摘要
目的：通过观察c-myc对胆囊癌细胞运动和侵袭能力的影响,以论证c-myc在肿瘤侵袭和转移中的作用,为胆囊癌侵袭和转移干预提供分子靶点。
方法：采用实时定量PCR和western blot检测亲代和高转移胆囊癌细胞系中c-myc基因表达,设计靶向c-myc的小RNA[small interference RNA,siRNA]转染高转移的胆囊癌细胞,通过体外侵袭实验研究c-myc siRNA对胆囊癌高转移细胞迁移和侵袭能力的影响。此外,通过western blot方法检测转染c-myc siRNA后c-myc下游部分信号通路的变化,进一步研究c-myc siRNA抑制胆囊癌细胞转移的机制。
结果：通过实时定量PCR检测发现,c-myc的RNA水平在GBC-SD/M3细胞中是其在GBC-SD细胞中的2.18倍(p=0.0019)；在蛋白水平上,c-myc在GBC-SD/M3中的表达也高于其在GBC-SD中的表达。靶向c-myc的siRNA转染GBC-SD/M3细胞后,与对照相比,c-myc siRNA能够有效地抑制c-myc蛋白在GBC-SD/M3细胞中的表达。与对照相比,c-myc siRNA可以有效的抑制GBC-SD/M3细胞的迁移(p=0.0168)。对GBC-SD/M3细胞的侵袭能力检测后发现,c-myc siRNA同样也能够有效的抑制GBC-SD/M3细胞的侵袭(p=0.0005)。进一步检测c-myc siRNA对LIN28蛋白水平的影响,表明c-myc siRNA转染后,LIN28的蛋白水平也相应下调,let-7a的表达水平有所上升,从而抑制肿瘤细胞的转移。
结论：c-myc在高转移的胆囊癌细胞中表达上调。通过siRNA下调其表达后可以抑制胆囊癌细胞的转移和侵袭,这种抑制作用可能与LIN28的下调有关。
objective The role of c-myc gene in metastasis of gallbladder cancer was investigated by observation of the mobility and invasion of gallbladder cancer cells in vitro, in order to provide the evidences for selecting molecular targets in interfering gallbladder cancer metastasis.
Methods the expression of c-myc gene was determined by real time PCR and western blot in parental gallbladder cancer cell line GBC-SD and its highly metastatic subpopulation GBC-SD/M3. The siRNA targeted to c-myc were transfected into GBC-SD/M3, and then the migration and invasion abilities of GBC-SD/M3 cells were detected by in vitro transwell assays. The changes of the downstream signal of c-myc were determined by western blot assays.
Results the expression of c-myc gene was up-regulated in highly metastatic subpopulation, compared with that in its parental gallbladder cancer cell line. After the transfection of the c-myc siRNA in highly metastatic subpopulation, the RNA and protein levels were significantly reduced. Meanwhile, the migration and invasion abilities of the GBC-SD/M3 cells were inhibited. The detection of the downstream signal pathway of c-myc indicated LIN28 was reduced after the transfection of c-myc siRNA.
Conclusion c-myc was up-regulated in the GBC-SD/M3 compared with GBC-SD. c-myc siRNA reduced the migration and invasion of gallbladder cancer cells. The inhibition may involve the down-regulation of LIN28.
引文
[1]Kaye M. Reid & Antonio Ramos-De la Medina &John H. Donohue. Diagnosis and Surgical Management of Gallbladder Cancer:A Review. J Gastrointest Surg 2007; 11:671-681.
[2]Shibata K, Uchida H, Iwaki K, Kai S, Ohta M, Kitano S. Lymphatic invasion:an important prognostic factor for stages Tlb-T3 gallbladder cancer and an indication for additional radical resection of incidental gallbladder cancer. World J Surg.2009; 33[5]:1035-41.
[3]Meyer N, Penn LZ. Reflecting on 25 years with MYC. Nat Rev Cancer. 2008;8[12]:976-90.
[4]Wolfer A, Wittner BS, Irimia D, Flavin RJ, Lupien M, Gunawardane RN, Meyer CA, Lightcap ES, Tamayo P, Mesirov JP, Liu XS, Shioda T, Toner M, Loda M, Brown M, Brugge JS, Ramaswamy S. MYC regulation of a "poor-prognosis" metastatic cancer cell state. Proc Natl Acad Sci U S A.2010. Epub ahead of print
[5]王耀辉,孔凡民,佟玉兰.c-myc基因与胆管癌关系的研究[J].中国医科大学学报,2005,34(3)：248-249.
[6]王少军,赵屹,张峰,等.c-myc在肝细胞肝癌中的表达及其临床意义[J].实用临床医药杂志,2006,10(4)：49-51.
[7]许宁,郑学清,石爱平,等.肾癌细胞c-myc的表达分布及其临床意义[J].临床泌尿外科杂志,1999,14(9)：403-405.
[8]李娟,王沁,严祥.食管癌组织hTERT和c-myc的表达[J].第四军医大学学报,2003,24(18)：1662-1664
[9]Brablelz J, Herrmann K, Jung A, et al. Expression of nuclear beta catenin and C—myc is correlated with tumor size but not with proliferative activity of colorectal adenomas[J]. Am J Pathol,2000,156:865
[10]张小化唐兰谭毅.胆囊癌P53、c—myc mRNA异常表达与TNM分期相关研究,齐鲁肿瘤杂志,1996,3(2)：108-109
[11]崔勇陈玉丙翁公羽唐焕峰.survivin和c—myc在胆囊癌组织中的表达及意义,中国煤炭工业医学杂志,2007,10(8)：888-889
[12]王江窦科峰.胆囊癌组织hPTTG1与c-Myc的表达及意义,第四军医大学学报,2004,25(4)：365-367
[13]刘智敏蒋莉莉阳斌廖殿英.VEGF和C—myc表达在胆囊癌形成、发展和转移中的作用生物医学工程学杂志,2003,20(1)：68-70
[14]Roa I, Araya JC, Shiraishi T, Yatani R, Wistuba I, Villaseca M, de Aretxabala X. Gallbladder carcinoma:expression of the c-myc and ras-p-21 oncogene products. Rev Med Chil.1994;122[7]:754-9.
[15]刘颖斌何小伟王建伟等。胆囊癌肝转移模型建立及高转移亚群细胞的筛选中华医学杂志2006,86(30)；2117-2121.
[16]ElbashirS M, HarborthJ, LendeckelW, et al. Duplexes of21-nucleotide RNA smediateRNA interference in cultured mainmalian cells. Nature,2001,411(6836): 494-498
[17]Brummelkamp T R, Bernards R, Agami R. A system for stable expression of short inmrfedng RNAs in mammalian cells. Science,2002.296:550-553
[18]Yu J Y, DeRuiter S L, TuFiler D L. RNA interference by expression of short-interfering RNAs and hairpin RNAS inmammalian cells. Proc Natl Acad Sci USA.2002,99:6047-6052
[19]SuiGC, SoohooC, AfarEIB. ADNAvector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA,2002,99: 5515-5520
[20]Rowley P T. RNA interference. N Engl Jmed,2003,348(6):564
[21]Pickford A S, Cogoni C. RNA mediated gene silencing. Cell Mol Life Sci,2003, 60(5):871-882
[22]岳枫,马文丽,宋艳斌,石嵘等,RNAi技术沉默K562细胞中c-myc基因的初步研究,第一军医大学学报,2005；25(6),647-650
[23]黄明德,陶敏,李振江,岑建农等,小干扰RNA抑制c—Myc表达对MCF—7细胞增殖和凋亡的影响,苏州大学学报(医学版)2006；26(6),936-938
]24]王林,潘晓涛,耿智敏,RNA干扰Bcl—2基因表达对人胆囊癌细胞株GBC—SD裸鼠移植瘤生长的影响,西安交通大学学报(医学版),2009,30(1),93-96
[25]Dangi-Garimella S, Yun J, Eves EM, Newman M, Erkeland SJ, Hammond SM, Minn AJ, Rosner MR.Raf kinase inhibitory protein suppresses a metastasis signalling cascade involving LIN28 and let-7.EMBO J.2009;28[4]:347-58.
[26]Takamizawa J, Konishi H, Yanagisawa K, et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival[J]. Cancer Res,2004,64(11):3753-3756.
[27]Bussing I, Slack FJ, Grosshans HL, et al. Let-7 microRNAs in development, stem cells and cancer[J]. Mel Med,2008,14(9):400-409.
[28]Piskounova E, Viswanathan SR, Janas M, et al. Determinants of microRNA processing inhibition by the developmentally regu- lated RNA—binding protein Lin28[J]. J Biol Chem,2008,283(31):21310_21314.
[29]Klemke M, Meyer A, Hashemi Nezhad M, Belge G, Bartnitzke S, Bullerdiek J. Loss of let-7 binding sites resulting from truncations of the 3' untranslated region of HMGA2 mRNA in uterine leiomyomas.Cancer Genet Cytogenet.2010 Jan 15;196[2]:119-23.
[30]Rahman MM, Qian ZR, Wang EL, Sultana R, Kudo E, Nakasono M, Hayashi T, Kakiuchi S, Sano T. Frequent overexpression of HMGA1 and 2 in gastroenteropancreatic neuroendocrine tumours and its relationship to let-7 downregulation.Br J Cancer.2009;100[3]:501-10.
[31]He XY, Chen JX, Zhang Z, Li CL, Peng QL, Peng HM. The let-7a microRNA protects from growth of lung carcinoma by suppression of k-Ras and c-Myc in nude mice.[J],J Cancer Res Clin Oncol,2010,136:1023-1028.
[1]Brablelz J, Herrmann K, Jung A, et al. Expression of nuclear beta catenin and C—myc is correlated with tumor size but not with proliferative activity of colorectal adenomas[J]. Am J Pathol,2000,156:865
[2]Sauve S,Tremblay L, Lavigne P.The NMR solution structure of a mutant of the Max b/ HL H/ LZf ree of DNA:insights into the specific and reversible DNA binding mechanism of dimerict ranscription factors [J]. J Mol Biol,2004,342 (3): 813-832.
[3]Evan GI et al. Cell,1992,69:119
[4]Prall OW, Rogan EM,Musgrove EA,et al.Mol Cel 1 Biol,1998,18 (8):4499.
[5]Yuman Fong, Lawrence Wagman, Mithat Gonen, et al.Evidence-Based gallbladder cancer staging changing cancer staging by analysis of data from the National Cancer Database.Ann Surg,2006,243:161-774. 6, Ito M, Mishima Y, Sato T. An anatomical study of the lymphatic drainage of the gallbladder [J].Surg Radiol Anat,1991,13 (2):89
[7]Rolf J.Schauer, Guenter Meyer, et al.Prognostic factors and long-term results after surgery for gallbladder carcinoma:a retrospective study of 127 patients.Langenbeck's Arch Surg,2001,386:110-117.
[8]Palat Balachandran, Shaleen Agarwal, Narendra Krishnani, et al.Predictors of long-term survival in patients with gallbladder cancer.J Gastroiniest Surg,2006, 10:848-854.
[9]Xabier de Aretxabala, Ivan Roa, Luis Burgos, et al.Gallbladder cancer:an analysis of a series of 139 patients with invasion restricted to the subserosal layer.J Gastroiniest Surg,2006,10:186-192.
[10]Itaru Endo, Hiroshi Shimada, Mikiko Tanabe, et al.Prognostic significance of the number of positive lymph nodes in gallbladder cancer. J Gastrointest Surg,2006, 10:999-1007.
[11]建石,郑建勇.胆囊癌394例回顾性分析,第四军医大学学报,2002；23(增刊)：8-9.
[12]Hatakeyama S, Watanabe M, Fujii Y, et al. Targeted Destruction of c-Myc by an Engineered Ubiquitin Ligase Suppresses Cell Transformation and Tumor Formation[J].CancerRes,2005,65(17):7874-7879.
[13]Hemann MT,Bric A, Teruya2Feldstein J, et al. Evasion of the p53 tumour surveillance network by tumour2derived MYC mutant s[J]. Nature,2005,436 (7052):787-789.
[14]张小化唐兰谭毅.胆囊癌P53、c—myc mRNA异常表达与TNM分期相关研究,齐鲁肿瘤杂志,1996,3(2)：108-109
[15]陈澎周陈玉泉等.胆管良恶性病变组织内P62 C-myc表达的临床意义,肝胆胰外科杂志,1997,9(1)：19-20
[16]崔勇陈玉丙翁公羽唐焕峰.survivin和c—myc在胆囊癌组织中的表达及意义,中国煤炭工业医学杂志,2007,10(8)：888-889
[17]王江窦科峰.胆囊癌组织hPTTGl与c-Myc的表达及意义,第四军医大学学报,2004,25(4)：365-367
[18]刘智敏蒋莉莉阳斌廖殿英.VEGF和C—myc表达在胆囊癌形成、发展和转移中的作用生物医学工程学杂志,2003,20(1)：68-70
[19]靳斌王可新胡三元.胆囊癌hTERT、c—myc的表达及相关性的研究,中华肝胆外科杂志,2006,12(8)：568-569
[20]Roa I, Araya JC, Shiraishi T, Yatani R, Wistuba I, Villaseca M, de Aretxabala X. Gallbladder carcinoma:expression of the c-myc and ras-p-21 oncogene products. Rev Med Chil.1994;122[7]:754-9.