摘要
目的
分析研究高迁移率族蛋白1(HMGB1)和晚期糖基化终产物受体(RAGE)基因在宫颈鳞癌、癌旁、正常宫颈组织中的表达,探讨它们与宫颈鳞癌发生、侵袭和转移的相关性。并进一步研究HMGB1在宫颈癌细胞中的表达,为进一步探讨HMGB1参与宫颈肿瘤生物学行为的具体机制奠定基础。
方法
组织:宫颈鳞癌根治术后冻存癌及癌旁组织各60例,正常宫颈组织30例。通过SYBR Green I嵌合荧光实时定量PCR技术检测HMGB1及RAGE基因在宫颈鳞癌、癌旁、正常宫颈组织及宫颈鳞癌临床参数指标中的定性,定量表达。细胞:选择人宫颈癌细胞及人成纤维细胞,应用SYBR Green I嵌合荧光实时定量PCR及细胞免疫组化技术进一步检测HMGB1基因及蛋白的表达。
结果
1.HMGB1 mRNA在宫颈鳞癌、癌旁组织表达水平明显高于在正常宫颈组织的表达,HMGB1mRNA在宫颈鳞癌、癌旁组织的表达水平无明显的统计学差异。RAGE mRNA在宫颈鳞癌、癌旁及正常宫颈组织中的表达水平均无明显的统计学差异。
2.宫颈鳞癌组织中HMGB1mRNA的表达与肿瘤分期、淋巴转移相关,与肿瘤直径、分级无关。RAGE mRNA在宫颈鳞癌组织中的表达与肿瘤直径、分级和分期均无关,仅与淋巴转移相关。在宫颈鳞癌组织中,HMGB1 mRNA及RAGE mRNA表达水平行相关分析,淋巴转移组中显示二者表达水平存在显著相关性(r=0.883,P＜0.05)。
3.从细胞水平研究HMGB1在人宫颈癌细胞及正常人成纤维细胞中的表达情况,发现HMGB1基因及蛋白在人宫颈癌细胞中的表达水平明显高于正常人成纤维细胞。
结论
1.HMGB1 mRNA可能与宫颈鳞癌发生、发展、侵袭、转移密切相关,从基因水平确定HMGB1为宫颈癌相关癌基因。
2.RAGE mRNA可能增强了宫颈鳞癌淋巴转移能力。RAGE mRNA的表达与HMGB1 mRNA的表达在宫颈癌淋巴转移组中的正相关性分析结果,提示可能在宫颈鳞癌的转移过程中,RAGE mRNA与HMGB1mRNA起协同作用,增强了下游基因的信号传导,起到促进转移的作用。HMGB1/RAGE轴可能是促进宫颈癌转移发生的重要信号通路。
3.从细胞水平上检测HMGB1基因、蛋白在人宫颈癌细胞中的高表达,提示HMGB1与人宫颈癌细胞恶性生物学行为的相关性。
Objective
To investigate expression and clinico-pathological findings of high mobility group box1(HMGB1)mRNA and receptor for advanced glycation end products (RAGE)mRNA in cervical squamous carcinoma,to explore their roles during carcinoma process,invasion and metastasis,to study the expression of HMGB1 in cell of cervical cancer,At both of gene and protein level determined HMGB1 as a novel oncogene correlative with cervical cancer.
Methods
Tissue:60 cervical squamous carcinoma samples,60 paraneoplastic samples and 30 normal cervical samples.Upon collection,the samples were frozen immediately at -80℃for future analysis.Real time Quantitative polymerase chain reaction was employed to detect the expression of HMGB1 mRNA and RAGE mRNA in cervical squamous carcinoma and nomal cervical tissues.Cell:choose cervical carcinoma cell and fibroblast cell.Real time Quantitative polymerase chain reaction and immunohistochemistry were employed to detect the expression of HMGB1.
Results
1.The expression of HMGB1 mRNA in cervical squamous carcinoma and their paraneoplastic tissues was higher than that in normal cervix tissues.And expression of RAGE was no significantly differences between cervical squamous carcinoma,paraneoplastic and normal cervical tissues.
2.The overexpression of HMGB1 mRNA in the cancer tissues was significantly correlated with the stage,the presence of metastasis,but not correlated with the diameter and grade of tumor.Statistical analysis showed that RAGE mRNA has a relation with the metastasis.But no relation in stage,diameter and grade of tumor.
3.At both of gene and protein level determined HMGB1 as a novel oncogene correlative with cervical cancer cell.
Conclusion
1.HMGB1 could be a prognostic predictor of growth、invasion、malignant degree in the evaluation of cervical cancer.HMGB1 is the main mechanism correlated with the metastasis of cervical cancer and they may become new targets for treating lyphatic metastasis.
2.RAGE expression was not significantly different among these tissue types,and showed no significant correlation with the different clinical parameters.However there was a significant positive correlation betwwen the expression of HMGB1 and RAGE in the cervical cancer metastasis group.
3.The expression of HMGB1 mRNA in the cervical cancer cell was higher than that in normal cell.and the The expression of HMGB1 protein in the cervical cancer cell was positive.This will help us to find out the relationship between HMGB1 and biological behaviour of cervical cancer.
引文
[1]乐杰.妇产科学[M].北京:人民卫生出版社,2004:288-290.
[2]曹泽毅.中华妇产科学[M].北京:人民卫生出版社,1999:174-179.
[3]ho T,shizuka T,Suzuki K et al Cervical cancer in young japanese women[J].Arch Gynecol Obstet,2000,264(2):69-70.
[4]Frega A,Stentella P,De Ioris et al.Young wwomen cervical intraepithwlial neoplasia and papillomarus risk factors for persistence and reccurence[J].Canor Leters 2003,196(2):127-1340.
[5]Chan PG,Sung HY,Sauaya GF et al.Changes in cervical cancer incidence afrer three decades of screeing is women less than 30years old[J].obstet gynecol,2003,102(4):765-773.
[6]Yuan F,Gu L,Guo S,et al.Evidence for involvement of HMGB1 protein in human DNA mismatch repai[J].J Biol Chem,2004,279:20935-20940.
[7]Bianchi ME,Manfredi A.Chromatin and cell death[J].Biochim Biophys Acta.2004,1677:181-186.
[8]Taguchi A,Blood DC,del Toro G,et al.Blockade of RAGE2HMGB1 signalling suppresses turnour growth and metastases[J].Nature,2000,405(6784):354-360.
[9]Kawahara N,Tanaka T,Yokomizo A et al.Enhanced co-expression of thioredoxin and high mobility group protein 1 genes in human hepatocellular carcinoma and the possible association with decreased sensitivity to cisplatin[J].Cancer Res 1996;56:5330-5333.
[10]Xiang YY,Wang DY,Tanaka M et al.Expression of highmobility group-1mRNA in human gastrointestinal adenocarcinoma and corresponding non-cancerous mucosa[J].Int J Cancer,1997,74:1-6.
[11]Yon Rak Choi,Hyunki Kim,Hyun Ju Kang,et al.Overexpression of High Mobility Group Box 1 in Gastrointestinal Stromal Tumors with KIT Mutation[J].Cancer Res,2003,63(9):2188-2193.
[12]Pardo M,Garcia A,Thomas B,et al.The characterization of the invasion phenotype of uveal melanoma tumour cells shows the presence of MUC18 and HMG-1 metastasis markers and leads to the identification of DJ-1 as a potential serum biomarker[J]. Int J Cancer. 2006,119(5):1014-1022.
[13] Hirata K, Takada M, Suzuki Y, et al. Expression of receptor for advanced glycation end products (RAGE) in human biliary cancer cells[J]. Hepatogastroenterology. 2003 ,50(53):1205-1207.
[14] Evans A, Lennard TW, Davies BR. et al.High-mobility group protein 1(Y): metastasis-associated or metastasis-inducing[J]? J Surg Oncol. 2004,88(2):86-99.
[15] Volp, K,Brezniceanu ML, Bosser S,et al. Increased expression of high mobility group box 1 (HMGB1) is associated with an elevated level of the antiapoptotic C-IAP2 protein in human colon carcinomas[J]. Gut,2006,55(2):234-242.
[16] Huang QX, Sun NF, Wang GB, et al. Expression of HMGB1 Protein in Human pancreatic carcinoma[J].The Practical Journal of Cancer, 2004,19(1): 19-23.
[17] AM Schlnidt,Mvianna,Mgerlach,et al. Isolation and characterization of binding proteins for advanced glycosylation end Products from bovine lung which are present on the endothelialcellsurface[J].Blol.Chem.l992,267:14987-14997.
[18] Porifier o,micaud V,Vionnet N,et al.Polymorphism screening of four genes encoding advanced glycation end-product putative receptors.Association study with nephtropathy in type 1 diabetic patients[J].Diarates,2001,50(5):1214-1228.
[19] H Kuniyasu, Y Chihara, and T Takahashi.Co-expression of recepto for advanced glycation end products and the ligand amphoter inassociates closely with metastasis of colorectalcancer[J].oncol Rep, 2003,10(2):445-448.
[20] Livak KJ, Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2- A A CT method[J]. Methods, 2001,25,402-408.
[21]Nathanson SD.Insights into the mechanisms of lymph node metastasis[J].cancer,2003,98(2):413-423.
[22]Herzog TJ.New approaches for the management of cervical cancer[J].Genecol,2003,90(3): 22-27.
[23] Gullo CA, Chuah CT, Hwang WYD,et al.ection and quantification of the abelson tyrosine kinase domains of the BCR-ABL gene translocation in chronic myeloid leukaemia using genomic quantitative real-time polymerase chain reaction[J].Ann Acad Med Singapore.2006,35(10):680-687.
[24]Baigent S,Nair V,Currie R.Real-time quantitative PCR for Marek's disease vaccine virus in feather samples:applications and opportunities[J].Dev Biol (Base1).2006,126:271-81.
[25]De Preter K,Vandesompele J,Heimann P,Application of laser capture microdissection in genetic analysis of neuroblastoma and neuroblastoma precursor cells.Cancer Lett.2003,197(1-2),53-61.
[26]Cabart P,Kalousek I,Jandova D,Hrkal Z.Differential expression of nuclear HMG1,HMG2 proteins and H10 histone in various blood cells[J].Cell Biochem Funct 1995;13:125-133.
[27]Bustin M.Revised nomenclature for high mobility group(HMG)chromosomal proteins[J].Trends Biochem Sci 2001,26:152-153.
[28]M.Eetal.The C-Terminal extension(CTE)of the nuclear hormone receptor DNA binding domain determines interactions and function response to the HMGB1/2co-regulatory proteins[J].Biol Chem,2002,277(28):2511-2512.
[29]BustinM.Regulation of DNA-dependent activities by the functionalmotifs of the high mobility group chromosomal proteins[J].Mol Cell,1999,19(8):5237-5246.
[30]Lum HK,Lee KL.The human HMGB1 promoter is modulated by a silencer and an enhancer-containing intron[J].Biochim Biophys Acts,2001,1520(1):79-84.
[31]Rouhiainen A,Imai S,Rauvala H,et al.Occurrence of amphoterin(HMG1)as an endogenous protein of human platelets that is exportedto the cell surface upon platelet activation[J].Thromb Haemost,2000,84(6):1087-1094.
[32]Henri J,Huttunen,Carole Fages,et al.Receptor for adcanced glycation end products-binding COOH-terminal motif of amphoterin inhibits invasive migration and metastasis[J].Cancer Res,2002,62(16):4805-4811.
[33]黄庆先,孙念峰,王国斌.高迁移率族蛋白1基因在胰腺癌组织中的表达及其临床意义[J],实用癌症杂志,2004,19(1):19-23.
[34]黄庆先,王国斌,孙念峰,高迁移率族蛋白框1反义抑制人胰腺癌细胞系PCNA-1侵袭的研究[J],癌症,2004,23(9):1036-1040.
[35]Dolde CE,Mukherjee M,Cho C,et al.:HMGB1/Y in human breast cancer cell lines.Breast Cancer Res Treat 2002;71:181-191.
[36]Porifier O,micaud V,Vionnet N,et al.Polymorphism screening of four genes encoding advanced glycation end-product putative receptors.Association study with nephtropathy in type 1 diabetic patients[J].Diarates,2001,50(5):1214-1228.
[37] G Srikrishna,HJ Huttunen,Ljohansson,et al.N-Glycans on the receptor for advanced glycation end Products influence HMGB1 binding and neurite outgrowth[J].Neurochem,2002,80(6):998-1008.
[38] Schmidt AM,Yan SD,Yan SF,et al. The multiligand receptor RAGE as a progression factor amplifying immune and inflammatory responses[ J]. Clin Invest ,2001,108 :949-955.
[39] Valencia JV,Mone M,Zhang J ,et al.Divergent pathways of gene expression are activated by the RAGE ligands S100b and AGE-BSA[J]. Diabetes , 2004,53:743-751.
[40] Taguchi A ,Blood DC ,del Toro G, et al .Blockade of RAGE2HMGB1 signalling suppresses tumour growth and metastases[J].Nature ,2000 ,405(6784) :354-360.
[41] Zill H,Bek S,Hofman T,et al.RAGE-mediated MAPK activationbyfocd-derived AGE and non—AGE products.Biochem Biophys ResCornun, 2003,300(2):311-315.
[42] Fages C,Nolo R,Huttunen HJ,et al .Regulation of cell migration by amphoterin [J].Cell Science,2000,113:611-620.
[43] Huttunen HJ,Fages C,luvala H.Receptor fbr advanced glycation end products(RAGE)-mediated neurite ontg-mv,h and activation of NF-KB require the cytoplasmic doman of the receptor but dife,ent donstream signaling pathwaya [J] .Biol Chem, 1999,274:19919-19924.
[44] Yan SD,Schmidt AM,Anderson GM,et al .Enhanced cellular oxidant stress by the interaction of advanced glycation end products with their receptors / binding proteins[J].Bid Chem, 1994, 269: 9 889-9897
[45] Lj JF,Qu XQ,Sclunidt AM,et al. Sp1 -binding elements in thepromoter of RAGE are essential for amphoterin-mediated gene expression in cultured neuroblastoma cells[J]. Bilo Chem,1998,273: 30 870-30871.
[46] Ruderman NB, WilliamsonJR, Brownlee M,Glucose and diabetic vascular disease[J].FASEB J, 1992;6:2905-2914
[47]Kuniyasu H,Chihara Y,Takahashi T.Co-expression of recepto for advanced glycation end products and the ligand amphoter inassociates closely with metastasis of colorectalcancer[J].oncol Rep,Marchl,2003,10(2):445-458.
[48]Kuniyasu H,Chihare Y,Kondo H.Differential effects between amphoterin and RAGE on colon cancer cells[J].Cancer,2003,104(6)722-726..
[1]徐佳,刘志锋,姜勇.高迁移率族蛋白与真核基因表达调控[J].生物化学与生物物理进展,2005,32(5):397-340.
[2]Bustin M.Revised nomenclature for high mobility group(HMG)chromosomal proteins[J].Trends Biochem Sci 2001,26:152-153.
[3]M.Eetal.The C-Terminal extension(CTE)of the nuclear hormone receptor DNA binding domain determines interactions and function response to the HMGB1/2co-regulatory proteins[J].Biol Chem,2002,277(28):2511-2512.
[4]BustinM.Regulation of DNA-dependent activities by the functionalmotifs of the high mobility group chromosomal proteins[J].Mol Cell,1999,19(8):5237-5246.
[5]Lum HK,Lee KL.The human HMGB1 promoter is modulated by a silencer and an enhancer-containing intron[J].Biochim Biophys Acts,2001,1520(1):79-84.
[6]Rouhiainen A,Imai S,Rauvala H,et al.Occurrence of amphoterin(HMG1)as an endogenous protein of human platelets that is exportedto the cell surface upon platelet activation[J].Thromb Haemost,2000,84(6):1087-1094.
[7]Henri J,Huttunen,Carole Fages,et al.Receptor for adcanced glycation end products-binding COOH-terminal motif of amphoterin inhibits invasive migration and metastasis[J].Cancer Res,2002,62(16):4805-4811.
[8]AM Schlnidt,Mvianna,Mgerlach,et al.Isolation and characterization of binding proteins for advanced glycosylation end Products from bovine lung which are present on the endothelialcellsurface[J].Blol Chem,1992,267:14987-14997.
[9]Porifier O,micaud V,Vionnet N,et al.Polymorphism screening of four genes encoding advanced glycation end-product putative receptors.Association study with nephtropathy in type 1 diabetic patients[J].Diarates,2001,50(5):1214-1228.
[10]G Srikrishna,HJ Huttunen,Ljohansson,et al.N-Glycans on the receptor for advanced glycation end Products influence HMGB1 binding and neurite outgrowth[J].Neurochem,2002,80(6):998-1008.
[11]Schmidt AM,Yan SD,Yan SF,et al.The multiligand receptor RAGE as a progression factor amplifying immune and inflammatory responses[J].Clin Invest,2001,108:949-955.
[12] Valencia JV,Mone M,Zhang J ,et al.Divergent pathways of gene expression are activated by the RAGE ligands S100b and AGE-BSA[J]. Diabetes, 2004,53:743-751.
[13] Huttunen HJ ,Fages C,Rauvala H,et al. Receptor for advanced glyca-tion end products (RAGE)-mediated neurite outgrowth and activation of NF-kappaB require the cytoplasmic domain of the receptor but different downstream signaling pathways [J] J Biol Chem,1999,274 (28): 19919-19924.
[14] Taguchi A ,Blood DC ,del Toro G, et al .Blockade of RAGE-HMGB1 signalling suppresses tumour growth and metastases[J ]. Nature,2000, 405(6784):354-360.
[15] Zill H,Bek S,Horman T,et al.RAGE-mediated MAPK activationbyfocd-derived AGE and non—AGE products.Biochem Biophys ResCornun, 2003,300(2):311-315.
[16] Fages C,Nolo R,Huttunen HJ,et al .Regulation of cell migration by amphoterin [J].Cell Science,2000,l 13:611-620.
[17] Huttunen HJ,Fages C,luvala H.Receptor for advanced glycation end products(RAGE)-mediated neurite ontg-mvh and activation of NF-KB require the cytoplasmic doman of the receptor but dife,ent donstream signaling pathwaya [J].Biol Chem,1999,274:19919-19924.
[18] Yan SD,Schmidt AM,Anderson GM,et al .Enhanced cellular oxidant stress by the interaction of advanced glycation end products with their receptors / binding proteins[J].BidChem,1994,269:9889-9897
[19] Lj JF,Qu XQ,Sclunidt AM,et al.Sp1-binding elements in thepromoter of RAGE are essential for amphoterin-mediated gene expression in cultured neuroblastoma cells[J]. Bilo Chem,1998,273: 30 870-30871.
[20] Huttunen HJ,Panula JK,Sorci G,et al .Coregulation of neurite out gowth and cell survival by amphoterin and S100 protein through receptor for advanced glycation end products(RAGE)activation[J]. BiolChem,000,275(51):40096-40105.
[21] J Murphy G & Gavrilovic.Proteolysis and cell migration:creating apath?Curt Opin Cell Biol,1999,11(5):614-621.
[22]Kuniyasu H,Oue N,Wakikawa A,et al.Expression of receptors for advanced glycation end-productsl(RACG)is closely associated with the invasive and metstatic activity of gastric cancer[J].Pathology,2002,196(2):103-108.
[23]Abe N,Watanabe T,Izumisato Y,et al.Diagnostic significance ofhigh mobility group 1(Y)protein expression in intraductal papillarymucinous tumors of the pancreas[J].Pancreas,2002,25:198-200.
[24]Nestl A,on Stein OD,Zatloukal K,et al.Gene expression patterns associated with the metastatic phenotype in rodent and human tumours[J].Cancer Res,2001;61:15691571.
[25]Tarbe N,Evtimova V,Burtscher H,et al.:Transcriptional profiling of cell lines derived from an orthotopic pancreatic tumour modelreveals metastasis-associated genes[J].Anticancer Res 2001;21:3221-3225.
[26]M Takada,K Hirata,T Ajiki,et al.Expression of receptor for advanced glycation end products(RAGE)and MMP-9-inhuman pancreatic cancer cells[J].Hepatogastroenterology,2004;51(58):928-930.
[27]黄庆先,孙念峰,王国斌,等.高迁移率族蛋白1基因在胰腺癌组织中的表达及其临床意义[J].实用癌症杂志,2004,19(1):19-23.
[28]黄庆先,王国斌,孙念峰,等.高迁移率族蛋白框1反义抑制人胰腺癌细胞系PCNA-1侵袭的研究[J].癌症,2004,23(9):1036-1040.
[29]李焱,蒋凯,卓文磊.HMGB1在结肠癌中的表达及对VEGF-C的影响[J].临床肿瘤学杂志,2007,12(6):417-420.
[30]Kuniyasu H,Chihara Y,Kondo H.Diferential efects between amphoterin and advanced Slycation end products on colon cancel-ceils[J].Cancer,2003,104(6):722-727.
[31]Kuniyasu H,Chihara Y,Takahashi.T Co-expression of recepto for advanced glycation end products and the ligand amphoter inassociates closely with metastasis of colorectalcancer[J].oncol Rep,2003,10(2):445-458.
[32]Palumbo R,Sampaolesi M,De Marchis F,et al.Extracellular HMGB1,a signal of tissue damage,induces mesoangioblast migration and proliferation[J].Cell Biol,2004,164(3):441-449.
[33]上官翰京,李志春,张晖萍.喉鳞状细胞癌中RAGE与HMGB1的表达[J].山东大学耳鼻喉眼学报,2006,20(3):204-205.
[34]Dolde CE,Mukherjee M,Cho C,et al.:HMGB1/Y in human breast cancer cell lines.Breast Cancer Res Treat,2002,71:181-191.
[35]Taguchi A,Blood DC,del Toro G,et al.Blockade of RAGE2HMGB1 signalling suppresses tumour growth and metastases[J].Nature,2000,405(6784):354-360.
[36]Leman ES,Madigan MC,Brunagel G,et al.Nuclear matrix localization of high mobility group protein 1(Y)in a transgenic mouse model for prostate cancer[J].Cell Biochem,2003,88:599-608.
[37]H Lishiguro,N Nakaigawa,Y Miyoshi,et al.Receptor for advanced glycation end products(RAGE)and its ligand HMGB1 are over expressed and associated with prostate cancerdevelopment[J].Prostate,2005,64(1):92-100.
[38]Antonella Bandiera,Daniela Bonifacio,Guidalberto Manfioletti,et al.Expression of HMGI(Y)Proteins in Squamous Intraepithelial and invasive Lesions of the Uterine Cervix[J].Cancer,1998,58,426-431.
[39]Wood LJ,Maher JF,Bunton TE,et al.The oncogenic properties of the HMGB1family[J].Cancer Res,2000,60:4256-4261.
[40]Bussemakers MJG,Van de Ven WJM,Debruye FM,et al.Identification of high mobility group protein 1(Y)as a potential progression marker for prostate cancer by differential hybridization analysis[J].Cancer Res,1991,51:606-611.
[41]Poser I,Golob M,Buettner R,Bosserhoff AK.Upregulation of HMG1 leads to melanoma inhibitory activity expression in malignant melanoma cells and contributes to their malignancy phenotype[J].Mol Cell Biol,2003,23:2991-2998.
[42]曾益新.肿瘤学[M].北京:人民卫生出版社,2003:1
[43]刘瑛,姜浩.Amphoterin—RAGE轴与肿瘤[J].美国中华临床医学杂志,2004,6(3),270-273.
[44]Meyer B,Murna Escobar H,Hauke S,et al.Expression pattern of the HMGB1gene in sarcomas of the dog[J].Anticancer Res,2004,24(2),707-710
[45]Reeves R,Beckerbauer L M.HMGA proteins as therapeutic drug targets[J],Prog Cell Cycle Res,2003,5:279-286.
[46] Riuzzi F, Sorci G, Donato R. The amphoterin (HMGB1)/receptor for advanced glycation end products (RAGE) pair modulates myoblast proliferation, apoptosis, adhesiveness, migration, and invasiveness. Functional inactivation of RAGE in L6 myoblasts results in tumor formation in vivo[J]. J Biol Chem. 2006;281(12):8242-8253.
