摘要
膀胱癌是泌尿系统最常见的恶性肿瘤。Ezrin被研究发现同许多恶性肿瘤的发生、发展、浸润、转移有关。但是ezrin同膀胱移行细胞的作用不清楚,并且未见有用RNAi技术研究ezrin同膀胱移行细胞癌关系的研究。本实验我们首次使用siRNA抑制膀胱移行细胞癌细胞株T24中ezrin基因的表达,研究ezrin在膀胱癌发生、发展、侵袭中的作用.本实验同时探讨了RhoA与ezrin在膀胱癌中的关系。为探讨膀胱癌基因治疗新途径提供理论基础。
第一部分膀胱移行细胞癌组织中ezrin、CD44的表达
目的探讨ezrin、CD44在膀胱移行细胞癌中的表达与意义
方法1、采用免疫组织化学检测60例膀胱移行细胞癌与14例正常膀胱组织中ezrin、CD44的蛋白表达。2、采用RT-PCR方法检测40例膀胱移行细胞癌与10例正常膀胱组织中ezrin、CD44的mRNA表达.
结果1、ezrin、CD44在膀胱移行细胞癌组织中的蛋白表达明显高于正常膀胱组织(P<0.05), ezrin、CD44的蛋白表达与膀胱移行细胞癌组织的病理分级、临床分期密切相关,ezrin、CD44蛋白表达的阳性表达随着膀胱肿瘤病理分级、临床分期的增高而增高(P<0.05)。2、ezrin、CD44在膀胱移行细胞癌组织中的mRNA表达明显高于正常膀胱组织(P<0.05), ezrin、CD44的mRNA表达与膀胱移行细胞癌组织的病理分级、临床分期密切相关,ezrin、CD44mRNA的阳性表达随着膀胱肿瘤病理分级、临床分期的增高而增高(P<0.05)。3、ezrin、CD44的蛋白表达在膀胱移行细胞癌中有正相关性。
结论ezrin、CD44蛋白均在BTCC中表达异常增强,在BTCC发生、发展过程中起重要作用。ezrin可作为诊断、判断预后、指导治疗、随访检测的指标。ezrin基因可能成为BTCC新的治疗靶点。
第二部分应用RNAi沉默ezrin基因对膀胱移行细胞癌株生物学行为的影响
目的探讨ezrin基因表达与膀胱移行细胞癌株生物学行为的关系。
方法1、设计siRNA ezrin,用于干扰并下调T24细胞中目的基因的表达。2、采用RT-PCR以及western-blot方法检测实施siRNA干扰后,T24细胞中ezrin的mRNA以及蛋白量。3、采用绘制细胞生长曲线、MTT实验、流式细胞检测、细胞划痕实验以及细胞迁徙实验、细胞粘附实验,观察siRNA干扰后,细胞生物学行为变化。
结果1、设计的siRNA能够很好的对目的基因产生沉默效应。2、转染siRNA后的实验组T24细胞,ezrin的mRNA表达量较对照组明显下调,差异有统计学意义(P<0.05)。ezrin蛋白表达量显著下调,差异有统计学意义。3、转染siRNA后的实验组T24细胞,细胞生长速度较对照组明显减慢,差异有统计学意义(P<0.05)；实验组T24细胞凋亡率为(30.37±8.37)%,实验组细胞凋亡率同空白组、阴性对照组比较,凋亡率明显增加,(p<0.05)。24小时后,实验组T24细胞迁移到划痕区的细胞明显低于空白组和阴性对照组。实验组发生迁移细胞数目明显降低,迁移能力明显下降,与对照组比较差异有显著性(P<0.05)。siRNA干扰可以降低细胞的粘附能力,空白组、阴性对照组、实验组细胞粘附率分别为72.3±5.1%,70.5±5.7%以及39.2±4.6%,差异有统计学意义(p<0.05)
结论siRNA可以明显下调T24细胞中ezrin的mRNA以及蛋白表达量,且导致细胞生物学行为的改变,表现为细胞的生长减慢、凋亡增加,细胞的迁移能力及粘附能力降低。
第三部分ezrin同RhoA信号传导通路关系的研究
目的探讨RhoA及ezrin在膀胱癌中的关系。
方法1)EGF作用于膀胱移行细胞癌T24细胞,采用Western-blot检测ezrin、RhoA、磷酸化RhoA的蛋白含量变化。2)将EGF及RhoA激酶抑制剂Y-27632作用于膀胱移行细胞癌T24细胞,检测ezrin、磷酸化RhoA的蛋白含量变化。
结果Western-blot结果显示,在给予EGF刺激后,p-RhoA水平逐渐升高,于10、30、60、90min时分别升高了70±11%,250±23%,420±47%以及490±43%。在EGF刺激下RhoA蛋白表达无明显变化。Western-blot结果显示,在给予EGF刺激后,ezrin水平逐渐升高,于8、16、24小时分别升高了170±60%,310±73%,750±47%。用Y-27632预处理T24细胞,EGF诱导的RhoA磷酸化水平明显受到抑制,同时ezrin蛋白表达水平也受到抑制。
结论RhoA可能作为ezrin的上游调控元件。
Bladder cancer was the most common cancer in urology.Ezrin has been thought that it may play an important role in genesis、development、invasion metastasis of variety tumor. What kind of role ezrin playing was not yet clear in development of BTCC..Experimental study on siRNA of ezrin transfecting BTCC cell lines was not yet reported at home and abroad. The first time we used siRNA to inhibit BTCC cell line T24 to reduced the ezrin gene expression,to study fuction of ezrin in BTCC genesis、development、invasion.The relationship of RhoA and ezrin in BTCC also was studied in this study. Providing foundation of theory which can explore a new way of gene therapy in BTCC.
PartⅠThe expression of ezrin and CD44 in bladder transitional cell carcinoma
Objective
To explore the expression of ezrin and CD44 in bladder transitional cell carcinoma.
Methods
Immunohistochemical method was used to assess the expression of ezrin and CD44 in 60 cases of bladder transitional cell carcinoma and 14 cases of control. RT-PCR was used to determine the mRNA of ezrin and CD44 in 40 cases of bladder transitional cell carcinoma of and 10 cases of control.
Results
The protein expression of ezrin and CD44 were significantly higher in BTCC than that of the controls (P<0.05). The protein expression of ezrin and CD44 were positively correlated with the histological grade and clinical stage of the BTCC(P<0.05). The mRNA expression of ezrin and CD44 were significantly higher in BTCC than that of controls. The mRNA level of ezrin and CD44 were positively correlated with the histological grade and clinical stage of the BTCC(P<0.05).
Conclusion
Both expression of ezrin and CD44 were higher in BTCC. The ezrin and CD44 play important role in the development of BTCC. The ezrin can serve as indicators of diagnosis,prognosis and guide treatment and the new target of the BTCC therapy.
PartⅡsiRNA on ezrin effect on biological behavior of bladder carcinoma cell
Objective
To explore the relationship between the ezrin gene expression in BTCC and the biological behavior of strain.
Methods
The siRNA ezrin probe was designed to interfere and reduce the ezrin gene expression in T24 cell. The RT-PCR and western-blot were used to determined the mRNA and protein of ezrin in the cells, which were treated by siRNA ezrin. Cell growth curve,MTT assay,The flow cytometry,cell scratch experiments and cell migration experiments cell adhere experiments were used to test the biological behavior of cells, which were treated by siRNA ezrin.
Results
The siRNA silencing effect was observed by the treatment of siRNA ezrin. After the siRNA transfection, the mRNA expression of ezrin in experimental group was significantly lower than that of control (P<0.05). The ezrin protein expression was significantly reduced by the transfection (P<0.05).The growth rate of T24 cells, which was treated by siRNA was much slower than the control group, the difference was statistically significant. The apoptosis rate of experimental group was 30.37±8.37%, which was signicantly increased than those of blank control and negative control(P<0.05). The migrating cells was significantly lower than the control group and negative control group. The number of migrating cells in the experimental group was signicantly smaller than those of control (P<0.05). The treatment of siRNA can reduce the cell adhesion capacity.The adhesion rate of the blank control and negative control and experiment group were 72.3±5.1%,70.5±5.7% and 39.2±4.6% respectively. The difference was statistically different (P<0.05).
Conclusion
The siRNA can signicantly decreased the mRNA and protein expression of ezrin in T24 cells and cause the different behavioral. The decreased growth rate, adhesion and migrating capacity and increased apoptosis can be observed after the siRNA treatment.
PartⅢThe study of the relationship of ezrin and RhoA in BTCC
Objective
To explore the the relationship of ezrin and RhoA in BTCC
Method
Western-blot was used to determine the protein expression of Ezrin and RhoA and p-RhoA after the treatment of EGF. The protein expression of ezrin and p-RhoA after the treamtment of EGF and Y-27632.
Results
The p-RhoA protein expression was increased by 70±11%, 250±23%,420±47% and 490±43%, after the treatment of EGF for 10、30、60、90mins. No difference of the RhoA protein expression was observed after the treatment of EGF. The ezrin protein expression was significantly increased by 170±60%,310±73%,750±47% after the treatment of EGF for 8、16、24 hours. The Y-27632 treatment can decreased the effect of EGF on the expression of p-RhoA and ezrin.
Conclusion
RhoA may regulatd ezrin as a upstream regulatory signal.
引文
[1]吴阶平主编。吴阶平泌尿外科学(上册)。山东科技出版社,2004
[2]Cote RJ. Datar RH. Therapeutic approaches to bladder cancer:identilying targets and mechanisms. Crit Rev Oncol Hematol.2003 Jun 27; 46 Suppl:S67-83。
[3]Irie A.Advances in gene therapy for bladder cancer.Curr Gene Ther,2003,3(1):1-11.
[4]Valdman A,Fang X,Pang ST, et al. Ezrin expression in prostate cancer and benign prostatic tissue. Eur Urol,2005,48 (5):852-857
[5]Bal N, Yildirim S, Nursal TZ,et al. Association of ezrin expression in intestinal and diffuse gastric carcinoma with clinicopathological parameters and tumor type. World J Gastroenterol.2007;13(27):3726-3729.
[6]Khanna C,Wan X,Bose S,et al:The membrane-cytoskeleton linker ezrin isnecessary for osteosarcoma metastasis.Nat Med.2004,10(2):182-6。
[7]Yu Y,Khan J,Khanna C,et al.Expression profiling identifies the cytoskeletalorganizer ezrin and the developmental homeoprotein Six-1 as key metastatic regulators.Nat Med.2004,10(2):175-81
[8]Xie JJ Xu LY, Xie YM,et al. Roles of ezrin in the growth and invasiveness of esophageal squamous carcinoma cells. Int J Cancer.2009,124(11):2549-2558.
[9]Wagener N, Holland D, Bulkescher J,et al. The enhancer of zeste homolog2gene contributes to cell proliferation and apoptosis resistance in renal cell carcinoma cells. Int J Cancer.2008,123(7):1545-1550.
[10]Martins VL, Vyas JJ, Chen M,et al. Increased invasive behaviour in cutaneous squamous cell carcinoma with loss of basement-membrane type VII collagen. J Cell Sci.2009,122(Pt 11):1788-1799.
[11]kivela T,Jaskelainen J,Valeri A,et al.Ezrin,a membrane-organizing protein,as a polarization marker of the retinal pigment epithelium in vertebrates.Cell Tissue Res,2003,301(2):217-223.
[12]Bretscher A.Regulation of corrtical structure by the ezrin-radixin-moesin protein family.Curr Opin Cell Biol.1999,11(1):109-112.
[13]Ohtani K,Sakamoto H,Rutherford,et al.Ezrin,a membrance-cytoskeletal linking protein,is highly expressed in atypical endometrial hyperplasia and uterine endometrioid adenocarcinoma. Cancer Lett.2002,179(1):79-86.
[14]Kobel M,Gradhand E,Zeng K, et al. Ezrin promotes ovarian carcinoma cell invasion and its retained expression predicts poor prognosis in ovarian carcinoma. Int J Gynecol Pathol,2006,25 (2):121-130.
[15]Shen ZY,Xu L Y, Chen MH,et al. Upregulated expression of Ezrin and invasive phenotype in malignantly transformed esophageal epithlial cells. World J Gastroenterol,2003,9(6):1182-1186.
[16]Yeh TS.Significance of cellular distribution of Ezrin in pancreatic cystic neoplasms and ductal adenocarcinoma.Arch Surg,2005,140(12):1184-1190.
[17]Katagiri YU, Sleeman J, Fujii H, et al. CD44 variants but not CD44s cooperate with beta 1-containing integrins to permit cells to bind to os2 teopontin independently of arginine-glycine-aspartic acid, thereby stimulating cell motility and chemotaxis. Cancer Res,1999,59(1):219-226.
[18]Kan M, FurukawaA, AkiM, et al. Expression of CD44 splice variants in bladder cancer. Int J Urol,1995,2:295-297.
[19]Chen JQ, Zhan WH, He YL, et al. Expression of heparanase gene, CD44v6,MMP27 and nm23 protein and their relationship with the invasion and metastasis of gastric carcinomas. World J Gastroenterol,2004,10 (56):776-782.
[20]Martin TA, Harrison G,Mansel RE,et al. The role of theCD44/ezrin complex in cancer metastasis. Crit Rev Onco Hematol,2003,46 (2):165-186
[21]Hefler L A, Concin N, Mincham D, et al. The prognostic value of immunohistochemically detected CD44-V3 and CD44-V6 expression in patients with surgically staged vulvar carcinoma:a multicenter study Cancer,2002,94 (1): 125-130.
[22]Harrison GM,Davies G,Martin TA, et al. Distribution and expression of CD44 isoforms and Ezrin during prostate cancer-endothelium interaction. Int J Oncol 2002;21(5):935-940.
[23]Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature,1998,391 (6669):806-811
[24]Matzke M,Matzke AJ.Plants,RNAi,and the Nobel Prize. Science, 2006,314 (5803):1242-1243
[25]Zamore PD.Ancient pathways programmed by smallRNAs. Science,2002, 296:1265-1269.
[26]KnudsenH, Nielsen PE. Antisense properties of duplex-and triplexforming PNAs. Nucleic Acids Res,1996,24 (3):495-500
[27]Thompson JD. Applications of antisense and siRNAs during preclinical drug development. Drug Discovery Today,2002,7(17):912-917
[28]Sabine B. Antisense-RNA regulation and RNA interference.Bioc hemicaet.Biophysica Acta,2002,1575(1-3):15-25.
[29]Elbashir S M, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAsmediate RNA interference in cultured mammalian cells. Nature,2001; 411(6836):494-498.
[30]Micura R.Small interfering RNAs and their chemical synthesis.Angew Chem Int Ed Engl,2002,41(13):2265-2269.
[31]Brunmelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells.Science,2002,296(5567): 550-553.
[32]Sui GC. Soohoo Ce Afar EB. et al.A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.PNAS,2002; 99(6):5515-5520
[33]Castanotto D, Li H, Rossi JJ. Functional siRNA expression from transfected PCR products.RNA,2002,8(11):1454-1460
[34]Corey DR,Telomerase inhibition,oligonucleotides,and clinical trial. Oncogene,2002;21:631-637。
[35]Zhang Y,Hu MY,Chen BH,et al.The effects of ezrin on the proliferation and the invasiveness of cells of different hepatocellular carcinoma cell lines.Zhonghua Gan Zang Bing Za Zhi,2006,14(7):489-494.
[36]SioudM.TheraPeuticsiRNAs.TrendsPhannaeolSei,2004,25(1):22-28.
[37]Carey M,Smale ST.Transcription regulation in Eukaryotes:Concepts, Strategies and Techniques.New York,Cold Spring Harbor Laboratory Press.2000.
[38]Nogawa M,Yuasa T,Kimura S,et al.Intravesical administration of small interfering RNA targeting PLK-1 successfully prevenes the growth of bladder cancer. J clin Invest,2005,115(4):978-985.
[39]wu F,Chen Y,Li Y,et al.RNA interference-mediaed Cdc42 silenceing downregulates phosphorylation of STAT3 and suppresses growth in human bladder cancer cells.Biotechol Appl Biochem,2008,49(pt 2):121-128.
[40]Chung LW, Baseman A, Ass ikis V,et al.Moleeular insights into Prostate cancer Progression:the missing link of tumor microenvironment. J Urol,2005, 173(6Ptl):10-20.
[41]Pujuguet P,Del M,Cautreau A,et al.Ezrin regulates E-cadherin-dependent adherens junction assembly through Racl activation.Mol Biol Cell,2003; 14:2181-2191.
[42]Crepaldi T,Gautreau A,Comoglio PM,et al.Ezrin is an effortor of hepatoctye growth factor-mediated migration and morphogenesis in epithelial cells.J Cell Biol,1997;138:423-434.
[43]kisawa N, Nishimori I, Iwamuira T, et al. High levels of ezrin expressed byhuman pancreatic adenocarcinoma cell line with metastasis potential. Biochem Biophys Res Commun,1999,258(2):395-400.
[44]Wan X, Mendoza A, Khanna C, et al. Rapamycin inhibits ezrin-mediated metastasis behavior in a murine model of osteosarcoma. Cancer Res,2005,65(6): 2406-2411
[45]Sahai E,Marshall CJ.RHO-GTPases and cancer.Nat Rev Cancer,2002; 2(2):133-142
[46]Jian M,Jie Z,songqing F,et al.Role of a novel EGF-like domain-containing gene NGX6 in cell adhesion modulation in nasopharyngeal carcinoma cell.Carcinogenesis,2005,26:(2):281-291.
[47]Fukata M, Nakagawa M, Kaibuchi K. Roles of Rho-family GTPases in cell polarisation and directional migration. Curr Opin Cell Biol,2003,15(5):590-597.
[48]Ridley AJ. Rho GTPases and cell migration. J Cell Sci,2001,114(Pt 15): 2713-2722.
[49]Han S, Sidell N, Roman J. Fibronectin stimulates human lung carcinoma cell proliferation by suppressing p21 gene expression via signals involving Erk and Rho kinase. Cancer Lett,2005,219(1):71-81.
[50]Gadea G, de Toledo M, Anguille C, et al.Loss of p53 promotes RhoA-ROCK-dependent cell migration and invasion in 3D matrices J Cell Biol, 2007,178 (1):23-30.
[1]Wiold D,Michal QAleksander F.Protein 4.1,a component of the erythrocyte membrane skeleton and its related homologue proteins forming the protein 4.1/FERN superfamily.Folia Histochem Cytobiol,2006 44(4):231-248.
[2]Krieg J,Hunter T. Identification of the two major epidermal growth factor-induced tyrosine phosphorylation sites in the microvillar core protein ezrin. J Biol Chem,_1992;267(27):19258-19265.
[3]Fievet BT,Gautreau A,Roy C et al.Phosphoinositide binding and phosphorylation act sequentially in the activation mechanism of ezrin. J Cell Biol, 2004;164(5):653-659.
[4]Martin TA, Harrison G Mansel RE et al. The role of the CD44/ezrin complex in cancer metastasis._Crit Rev Oncol Hematol.2003,46(2):165-186
[5]Hiscox S, Jiang WQet al. Ezrin regulates cell-cell and cell-matrix adhesion, a possible role with E-cadherin/beta-catenin._J Cell Sci.1999;112 Pt 18:3081-3090
[6]Vaheri A,Carpen O Heiska Let al. The ezrin protein family: membrane-cytoskeleton interactions and disease associations._Curr Opin Cell Biol.1997;9(5):659-666.
[7]Ingraffea J, Reczek D, Bretscher A et al. Distinct cell type-specific expression of scaffolding proteins EBP50 and E3KARP:EBP50 is generally expressed with ezrin in specific epithelia, whereas E3KARP is not.Eur J Cell Biol.2002;81(2):61-68.
[8]Tran YK, Bogler O, Gorse KM et al. A novel member of the NF2/ERM/4.1 superfamily with growth suppressing properties in lung cancer..__Cancer Res.1999;59(1):35-43.
[9]Lugini L Lozupone F, Matarrese P et al. Potent phagocytic activity discriminates metastatic and primary human malignant melanomas:a key role of ezrin. Lab Invest.2003;83(11):1555-1567.
[10]Kishore R, Qin G, Luedemann C,et al. The cytoskeletal protein ezrin regulates EC proliferation and angiogenesis via TNF-alpha-induced transcriptional repression of cyclin A.J Clin Invest.2005;115(7):1785-1796
[11]Thiery JP. Epithelial-mesenchymal transitions in tumor progression.Nat Rev Cancer.2002;2(6):442-454.
[12]Moilanen J, Lassus H, Leminen A et al. Ezrin immunoreactivity in relation to survival in serous ovarian carcinoma patients.___Gynecol Oncol. 2003,90(2):273-281.
[13]Sarrio D, Rodriguez2Pinilla SM, Dotor A, et al. Abnormal ezrinlocalization is associated with clinicopathological features in inva2sive breast carcinomas. Breast Cancer Res Treat,2006,98:71-79.
[14]Yu Y, Khan J, Khanna C, et al. Exp ression p rofiling identifiesthe cytoskeletal organizer ezrin and the developmental homeop rotein Six-1 as keymetastasic regulators. Nat Med,2004,10:175-181.
[15]Elliott BE, Meens JA, SenGup ta SK, et al. The membrane cytoskeletal crosslinker ezrin is required formetastasis of breast carcinoma cells. Breast Cancer Res,2005,7:365-373.
[16]Stapleton G,Malliri A,Ozanne BW.Downregulated AP-1 activity is as-sociatedwith inhibition of Protein-Kinase-C-dependent CD44and ezrinlocalisation and upregulation of PKC theta in A431 cells.Cell Sci,2002,115(13):2713-2724.
[17]Harrison GM,Davies G,Martin TA, et al. Distribution and expression of CD44 isoforms and Ezrin during prostate cancer-endothelium interaction. Int J Oncol 2002;21(5):935-940.
[18]Pujuguet P, DelMaestro L, Gautreau A, et al. Ezrin regulates E-cadherin-dependent adherens junction assembly through Racl activation. Mol Biol Cell, 2003,14 (5):2181-2191.
[19]Khanna C, Wan X, Bose S, et al. The membrane cytoskeleton linker ezrin is necessary for osteosarcoma metastasis. NatMed,2004,10 (2):182-186.
[20]Lozupone F, Lugini L, Matarrese P,et al. Identification and relevance of the CD95-binding domain in the N-terminal region of ezrin..__J Biol Chem. 2004;279(10):9199-9207.
[21]Luciani F, Matarrese P, Giammarioli AM, et al. CD95/phosphorylated ezrin association underlies HIV-1 GP120/IL-2-induced susceptibility to CD95(APO-1/Fas)-mediated apoptosis of human resting CD4(+)T lymphocytes._Cell Death Diffe.2004;11(5):574-582.
[22]Clark EA, Golub TR, Lander ES, et al. Genomic analysis of metastasis reveals an essential role for RhoC. Nature,2000,406 (6795):532-535.
[23]Fiorentini C, Falzano L, FabbriA, et al. Activation of Rho GT2 Pases by cytotoxic necrotizing factor 1 induces macrop ino cytosis and scavenging activity in ep ithelial cells. Mol Biol Cell,2001,12 (7):2061-2073.
[24]Xie JJ, Xu LY, Xie YM,et al. Roles of ezrin in the growth and invasiveness of esophageal squamous carcinoma cells. Int J Cancer 2009;124(11):2549-2558.
[25]Okamura D, Ohtsuka M, Kimura F,et al. Ezrin expression is associated with hepatocellular carcinoma possibly derived from progenitor cells and early recurrence after surgical resection. Mod Pathol.2008;21(7):847-855.
[26]Bal N, Yildirim S, Nursal TZ,et al. Association of ezrin expression in intestinal and diffuse gastric carcinoma with clinicopathological parameters and tumor type. World J Gastroenterol.2007;13(27):3726-3729.
[27]TokunouM,Niki T,Saitoch Y, et al. Altered expression of the ERM proteins in lung adenocarcinoma. Lab Invest,2000,80 (11):1643-1650.
[28]Deng X, Tannehill-Gregg SH, Nadella MV,et al. Parathyroid hormone-related protein and ezrin are up-regulated in human lung cancer bone metastases. Clin Exp Metastasis.2007;24(2):107-119.
[29]Kobel M,Gradhand E,Zeng K, et al. Ezrin promotes ovarian carcinoma cell invasion and its retained expression predicts poor prognosis in ovarian carcinoma. Int J Gynecol Pathol,2006,25 (2):121-130.
[30]Kobel M, Langhammer T, Huttelmaier S, et al. Ezrin expression is related to poor prognosis in FIGO stage I endometrioid carcinomas. Mod Pathol.2006; 19(4):581-587.
[31]马力,张祥宏,邢玲霄等。Ezrin与乳腺浸润性导管癌发生发展及预后的关系。中华肿瘤杂志。2008；30(4)：279-283.
[32]Valdman A,Fang X,Pang ST, et al. Ezrin expression in prostate cancer and benign prostatic tissue. Eur Urol,2005,48 (5):852-857
[33]Madan R, Brandwein-Gensler M, Schlecht NF,et al. Differential tissue and subcellular expressionof ERM proteins in normal and malignant tissues:cytoplasmic ezrin expression has prognostic signficance for.head and neck squamous cell carcinoma. Head Neck(?)2006;28(11):1018-1027.
[1]Guo S and Kemphues KJ. par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/thr kinase that is asymmetrically distributed. Cell,1995,81 (4):611-620.
[2]Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature,1998,391 (6669): 806-811
[3]Dykxhoorn DM, Novi CD, Sharp PA. Killing t he messenger:short RNAs t hat silence gene expression. Nat Rev Mol Cell Biol,2003,4 (6):457-467.
[4]Harper SQ, Davidson BL. Plasmid-based RNA interference:construction of small-hairpin RNA expression vectors Methods Mol Biol,2005,309:219-235.
[5]Sabine B. Antisense-RNA regulation and RNA interference.Bioc hemicaet. Biophysica Acta,2002,1575(1-3):15-25.
[6]Clemens JC, Worby CA, Simonson LeffN, et al. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc Natl Acad Sci USA,2000,97 (12):6499-6503.
[7]Elbashir S M, Harborth J, Lendeckel W, et al. Duplexes of 21 - nucleotide RNAsmediate RNA interference in cultured mammalian cells. Nature,2001; 411(6836):494-498.
[8]Micura R.Small interfering RNAs and their chemical synthesis.Angew Chem Int Ed Engl,2002,41(13):2265-2269.
[9]Brunmelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells.Science,2002,296(5567): 550-553.
[10]Sui GC. Soohoo Ce Afar EB. et al.A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.PNAS,2002; 99(6):5515-5520
[11]Castanotto D, Li H, Rossi JJ. Functional siRNA expression from transfected PCR products.RNA,2002,8(11):1454-1460
[12]Kin SK. Functional genomics the worm scores a knockoat. Curt Biol,2001,11(3):85-87.
[13]Harbath J, Elbashir SM, Becheer K, et al. Identification of essential genesincultured mammaliam cells using small interfering RNAs. J Cell Sci,2001,114 (24):4557-4565.
[14]CarpenterAE, Sabatini DM. Systematic genome-wide screens of gene function. Nature Rev Genet,2004,5 (1):11-22.
[15]Shen C,Buck AK,Liu X, et al. Gene silencing by a denovirus-deliv-ered siRNA. FEBS Lett,2003,539 (13):111-114.
[16]Hammond SM,Bernstein E,Beach D, et al. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells Nature,2000,404 (6775):293-296.
[17]Jacque JM, Triques K, StevensonM. Modulation of HIV-1 replication by RNA interference. Nature,2002,418 (6896):379-380.
[18]Mette MF.Aufsatz W, vander Windern J, et al. Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J,2000, 19(19):5194-5201.
[19]Davidson BL, Paulson HL. Molecular medicine for the brain:silencing of disease genes with RNA interference. Lancet Neurol,2004,3(3):145-149.
[20]Boudreau RL,Davidson BL. RNAi therapy for neurodegenerative diseases. Curr Top Dev Biol,2006,75:73-92.
[21]Xie JJ Xu LY, Xie YM,et al. Roles of ezrin in the growth and invasiveness of esophageal squamous carcinoma cells. Int J Cancer.2009,124(11):2549-2558.
[22]Li J, Piao YF, Jiang Z,et al. Silencing of signal transducer and activator of transcription 3 expression by RNA interference suppresses growth of human hepatocellular carcinoma in tumor-bearing nude mice. World J Gastroenterol. 2009,15(21):2602-2608.
[23]Anders M, Vieth M, Rocken C, et al. Loss of the coxsackie and adenovirus receptor contributes to gastric cancer progression. Br J Cancer.2009,100(2):352-359.
[24]Ohtaka K, Kohya N, Sato K,et al. Ribonucleotide reductase subunit M1 is a possible chemoresistance marker to gemcitabine in biliary tract carcinoma. Oncol Rep. 2008,20(2):279-286.
[25]Bulk E, Sargin B, Krug U, et al. S100A2 induces metastasis in non-small cell lung cancer. Clin Cancer Res.2009,15(1):22-29.
[26]Sun Y, Liu M, Yang B, et al. Inhibition of laryngeal cancer cell invasion and growth with lentiviral-vector delivered short hairpin RNA targeting human MMP-9 gene. Cancer Invest.2008,26(10):984-989.
[27]Lee MW, Kim DS, Min NY,et al. Aktl inhibition by RNA interference sensitizes human non-small cell lung cancer cells to cisplatin. Int J Cancer. 2008,122(10):2380-2384.
[28]Wagener N, Holland D, Bulkescher J,et al. The enhancer of zeste homolog 2 gene contributes to cell proliferation and apoptosis resistance in renal cell carcinoma cells. Int J Cancer.2008,123(7):1545-1550.
[29]Possner M, Heuser M, Kaulfuss S,et al. Functional analysis of NKX3.1 in LNCaP prostate cancer cells by RNA interference. Int J Oncol.2008,32(4):877-884.
[30]Wu F, Chen Y, LI Y, et al.RNA interference-mediated Cdc42 silencing downregulates phosphorylation of STAT3 and suppresses growth in human bladder cancer cells.Biotechnol Appl Biochem,2008,49(Pt2):121-128.
[31]Dong PX, Jia N, Xu ZJ, et al. Silencing of IQGAP1 by shRNA inhibits the invasion of ovarian carcinoma HO-8910PM cells in vitro. J Exp Clin Cancer Res. 2008 Nov 27;27:77.
[32]Sanceau J, Truchet S, Bauvois B,et al. Matrix metalloproteinase-9 silencing by RNA interference triggers the migratory-adhesive switch in Ewing's sarcoma cells. J Biol Chem,2003,278(38):36537-36546.
[33]Martins VL, Vyas JJ, Chen M,et al. Increased invasive behaviour in cutaneous squamous cell carcinoma with loss of basement-membrane type Ⅶ collagen. J Cell Sci.2009,122(Pt 11):1788-1799.