摘要
目的：研究滑膜肉瘤融合基因SYT-SSX的下游基因,筛选并明确其中部分基因在滑膜肉瘤发生发展中的功能与内在联系,从融合基因及下游基因的角度阐明滑膜肉瘤的发生发展机制,为临床治疗提供新的线索。
方法：选取滑膜肉瘤细胞系SYO-1,构建特异性封闭融合基因SYT-SSX的siRNA真核载体,采用脂质体转染的方法,干扰并封闭融合基因的表达。利用Realtime RT-PCR的方法验证封闭效果。选取其中封闭效果良好的样本进行人类全基因组表达谱基因芯片分析。实验所得所有变化基因使用基于网络的基因分析工具,进行分类注释分析及代谢通路分析。选取其中一条与SYT-SSX密切相关的细胞代谢通路,Realtime RT-PCR验证其关键位点基因,在SYT-SSX封闭后mRNA表达水平的变化。Western Blot方法验证融合基因被特异性siRNA封闭后,细胞代谢通路关键位点的基因蛋白水平的变化。采用MTT染色活细胞数,评估细胞增殖活力,验证封闭SYT-SSX后,对于细胞的增殖的影响。PI染色,流式细胞仪检测细胞周期,同时检测细胞周期相关蛋白水平表达变化,验证SYT-SSX及所选通路对于细胞周期的影响。Annexin V-FITC和PI双染色,流式细胞仪检测细胞坏死与凋亡,初步探讨SYT-SSX与所选通路对于细胞凋亡的影响。同时,选取表达融合基因SYT-SSX及不表达融合基因的人体滑膜肉瘤及其它组织学类型肉瘤共74例,以免疫组织化学法及TUNEL染色方法,在蛋白水平验证体内环境中,SYT-SSX表达及所选通路蛋白与其下游功能蛋白之间的联系。应用统计学分析方法对所有结果进行计数资料的正态性检验、方差齐性检验,方差分析和t检验等,以P<0.05,差别具有统计学意义。
结果：(1)构建的siRNA真核表达载体,在体外环境中可以特异性封闭融合基因SYT-SSX,并且在72h达到93.7%抑制率；(2)基因芯片检测结果全基因组表达谱基因芯片中375个基因上调,418个基因下调,所有变化的793个基因中653个可以进行基因功能分类,其中涉及蛋白磷酸化,细胞分裂,细胞周期,细胞转运,RNA结合,离子代谢,核蛋白代谢等。对所有变化基因进行细胞通路分析,5条通路被选中,其中ERK通路与SYT-SSX相关性最为显著(P<0.05)；(3)mRNA水平及蛋白水平验证ERK表达量在封闭融合基因SYT-SSX后,有明显下降；(4)封闭融合基因SYT-SSX后,MTT法检测活细胞数封闭组明显低于未处理组,封闭组细胞周期调控出现明显GO/G1期阻滞,处于DNA合成期(S期)细胞明显减少。Western Blot结果显示细胞周期相关蛋白,CyclinD1表达量明显下降,CDK4表达变化不明显；(5)融合基因封闭后, Annexin V-FITC和PI双染色,流式细胞仪测定,凋亡细胞数明显高于阴性对照siRNA组及仅使用脂质体处理组；(6)免疫组化染色结果显示,表达SYT-SSX融合基因患者,具有较高的ki-67染色指数,体内环境中表达较高的p-ERK蛋白及CyclinDl及CDK4蛋白,而TUNEL染色显示凋亡指数组间差别无统计学意义；(7)免疫组化结果显示,表达较高水平p-ERK的患者,同时表达较高水平的ki-67染色指数,CyclinD1和CDK4蛋白表达量高于p-ERK表达水平较低的患者,而TUNEL染色凋亡指数组间差别无统计学意义。
结论：(1)构建siRNA真核载体,在体外环境中可以高效率,特异性的抑制融合基因SYT-SSX的表达；(2)融合基因可影响细胞增殖,凋亡,分化发育的多种基因,其中ERK可能为其中重要的通路之一,融合基因通过ERK通路促进细胞增殖,调控细胞周期,在一定程度上抑制细胞凋亡,从而在滑膜肉瘤的发生发展中起重要作用。(3)体内环境中,SYT-SSX可以上调p-ERK蛋白表达,进而提高细胞增殖活性,对细胞周期发挥正向调控作用,与体外实验结果一致。
OBJECTIVE:This study is designed to gain more insight into the function of synovial sarcoma (SS) fusion gene. Screening part of genes on downstream of SYT-SSX, the aim was to explain the relationship of SYT-SSX and other pathways. These results would be hoped to elicit a novel path to discovery the function of the fusion genes in SS.
METHODS:Two distinct siRNA duplexes for SYT-SSX carried by vector were synthesized, transfected by LipofectamineTM 2000. Total cellular RNAs were isolated using Trizol reagent. Then quantitative real-time PCR (qRT-PCR) was performed using SYBR Green Premix Ex TaqTM in order to manifest the inhibiting effect of SYT-SSX. The study evaluated the whole genome expression in SYO-1 cells inhibited as a result of specific small interfering RNA for SYT-SSX. All microarray data were submitted to the Database of Annotation, Visualization and Integrated Discovery (DAVID) 2008. Some down-stream genes of SYT-SSX chosen from the microarray analysis and involved in cell proliferation were further verified. Their expression was compared in SYT-SSX-blocked SYO-1 and control SYO-1 cells by qRT-PCR. The protein expression of some related down-stream genes was detected by western blot. Cell proliferation was measured using the methyl thiazolyl blue tetrazolium bromide colorimetric dye assay. Cell cycle analysis was conducted by fluorescence-activated cell sorting with cells stained by propidiumiodide. Apoptotic cells were measured with an Annexin V/FITC kit. and analyzed by flow cytometer after transfection. Seventy-four cases were selected into the study, including 54 SYT-SSX positive SSs tested by RT-PCR,4 SYT-SSX negative SSs (diagnosed as SS according to typical clinical context, histologic aspect and immunohistochemical profile), and 16 non-SSs (4 malignant melanoma,4 Ewing's sarcomas,4 malignant peripheral nerve sheath tumor and 4 hemangio-peritheliomas). A tissue microarray (TMA) was constructed. Visualization of DNA fragmentation, a marker of apoptosis, was performed by the TUNEL method using the In Situ Apoptosis Detection Kit. Immunohistochemistry staining was performed on TMA. The expressions of Cyclin D1, CDK4, and p-ERK were analyzed in considering the staining intensity and area extent. Statistical comparisons were made by using ANOVA with subsequent application or t test where appropriate. P value of less than 0.05 was considered statistically significant.
RESULTS:1 Compared with the cells only treated with lipofectamineTM, the expression of SYT-SSX reduced about 93.7%in the cells transfected with SYT-SSX-specific siRNA1.2. Compared with gene expression in cells transfected with negative control siRNA, the expression of 375 genes was up-regulated and of 418 genes was down-regulated in cells transfected with SYT-SSX-specific siRNAl. Among the 793 genes with significant change,653 genes could be assigned into different functional categories with DAVID gene functional category analysis. Subsequently,5 pathways hit by biocarta pathway analysis were indicated, including ERK1/2 pathway (P=0.043).3.The expression of ERK mRNA was confirmed to be significantly deceased after blocking SYT-SSX. Moreover, protein expression of ERK and p-ERK detected by western blot was equal to the results of qRT-PCR.4. The survival rate of SYO-1 cells transfected with specific siRNAl was most significantly reduced compared with that of SYO-1 cells only treated with lipofectamineTM.5. In comparison with control SYO-1 cells treated with lipofectamineTM, SYT-SSX-specific siRNAl and siRNA2 caused an increase in the percentage of G1/G0 phase cells, accompanied by a significant decrease in the percentage of S phase cells, cell cycle related proteins, Cyclin D1 and CDK4 were detected in SYO-1 cells after transfection with specific siRNAl and negative control siRNA. The expression of CyclinDl in siRNA1 transfected SYO-1 cells was significantly lower than that in negative control siRNA transcripted cells and cells untreated. But there was no notable change of CDK4 expression in SYO-1 cells treated with different siRNAs. It was shown the apoptotic rate in SYT-SSX-specific siRNAs transfected cells obviously increased.6. Cases positive for SYT-SSX showed higher ki-67 LI than SYT-SSX negative ones. In comparison of cases not detected SYT-SSX, expression of CyclinDl, CDK4 and p-ERK showed a trend to be more expressed in cases with SYT-SSX. Although the AI means in SYT-SSX positive SSs was higher than that in SYT-SSX negative cases, there were not significant difference between TUNEL AI according to expression of SYT-SSX.7. Higher ki-67 LI were shown in p-ERK positive group. Moreover, CyclinD1 and CDK4 were more expressed in p-ERK positive group than those in negative group.
CONCLUSIONS:1. vector-based siRNA to block the expression of SYT-SSX had high specificity and efficiency.2. All genes changed were involved in phosphoprotein, transcription regulation, alternative splicing, direct protein sequencing, nucleotide-binding, transport, cell cycle, kinase and so on. Then pathway analysis indicated the ERK pathway were included in. SYT-SSX might play an important role via ERK pathway. Therefore activation of ERK may affect a broad array of cellular functions, including proliferation, survival, apoptosis, motility, transcription, metabolism and differentiation, and is in part responsible for oncogenesis.3. The results in vivo also suggested that the fusion gene SYT-SSX should be considered to play important role on SS cell growth maybe via ERK pathway.
引文
[1]Weiss SW, Goldblum JR. Malignant soft tissue tumors of uncertain type.[R] Weiss SW, Goldblum JR. Enzingerand Weiss's soft tissue tumors. St. Louis: Mosby,2001:1483-1571.
[2]范嫏娣主编.王德延肿瘤病理诊断学.第二版[M].天津.天津科学技术出版社.1996：269-287.
[3]Ture-Carel C,Dal Cin P,Limon J,et al.Translocation X;18 in synovial sarcoma[J].Cancer Gene Cytogenel,1986,23(1):93
[4]Sreekantaiah C,landanyi M, Rodriguez E, et al.Chlomosomal aberrations in soft tissue tumors:relevances to diagnosis, classification, and molecular mechanisms[J].Am J Pathol.1994.144(6):1121-1134.
[5]Crew AJ.Clark J, Fisher C,et al. Fusion of SYT to two genes, SSX1 and SSX2,encoding proteins with homology to the Kruppel-associated box in human synovial sarcoma [J].EMBO J,1995,14(7):2333-2340.
[6]Thaete C, Brett D, Monaghan P, Whitehouse S, et al. Functional domains of the SYT and SYT-SSX synovial sarcoma translocation proteins and colocalization with the SNF protein BRM in the nucleus. [J] Hum Mol Genet 1999.8:585-591.
[7]Ladanyi, M., Fusions of the SYT and SSX genes in synovial sarcoma. [J]Oncogene,2001.20(40):p.5755-62.
[8]Guillou L, Coindre J, Gallagher G, et al. Detection of the synovial sarcoma translocation t(X;18) (SYT;SSX) in paraffin-embedded tissues using reverse transcriptase-polymerase chain reaction:a reliable and powerful diagnostic tool for pathologists. A molecular analysis of 221 mesenchymal tumors fixed in different fixatives. [J] Hum Pathol,2001,32:105-112.
[9]Nilsson G, Skytting B, Xie Y, et al. The SYT-SSX1 variant of synovial sarcoma is associated with a high rate of tumor cell proliferation and poor clinical outcome [J]Cancer Res,1999,59,3180-3184.
[10]Inagaki H,Nagasaka T,Otsuka T,et al. Association of SYT-SSX fusion types with proliferative activity and prognosis in synovial sarcoma..[J].Mod Pathol. 2000,13(5):482-8.
[11]Antonescu CR,Kawai A,Leung DH,et al. Strong association of SYT-SSX fusion type and morphologic epithelial differentiation in synovial sarcoma. [J]. Diagn Mol Pathol 2000,9(1):1-8.
[12]Yuntao Xie, Bjorn Skytting, Gunnar Nilsson et al.The SYT-SSX1 fusion type of synovial sarcoma is associated with increased expression of cyclin A and D1. A link between t(X;18)(p11.2; qll.2) and the cell cycle machinery.[J] Oncogen 2002,21:5791-5796.
[13]Yuntao Xie, Bjorn Skytting, Gunnar Nilsson,et al. SYT-SSX Is Critical for Cyclin D1 Expression in Synovial Sarcoma Cells:A Gain of Function of the t(X;18)(p11.2;q11.2) Translocation.[J] Cancer Res 2002,62(1):3861-3867
[14]Thompson N, Lyons J. Recent progress in targeting the Raf/MEK/ERK pathwaywith inhibitors in cancerdrug discovery[J].CurrOpinPharmacol,2005, 5(4):350-356.
[15]Pai SI, Lin YY,Macaes B, et al. Prospects of RNA interference therapy for cancer [J]. Gene Ther,2006,13(6):464-477.
[16]Dei tos AP,Doglioni C,Calretinin:a novel tool for diagnostic immunohistochemistry.[J] Adv Anat Pathol 1998,5:61-66.
[17]徐治宽,杨堂斗,吴秉全,等.滑膜肉瘤的融合基因检测分析[J]中华病理学杂志,2001,30：431-433.
[18]李锋,李新霞,陆天才,等.34例滑膜肉瘤分子遗传学改变的诊断意义.[J]临床与实验病理学杂志,2003,19：571-575.
[19]Crew AJ.Clark J, Fisher C,et al. Fusion of SYT to two genes, SSX1 and SSX2,encoding proteins with homology to the Kruppel-associated box in human synovial sarcoma [J].EMBO J,1995,14(7):2333-2340.
[20]dos Santos NR, de Bruijn DR, van Kessel AG. Molecular mechanisms underlying human synovial sarcoma development. Genes Chromosomes [J]Cancer,2001,30:1-14.
[21]Ladanyi M, Antonescu CR, Leung DH, et al. Impact of SYT-SSX fusion type on the clinical behavior of synovial sarcoma:a multi-institutional retrospective study of 243 patients [J] Cancer Res,2002,62:135-140.
[22]Limon J, Babinska M,Denis A,et al. Parachordoma:a rare sarcoma with clonal chromosomal changes [J]. Cancer Genet Cytogenet.1998,102(1):78-80.
[23]Lee W,Han K,Harris CP,et al. Use of FISH to detect chromosomal translocations and deletions. Analysis of chromosome rearrangement in synovial sarcoma cells from paraffin-embedded specimens [J].Am J Pathol,1993,143(1):15-19.
[24]Nagao K,Ito H,Yoshida H. Chromosomal translocation t(X;18) in human synovial sarcomas analyzed by fluorescence in situ hybridization using paraffin-embedded tissue [J]. Am J Pathol.1996,148(2):601-9.
[25]Argani P,Zakowski MF,Klimstra DS,et al. Detection of the SYT-SSX chimeric RNA of synovial sarcoma in paraffin-embedded tissue and its application in problematic cases [J]. Mod Pathol.1998,11(1):65-71.
[26]Iwasaki H,Ishiguro M,Ohjimi Y,et al. Synovial sarcoma of the prostate with t(X;18)(p11.2;q11.2) [J]. Am J Surg Pathol.1999,23(2):220-6.
[27]Ishida S, Yoshida K, Kaneko Y, et al. The genomic breakpoint and chimeric transcripts in the EWSR1-ETV4/E1AF gene fusion in Ewing sarcoma [J]Cytogenet Cell Genet,1998,82:278-283.
[28]Kanoe H, Nakayama T, Hosaka T, et al. Characteristics of genomic breakpoints in TLS-CHOP translocations in liposarcomas suggest the involvement of Translin and topoisomerase II in the process of translocation [J]Oncogene,l 999,18:721-729.
[29]Kawai A, Woodru. J, Healey JH, Brennan MF, AntonescuCR and Ladanyi M. Establishment and characterization of a biphasic synovial sarcoma cell line, SYO-l.[J].Adv Exp Med Biol,2006,587:99-113
[30]H.Sonobe, Y. Manabe, M. Furihata, J. Iwata, T. Oka, Y.Ohtsuki, et al., Establishment and characterization of a new human synovial sarcoma cell line, HS-SY-II,[J] Lab. Invest.1992,67:498-505.
[31]P.J. Renwick, B.R. Reeves, P. Dal Cin, C.D.M. Fletcher, H.Kempski, R. Sciot, et al., Two categories of synovial sarcoma defined by divergent chromosome translocation breakpoints in Xp11.2, with implications for the histologic sub-classification of synovial sarcoma, Cytogenet. [J]Cell Genet.1995,70: 58-63.
[32]S. Noguchi, T. Ueki, S. Kawauchi, T. Fukuda, H. Matsuura, T.Sonoda, M. Tsuneyoshi, Establishment and characterization of a new synovial sarcoma cell line, SN-SY-1:special reference to bcl-2 protein and SYT-SSX1 hybrid transcripts [J]Cancer 1997,72:995-1002.
[33]Sonobe, T. Takeuchi, S.B. Liag, T. Taguchi, K. Yuri, K.Shimizu, et al., A new human synovial sarcoma cell line, HSSY-3, with a truncated form of hybrid SYT/SSX1 gene [J] Cancer 1999,82:459-464.
[34]T. Yakushiji, K. Yonemura, J. Tsuruta, K. Nishida, T. Kato, K.Takagi, Capacity for epithelial differentiation in synovial sarcoma:analysis of a new human cell line [J] J. Clin. Pathol.2000,53:525-531.
[35]J. Nishio, H. Iwasaki, M. Ishiguro, Y. Ohjimi, C. Fujita, T.Isayama, et al., Establishment of a new human synovial sarcoma cell line, FU-SY-1, that expresses c-Met receptor and its ligand hepatocyte growth factor [J]Int. J. Oncol.2002 Jul;21(1):17-23
[36]Kawai A, Naito N, Yoshida A, Morimoto Y, Ouchida M, Shimizu K, Beppu Y. Establishment and characterization of a biphasic synovial sarcoma cell line, SYO-1 [J]Cancer Lett.2004 Feb 10;204(1):105-13.
[37]Elbashir SM, Harborth J, LendeckelW et al.2001. Dup lexes of 21-nucleotide RNAsmediate RNA interference in cultured mammalian cells [J]Nature, 2001,411(6836):494-498
[38]Elbashir SM, Harborth J, Weber K, Tuschl T., Analysis of gene function in somatic mammalian cells using small interfering RNAs [J]Methods.2002 Feb;26(2):199-213.
[39]Pai SI, Lin YY,Macaes B, et al. Prospects of RNA interference therapy for cancer [J]. Gene Ther,2006,13(6):464-477
[40]Fedorov Y, Anderson EM, Birmingham A, Reynolds A, Karpilow J, Robinson K, Leake D, Marshall WS, Khvorova A. Off-target effects by siRNA can induce toxic phenotype [J] RNA.2006 Jul; 12(7):1188-96. Epub 2006 May 8.
[41]Schubert S. GrnnweHer A. Erdmann VA, et al. The ubiquitin-domain protein HERP forms a complex with components of the endoplasmic reticulum associated degradation pathway[J] J Mol Biol,2005,348(4):883-893,
[42]Clark, J., P. J. Rocques, et al. Identification of novel genes, SYT and SSX, involved in the t(X;18)(p11.2;q11.2) translocation found in human synovial sarcoma.[J] Nat Genet,19947(4):502-8.
[43]dos Santos NR, de Bruijn DR, van Kessel AG. Molecular mechanisms underlying human synovial sarcoma development. [J]Genes Chromosomes Cancer,2001 Jan;30(1):1-14.
[44]de Bruijn DRH, Baats E, Zechner U, et al. Isolation and characterization of the mouse homolog of SYT, a gene implicated in the development of human synovial sarcomas. [J]Oncogene,1996,13:643-648
[45]Perani, M., et al., The proto-oncoprotein SYT interacts with SYT-interacting protein/co-activator activator (SIP/CoAA), a human nuclear receptor co-activator with similarity to EWS and TLS/FUS family of proteins. [J]J Biol Chem,2005.280(52):p.42863-76.
[46]Perani M, Ingram CJ, Cooper CS, Garrett MD, Goodwin GH.2003.Conserved SNH domain of the proto-oncoprotein SYT interacts with components of the human chromatin remodelling complexes, while the QPGY repeat domain forms homo-oligomers.[J]Oncogene 22:8156-8167.
[47]Thaete, C, et al., Functional domains of the SYT and SYT-SSX synovial sarcoma translocation proteins and co-localization with the SNF protein BRM in the nucleus. [J]Hum Mol Genet,1999.8(4):p.585-91.
[48]de Bruijn DRH, dos Santos NR, Thijssen J, Balemans M, Debernardi S, Linder B, Young BD, Geurts van Kessel A.2001b. The synovial sarcoma associated protein SYT interacts with the acute leukemia associated protein AF10.[J] Oncogene 20:3281-3289.
[49]Eid, J.E., et al., p300 interacts with the nuclear proto-oncoprotein SYT as part of the active control of cell adhesion. [J]Cell,2000.102(6):p.839-48
[50]Ito, T., et al., SYT, a partner of SYT-SSX oncoprotein in synovial sarcomas, interacts with mSin3A, a component of histone deacetylase complex. [J]Lab Invest,2004.84(11):p.1484-90.
[51]dos Santos NR, de Bruijn DRH, Kater-Baats E, et al. Delineation of the protein domains responsible for SYT, SSX and SYT-SSX nuclear localization. [J]Exp Cell Res 2000.256:192-202.
[52]Gure AO,Wei IL Old Lj,et al. The SSX gene family:characterization of 9 complete gene.[J] Int J Cancer 2002.101 (5):448-453
[53]Tureci, ChenY T, SahinU, et al.Experssion of SSX genes in human tumors. [J]Int J Cancer,1998,77(1):19-23.
[54]Tureci O, Sahin U, Schobert I, et al. The SSX-2 gene, which is involved in the t(X,18) translocation of synovial sarcomas, codes for the human tumor antigen HOM-MEL 40. [J]Cancer Res,1996,56:4766-4772.
[55]Ladanyi M.Fusion of the SYT and SSX genes in synovial sarcoma [J].Oncogen,2001,20(40):5755-5762.
[56]dos Santos NR, de Bruijn DRH, Kater-Baats E, et al. Delineation of the protein domains responsible for SYT, SSX and SYT-SSX nuclear localization. [J]Exp Cell Res,2000,256:192-202.
[57]Satijn DPE, Otte AP. Polycomb group protein complexes:do different complexes regulate distinct target genes? [J]Biochim Biophys Acta,1999, 1447:1-16.
[58]Makris A,PowlesTJ,Kakolp4s S,et al.Ruduction in angiogenesis after neoadjuvant chemoendoctine therapy in patients with operable breast carcinoma[J] Cancer,199985(9):1996-2000.
[59]Thompson N, Lyons J. Recent progress in targeting the Raf/MEK/ERK pathway with inhibitors in cancerdrug discovery.[J].CurrOpinPharmacol,2005, 5(4):350-356.
[60]AisaY, MiyakawaY, NakazatoT, eta.l Fucoidan induces apoptosis of humanHS-sultan cells accompanied by activation ofcaspase-3 anddown-regulation of ERK pathways.[J].Am JHematol,2005,78(1):7-14.
[61]PohlG, Ho CL, Kurman RJ, et al. Inactivation of the mitogen——aetiva——ted protein kinase pathway as a potential target--based therapy in OvaHan serous tumors with KRAS or BRAF mutations.[J]Cancer Res,2005,65(5): 1994-2000.
[62]Mishima K, Inoue K, Hayashi Y. Overexpression of extra-cellular-signal regulated kinases on oral squamous cell carcinoma. Smalley KS. A piyotal role for ERK in the oncogenic be-haviour of malignant melanoma. [J]Int J Cancer,2003;104(5):527-532.
[63]Santen RJ, Song RX, McPherson R, et al. The role of mi-togen-activated protein (MAP) kinase in breast cancer. [J]J Steroid Biochem Mol Biol, 2002;80(2):239-256.
[64]Yan KX,Liu BC,Shi XL,et al.Role of cyclinDl and CDK4 in the carcinogenesis induced by silica [J] Biomed Environ Sci.2005 Oct;18(5):286-296.
[65]Bartkova J,Horejsi Z, Koed K,et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis [J]. Nature,2005,434 (7035):864-870.
[66]Sangkhthathat S,Kusafuka T,Miao J et al.In vitro RNA interference against beta-catenin inhibits the proliferation of pediatric hepatic tumors [J]Int J Oncol.2006 Mar;28(3):715-722.
[67]董秦,孔广忠,欧阳晓辉,等.胃癌及癌旁组织中CyclinDl, CDK4表达及意义[J]中华现代外科学杂志,2004,1(3)205-207.
[68]朱东望,赵秀兰,李世芬,等.口腔鳞状细胞癌细胞周期因子p27,CyclinD1及CDK4的表达与意义[J]中国肿瘤临床,2003,30(1)：40-44.
[69]Yu Q, Geng Y, Sicinski P, et al Specific protection againstbreast cancers by cyclin D1 ablation[J]. Nature,2001,411(6841):1071-1021
[70]Yuntao Xie, Bjorn Skytting, Gunnar Nilsson,et al. SYT-SSX Is Critical for Cyclin D1 Expression in Synovial Sarcoma Cells:A Gain of Function of the t(X;18)(p11.2;q11.2) Translocation.[J] Cancer Res 2002,62(1):3861-3867.
[71]Caraglia M,Tassone P,Marra M,et al.Targeting Raf-kinase:molecular rationales and translational issues[J].Ann Oncol,2006, Jun;17 Suppl 7.
[72]C.P. Han, M.Y. Lee, S.L. Tzeng, C.C. Yao, P.H. Wang, Y.W. Cheng, S.L. Chen, T.S. Wu, Y.S. Tyan, and L.F. Kok, Nuclear Receptor Interaction Protein (NRIP) expression assay using human tissue microarray and immunohistochemistry technology confirming nuclear localization [J] J Exp Clin Cancer Res,2008 Aug 2;27:25.
[73]Kawai A. WondruffJ, Healy JH. et al.SYT-SSX gene fusion as determinant of morphology and prognosis in synovial sarcoma [J] N Engl J Med.1998, 338(3):153-160
[74]Johannessen AL,Torp SH,et al. The clinical value of Ki-67/MIB-1 lableling index in human astrocytomas [J] Pathol Oncol Res 2006;12(3):143-147.
[75]Antonescu CR,Kawai A,Leung DH,et al. Strong association of SYT-SSX fusion type and morphologic epithelial differentiation in synovial sarcoma [J]. Diagn Mol Pathol 2000,9(1):1-8.
[76]Kahl C R, Maean s A R. Regu lation of cell cycle progression by caleium/calmedulin-dependent pathways[J] Endocr Rev,2003,24(6):719-736.
[77]Reeves H L, Narla G, Ogu nbiyi O, et al. Kruppel——like factor 6(KLF6)is a tumor suppressor gene frequently inaetived in colorectalcancer [J]. Gastroenterology,2004,126(4):1090-1103.
[78]Dajee M, Lazal'OV M, Zhang J Y, et al. NFKB blockade and oncogenicras trigger invasive human epidermal neoplasia[J]. Nature,2003,421 (6923):639-643.
[79]Whitson BA, Jacobson BA, Frizelle S, Patel MR, Yee D, Maddaus MA, Kratzke RA. Effects of insulin-like growth factor-1 receptor inhibition in mesothelioma,Thoracic Surgery Directors Association Resident Research Award[J]Ann Thorac Surg.2006 Sep;82(3):996-1002.
[1]Bergh P, Meis-Kindblom JM, Gherlinzoni F, et al.Synovial sarcoma: identification of low and high risk groups[J].Cancer,1999,85:2596-2607.
[2]Skytting B, Meis-Kindblom JM, Larsson O, et al. Synovial sarcoma--identification of favorable and unfavorable histologic types:a Scandinavian sarcoma group study of 104 cases.[J] Cancer Res,1999,59: 3180-3184.
[3]Sperling, B.L., et al., Synovial chondromatosis and chondrosarcoma:a diagnostic dilemma. Sarcoma,2003.7(2):p.69-73.
[4]Ladanyi, M., Fusions of the SYT and SSX genes in synovial sarcoma. [J] Oncogene,2001.20(40):5755-62.
[5]Okamoto, S., et al., Primary pulmonary synovial sarcoma:a clinicopathologic, immunohistochemical, and molecular study of 11 cases. [J] Hum Pathol,2004. 35(7):850-856.
[6]Suster S, Moran CA. Primary synovial sarcomas of the mediastinum:a clinicopathologic, immunohistochemical, and ultrastructural study of 15 cases.[J]. Am J Surg Pathol.,2005,29(5):569-78.
[7]Suster S, Moran CA. Primary synovial sarcomas of the mediastinum:a clinicopathologic, immunohistochemical, and ultrastructural study of 15 cases.[J]. Am J Surg Pathol.,2005,29(5):569-78.
[8]Dei tos AP, Doglioni C,et al,Calretinin:a novel tool for diagnostic immunohistochemistry. [J] Adv Anat Pathol 1998,5:61-66.
[9]张惠铭,等,中间丝蛋白在滑膜肉瘤中的表达[J].中华病理学杂志,1992,21(3)：159.
[10]熊敏,等.免疫组织化学和组织化学在滑膜肉瘤诊断中的意义[J]诊断病理学杂志,1994,1(1)：36-37.
[11]Ture-Carel C,Dal Cin P,Limon J,et al.Translocation X;18 in synovial sarcoma[J].Cancer Gene Cytogenel,1986,23(1):93.
[12]Sreekanlaiah C, landanyi M, Rodriguez E, et al.Chlomosomal aberrations in soft tissue tumors:relevances to diagnosis, classification, and molecular mechanisms[J].Am J Pathol.1994,144(6):1121-1134.
[13]Clark, J., P. J. Rocques, et al. (1994). "Identification of novel genes, SYT and SSX, involved in the t(X;18)(pll.2;qll.2) translocation found in human synovial sarcoma." [J] Nat Genet 7(4):502-508.
[14]Crew AJ.Clark J, Fisher C,et al. Fusion of SYT to two genes, SSX1 and SSX2,encoding proteins with homology to the Kruppel-associated box in human synovial sarcoma [J].EMBO J,1995,14(7):2333-2340.
[15]dos Santos NR, de Bruijn DR, van Kessel AG. Molecular mechanisms underlying human synovial sarcoma development. Genes Chromosomes [J] Cancer,2001,30:1-14.
[16]Ladanyi M, Antonescu CR, Leung DH, et al. Impact of SYT-SSX fusion type on the clinical behavior of synovial sarcoma:a multi-institutional retrospective study of 243 patients. [J] Cancer Res,2002,62:135-140.
[17]Limon J, Babinska M,Denis A,et al. Parachordoma:a rare sarcoma with clonal chromosomal changes.[J]. Cancer Genet Cytogenet.1998,102(1):78-80.
[18]Lee W,Han K,Harris CP,et al. Use of FISH to detect chromosomal translocations and deletions. Analysis of chromosome rearrangement in synovial sarcoma cells from paraffin-embedded specimens. [J].Am J Pathol,1993,143(1):15-19.
[19]Nagao K, Ito H,Yoshida H. Chromosomal translocation t(X;18) in human synovial sarcomas analyzed by fluorescence in situ hybridization using paraffin-embedded tissue.[J]. Am J Pathol.1996,148(2):601-9.
[20]Argani P, Zakowski MF, Klimstra DS,et al. Detection of the SYT-SSX chimeric RNA of synovial sarcoma in paraffin-embedded tissue and its application in problematic cases.[J]. Mod Pathol.1998,11(1):65-71.
[21]Iwasaki H,Ishiguro M,Ohjimi Y,et al. Synovial sarcoma of the prostate with t(X;18)(p11.2;q11.2).[J]. Am J Surg Pathol.1999,23(2):220-6.
[22]Kawai A, Woodru. J, Healey JH, Brennan MF, AntonescuCR and Ladanyi M.[J].Adv Exp Med Biol,2006,587:99-113.
[23]Guillou L, Coindre J, Gallagher G, et al. Detection of the synovial sarcoma translocation t(X;18) (SYT;SSX) in paraffin-embedded tissues using reverse transcriptase-polymerase chain reaction:a reliable and powerful diagnostic tool for pathologists. A molecular analysis of 221 mesenchymal tumors fixed in different fixatives. [J] Hum Pathol,2001,32:105-112.
[24]Finger LR, Harvey RC, Moore RC, et al. A common mechanism of chromosomal translocation in T-and B-cell neoplasia. [J]Science,1986,234 982-985.
[25]Aoki K, Suzuki K, Sugano T, et al. A novel gene, Translin, encodes a recombination hotspot binding protein associated with chromosomal translocation. [J] Nat Genet,1995,10:167-174.
[26]Bullock P, Champoux JJ, Botchan M. Association of crossover points with topoisomerase I cleavage sites:a model for nonhomologous recombination. [J] Science,1985,230:954-958.
[27]Broeker PL, Super HG, Thirman MJ, et al. Distribution of 11q23 breakpoints within the MLL breakpoint cluster region in de novo acute leukemia and in treatment-related acute myeloid leukemia:correlation with scaffold attachment regions and topoisomerase Ⅱ consensus binding sites. [J]Blood, 1996,87:1912-1922.
[28]Chen SJ, Chen Z, d'Auriol L, et al. Ph1+bcr-acute leukemias:implication of Alu sequences in a chromosomal translocation occurring in the new cluster region within the BCR gene. [J]Oncogene,1989,4:195-202.
[29]Boehm T, Mengle-Gaw L, Kees UR, et al. Alternating purinepyrimidine tracts may promote chromosomal translocations seen in a variety of human lymphoid tumours. [J] EMBO J,1989,8:2621-2631.
[30]Nicholls RD, Fischel-Ghodsian N, Higgs DR. Recombination at the human a-globin gene cluster:sequence features and topological constraints. [J] Cell, 1987,49:369-378.
[31]Camerini-Otero RD, Hsieh P. Parallel DNA triplexes, homologous recombination, and other homology-dependent DNA interactions. [J] Cell,1993,73:217-223.
[32]Chalk JG, Barr FG, Mitchell CD. Translin recognition site sequences flank chromosome translocation breakpoints in alveolar rhabdomyosarcoma cell lines. [J] Oncogene,1997,15:1199-1205.
[33]Panagopoulos I, Lassen C, Isaksson M, et al. Characteristic sequence motifs at the breakpoints of the hybrid genes FUS/CHOP, EWS/CHOP and FUS/ ERG in myxoid liposarcoma and acute myeloid leukemia. [J]Oncogene,1997, 15:1357-1362.
[34]Barr FG, Nauta LE, Hollows JC. Structural analysis of PAX3 genomi rearrangements in alveolar rhabdomyosarcoma. [J] Cancer Genet Cytogenet, 1998,102:32-39.
[35]Ishida S, Yoshida K, Kaneko Y, et al. The genomic breakpoint and chimeric transcripts in the EWSR1-ETV4/E1AF gene fusion in Ewing sarcoma. [J] Cytogenet Cell Genet,1998,82:278-283.
[36]Kanoe H, Nakayama T, Hosaka T, et al. Characteristics of genomic breakpoints in TLS-CHOP translocations in liposarcomas suggest the involvement of Translin and topoisomerase II in the process of translocation. [J] Oncogene,1999,18:721-729.
[37]Perani, M., et al., The proto-oncoprotein SYT interacts with SYT-interacting protein/co-activator activator (SIP/CoAA), a human nuclear receptor co-activator with similarity to EWS and TLS/FUS family of proteins. [J] J Biol Chem,2005.280(52):42863-76.
[38]Perani M, Ingram CJ, Cooper CS, Garrett MD, Goodwin GH.2003.Conserved SNH domain of the proto-oncoprotein SYT interacts with components of the human chromatin remodelling complexes, while the QPGY repeat domain forms homo-oligomers. [J] Oncogene 22:8156-8167.
[39]Thaete, C, et al., Functional domains of the SYT and SYT-SSX synovial sarcoma translocation proteins and co-localization with the SNF protein BRM in the nucleus. [J] Hum Mol Genet,1999.8(4):585-591.
[40]de Bruijn DRH, dos Santos NR, Thijssen J, Balemans M, Debernardi S, Linder B, Young BD, Geurts van Kessel A.2001b. The synovial sarcoma associated protein SYT interacts with the acute leukemia associated protein AF10. [J] Oncogene 20:3281-3289.
[41]Eid, J.E., et al., p300 interacts with the nuclear proto-oncoprotein SYT as part of the active control of cell adhesion. [J] Cell,2000.102(6):839-48.
[42]Ito, T., et al., SYT, a partner of SYT-SSX oncoprotein in synovial sarcomas, interacts with mSin3a, a component of histone deacetylase complex. [J] Lab Invest,2004.84(11):1484-90.
[43]dos Santos NR, de Bruijn DRH, Kater-Baats E, et al. Delineation of the protein domains responsible for SYT, SSX and SYT-SSX nuclear localization. [J] Exp Cell Res 2000.256:192-202.
[44]Gure AO, Wei IL Old Lj,et al. The SSX gene family:characterization of 9 complete gene.[J] Int J Cancer 2002.101 (5):448-453.
[45]Tureci, ChenY T, SahinU, et al.Experssionof SSX genes in human tumors [J]. IntJ Cancer,1998,77(1):19-23.
[46]Panagopoulos,I.,Merten,F,et al.Clinical impact of molecular and cytogenetic findings in synovial saracoma.[J]Genes Chromosomes Cancer,2001, 31:362-372
[47]Ladanyi M.Fusion of the SYT and SSX genes in synovial sarcoma [JJ.Oncogen,2001,20(40):5755-5762.
[48]Makris A,PowlesTJ,Kakolp4s S,et al.Ruduction in angiogenesis after neoadjuvant chemoendoctine therapy in patients with operable breast carcinoma[J] Cancer,1999 85(9); 1996-2000.
[49]Johannessen AL, Torp SH, et al. The clinical value of Ki-67/MIB-1 labeling index in human astrocytomas. Pathol Oncol Res.2006; 12(3):143-147.
[50]Nilsson G, Skytting B, Xie Y, et al. The SYT-SSX1 variant of synovial sarcoma is associated with a high rate of tumor cell proliferation and poor clinical outcome [J]Cancer Res,1999,59,3180-3184.
[51]Inagaki H,Nagasaka T,Otsuka T,et al. Association of SYT-SSX fusion types with proliferative activity and prognosis in synovial sarcoma..[J].Mod Pathol. 2000,13(5):482-8
[52]Antonescu CR,Kawai A,Leung DH,et al. Strong association of SYT-SSX fusion type and morphologic epithelial differentiation in synovial sarcoma. [J]. Diagn Mol Pathol 2000,9(1):1-8
[53]中华病理学杂志委员会.全国端粒酶、p53和细胞凋亡的研究应用[J].中华病理学杂志,2000,29(1)：9.
[54]David L. Awakening angels [J] Nature,1998;394:616-617
[55]Yan KX,Liu BC,Shi XL,et al.Role of cyclinDl and CDK4 in the carcinogenesis induced by silica.[J]. Biomed Environ Sci.2005 Oct; 18(5):286-296.
[56]Bartkova J,Horejsi Z,Koed K,et al.DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. [J]. Nature,2005,434 (7035):864-870.
[57]Kim DH,Muto M Kuwahara Y,et al.Array-based comparative genomic hybridization of circulating esophageal tumor cells. Oncol Rep.2006 Nov;16(5):1053-1059
[58]陆劲松,沈镇宙,吴灵等.细胞周期蛋白D1、CDK4在乳腺癌中的表达及其临床意义.[J]中国癌症杂志,2001,11(2)105-108.
[59]Sangkhthathat S,Kusafuka T,Miao J et al.In vitro RNA interference against beta-catenin inhibits the proliferation of pediatric hepatic tumors.Int J Oncol. 2006 Mar;28(3):715-722.
[60]董秦,孔广忠,欧阳晓辉,等.胃癌及癌旁组织中CyclinD1,CDK4表达及意义.[J]中华现代外科学杂志,2004,1(3)205-207.
[61]朱东望,赵秀兰,李世芬,等.口腔鳞状细胞癌细胞周期因子p27,CyclinD1及CDK4的表达与意义.[J]中国肿瘤临床,2003,30(1)：40-44.
[62]Sakaguchi T, Watanabe A, Sawada H, et al. Prognostic value of cyclin E and p53 expression in gastric carcinoma. Cancer,1998,82:1238-1243.
[63]Mishina T, Dosaka2Akita H, Hmmura F, et al. CyclinE expression, a potential prognostic marker for non2small cell lung cancer. Cancer Res 2000,6:11-16.
[64]Iseki H, Ko TC, Xue XY, et al. Cyclin2dependent kinase inhibitors block proliferation of human gastric cancer cells. Surgery,1997,122:187-194.
[65]Resnitzky D, Reed SI. Different roles for cyclins D1 and E in regulation of the Gl-S transition. Mol Cell Biol,1995,15:3463-3469.
[66]Yu HY, Shi CG, Zhu JJ, et al. Cyclin A expression in non2small cell lung carcinoma as related to proliferative activity and prognosis. Chin J Cancer, 2001,20:38-40.
[67]张益,彭志海,裘国强,等1周期蛋白A在原发性肝癌中的表达及意义1中华肿瘤杂志,2002,24：353-355.
[68]朱明华,倪灿荣,祝峙,等.原发性肝细胞癌中5种细胞周期蛋白的表达及其临床意义,中华病理学杂志,2003,32：440-443.
[69]Yu Q, Geng Y, Sicinski P, et al Specific protection againstbreast cancers by cyclin D1 ablation[J]. Nature,2001,411(6841):1071-1021.
[70]Yuntao Xie, Bjorn Skytting, Gunnar Nilsson et al.The SYT-SSX1 fusion type of synovial sarcoma is associated with increased expression of cyclin A and D1. A link between t(X;18)(p11.2; q11.2) and the cell cycle machinery.[J] Oncogen 2002,21:5791-5796.
[71]Yuntao Xie, Bjorn Skytting, Gunnar Nilsson,et al. SYT-SSX Is Critical for Cyclin D1 Expression in Synovial Sarcoma Cells:A Gain of Function of the t(X;18)(p11.2;q11.2) Translocation.[J] Cancer Res 2002,62(1):3861-3867.
[72]Garber K. Beyond the Nobel Price:cell cycle researcs new view on cancer. Nat Cancer Inst,2001,93:1766-1768.
[73]Sugimoto M,Martin N,Wilks DP,et al. Activation of cyclin D1-kinase in murine fibroblasts lacking both p21(Cipl) and p27(Kip1).[J] Oncogene.2002, 21(53):8067-74.
[74]Langner C, Von WR, Ratschek M, et al. Biological significance of p27and Skp2 expression in renal cell carcinoma. A systematic analysis of primary and metastatic tumour tissues using a tissue microarray technique. [J].Virchows Arch,2004,445 (6):631-636.
[75]Xia M,Land H,et al. Tumor suppressor p53 restricts Ras stimulation of RhoA and cancer cell motility.[J], Nat Struct Mol Biol.2007,14(3):215-223.
[76]Matsuta M,Kon S,Sasaki K,et al. Immunohistochemical detection of p21WAF1/CIP1 and p53 proteins in formalin-fixed paraffin-embedded tissue sections of squamous cell carcinoma of the skin.[J], J Dermatol Sci. 1997,14(3):233-239.
[77]Kalra N, Arora A,Shukla Y,et al. Involvement of multiple signaling pathways in diallyl sulfide mediated apoptosis in mouse skin tumors. [J], Asian Pac J Cancer Prev.2006,7(4):556-562.
[78]Ciccarelli C,Marampon F,Scoglio A,et al. p21WAF1 expression induced by MEK/ERK pathway activation or inhibition correlates with growth arrest, myogenic differentiation and onco-phenotype reversal in rhabdomyosarcoma cells.[J], Mol Cancer.2005,13(4):41.
[79]Sato F,Kurokawa M,Yamauchi N,et al. Gene silencing of myostatin in differentiation of chicken embryonic myoblasts by small interfering RNA.[J], Am J Physiol Cell Physiol.2006 Sep;291(3):C538-545.
[80]Masumi A,FuKazawa H,Shimazu T,et al. Nucleolin is involved in interferon regulatory factor-2-dependent transcriptional activation.[J], Oncogene.2006 24;25(37):5113-5124.
[81]Millar EK, Trank,Marr P,Graham Ph,et al. p27KIP-l, cyclin A and cyclin D1 protein expression in ductal carcinoma in situ of the breast:p27KIP-l correlates with hormone receptor status but not with local recurrence.[J], Pathol Int.2007,57(4):183-189.
[82]Shapira M,Ben-Izhak O,Slotky M,et al. Expression of the ubiquitin ligase subunit cyclin kinase subunit 1 and its relationship to S-phase kinase protein 2 and p27Kip1 in prostate cancer. [J],J Urol.2006 Nov;176(5):2285-2289.
[83]Kossatz U, Vervoorts J,Nickeleit I,et al. C-terminal phosphorylation controls the stability and function of p27kip1. EMBO J.2006,25(21):5159-5170.
[84]Langner C,von wasielewski R,Ratschek M,et al. Biological significance of p27 and Skp2 expression in renal cell carcinoma. A systematic analysis of primary and metastatic tumour tissues using a tissue microarray technique. [J], Virchows Arch.2004,445(6):631-636.
[85]Li Q,Murphy M,Ross J, et al. Skp2 and p27kipl expression in melanocytic nevi and melanoma:an inverse relationship.[J]. J Cutan Pathol.2004 31(10):633-642.
[86]Li W, Sanki A, Karim RZ,et al. The role of cell cycle regulatory proteins in the pathogenesis of melanoma. [J]. Pathology.2006,38(4):287-301.
[87]Kawauchi S, Goto Y, Liu XP,et al. Low expression of p27(Kipl), a cyclin-dependent kinase inhibitor, is a marker of poor prognosis in synovial sarcoma.[J].Cancer,2001,91(5):1005-1012.
[88]Inagaki H,Nagasaka T,Otsuka T,et al. Association of SYT-SSX fusion types with proliferative activity and prognosis in synovial sarcoma..[J]. Mod Pathol. 2000,13(5):482-8.
[89]Loda M,et al. p27KIP1:androgen regulation and prognostic significance in prostate cancer.[J]. Adv Clin Path.2000,4(4):226-32.
[90]ReikW, Dean W, WalterJ.Epigenetic reprogramming in mammalian development. [J].Science,2001,293(5532):1089-1093.
[91]BartovaE, Pachemik J, Hamicarova A, et al. Nuclear levels and patterns Of histone H3 mdoification and HP1 proteins after inhibition of histone deacetylase. [J] J Ceu sci,2005,118(Pt21):5035-5046.
[92]Baker E K, EI-osta A.MDR1, chemotherapy and chromatin remodeling[J] Cancer Bio 1 Ther,2004,3(9):819-824
[93]Allander, S.V., et al., Expression profiling of synovial sarcoma by cDNA microarrays:association of ERBB2, IGFBP2, and ELF3 with epithelial differentiation. Am J Pathol,2002.161(5):p.1587-1595.
[94]Ito, T., et al., Significant growth suppression of synovial sarcomas by the histone deacetylase inhibitor FK228 in vitro and in vivo. Cancer Lett,2005. 224(2):p.311-319.