. Genomics.2008, 48(3):330-340.
[16] PandoMP, et al, Signal-dependent and -independent degradation of free and NF-kappa B-bound IkappaBalpha [J] biol chem, 2000,275(28):21278-86.
[17] Choi BH , Choi M , J eon HY, et al . Hepatitis B viral X protei2novercomes inhibition of E2F21 activity by pRb on t he human Rb genepromoter [ J ] .DNA Cell Biol , 2001 , 20 ( 2 ) : 75280.
[18] Krek W, Xu G, Livingston DM. Cyclin A2kinase regulation of E2 F21 DNA binding function underlies suppression of a S phase checkpoint [J ] . Cell , 1995 , 29,83 (7) :1149-58
[19] Puga A , Barnes SJ , Dalton TP , et al . Aromatic hydrocarbonreceptorint- eraction wit h t he retinoblastoma protein potentiatesrepression of E2 F2dependent t ranscription and cell cycle arrest[J ] . J Biol Chem , 2000 ,275 (4) :2943-2950.
[20] Pail F, Bibud hendra S. Metal replacement in Zinc .Finger and its Effect on DNA Binding.[J].Environmental health perspectives,1994.102 ( 3):195-198.
[1] Chao Qi, Jeffrey Chang, Yiwei Zhu et al. Identification of Protein Arginine Methyltransferase 2 as a Coactivator for Estrogen Receptor[J]. Biol.chem.2002,277(32):28624-28630.
[2] Meyer R, Wolf SS , Obendorf M. PRMT2, a member of the protein arginine methyltransferasefamily, is a coactivator of the androgen receptor[J]Steroid Biochem Mol Biol.2007, (1-2):1–14.
[3] Yoshimoto T, Boehm M, OliveM,etal.The arginine methyltransferase PRMT- bindsRBandregulatesE2Ffunction.[J].ExpCellRes .Res .2006,312(11):2040-2053.
[4] Ganesh L, Yoshimoto T, Moorthy NC et al. Protein Methyltransferase 2 Inhibits NF-B Functionand Promotes Apoptosis. [J]. Mol. Cell. Res. 2006 ,312 (11): 2040-2053.
[5] Scott HS,Antonarakis SE, Lalioti MD et al.Identification and characterization of two putative human arginine methyltransferases (HEMT1L1 and HRMT1L2).[J]. Genomics.1998, 48(3):330-340.
[6] Katsanis N,Yaspo ML,Fisher EM. Identification and mapping of a novel human gene, HRMTL1,homologous to the rat protein argininr N-methyltransferase. [J]. Mamm Genome.1997 ,8(7):526-529.
[7] Ted M. LAKOWSKI and Adam FRANKEL1. Kinetic Analysis of Human Protein Arginine N-Methyltransferase 2: Formation ofMonomethyl- and Asymmetric Dimethylarginine Residues on Histone H4.[J] Biochemical Journal Immediate Publication. 2009,(2)68-52
[8] PandoMP, et al, Signal-dependent and -independent degradation of free and NF-kappa B-bound IkappaBalpha [J] biol chem, 2000,275(28):21278-86.
[9] Kraus WL,Mcinerney EM,Katzeneuenenbogen BS. Ligand-dependent transcriptionally productive association of the amino-and carboxy-terminal regions of a steroid hormone nuclear receptor[J]:Natl Acad sci USA, 1995,92:12314-12318.
[10] Pail F, Bibud hendra S. Metal replacement in Zinc .Finger and its Effect on DNA Binding.[J].Environmental health perspectives,1994.102 (3):195-198
[11] Evans RM. The steroid and thyroid hormone surperfamily [J]science 1988, 240:889-895.
[12] Palvimo J J , Kallio P J , J konen T , et al . Dominant negative regu2lation oftrans2activation by the rat androgen receptor : roles of theN2terminal domain and heterodimer formation. Mol Endocrinol ,1993 , 7 (11) : 1399～1407.
[13] Puga A , Barnes SJ , Dalton TP , et al . Aromatic hydrocarbonreceptorint- eraction wit h t he retinoblastoma protein potentiatesrepression of E2 F2dependent t ranscription and cell cycle arrest[J ] . J Biol Chem , 2000 ,275 (4):2943-2950.
[14] Choi BH , Choi M , J eon HY, et al . Hepatitis B viral X protei2novercomes inhibition of E2F21 activity by pRb on t he human Rb genepromoter [ J ] . DNA Cell Biol , 2001 , 20 ( 2 ) : 75280.
[15] Krek W, Xu G, Livingston DM. Cyclin A2kinase regulation of E2 F21 DNA binding function underlies suppression of a S phase checkpoint [J ] . Cell , 1995 , 83 (7) :1149-1158.