小干扰RNA靶向抑制DcR3基因对肝癌细胞凋亡和迁移的影响
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摘要
目的:应用RNA干扰技术抑制人肝细胞癌细胞系HepG2细胞DcR3表达,并研究DcR3基因沉默后对HepG2细胞凋亡和迁移能力的影响.方法:设计并合成特异性DcR3-siRNA 4条和阴性对照siRNA 1条,转染HepG2细胞,通过半定量反转录-PCR(RT-PCR)及免疫细胞化学方法检测其对DcR3表达的抑制作用并筛选出干扰效果最强的siRNA,应用流式细胞仪、DNA ladder方法检测细胞的凋亡情况,应用划痕实验检测细胞体外迁移能力的变化.结果:各特异性DcR3-siRNA均能抑制DcR3mRNA表达(P<0.05),其中以DcR3-siRNA4的抑制作用最明显,抑制作用达62.9%;将DcR3-siRNA4转染HepG2细胞能明显抑制HepG2细胞DcR3蛋白的表达(P<0.01).流式细胞仪检测结果显示,特异性DcR3-siRNA转染组细胞的凋亡率(22.97%±2.10%)明显高于空白对照组(1.17%±0.32%)、脂质体转染组(1.44%±0.43%)和非特异性siRNA转染组(1.22%±0.40%)(均P<0.001);DNA ladder实验发现特异性DcR3-siRNA转染组出现DNAladder,而空白对照组、脂质体转染组和非特异性siRNA转染组均未出现DNA ladder.划痕实验结果显示,转染后24、48和72 h,特异性DcR3-siRNA转染组细胞的相对迁移率均低于空白对照组、脂质体转染组和非特异性siRNA转染组.结论:DcR3在肝癌细胞的凋亡及迁移中发挥重要作用,抑制DcR3的表达能诱导肝癌细胞凋亡并降低肝癌细胞的迁移能力.
AIM:To investigate the effect of siRNA-mediated inhibition of DcR3 gene expression on apoptosis and migratory ability of HepG2 cells.METHODS:Four DcR3-specific siRNAs and one random siRNA were designed,synthesized,and transfected into HepG2 cells using LipofectamineTM2000.Semi-quantitative RT-PCR was used to screen the siRNA that had the best interfering effect.Immunocytochemistry was used to assess the expression of DcR3 protein.Cell apoptosis was assessed by flow cytometry,and cell migratory ability was determined by wound healing assay.RESULTS:All four DcR3-specific siRNAs could decrease the expression of DcR3 mRNA,and siRNA4 had the best interfering effect,which could silence the mRNA expression by 62.9%and inhibit the expression of DcR3 protein.The apoptosis rate was significantly higher in the specific interference group than in the three control groups(22.97% ± 2.10% vs 1.17% ± 0.32%,1.44% ± 0.43%,1.22% ± 0.40%,all P < 0.001).The relative migratory ability of HepG2 cells in the specific interference group at 24,48 and 72 h after transfection was significantly lower than that in control groups(all P < 0.001).CONCLUSION:DcR3 plays an important role in the apoptosis and migration of HepG2 cells.Inhibition of expression of DcR3 can induce apoptosis and repress migration of HepG2 cells.
AIM:To investigate the effect of siRNA-mediated inhibition of DcR3 gene expression on apoptosis and migratory ability of HepG2 cells.METHODS:Four DcR3-specific siRNAs and one random siRNA were designed,synthesized,and transfected into HepG2 cells using LipofectamineTM2000.Semi-quantitative RT-PCR was used to screen the siRNA that had the best interfering effect.Immunocytochemistry was used to assess the expression of DcR3 protein.Cell apoptosis was assessed by flow cytometry,and cell migratory ability was determined by wound healing assay.RESULTS:All four DcR3-specific siRNAs could decrease the expression of DcR3 mRNA,and siRNA4 had the best interfering effect,which could silence the mRNA expression by 62.9%and inhibit the expression of DcR3 protein.The apoptosis rate was significantly higher in the specific interference group than in the three control groups(22.97% ± 2.10% vs 1.17% ± 0.32%,1.44% ± 0.43%,1.22% ± 0.40%,all P < 0.001).The relative migratory ability of HepG2 cells in the specific interference group at 24,48 and 72 h after transfection was significantly lower than that in control groups(all P < 0.001).CONCLUSION:DcR3 plays an important role in the apoptosis and migration of HepG2 cells.Inhibition of expression of DcR3 can induce apoptosis and repress migration of HepG2 cells.
引文
1 Pitti RM,Marsters SA,Lawrence DA,Roy M,Kischkel FC,Dowd P,Huang A,Donahue CJ,Sherwood SW,Baldwin DT,Godowski PJ,Wood WI,Gurney AL,Hillan KJ,Cohen RL,Goddard AD,Botstein D,Ashkenazi A.Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer.Nature 1998;396:699-703[PMID:9872321 DOI:10.1038/25387]
2 Yu KY,Kwon B,Ni J,Zhai Y,Ebner R,Kwon BS.A newly identified member of tumor necrosis factor receptor superfamily(TR6)suppresses LIGHT-mediated apoptosis.J Biol Chem 1999;274:13733-13736[PMID:10318773 DOI:10.1074/jbc.274.20.13733]
3 Migone TS,Zhang J,Luo X,Zhuang L,Chen C,Hu B,Hong JS,Perry JW,Chen SF,Zhou JX,Cho YH,Ullrich S,Kanakaraj P,Carrell J,Boyd E,Olsen HS,Hu G,Pukac L,Liu D,Ni J,Kim S,Gentz R,Feng P,Moore PA,Ruben SM,Wei P.TL1A is a TNF-like ligand for DR3 and TR6/DcR3 and functions as a T cell costimulator.Immunity 2002;16:479-492[PMID:11911831 DOI:10.1016/S1074-7613(02)00283-2]
4 Roth W,Isenmann S,Nakamura M,Platten M,Wick W,Kleihues P,B hr M,Ohgaki H,Ashkenazi A,Weller M.Soluble decoy receptor 3 is expressed by malignant gliomas and suppresses CD95 ligandinduced apoptosis and chemotaxis.Cancer Res 2001;61:2759-2765[PMID:11289159]
5 Shi G,Wu Y,Zhang J,Wu J.Death decoy receptor TR6/DcR3 inhibits T cell chemotaxis in vitro and in vivo.J Immunol 2003;171:3407-3414[PMID:14500635]
6 Wan X,Shi G,Semenuk M,Zhang J,Wu J.DcR3/TR6 modulates immune cell interactions.J Cell Biochem 2003;89:603-612[PMID:12761893 DOI:10.1002/jcb.10523]
7 刘忠臣,罗芳洪,陈彩霞,杨东海,范鑫,程小峰,庄国洪.肝癌发展中FasL及其受体Fas/DcR3的表达分析.免疫学杂志2010;26:242-245
8 Yang CR,Hsieh SL,Teng CM,Ho FM,Su WL,Lin WW.Soluble decoy receptor 3 induces angiogenesis by neutralization of TL1A,a cytokine belonging to tumor necrosis factor superfamily and exhibiting angiostatic action.Cancer Res 2004;64:1122-1129[P M I D:1 4 8 7 1 8 4 7 D O I:1 0.1 1 5 8/0 0 0 8-5 4 7 2.CAN-03-0609]
9 Yang CR,Hsieh SL,Ho FM,Lin WW.Decoy receptor 3 increases monocyte adhesion to endothelial cells via NF-kappa B-dependent up-regulation of intercellular adhesion molecule-1,VCAM-1,and IL-8 expression.J Immunol 2005;174:1647-1656[PMID:15661928]
10 陈张铭,任四兰,张蕾.陷阱受体3在肝细胞性肝癌中的表达及其对血管生成的影响.中国现代医学杂志2009;19:1792-1792
11 陈张铭,张阳德,任四兰,张蕾.肝细胞性肝癌中DcR3的表达及其对微血管密度的影响.长沙医学院学报2007;12:18-23
12 Wu Y,Han B,Sheng H,Lin M,Moore PA,Zhang J,Wu J.Clinical significance of detecting elevated serum DcR3/TR6/M68 in malignant tumor patients.Int J Cancer 2003;105:724-732[PMID:12740925DOI:10.1002/ijc.11138]
13 Takahama Y,Yamada Y,Emoto K,Fujimoto H,Takayama T,Ueno M,Uchida H,Hirao S,Mizuno T,Nakajima Y.The prognostic significance of overexpression of the decoy receptor for Fas ligand(DcR3)in patients with gastric carcinomas.Gastric Cancer2002;5:61-68[PMID:12111580 DOI:10.1007/s101200200011]
14 Shen HW,Gao SL,Wu YL,Peng SY.Overexpression of decoy receptor 3 in hepatocellular carcinoma and its association with resistance to Fas ligandmediated apoptosis.World J Gastroenterol 2005;11:5926-5930[PMID:16273601]
15 Shen HW,Wu YL,Peng SY.[Overexpression and genomic amplification of decoy receptor 3 in hepatocellular carcinoma and significance thereof].Zhonghua Yixue Zazhi 2003;83:744-747[PMID:12899749]
16 陈罡,罗殿中,王云,廖芝玲,张美艳.肝细胞癌中DcR3表达和癌细胞凋亡的关系研究.中华病理学杂志2007;36:113-117
17 陈罡,罗殿中.原发性肝细胞癌中诱捕受体3的过度表达及临床意义.广东医学2006;27:1438-1440
18 杨梅松竹,陈罡,党裔武,陈霜,罗殿中.肝细胞癌患者血清DcR3水平与临床意义.世界华人消化杂志2009;17:2042-2047
19 Fire A,Xu S,Montgomery MK,Kostas SA,Driver SE,Mello CC.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature 1998;391:806-811[PMID:9486653DOI:10.1038/35888]
20 Hutvagner G.Small RNA asymmetry in RNAi:function in RISC assembly and gene regulation.FEBS Lett 2005;579:5850-5857[PMID:16199039DOI:10.1016/j.febslet.2005.08.071]
21 Scherer LJ,Rossi JJ.Approaches for the sequencespecific knockdown of mRNA.Nat Biotechnol 2003;21:1457-1465[PMID:14647331 DOI:10.1038/nbt915]
22 Ait-Si-Ali S,Guasconi V,Harel-Bellan A.[RNA interference and its possible use in cancer therapy].Bull Cancer 2004;91:15-18[PMID:14975801]
23 Hannon GJ.RNA interference.Nature 2002;418:244-251[PMID:12110901 DOI:10.1038/418244a]
24 Elbashir SM,Harborth J,Lendeckel W,Yalcin A,Weber K,Tuschl T.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature 2001;411:494-498[PMID:11373684 DOI:10.1038/35078107]
25 Czauderna F,Fechtner M,Dames S,Aygün H,Klippel A,Pronk GJ,Giese K,Kaufmann J.Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells.Nucleic Acids Res 2003;31:2705-2716[PMID:12771196 DOI:10.1093/nar/gng066]
26 Zou GM,Wu W,Chen J,Rowley JD.Duplexes of 21-nucleotide RNAs mediate RNA interference in differentiated mouse ES cells.Biol Cell2003;95:365-371[PMID:14519553 DOI:10.1016/S0248-4900(03)00079-0]
27 Elbashir SM,Harborth J,Weber K,Tuschl T.Analysis of gene function in somatic mammalian cells using small interfering RNAs.Methods2002;26:199-213[PMID:12054897 DOI:10.1016/S1046-2023(02)00023-3]
28 吴泰璜,段伟宏,苏忠学,吴亚光.反义DcR3 RNA转染诱导肝癌细胞凋亡的作用.中华微生物学和免疫学杂志2005;25:799-802
29 段伟宏,苏忠学,吴泰璜.DcR3反义RNA与顺铂对肝癌细胞凋亡影响的比较.中国现代普通外科进展2005;8:90-92
30 段伟宏,苏忠学,吴泰璜.转染反义DcR3真核表达载体协同氟尿嘧啶诱导肝癌细胞凋亡.中华肝胆外科杂志2006;12:98-100
31 陈罡,党裔武,罗殿中.诱捕受体3中和性抗体在HepG2细胞凋亡中的作用.中华肝脏病杂志2008;16:791-792
32 陈罡,罗殿中,党裔武.DcR3对肝癌细胞生长迁移的影响.陕西医学杂志2009;38:344-347
2 Yu KY,Kwon B,Ni J,Zhai Y,Ebner R,Kwon BS.A newly identified member of tumor necrosis factor receptor superfamily(TR6)suppresses LIGHT-mediated apoptosis.J Biol Chem 1999;274:13733-13736[PMID:10318773 DOI:10.1074/jbc.274.20.13733]
3 Migone TS,Zhang J,Luo X,Zhuang L,Chen C,Hu B,Hong JS,Perry JW,Chen SF,Zhou JX,Cho YH,Ullrich S,Kanakaraj P,Carrell J,Boyd E,Olsen HS,Hu G,Pukac L,Liu D,Ni J,Kim S,Gentz R,Feng P,Moore PA,Ruben SM,Wei P.TL1A is a TNF-like ligand for DR3 and TR6/DcR3 and functions as a T cell costimulator.Immunity 2002;16:479-492[PMID:11911831 DOI:10.1016/S1074-7613(02)00283-2]
4 Roth W,Isenmann S,Nakamura M,Platten M,Wick W,Kleihues P,B hr M,Ohgaki H,Ashkenazi A,Weller M.Soluble decoy receptor 3 is expressed by malignant gliomas and suppresses CD95 ligandinduced apoptosis and chemotaxis.Cancer Res 2001;61:2759-2765[PMID:11289159]
5 Shi G,Wu Y,Zhang J,Wu J.Death decoy receptor TR6/DcR3 inhibits T cell chemotaxis in vitro and in vivo.J Immunol 2003;171:3407-3414[PMID:14500635]
6 Wan X,Shi G,Semenuk M,Zhang J,Wu J.DcR3/TR6 modulates immune cell interactions.J Cell Biochem 2003;89:603-612[PMID:12761893 DOI:10.1002/jcb.10523]
7 刘忠臣,罗芳洪,陈彩霞,杨东海,范鑫,程小峰,庄国洪.肝癌发展中FasL及其受体Fas/DcR3的表达分析.免疫学杂志2010;26:242-245
8 Yang CR,Hsieh SL,Teng CM,Ho FM,Su WL,Lin WW.Soluble decoy receptor 3 induces angiogenesis by neutralization of TL1A,a cytokine belonging to tumor necrosis factor superfamily and exhibiting angiostatic action.Cancer Res 2004;64:1122-1129[P M I D:1 4 8 7 1 8 4 7 D O I:1 0.1 1 5 8/0 0 0 8-5 4 7 2.CAN-03-0609]
9 Yang CR,Hsieh SL,Ho FM,Lin WW.Decoy receptor 3 increases monocyte adhesion to endothelial cells via NF-kappa B-dependent up-regulation of intercellular adhesion molecule-1,VCAM-1,and IL-8 expression.J Immunol 2005;174:1647-1656[PMID:15661928]
10 陈张铭,任四兰,张蕾.陷阱受体3在肝细胞性肝癌中的表达及其对血管生成的影响.中国现代医学杂志2009;19:1792-1792
11 陈张铭,张阳德,任四兰,张蕾.肝细胞性肝癌中DcR3的表达及其对微血管密度的影响.长沙医学院学报2007;12:18-23
12 Wu Y,Han B,Sheng H,Lin M,Moore PA,Zhang J,Wu J.Clinical significance of detecting elevated serum DcR3/TR6/M68 in malignant tumor patients.Int J Cancer 2003;105:724-732[PMID:12740925DOI:10.1002/ijc.11138]
13 Takahama Y,Yamada Y,Emoto K,Fujimoto H,Takayama T,Ueno M,Uchida H,Hirao S,Mizuno T,Nakajima Y.The prognostic significance of overexpression of the decoy receptor for Fas ligand(DcR3)in patients with gastric carcinomas.Gastric Cancer2002;5:61-68[PMID:12111580 DOI:10.1007/s101200200011]
14 Shen HW,Gao SL,Wu YL,Peng SY.Overexpression of decoy receptor 3 in hepatocellular carcinoma and its association with resistance to Fas ligandmediated apoptosis.World J Gastroenterol 2005;11:5926-5930[PMID:16273601]
15 Shen HW,Wu YL,Peng SY.[Overexpression and genomic amplification of decoy receptor 3 in hepatocellular carcinoma and significance thereof].Zhonghua Yixue Zazhi 2003;83:744-747[PMID:12899749]
16 陈罡,罗殿中,王云,廖芝玲,张美艳.肝细胞癌中DcR3表达和癌细胞凋亡的关系研究.中华病理学杂志2007;36:113-117
17 陈罡,罗殿中.原发性肝细胞癌中诱捕受体3的过度表达及临床意义.广东医学2006;27:1438-1440
18 杨梅松竹,陈罡,党裔武,陈霜,罗殿中.肝细胞癌患者血清DcR3水平与临床意义.世界华人消化杂志2009;17:2042-2047
19 Fire A,Xu S,Montgomery MK,Kostas SA,Driver SE,Mello CC.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature 1998;391:806-811[PMID:9486653DOI:10.1038/35888]
20 Hutvagner G.Small RNA asymmetry in RNAi:function in RISC assembly and gene regulation.FEBS Lett 2005;579:5850-5857[PMID:16199039DOI:10.1016/j.febslet.2005.08.071]
21 Scherer LJ,Rossi JJ.Approaches for the sequencespecific knockdown of mRNA.Nat Biotechnol 2003;21:1457-1465[PMID:14647331 DOI:10.1038/nbt915]
22 Ait-Si-Ali S,Guasconi V,Harel-Bellan A.[RNA interference and its possible use in cancer therapy].Bull Cancer 2004;91:15-18[PMID:14975801]
23 Hannon GJ.RNA interference.Nature 2002;418:244-251[PMID:12110901 DOI:10.1038/418244a]
24 Elbashir SM,Harborth J,Lendeckel W,Yalcin A,Weber K,Tuschl T.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature 2001;411:494-498[PMID:11373684 DOI:10.1038/35078107]
25 Czauderna F,Fechtner M,Dames S,Aygün H,Klippel A,Pronk GJ,Giese K,Kaufmann J.Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells.Nucleic Acids Res 2003;31:2705-2716[PMID:12771196 DOI:10.1093/nar/gng066]
26 Zou GM,Wu W,Chen J,Rowley JD.Duplexes of 21-nucleotide RNAs mediate RNA interference in differentiated mouse ES cells.Biol Cell2003;95:365-371[PMID:14519553 DOI:10.1016/S0248-4900(03)00079-0]
27 Elbashir SM,Harborth J,Weber K,Tuschl T.Analysis of gene function in somatic mammalian cells using small interfering RNAs.Methods2002;26:199-213[PMID:12054897 DOI:10.1016/S1046-2023(02)00023-3]
28 吴泰璜,段伟宏,苏忠学,吴亚光.反义DcR3 RNA转染诱导肝癌细胞凋亡的作用.中华微生物学和免疫学杂志2005;25:799-802
29 段伟宏,苏忠学,吴泰璜.DcR3反义RNA与顺铂对肝癌细胞凋亡影响的比较.中国现代普通外科进展2005;8:90-92
30 段伟宏,苏忠学,吴泰璜.转染反义DcR3真核表达载体协同氟尿嘧啶诱导肝癌细胞凋亡.中华肝胆外科杂志2006;12:98-100
31 陈罡,党裔武,罗殿中.诱捕受体3中和性抗体在HepG2细胞凋亡中的作用.中华肝脏病杂志2008;16:791-792
32 陈罡,罗殿中,党裔武.DcR3对肝癌细胞生长迁移的影响.陕西医学杂志2009;38:344-347