MiRNA-99a通过靶向调节EIF2C2/PTEN信号通路抑制肝癌细胞的生长
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摘要
目的:研究miR-99a对肝癌的细胞的抑制作用和机制，提供一种腺病毒介导的miRNA治疗肿瘤的新方法。
方法：qRT-PCR检测肝癌和对应正常肝组织中miR-99a的表达；通过统计分析检验构miR-99a与肝癌患者预后的关系；在miR-99a作用机制的研究方面，重组5型腺病毒Ad5-miR-99a，通过体外和体内实验验证miR-99a对肝癌细胞的抑制作用；通过双荧光素酶报告基因系统验证miR-99a调控的新靶基因EIF2C2；在体内、体外实验和临床组织中，通过qRT-PCR、Western blot、免疫荧光和免疫组化检测EIF2C2及其下游基因PTEN的表达。
结果：miR-99a在肝癌组织中低表达，与肝癌患者的预后相关；体内体外实验显示过表达miR-99a可抑制HCC的生长；miR-99a靶向抑制EIF2C2的表达，从而促进PTEN的表达。
结论：miR-99a与肝癌患者的预后相关，通过靶向调控EIF2C2/PTEN信号通路抑制HCC的生长，在将来很有可能成为治疗肝癌的新方法。
Objective:
In order to further research the suppression of has-miR-99a on proliferation ofhepatocellular carcinoma cells through constructing adenovirus vector.
Methods:
Expression of miR-99a was detected by quantitative real-time polymerase chainreaction (qRT-PCR) in HCC. The adenovirus vector Ad5-miR-99a were constructed.Through the Luciferase Reporter Assay found that miR-99a regulation of the new target genesEIF2C2. Then we detected EIF2C2and downstream PTEN gene expression by qRT-PCR、Western blot、immunofluorescence and Immunohistochemistry in vitro and in vivo.
Results: MicroRNA-99a markedly decreases in HCC.miR-99a expression correlateswith HCC patients’ survival, and miR-99a restoration suppresses HCC growth invitro and in vivo by targeting EIF2C2/PTENpathway. MiR-99a could stable highexpression in HCC after transfected Ad5-miR-99a for48hours.
Conclusion:Ad5-miR-99a successfully suppresses HCC growth and could be aprospective therapeutic approach for HCC in the near future.
方法：qRT-PCR检测肝癌和对应正常肝组织中miR-99a的表达；通过统计分析检验构miR-99a与肝癌患者预后的关系；在miR-99a作用机制的研究方面，重组5型腺病毒Ad5-miR-99a，通过体外和体内实验验证miR-99a对肝癌细胞的抑制作用；通过双荧光素酶报告基因系统验证miR-99a调控的新靶基因EIF2C2；在体内、体外实验和临床组织中，通过qRT-PCR、Western blot、免疫荧光和免疫组化检测EIF2C2及其下游基因PTEN的表达。
结果：miR-99a在肝癌组织中低表达，与肝癌患者的预后相关；体内体外实验显示过表达miR-99a可抑制HCC的生长；miR-99a靶向抑制EIF2C2的表达，从而促进PTEN的表达。
结论：miR-99a与肝癌患者的预后相关，通过靶向调控EIF2C2/PTEN信号通路抑制HCC的生长，在将来很有可能成为治疗肝癌的新方法。
Objective:
In order to further research the suppression of has-miR-99a on proliferation ofhepatocellular carcinoma cells through constructing adenovirus vector.
Methods:
Expression of miR-99a was detected by quantitative real-time polymerase chainreaction (qRT-PCR) in HCC. The adenovirus vector Ad5-miR-99a were constructed.Through the Luciferase Reporter Assay found that miR-99a regulation of the new target genesEIF2C2. Then we detected EIF2C2and downstream PTEN gene expression by qRT-PCR、Western blot、immunofluorescence and Immunohistochemistry in vitro and in vivo.
Results: MicroRNA-99a markedly decreases in HCC.miR-99a expression correlateswith HCC patients’ survival, and miR-99a restoration suppresses HCC growth invitro and in vivo by targeting EIF2C2/PTENpathway. MiR-99a could stable highexpression in HCC after transfected Ad5-miR-99a for48hours.
Conclusion:Ad5-miR-99a successfully suppresses HCC growth and could be aprospective therapeutic approach for HCC in the near future.
引文
[1] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism,and function[J]. Cell.2004,116(2):281-297.
[2] Haybaeck J, Zeller N, Heikenwalder M. The parallel universe:microRNAs and their role in chronic hepatitis, liver tissue damage andhepatocarcinogenesis[J]. Swiss Med Wkly.2011,141: w13287.
[3] Jiang J, Gusev Y, Aderca I et al. Association of MicroRNAexpression in hepatocellular carcinomas with hepatitis infection,cirrhosis, and patient survival[J]. Clinical cancer research.2008,14(2):419-427.
[4] Hou J, Lin L, Zhou W et al. Identification of miRNomes inhuman liver and hepatocellular carcinoma reveals miR-199a/b-3p astherapeutic target for hepatocellular carcinoma[J]. Cancer cell.2011,19(2):232-243.
[5] Wong TS, Liu XB, Wong BYH et al. Mature miR-184aspotential oncogenic microRNA of squamous cell carcinoma of tongue[J].Clinical Cancer Research.2008,14(9):2588-2592.
[6] Yamada H, Yanagisawa K, Tokumaru S et al. Detailedcharacterization of a homozygously deleted region corresponding to acandidate tumor suppressor locus at21q11‐21in human lung cancer[J].Genes, Chromosomes and Cancer.2008,47(9):810-818.
[7] Nam EJ, Yoon H, Kim SW et al. MicroRNA expression profilesin serous ovarian carcinoma[J]. Clinical cancer research.2008,14(9):2690-2695.
[8] Doghman M, Wakil AE, Cardinaud B et al. Regulation ofinsulin-like growth factor–mammalian target of rapamycin signaling bymicroRNA in childhood adrenocortical tumors[J]. Cancer research.2010,70(11):4666.
[9] Sun D, Lee YS, Malhotra A et al. miR-99family of microRNAssuppresses the expression of prostate-specific antigen and prostatecancer cell proliferation[J]. Cancer research.2011,71(4):1313.
[10] Tan GS, Garchow BG, Liu X et al. Expanded RNA-bindingactivities of mammalian Argonaute2[J]. Nucleic acids research.2009,37(22):7533-7545.
[11] Roberts LR. Sorafenib in liver cancer—just the beginning[J].New England Journal of Medicine.2008,359(4):420-422.
[12] Poon RTP, Fan ST, Wong J. Risk factors, prevention, andmanagement of postoperative recurrence after resection ofhepatocellular carcinoma[J]. Annals of surgery.2000,232(1):10.
[13] Lo CM, Ngan H, Tso WK et al. Randomized controlled trialof transarterial lipiodol chemoembolization for unresectablehepatocellular carcinoma[J]. Hepatology.2002,35(5):1164-1171.
[14] Eulalio A, Huntzinger E, Izaurralde E. Getting to the root ofmiRNA-mediated gene silencing[J]. Cell.2008,132(1):9-14.
[15] Negrini M, Gramantieri L, Sabbioni S et al. microRNAinvolvement in hepatocellular carcinoma[J]. Anti-Cancer Agents inMedicinal Chemistry (Formerly Current Medicinal Chemistry.2011,11(6):500-521.
[16] Jin H, Lv S, Yang J et al. Use of MicroRNA Let-7to Controlthe Replication Specificity of Oncolytic Adenovirus in HepatocellularCarcinoma Cells[J]. PloS one.2011,6(7): e21307.
[17] Nathwani AC, Tuddenham EGD, Rangarajan S et al.Adenovirus-associated virus vector–mediated gene transfer inhemophilia B[J]. New England Journal of Medicine.2011,365(25):2357-2365.
[18] Zhang J, Yang Y, Yang T et al. microRNA-22, downregulatedin hepatocellular carcinoma and correlated with prognosis, suppressescell proliferation and tumourigenicity[J]. British journal of cancer.2010,103(8):1215-1220.
[19] Li D, Liu X, Lin L et al. MicroRNA-99a InhibitsHepatocellular Carcinoma Growth and Correlates with Prognosis ofPatients with Hepatocellular Carcinoma[J]. Journal of BiologicalChemistry.2011,286(42):36677-36685.
[20] Bartel DP. MicroRNAs:: Genomics, Biogenesis, Mechanism,and Function[J]. Cell.2004,116(2):281-297.
[21] Haybaeck J, Zeller N, Heikenwalder M. The parallel universe:microRNAs and their role in chronic hepatitis, liver tissue damage andhepatocarcinogenesis[J]. Swiss medical weekly.141: w13287.
[22] Davis-Dusenbery BN, Hata A. Mechanisms of control ofmicroRNA biogenesis[J]. Journal of biochemistry.148(4):381.
[23] Li W, Xie L, He X et al. Diagnostic and prognosticimplications of microRNAs in human hepatocellular carcinoma[J].International Journal of Cancer.2008,123(7):1616-1622.
[24] Huang S, He X. The role of microRNAs in liver cancerprogression[J]. British journal of cancer.2011,104(2):235-240.
[25] Ambros V. The functions of animal microRNAs[J]. Nature.2004,431(7006):350-355.
[26] Esquela-Kerscher A, Slack FJ. Oncomirs-microRNAs with arole in cancer[J]. Nature Reviews Cancer.2006,6(4):259-269.
[27] Kato M, Slack F. microRNAs: small molecules with bigroles-C. elegans to human cancer[J]. Biology of the Cell.2008,100:71-81.
[28] Giraldez AJ, Mishima Y, Rihel J et al. Zebrafish MiR-430promotes deadenylation and clearance of maternal mRNAs[J]. Science.2006,312(5770):75.
[29] Naguibneva I, Ameyar-Zazoua M, Polesskaya A et al. ThemicroRNA miR-181targets the homeobox protein Hox-A11duringmammalian myoblast differentiation[J]. Nature cell biology.2006,8(3):278-284.
[30] Kloosterman WP, Lagendijk AK, Ketting RF et al. Targetedinhibition of miRNA maturation with morpholinos reveals a role formiR-375in pancreatic islet development[J]. PLoS biology.2007,5(8):e203.
[31] Esau C, Kang X, Peralta E et al. MicroRNA-143regulatesadipocyte differentiation[J]. Journal of Biological Chemistry.2004,279(50):52361.
[32] Kutay H, Bai S, Datta J et al. Downregulation of miR-122inthe rodent and human hepatocellular carcinomas[J]. Journal of cellularbiochemistry.2006,99(3):671-678.
[33] Nohata N, Hanazawa T, Kikkawa N et al. Tumor suppressivemicroRNA-375regulates oncogene AEG-1/MTDH in head and necksquamous cell carcinoma (HNSCC)[J]. Journal of human genetics.2011,56(8):595-601.
[34] Liang L, Wong CM, Ying Q et al. MicroRNA-125bsuppressesed human liver cancer cell proliferation and metastasis bydirectly targeting oncogene LIN28B2[J]. Hepatology.2010.
[35] Huang S, He X. The role of microRNAs in liver cancerprogression[J]. British journal of cancer.2010,104(2):235-240.
[36] Bousquet M, Quelen C, Rosati R et al. Myeloid celldifferentiation arrest by miR-125b-1in myelodysplasic syndrome andacute myeloid leukemia with the t (2;11)(p21; q23) translocation[J]. TheJournal of experimental medicine.2008,205(11):2499.
[37] Calin GA, Cimmino A, Fabbri M et al. MiR-15a andmiR-16-1cluster functions in human leukemia[J]. Proceedings of theNational Academy of Sciences.2008,105(13):5166.
[38] Bonci D, Coppola V, Musumeci M et al. ThemiR-15a-miR-16-1cluster controls prostate cancer by targeting multipleoncogenic activities[J]. Nature medicine.2008,14(11):1271-1277.
[39] Slaby O, Svoboda M, Fabian P et al. Altered expression ofmiR-21, miR-31, miR-143and miR-145is related to clinicopathologicfeatures of colorectal cancer[J]. Oncology.2008,72(5-6):397-402.
[40] Takamizawa J, Konishi H, Yanagisawa K et al. Reducedexpression of the let-7microRNAs in human lung cancers in associationwith shortened postoperative survival[J]. Cancer research.2004,64(11):3753.
[41] Huang S, He X, Ding J et al. Upregulation of miR-3a-27a-24decreases transforming growth factor-beta-nduced tumor-suppressiveactivities in human hepatocellular carcinoma cells[J]. InternationalJournal of Cancer.2008,123(4):972-978.
[42] Zhou X, Ren Y, Moore L et al. Downregulation of miR-21inhibits EGFR pathway and suppresses the growth of humanglioblastoma cells independent of PTEN status[J]. LaboratoryInvestigation.2010,90(2):144-155.
[43] Eis PS, Tam W, Sun L et al. Accumulation of miR-155andBIC RNA in human B cell lymphomas[J]. Proceedings of the NationalAcademy of Sciences of the United States of America.2005,102(10):3627.
[44] Ventura A, Young AG, Winslow MM et al. Targeted DeletionReveals Essential and Overlapping Functions of the miR-17-92Family of miRNA Clusters[J]. Cell.2008,132(5):875-886.
[45] Mendell JT. miRiad roles for the miR-17-92cluster indevelopment and disease[J]. Cell.2008,133(2):217-222.
[46] Pall GS, Codony-Servat C, Byrne J et al.Carbodiimide-mediated cross-linking of RNA to nylon membranesimproves the detection of siRNA, miRNA and piRNA by northern blot[J].Nucleic acids research.2007,35(8): e60.
[47] Chen C, Ridzon DA, Broomer AJ et al. Real-timequantification of microRNAs by stem-oop RT-PCR[J]. Nucleic acidsresearch.2005,33(20): e179-e179.
[48] Budhu A, Jia HL, Forgues M et al. Microarray analysis ofmicroRNA expression in hepatocellular carcinoma and non‐tumoroustissues without viral hepatitis[J]. Hepatology.2008,47(3):897-907.
[49] Pedersen IM, Cheng G, Wieland S et al. Interferonmodulation of cellular microRNAs as an antiviral mechanism[J]. Nature.2007,449(7164):919-922.
[50] Trang P, Wiggins JF, Daige CL et al. Systemic delivery oftumor suppressor microRNA mimics using a neutral lipid emulsioninhibits lung tumors in mice[J]. Molecular Therapy.2011,19(6):1116-1122.
[51] Xie Z, Johansen LK, Gustafson AM et al. Genetic andfunctional diversification of small RNA pathways in plants[J]. PLoSbiology.2004,2(5): e104.
[52] Lagos-Quintana M, Rauhut R, Lendeckel W et al.Identification of novel genes coding for small expressed RNAs[J]. Science.2001,294(5543):853.
[2] Haybaeck J, Zeller N, Heikenwalder M. The parallel universe:microRNAs and their role in chronic hepatitis, liver tissue damage andhepatocarcinogenesis[J]. Swiss Med Wkly.2011,141: w13287.
[3] Jiang J, Gusev Y, Aderca I et al. Association of MicroRNAexpression in hepatocellular carcinomas with hepatitis infection,cirrhosis, and patient survival[J]. Clinical cancer research.2008,14(2):419-427.
[4] Hou J, Lin L, Zhou W et al. Identification of miRNomes inhuman liver and hepatocellular carcinoma reveals miR-199a/b-3p astherapeutic target for hepatocellular carcinoma[J]. Cancer cell.2011,19(2):232-243.
[5] Wong TS, Liu XB, Wong BYH et al. Mature miR-184aspotential oncogenic microRNA of squamous cell carcinoma of tongue[J].Clinical Cancer Research.2008,14(9):2588-2592.
[6] Yamada H, Yanagisawa K, Tokumaru S et al. Detailedcharacterization of a homozygously deleted region corresponding to acandidate tumor suppressor locus at21q11‐21in human lung cancer[J].Genes, Chromosomes and Cancer.2008,47(9):810-818.
[7] Nam EJ, Yoon H, Kim SW et al. MicroRNA expression profilesin serous ovarian carcinoma[J]. Clinical cancer research.2008,14(9):2690-2695.
[8] Doghman M, Wakil AE, Cardinaud B et al. Regulation ofinsulin-like growth factor–mammalian target of rapamycin signaling bymicroRNA in childhood adrenocortical tumors[J]. Cancer research.2010,70(11):4666.
[9] Sun D, Lee YS, Malhotra A et al. miR-99family of microRNAssuppresses the expression of prostate-specific antigen and prostatecancer cell proliferation[J]. Cancer research.2011,71(4):1313.
[10] Tan GS, Garchow BG, Liu X et al. Expanded RNA-bindingactivities of mammalian Argonaute2[J]. Nucleic acids research.2009,37(22):7533-7545.
[11] Roberts LR. Sorafenib in liver cancer—just the beginning[J].New England Journal of Medicine.2008,359(4):420-422.
[12] Poon RTP, Fan ST, Wong J. Risk factors, prevention, andmanagement of postoperative recurrence after resection ofhepatocellular carcinoma[J]. Annals of surgery.2000,232(1):10.
[13] Lo CM, Ngan H, Tso WK et al. Randomized controlled trialof transarterial lipiodol chemoembolization for unresectablehepatocellular carcinoma[J]. Hepatology.2002,35(5):1164-1171.
[14] Eulalio A, Huntzinger E, Izaurralde E. Getting to the root ofmiRNA-mediated gene silencing[J]. Cell.2008,132(1):9-14.
[15] Negrini M, Gramantieri L, Sabbioni S et al. microRNAinvolvement in hepatocellular carcinoma[J]. Anti-Cancer Agents inMedicinal Chemistry (Formerly Current Medicinal Chemistry.2011,11(6):500-521.
[16] Jin H, Lv S, Yang J et al. Use of MicroRNA Let-7to Controlthe Replication Specificity of Oncolytic Adenovirus in HepatocellularCarcinoma Cells[J]. PloS one.2011,6(7): e21307.
[17] Nathwani AC, Tuddenham EGD, Rangarajan S et al.Adenovirus-associated virus vector–mediated gene transfer inhemophilia B[J]. New England Journal of Medicine.2011,365(25):2357-2365.
[18] Zhang J, Yang Y, Yang T et al. microRNA-22, downregulatedin hepatocellular carcinoma and correlated with prognosis, suppressescell proliferation and tumourigenicity[J]. British journal of cancer.2010,103(8):1215-1220.
[19] Li D, Liu X, Lin L et al. MicroRNA-99a InhibitsHepatocellular Carcinoma Growth and Correlates with Prognosis ofPatients with Hepatocellular Carcinoma[J]. Journal of BiologicalChemistry.2011,286(42):36677-36685.
[20] Bartel DP. MicroRNAs:: Genomics, Biogenesis, Mechanism,and Function[J]. Cell.2004,116(2):281-297.
[21] Haybaeck J, Zeller N, Heikenwalder M. The parallel universe:microRNAs and their role in chronic hepatitis, liver tissue damage andhepatocarcinogenesis[J]. Swiss medical weekly.141: w13287.
[22] Davis-Dusenbery BN, Hata A. Mechanisms of control ofmicroRNA biogenesis[J]. Journal of biochemistry.148(4):381.
[23] Li W, Xie L, He X et al. Diagnostic and prognosticimplications of microRNAs in human hepatocellular carcinoma[J].International Journal of Cancer.2008,123(7):1616-1622.
[24] Huang S, He X. The role of microRNAs in liver cancerprogression[J]. British journal of cancer.2011,104(2):235-240.
[25] Ambros V. The functions of animal microRNAs[J]. Nature.2004,431(7006):350-355.
[26] Esquela-Kerscher A, Slack FJ. Oncomirs-microRNAs with arole in cancer[J]. Nature Reviews Cancer.2006,6(4):259-269.
[27] Kato M, Slack F. microRNAs: small molecules with bigroles-C. elegans to human cancer[J]. Biology of the Cell.2008,100:71-81.
[28] Giraldez AJ, Mishima Y, Rihel J et al. Zebrafish MiR-430promotes deadenylation and clearance of maternal mRNAs[J]. Science.2006,312(5770):75.
[29] Naguibneva I, Ameyar-Zazoua M, Polesskaya A et al. ThemicroRNA miR-181targets the homeobox protein Hox-A11duringmammalian myoblast differentiation[J]. Nature cell biology.2006,8(3):278-284.
[30] Kloosterman WP, Lagendijk AK, Ketting RF et al. Targetedinhibition of miRNA maturation with morpholinos reveals a role formiR-375in pancreatic islet development[J]. PLoS biology.2007,5(8):e203.
[31] Esau C, Kang X, Peralta E et al. MicroRNA-143regulatesadipocyte differentiation[J]. Journal of Biological Chemistry.2004,279(50):52361.
[32] Kutay H, Bai S, Datta J et al. Downregulation of miR-122inthe rodent and human hepatocellular carcinomas[J]. Journal of cellularbiochemistry.2006,99(3):671-678.
[33] Nohata N, Hanazawa T, Kikkawa N et al. Tumor suppressivemicroRNA-375regulates oncogene AEG-1/MTDH in head and necksquamous cell carcinoma (HNSCC)[J]. Journal of human genetics.2011,56(8):595-601.
[34] Liang L, Wong CM, Ying Q et al. MicroRNA-125bsuppressesed human liver cancer cell proliferation and metastasis bydirectly targeting oncogene LIN28B2[J]. Hepatology.2010.
[35] Huang S, He X. The role of microRNAs in liver cancerprogression[J]. British journal of cancer.2010,104(2):235-240.
[36] Bousquet M, Quelen C, Rosati R et al. Myeloid celldifferentiation arrest by miR-125b-1in myelodysplasic syndrome andacute myeloid leukemia with the t (2;11)(p21; q23) translocation[J]. TheJournal of experimental medicine.2008,205(11):2499.
[37] Calin GA, Cimmino A, Fabbri M et al. MiR-15a andmiR-16-1cluster functions in human leukemia[J]. Proceedings of theNational Academy of Sciences.2008,105(13):5166.
[38] Bonci D, Coppola V, Musumeci M et al. ThemiR-15a-miR-16-1cluster controls prostate cancer by targeting multipleoncogenic activities[J]. Nature medicine.2008,14(11):1271-1277.
[39] Slaby O, Svoboda M, Fabian P et al. Altered expression ofmiR-21, miR-31, miR-143and miR-145is related to clinicopathologicfeatures of colorectal cancer[J]. Oncology.2008,72(5-6):397-402.
[40] Takamizawa J, Konishi H, Yanagisawa K et al. Reducedexpression of the let-7microRNAs in human lung cancers in associationwith shortened postoperative survival[J]. Cancer research.2004,64(11):3753.
[41] Huang S, He X, Ding J et al. Upregulation of miR-3a-27a-24decreases transforming growth factor-beta-nduced tumor-suppressiveactivities in human hepatocellular carcinoma cells[J]. InternationalJournal of Cancer.2008,123(4):972-978.
[42] Zhou X, Ren Y, Moore L et al. Downregulation of miR-21inhibits EGFR pathway and suppresses the growth of humanglioblastoma cells independent of PTEN status[J]. LaboratoryInvestigation.2010,90(2):144-155.
[43] Eis PS, Tam W, Sun L et al. Accumulation of miR-155andBIC RNA in human B cell lymphomas[J]. Proceedings of the NationalAcademy of Sciences of the United States of America.2005,102(10):3627.
[44] Ventura A, Young AG, Winslow MM et al. Targeted DeletionReveals Essential and Overlapping Functions of the miR-17-92Family of miRNA Clusters[J]. Cell.2008,132(5):875-886.
[45] Mendell JT. miRiad roles for the miR-17-92cluster indevelopment and disease[J]. Cell.2008,133(2):217-222.
[46] Pall GS, Codony-Servat C, Byrne J et al.Carbodiimide-mediated cross-linking of RNA to nylon membranesimproves the detection of siRNA, miRNA and piRNA by northern blot[J].Nucleic acids research.2007,35(8): e60.
[47] Chen C, Ridzon DA, Broomer AJ et al. Real-timequantification of microRNAs by stem-oop RT-PCR[J]. Nucleic acidsresearch.2005,33(20): e179-e179.
[48] Budhu A, Jia HL, Forgues M et al. Microarray analysis ofmicroRNA expression in hepatocellular carcinoma and non‐tumoroustissues without viral hepatitis[J]. Hepatology.2008,47(3):897-907.
[49] Pedersen IM, Cheng G, Wieland S et al. Interferonmodulation of cellular microRNAs as an antiviral mechanism[J]. Nature.2007,449(7164):919-922.
[50] Trang P, Wiggins JF, Daige CL et al. Systemic delivery oftumor suppressor microRNA mimics using a neutral lipid emulsioninhibits lung tumors in mice[J]. Molecular Therapy.2011,19(6):1116-1122.
[51] Xie Z, Johansen LK, Gustafson AM et al. Genetic andfunctional diversification of small RNA pathways in plants[J]. PLoSbiology.2004,2(5): e104.
[52] Lagos-Quintana M, Rauhut R, Lendeckel W et al.Identification of novel genes coding for small expressed RNAs[J]. Science.2001,294(5543):853.