K-ras基因RNA干扰对胰腺癌细胞株增殖凋亡和信号通路的影响
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摘要
背景：胰腺癌是目前已知恶性程度最高的肿瘤之一,它生长迅速,早期症状隐匿,发现时多已发生转移,且对化疗药物敏感性不好,目前唯一证实有效的治疗方法是手术切除。然而,由于发现多为晚期,只有少数病人有手术机会,且即使是术后5年生存率也在12%～20%之间。在西方国家,胰腺癌已列死亡率最高的恶性肿瘤第4位。
胰腺癌的病因目前仍未研究清楚,多种环境因素和多个基因异常被认为在其发生过程中起作用。其中,癌基因的激活和抑癌基因的失活起到了重要作用。K-ras基因是胰腺癌发生发展中最为重要的原癌基因。K-ras蛋白是小分子G蛋白,当它与GTP结合时为活性状态,可进一步激活下游信号通路,调控细胞生长、增殖。正常情况下,Ras蛋白是一类在组织中广泛存在的信号通路中心分子,具有重要生理功能。当Ras蛋白突变时,构型改变,始终处于被激活的“锁定”状态,不需外界生长信号就可以持续激活下游信号分子,导致细胞无节制地异常增殖,最终发生肿瘤。本实验室多年来研究一个与K-ras基因具有同源性,而功能相反的抑癌基因ARHI,研究表明其作用可能通过抑制Ras经典信号通路实现,甚至可以抑制Ras基因的表达。其与Ras相互作用的机制有待进一步研究。
K-ras基因的突变类型多为点突变,尤以第一外显子的第12位密码子点突变最为多见。由于其为胰腺癌发生中的早期事件,又有突变频率高,突变类型固定的特点,临床上已将该位点作为胰腺癌早期诊断的新的分子标志物之一。此外,人们也希望将该位点作为胰腺癌早期治疗的新的靶点。RNA干扰技术的出现,给这种希望赋予了更大的可能。其高度特异性降解目标mRNA的特性,可特异性针对突变型的K-ras基因产生效应,而保留正常K-ras基因的功能。目前已有许多体外实验和动物体内实验进行这方面的尝试。
目的：对K-ras突变型胰腺癌细胞系,特异性敲除K-ras基因,观察对胰腺癌细胞的增殖、凋亡和细胞周期的影响,初步探讨K-ras下游信号通路的改变情况,同时观察ARHI表达的情况。
方法：应用RNA干扰技术,采用脂质体介导的转染小干扰RNA (siRNA)的方法,瞬时转染PANC-1、MiaPaCa-2两株细胞系,通过荧光显微镜和流式细胞仪测定转染率。优化条件后从RNA和蛋白水平验证K-ras基因的转录和表达水平。继而观察K-ras干扰前后胰腺癌细胞增殖、凋亡和细胞周期的变化,以及K-ras相关的下游经典信号通路Ras-Raf-ERK和Ras-PI-3K/AKT中的核心蛋白ERK和AKT的表达情况。以CCK-8细胞活力检测试剂检测细胞增殖；以流式细胞仪PI染色法测定细胞凋亡和细胞周期；以凋亡小体染色和吖啶橙染色观察干扰后细胞凋亡增多的形态学表现；以荧光实时定量PCR验证K-ras表达下调情况下细胞周期相关因子Cyclin A和Cyclin E以及ARHI等的mRNA相对定量情况；以Western Blot检测功能性ERK、功能性AKT和总AKT,以及与AKT调控有关的抑癌基因p53的蛋白水平和ARHI蛋白水平变化。结果：脂质体介导的siRNA转染效率在两个细胞株均可达90%以上。针对K-ras突变类型设计的siRNA可特异性敲除相应的K-ras基因,而不干扰其他突变类型。24h和48h提取的RNA均随siRNA浓度升高产生明显K-ras mRNA下调,siRNA终浓度达100nM时干扰效果最明显。干扰K-ras之后,MiaPaCa-2细胞增殖受到明显抑制,重复测量方差分析p<0.05, PANC-1细胞差异未达显著。两株细胞系的凋亡率均增加,干扰组PANC-1、MiaPaCa-2凋亡细胞的百分率分别为22.6%和32.1%,相对于对照组的9.7%和17.3%有显著性差异(p<0.05)。凋亡形态学观察可以得出相同的结论。细胞周期发生停滞,G1期增加,S期和G2期减少,PANC-1和MiaPaCa-2的G1期比例分别由50.4%、58.7%上升到65.1%和67.4%；S期由32.4%、26.5%下降至21.8%、21.7%；两期的变化有明显差异,p<0.05,而G2期差异不显著；细胞周期相关因子Cyclin A和Cyclin E在mRNA水平有下降趋势,除MiaPaCa-2的cyclin A相对水平降至0.4以外,其余下降幅度均未达50%。p-ERK、p-AKT蛋白表达明显下调,而总AKT无明显变化；p53表达明显上调。蛋白水平变化于96h时更为明显。ARHI蛋白在干扰后仍无表达,荧光实时定量PCR有差异,但因表达量太低,差异不明确。结论：对胰腺癌细胞株瞬时转染特异性siRNA可特异性干扰K-ras突变序列,使MiaPaCa-2细胞增殖受到抑制,两株细胞凋亡增加,细胞周期停滞；可明显下调K-ras下游关键信号蛋白的表达,抑制相关信号转导通路。K-ras与ARHI基因之间的关系仍需进一步研究明确。
BACKGROUND:Pancreatic Cancer is one of the most aggressive human solid tumors, with rapid growth, hidden symptoms and early metastatic, as well as resistance to chemotherapeutic drugs. The only effective therapy known is surgery. While it is difficult to discover this tumor in its early stage, few patients have the chance of surgery resection. The5-year survival rate among patients with pancreatic cancer, even after surgery, is about12-20%. The pancreatic cancer currently rank fourth among cancer-related deaths in western countries.
The causes of pancreatic cancer remain unknown. Several environmental factors and multiple genes abnormally expresstion have been implicated. Activation of oncogenes and inactivation of tumor suppressor genes are believed to be basic moleculer events. K-ras oncogene plays a vital role in pancreatic cancer's growth and maintenance. A K-ras mutation is present in up to80～95%of sporadic pancreatic cancers and is reported to be an early genetic event. K-ras encodes a small molecular GTP/GDP binding protein, when binds to GTP, it is in the active state and activates proteins necessary for the growth and proliferation of cells, and other cell signal factors. The protein product of the normal K-ras gene performs an essential function in normal tissue signaling. While mutated, this transformed protein is trapped in the activated state enables downstream signaling pathways without the activation of growth factors upstreams, which finally leads to unlimited cell proliferation and results in the invasive carcinoma.
Point mutation is the most common format in K-ras mutation, especially in the codon12. As the earliest and most common genetic mutation in pancreas cell transformation and tumor progression, as well as the central importance of ras signaling for cell function and survival, highlight that mutant K-ras is a new attractive molecular marker for the early diagnosis and target for the treatment of pancreatic cancer. The extraordinary sequence specificity of RNA interference (RNAi) offers a hope for cancer therapy that may differentiate the small difference between mutant and normal K-ras genes.
ARHI is a human suppressor gene that belongs to the Ras superfamily. Unlike Ras, It exerts opposite functions against most of its family member, which attracted us in its function in pancreatic cancer. It has been proved to be inactive in most pancreatic cancers. While reexpression, ARHI can inhibits the proliferation of PC cell lines, induces apoptosis, leads to cell cycle arrest and blocks the downstream signaling pathways of Ras. It even reduces the expression of Ras. The interaction between ARHI and Ras remains to be studied.
OBJECTS:Specific knockdown of activated K-ras via RNA interference in pancreatic cancer cell lines, then examine the proliferation, apoptosis and cell cycle and corresponding molecular consequences of K-ras cell signaling pathways. Determine whether the expression of ARHI is increased.
METHODS:Two human pancreatic carcinoma lines with activated K-ras (PANC-1and MiaPaCa-2) of point mutation in codon12were used. K-ras inhibition was accomplished by small interfering RNA (siRNA) knockdown of K-ras expression via transfected using lipofectmine2000. Tranfection effects were examined by fluorescence microscope and flow cytometry. K-ras mRNA and protein expression were analyzed by RT-PCR and western blot respectively. Cell proliferations were determined by CCK-8method. Apoptosis were analyzed by flow cytometry, as well as Hoechst staining and Acridine orange staining in morphology. Real-time PCR was used to analyzing the relative quantity of cyclin A and E,as well as ARHI, and the cell signaling associated proteins as p-ERK, p-AKT, pan-AKT and the tumor suppressor gene p53and ARHI were detected by western blot method.
RESULTS:The transfection effects using lipofectmine2000can be higher than90%in both celss lines. K-ras gene in both cell lines reduced the expression of K-ras mRNA and protein, which were significant24h or48h after transfection. The effect of reduction was strongest when using1OOnM siRNA concentration. The reduction was specific as oligonucleotides with a single bp alteration, while the oligonucleotides specific for the other cell line were ineffective. MiaPaCa-2cells showed significant proliferation after K-ras RNAi, p<0.05, and both PANC-1and MiaPaca-2cells showed increased apoptosis, p<0.05. Both cell lines showed alteration in the cell cycle, a reduction of S phase from32.4%to21.8%in PANC-1,26.5%to21.7%in MiaPaCa-2, and cellular arrest in the G1phase,50.4%to65.1%in PANC-1and58.7%to67.4%in MiaPaCa-2, statistically significant (p<0.05). mRNA levels of cyclin A and E tent to be dereaesed, but the realative quantities were higher than0.5, while the alteration of mRNA level of ARHI remained unclear. The levels of p-ERK, p-AKT protein expression decreased significantly while which of AKT remains. Furthermore, the tumor suppressor gene p53showed a increased level of protein expression, and ARHI protein band was not found after RNAi.
SUMMARY:Specific silencing mutant K-ras in human pancreatic cancer cell lines through RNAi by transient transfection can knockdown the specific mutant K-ras sequences, which results in reduced proliferation in MiaPaCa-2, increased apoptosis in both cell lines and cell cycle arrest. The protein levels of crucial downstream cell signaling associated proteins were reduced thus inhibit the signal transduction. The interaction between ARHI and Ras needs futher research.
胰腺癌的病因目前仍未研究清楚,多种环境因素和多个基因异常被认为在其发生过程中起作用。其中,癌基因的激活和抑癌基因的失活起到了重要作用。K-ras基因是胰腺癌发生发展中最为重要的原癌基因。K-ras蛋白是小分子G蛋白,当它与GTP结合时为活性状态,可进一步激活下游信号通路,调控细胞生长、增殖。正常情况下,Ras蛋白是一类在组织中广泛存在的信号通路中心分子,具有重要生理功能。当Ras蛋白突变时,构型改变,始终处于被激活的“锁定”状态,不需外界生长信号就可以持续激活下游信号分子,导致细胞无节制地异常增殖,最终发生肿瘤。本实验室多年来研究一个与K-ras基因具有同源性,而功能相反的抑癌基因ARHI,研究表明其作用可能通过抑制Ras经典信号通路实现,甚至可以抑制Ras基因的表达。其与Ras相互作用的机制有待进一步研究。
K-ras基因的突变类型多为点突变,尤以第一外显子的第12位密码子点突变最为多见。由于其为胰腺癌发生中的早期事件,又有突变频率高,突变类型固定的特点,临床上已将该位点作为胰腺癌早期诊断的新的分子标志物之一。此外,人们也希望将该位点作为胰腺癌早期治疗的新的靶点。RNA干扰技术的出现,给这种希望赋予了更大的可能。其高度特异性降解目标mRNA的特性,可特异性针对突变型的K-ras基因产生效应,而保留正常K-ras基因的功能。目前已有许多体外实验和动物体内实验进行这方面的尝试。
目的：对K-ras突变型胰腺癌细胞系,特异性敲除K-ras基因,观察对胰腺癌细胞的增殖、凋亡和细胞周期的影响,初步探讨K-ras下游信号通路的改变情况,同时观察ARHI表达的情况。
方法：应用RNA干扰技术,采用脂质体介导的转染小干扰RNA (siRNA)的方法,瞬时转染PANC-1、MiaPaCa-2两株细胞系,通过荧光显微镜和流式细胞仪测定转染率。优化条件后从RNA和蛋白水平验证K-ras基因的转录和表达水平。继而观察K-ras干扰前后胰腺癌细胞增殖、凋亡和细胞周期的变化,以及K-ras相关的下游经典信号通路Ras-Raf-ERK和Ras-PI-3K/AKT中的核心蛋白ERK和AKT的表达情况。以CCK-8细胞活力检测试剂检测细胞增殖；以流式细胞仪PI染色法测定细胞凋亡和细胞周期；以凋亡小体染色和吖啶橙染色观察干扰后细胞凋亡增多的形态学表现；以荧光实时定量PCR验证K-ras表达下调情况下细胞周期相关因子Cyclin A和Cyclin E以及ARHI等的mRNA相对定量情况；以Western Blot检测功能性ERK、功能性AKT和总AKT,以及与AKT调控有关的抑癌基因p53的蛋白水平和ARHI蛋白水平变化。结果：脂质体介导的siRNA转染效率在两个细胞株均可达90%以上。针对K-ras突变类型设计的siRNA可特异性敲除相应的K-ras基因,而不干扰其他突变类型。24h和48h提取的RNA均随siRNA浓度升高产生明显K-ras mRNA下调,siRNA终浓度达100nM时干扰效果最明显。干扰K-ras之后,MiaPaCa-2细胞增殖受到明显抑制,重复测量方差分析p<0.05, PANC-1细胞差异未达显著。两株细胞系的凋亡率均增加,干扰组PANC-1、MiaPaCa-2凋亡细胞的百分率分别为22.6%和32.1%,相对于对照组的9.7%和17.3%有显著性差异(p<0.05)。凋亡形态学观察可以得出相同的结论。细胞周期发生停滞,G1期增加,S期和G2期减少,PANC-1和MiaPaCa-2的G1期比例分别由50.4%、58.7%上升到65.1%和67.4%；S期由32.4%、26.5%下降至21.8%、21.7%；两期的变化有明显差异,p<0.05,而G2期差异不显著；细胞周期相关因子Cyclin A和Cyclin E在mRNA水平有下降趋势,除MiaPaCa-2的cyclin A相对水平降至0.4以外,其余下降幅度均未达50%。p-ERK、p-AKT蛋白表达明显下调,而总AKT无明显变化；p53表达明显上调。蛋白水平变化于96h时更为明显。ARHI蛋白在干扰后仍无表达,荧光实时定量PCR有差异,但因表达量太低,差异不明确。结论：对胰腺癌细胞株瞬时转染特异性siRNA可特异性干扰K-ras突变序列,使MiaPaCa-2细胞增殖受到抑制,两株细胞凋亡增加,细胞周期停滞；可明显下调K-ras下游关键信号蛋白的表达,抑制相关信号转导通路。K-ras与ARHI基因之间的关系仍需进一步研究明确。
BACKGROUND:Pancreatic Cancer is one of the most aggressive human solid tumors, with rapid growth, hidden symptoms and early metastatic, as well as resistance to chemotherapeutic drugs. The only effective therapy known is surgery. While it is difficult to discover this tumor in its early stage, few patients have the chance of surgery resection. The5-year survival rate among patients with pancreatic cancer, even after surgery, is about12-20%. The pancreatic cancer currently rank fourth among cancer-related deaths in western countries.
The causes of pancreatic cancer remain unknown. Several environmental factors and multiple genes abnormally expresstion have been implicated. Activation of oncogenes and inactivation of tumor suppressor genes are believed to be basic moleculer events. K-ras oncogene plays a vital role in pancreatic cancer's growth and maintenance. A K-ras mutation is present in up to80～95%of sporadic pancreatic cancers and is reported to be an early genetic event. K-ras encodes a small molecular GTP/GDP binding protein, when binds to GTP, it is in the active state and activates proteins necessary for the growth and proliferation of cells, and other cell signal factors. The protein product of the normal K-ras gene performs an essential function in normal tissue signaling. While mutated, this transformed protein is trapped in the activated state enables downstream signaling pathways without the activation of growth factors upstreams, which finally leads to unlimited cell proliferation and results in the invasive carcinoma.
Point mutation is the most common format in K-ras mutation, especially in the codon12. As the earliest and most common genetic mutation in pancreas cell transformation and tumor progression, as well as the central importance of ras signaling for cell function and survival, highlight that mutant K-ras is a new attractive molecular marker for the early diagnosis and target for the treatment of pancreatic cancer. The extraordinary sequence specificity of RNA interference (RNAi) offers a hope for cancer therapy that may differentiate the small difference between mutant and normal K-ras genes.
ARHI is a human suppressor gene that belongs to the Ras superfamily. Unlike Ras, It exerts opposite functions against most of its family member, which attracted us in its function in pancreatic cancer. It has been proved to be inactive in most pancreatic cancers. While reexpression, ARHI can inhibits the proliferation of PC cell lines, induces apoptosis, leads to cell cycle arrest and blocks the downstream signaling pathways of Ras. It even reduces the expression of Ras. The interaction between ARHI and Ras remains to be studied.
OBJECTS:Specific knockdown of activated K-ras via RNA interference in pancreatic cancer cell lines, then examine the proliferation, apoptosis and cell cycle and corresponding molecular consequences of K-ras cell signaling pathways. Determine whether the expression of ARHI is increased.
METHODS:Two human pancreatic carcinoma lines with activated K-ras (PANC-1and MiaPaCa-2) of point mutation in codon12were used. K-ras inhibition was accomplished by small interfering RNA (siRNA) knockdown of K-ras expression via transfected using lipofectmine2000. Tranfection effects were examined by fluorescence microscope and flow cytometry. K-ras mRNA and protein expression were analyzed by RT-PCR and western blot respectively. Cell proliferations were determined by CCK-8method. Apoptosis were analyzed by flow cytometry, as well as Hoechst staining and Acridine orange staining in morphology. Real-time PCR was used to analyzing the relative quantity of cyclin A and E,as well as ARHI, and the cell signaling associated proteins as p-ERK, p-AKT, pan-AKT and the tumor suppressor gene p53and ARHI were detected by western blot method.
RESULTS:The transfection effects using lipofectmine2000can be higher than90%in both celss lines. K-ras gene in both cell lines reduced the expression of K-ras mRNA and protein, which were significant24h or48h after transfection. The effect of reduction was strongest when using1OOnM siRNA concentration. The reduction was specific as oligonucleotides with a single bp alteration, while the oligonucleotides specific for the other cell line were ineffective. MiaPaCa-2cells showed significant proliferation after K-ras RNAi, p<0.05, and both PANC-1and MiaPaca-2cells showed increased apoptosis, p<0.05. Both cell lines showed alteration in the cell cycle, a reduction of S phase from32.4%to21.8%in PANC-1,26.5%to21.7%in MiaPaCa-2, and cellular arrest in the G1phase,50.4%to65.1%in PANC-1and58.7%to67.4%in MiaPaCa-2, statistically significant (p<0.05). mRNA levels of cyclin A and E tent to be dereaesed, but the realative quantities were higher than0.5, while the alteration of mRNA level of ARHI remained unclear. The levels of p-ERK, p-AKT protein expression decreased significantly while which of AKT remains. Furthermore, the tumor suppressor gene p53showed a increased level of protein expression, and ARHI protein band was not found after RNAi.
SUMMARY:Specific silencing mutant K-ras in human pancreatic cancer cell lines through RNAi by transient transfection can knockdown the specific mutant K-ras sequences, which results in reduced proliferation in MiaPaCa-2, increased apoptosis in both cell lines and cell cycle arrest. The protein levels of crucial downstream cell signaling associated proteins were reduced thus inhibit the signal transduction. The interaction between ARHI and Ras needs futher research.
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21. Landi S. Genetic predisposition and environmental risk factors to pancreatic cancer:a review of the literature. Mutat Res 2009;681:299-307.
22. Shi C, Hruban RH, Klein AP. Familial pancreatic cancer. Arch Pathol Lab Med 2009; 133:365-74.
23.王文耀.K-ras基因在胰腺癌诊断中的应用(文献综述).国外医学外科学分册.2001,28-2
24. Thomas B. Brunner, Keith A. Cengel, Stephen M. Hahn, Junmin Wu, Douglas L. Fraker, W. Gillies McKenna, and Eric J. Bernhard Pancreatic Cancer Cell Radiation Survival and Prenyltransferase Inhibition:The Role of K-Ras. Cancer Res 2005; 65: (18). September 15,2005
25. Adjei AA. Blocking oncogenic Ras signaling for cancer therapy. J Natl Cancer Inst 2001; 93:1062-1074
26. Thomas GR, Forbes JR. et al, The Wilson disease gene:spectrum of mutations and their consequences, Nat Genet,1995,9:210-217.
27. Semba S, Moriya T, Kimura W, et al. Phosphorylated AKT/PKB controls cell growth and apoptosis in intraductal papillary-mucinous tumor and inva sive ductal adenocarcinoma of the pancreas. Pancreas.2003; 26:250-257.
28. Asano T,Yao Y, Zhu J, Li D, Abbruzzese JL, Reddy SA. The PI 3-kinase/AKT signaling pathway is activated due to aberrant Pten expression and targets transcription factors NF-kappaB and c-Myc in pancreatic cancer cells. Oncogene 2004; 23:8571-8580.
29. Wheeler AP, Ridley AJ. Why three Rho Proteins? RhoA, RhoB, RhoC and cell motility. Exp CeⅡ Res,2004,30(1):43-49.
30. Sotiropoulos A, Gineitis D, Copeland J, Treisman R. Signal-regulated activation of serum response factor is mediated by changesin actin dynamics. Cell.1999; 98:159-169.
31. Fujioka S, Sclabas GM, Schmidt C, et al. Function of nuclear factor kappaB in pancreatic cancer metastasis. Clin Cancer Res.2003; 9:346-354.)
32. Mayo, L.D. and Donner, D.B. A phosphatidylinositol 3-kinase/AKT pathway promotes translocation of MDM2 from the cytoplasm to the nucleus. Proc. Natl. Acad. Sci. USA,2001; 98,11598-11603.
33. Xiong, Y, Hannon, GJ., Zhang, H., Casso, D., Kobayashi, R., and Beach, D. p21WAF1 is a universal inhibitor of cyclin kinases. Nature,1993; 366,701-704.
34. Ashcroft M, Ludwig RL, Woods DB, et al. Phosphorylation of MDM2 by AKT. Oncogene 2002; 21:1955-62.
35. Ogawara Y, Kishishita S, Obata T, et al. AKT enhances MDM2-mediated ubiquitination and degradation of p53. J Biol Chem2002; 277:21843-50.
36. Pan MH, Chen WJ, Lin-Shiau SY, Ho CT and Lin JK. Tangeretin induces cell-cycle G1 arrest through inhibiting cyclin-dependent kinases 2 and 4 activities as well as elevating Cdk inhibitors p21 and p27 in human colorectal carcinoma cells. Carcinogenesis 2002,23; 1677-1684.
37. Zhou B P, Liao Y, Xia, W., Zou, Y, Spohn, B. and Hung, M.C. HER-2/neu induces p53 ubiquitination via AKT-mediated MDM2 phosphorylation. Nat. Cell Biol., 2001; 3,973-982
1. M. Malumbres, M. Barbacid RAS oncogenes:the first, Nat. Rev., Cancer 3 (2003) 459-465.
2. Adjei AA. Blocking oncogenic Ras signaling for cancer therapy. J Natl Cancer Inst 2001; 93:1062-1074
3. Bos JL. ras oncogenes in human cancer:a review. Cancer Res.1989 Sep 1;49(17):4682-9.
4. Thomas GR, Forbes JR, et al, The Wilson disease gene:spectrum of mutations and their consequences, Nat Ge net,1995,9:210-217.
5. Semba S, Moriya T, Kimura W, et al. Phosphorylated AKT/PKB controls cell growth and apoptosis in intraductal papillary-mucinous tumor and inva sive ductal adenocarcinoma of the pancreas. Pancreas.2003; 26:250-257.
6. Wheeler AP, Ridley AJ. Why three Rho Proteins? RhoA, RhoB, RhoC and cell motility. Exp Ce Ⅱ Res,2004,30(1):43-49.
7. Sotiropoulos A, Gineitis D, Copeland J, Treisman R. Signal-regulated activation of serum response factor is mediated by changesin actin dynamics. Cell.1999; 98:159-169.
8. Yu Y, Xu F, Peng H, Fang X, Zhao S, Li Y, Cuevas B, Kuo W L, Gray, J W, Siciliano M, Mills G B, and Bast R C Jr NOEY2 (ARHI), an imprinted putative tumor suppressor gene in ovarian and breast carcinomas. Proc. Natl. Acad. Sci. USA 1999,96,214-219.
9. Bourne H R, Sanders D A, and McCormick F. The GTPase superfamily:Conserved structure and molecular mechanism. Nature.1991,349,117-127.
10. Luo R Z,Peng H Q,Xu F J, Bao J J,Pang Y,Pershad R,Issa J P J,Liao W S L, Bast RC Jr, and Yu Y H. Genomic structure and promoter characterization of an imprinted tumor suppressor gene ARHI. Biochim Biophys Acta.2001,1519,216-222.
11. Fujii S, Luo RZ, Yuan J, Kadota M, Oshimura M, Dent SR, Kondo Y, Issa JP, Bast RC Jr, and Yu Y. Reactivation of the silenced and imprinted alleles of ARHI is associated with increased histone H3 acetylation and decreased histone H3 lysine 9 methylation. Hum Mol Genet.2003,12,1791-1800.
12. Yuan J, Luo RZ, Fujii S, Wang L, Hu W, Andreeff M, Pan Y, Kadota M, Oshimura M, Sahin, AA, Issa JP, Bast RC Jr, and Yu Y. Aberrant methylation and silencing of ARHI,an imprinted tumor suppressor gene in which the function is lost in breast cancers. Cancer Res.2003,63,4174-4180.
13. Lu ZH, Chen J, Gu LJ, Luo YF, and Gu CF. ARHI mRNA and protein expression in pancreatic cancers. Zhong guo Yi Xue Ke Xue Yuan Xue Bao 2001.23,324-327.
14.杨红,陈原稼,徐峰极等.新的候选抑癌基因NOEY2在胰腺肿瘤中的表达.胰腺病学.2002,2(1)：28-30.
15. Bao J J, Le X F, Wang R Y, Yuan J, Wang L, Atkinson E N, LaPushin R, Andreeff M, Fang B, Yu Y, and Bast R C Jr. Reexpression of the tumor suppressor gene ARHI induces apoptosis in ovarian and breast cancer cells through a caspase-independent calpain-dependent pathway. Cancer Res.2002,62,7264-7272.
16. 胡益群等.ARHI基因在胰腺肿瘤中的抑癌作用及其可能细胞内信号传导的机制初探.2005.博士论文
17.路新卿等.ARHI基因对胰腺癌细胞体个增殖、凋亡和侵袭转移的影响及其相关机制研究.2009.博士论文
18.鲁嘉等,ARHI基因在胰腺癌中的抑癌作用及其对EGF-Ras-Raf-MAPK/ERK信号转导通路的影响.博士论文,2007
19. C.J. Der, T.G Krontiris, GM. Cooper, Transforming genes of human bladder and lung carcinoma cell lines are homologous to the ras genes of Harvey and Kirsten sarcoma viruses, Proc. Natl. Acad. Sci. U. S. A.79(1982) 3637-3640.
20. 苏震东.骆明德.K-ras基因突变在胰腺癌中的作用研究进展.期刊论文.国外医学(消化系疾病分册)2005(1)
21. Histopher A, ScoRe E. Molecular genetics of pancreatic carcinoma. In:Reher HA. Pancretie cancer pathogenesis, diagnosis and treatmen.1999:3—15.
22. Torrisani J, Bournet B, Cordelier P, Buscail L. New molecular targets in pancreatic cancer. Bull Cancer.2008 May;95(5):503-12.
23. 王文耀.K-ras基因在胰腺癌诊断中的应用(文献综述).国外医学外科学分册.2001,28-2
24. Yoshida T, Ohnami S, Aoki K. Development of gene therapy to target pancreatic cancer[J]. Cancer Sci,2004,95(4):283—289.
25. Jason B. Fleming, Guo-Liang Shen, Shane E. Holloway, Mishel Davis, and Rolf A. Brekken. Molecular Consequences of Silencing Mutant K-ras in Pancreatic Cancer Cells: Justification for K-ras-Directed Therapy
26. Morioka CY, Costa FP, Lima EMR, Saito S, Watanabe A, Huang CC, Arai H, Sakamoto T. Canantisense oligonucleotides specific to mutated K-ras gene inhibit the tumor growth, invasiveness, and MMP-2 and MMP-9 expression in hamster pancreatic cancer model in vitro and in vivo? EJC suppl 2007; 5:73
27. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002;296:550-553.
28. Caldas C, Kern SE. K-ras mutation and pancreatic adenocarcinoma. Int J Pancreatol 1995;18:1-6.
2. Ferrone CR,Brennan MF,Gonen M,et al:Pancreatic adenocarcinoma:the actual 5-year survivors[J].J Gastrointest Surg 2008,12:701-706,
3. Niederhuber JE, Brennan MF, Menck HR. The national cancer data base report on pancreatic cancer. Cancer,1995,76:1671-1677
4. Jemal A, Siegel 1. R, Ward E, et al. Cancer statistics,2008. CA Cancer J Clin2008;58:71-96.
5. Thijn R. Brummelkamp,Rene'Bernards, and Reuven Agam. Stable suppression of tumorigenicity by virus-mediated RNAinterference
6. Caldas C, Kern SE. K-ras mutation and pancreatic adenocarcinoma. Int J Pancreatol 1995; 18:1-6.
7. Fisher, GH., Wellen, S.L., Klimstra, D., Lenczowski, J.M., Tichelaar, J.W., Lizak, M.J., Whitsett, J.A., Koretsky, A., and Varmus, H.E. (2001). Induction and apoptotic regression of lung adenocarcinomas by regulation of a K-Ras after 3 weeks. transgene in the presence and absence of tumor suppressor genes. Genes Dev.15, 3249-3262.
8. Howe JR, Cordon KC. The molecular genetics of pancreatic cancer[J]. Surg Oncol, 1997,6(1):1-18.
9. Torrisani J, Bournet B, Cordelier P, Buscail L. New molecular targets in pancreatic cancer. Bull Cancer.2008 May;95(5):503-12.
10.王文耀.K-ras基因在胰腺癌诊断中的应用(文献综述).国外医学外科学分册.2001,28-2
11. Yoshida T, Ohnami S, Aoki K. Development of gene therapy to target pancreatic cancer[J]. Cancer Sci,2004,95(4):283—289.
12. Morioka CY, Costa FP, Lima EMR, Saito S, Watanabe A, Huang CC, Arai H, Sakamoto T. Canantisense oligonucleotides specific to mutated K-ras gene inhibit the tumor growth, invasiveness, and MMP-2 and MMP-9 expression in hamster pancreatic cancer model in vitro and in vivo? EJC suppl 2007; 5:73
13. Yu Y, Xu F, Peng H, Fang X, Zhao S, Li Y, Cuevas B, Kuo W L, Gray, J W, Siciliano M, Mills G B, and Bast R C Jr (1999). NOEY2 (ARHI), an imprinted putative tumor suppressor gene in ovarian and breast carcinomas. Proc. Natl. Acad. Sci. US A 96,214-219.
14. Hisatomi H, Nagao K, Wakita K, and Kohno N.(2002).ARHI/NOEY2 inactivation may be important in breast tumor pathogenesis. Oncology 62,136-140.
15.杨红,陈原稼,徐峰极等.新的候选抑癌基因NOEY2在胰腺肿瘤中的表达.胰腺病学.2002,2(1)：28-30
16.胡益群等.ARHI基因在胰腺肿瘤中的抑癌作用及其可能细胞内信号传导的机制初探.2005.博士论文
17.路新卿等.ARHI基因对胰腺癌细胞体个增殖、凋亡和侵袭转移的影响及其相关机制研究.2009.博士论文
18.王长印,董振芳,宋晓斐.RNA干扰技术及其在临床医学中的应用进展[J].山东医药.2005.45(5)：69-71.
19. Jason B. Fleming, Guo-Liang Shen, Shane E. Holloway, Mishel Davis, and Rolf A. Brekken. Molecular Consequences of Silencing Mutant K-ras in Pancreatic Cancer Cells:Justification for K-ras-Directed Therapy
20. Hassan MM, Bondy ML, Wolff RA, et al. Risk factors for pancreatic cancer: casecontrol study. Am J Gastroenterol 2007; 102:2696-707.
21. Landi S. Genetic predisposition and environmental risk factors to pancreatic cancer:a review of the literature. Mutat Res 2009;681:299-307.
22. Shi C, Hruban RH, Klein AP. Familial pancreatic cancer. Arch Pathol Lab Med 2009; 133:365-74.
23.王文耀.K-ras基因在胰腺癌诊断中的应用(文献综述).国外医学外科学分册.2001,28-2
24. Thomas B. Brunner, Keith A. Cengel, Stephen M. Hahn, Junmin Wu, Douglas L. Fraker, W. Gillies McKenna, and Eric J. Bernhard Pancreatic Cancer Cell Radiation Survival and Prenyltransferase Inhibition:The Role of K-Ras. Cancer Res 2005; 65: (18). September 15,2005
25. Adjei AA. Blocking oncogenic Ras signaling for cancer therapy. J Natl Cancer Inst 2001; 93:1062-1074
26. Thomas GR, Forbes JR. et al, The Wilson disease gene:spectrum of mutations and their consequences, Nat Genet,1995,9:210-217.
27. Semba S, Moriya T, Kimura W, et al. Phosphorylated AKT/PKB controls cell growth and apoptosis in intraductal papillary-mucinous tumor and inva sive ductal adenocarcinoma of the pancreas. Pancreas.2003; 26:250-257.
28. Asano T,Yao Y, Zhu J, Li D, Abbruzzese JL, Reddy SA. The PI 3-kinase/AKT signaling pathway is activated due to aberrant Pten expression and targets transcription factors NF-kappaB and c-Myc in pancreatic cancer cells. Oncogene 2004; 23:8571-8580.
29. Wheeler AP, Ridley AJ. Why three Rho Proteins? RhoA, RhoB, RhoC and cell motility. Exp CeⅡ Res,2004,30(1):43-49.
30. Sotiropoulos A, Gineitis D, Copeland J, Treisman R. Signal-regulated activation of serum response factor is mediated by changesin actin dynamics. Cell.1999; 98:159-169.
31. Fujioka S, Sclabas GM, Schmidt C, et al. Function of nuclear factor kappaB in pancreatic cancer metastasis. Clin Cancer Res.2003; 9:346-354.)
32. Mayo, L.D. and Donner, D.B. A phosphatidylinositol 3-kinase/AKT pathway promotes translocation of MDM2 from the cytoplasm to the nucleus. Proc. Natl. Acad. Sci. USA,2001; 98,11598-11603.
33. Xiong, Y, Hannon, GJ., Zhang, H., Casso, D., Kobayashi, R., and Beach, D. p21WAF1 is a universal inhibitor of cyclin kinases. Nature,1993; 366,701-704.
34. Ashcroft M, Ludwig RL, Woods DB, et al. Phosphorylation of MDM2 by AKT. Oncogene 2002; 21:1955-62.
35. Ogawara Y, Kishishita S, Obata T, et al. AKT enhances MDM2-mediated ubiquitination and degradation of p53. J Biol Chem2002; 277:21843-50.
36. Pan MH, Chen WJ, Lin-Shiau SY, Ho CT and Lin JK. Tangeretin induces cell-cycle G1 arrest through inhibiting cyclin-dependent kinases 2 and 4 activities as well as elevating Cdk inhibitors p21 and p27 in human colorectal carcinoma cells. Carcinogenesis 2002,23; 1677-1684.
37. Zhou B P, Liao Y, Xia, W., Zou, Y, Spohn, B. and Hung, M.C. HER-2/neu induces p53 ubiquitination via AKT-mediated MDM2 phosphorylation. Nat. Cell Biol., 2001; 3,973-982
1. M. Malumbres, M. Barbacid RAS oncogenes:the first, Nat. Rev., Cancer 3 (2003) 459-465.
2. Adjei AA. Blocking oncogenic Ras signaling for cancer therapy. J Natl Cancer Inst 2001; 93:1062-1074
3. Bos JL. ras oncogenes in human cancer:a review. Cancer Res.1989 Sep 1;49(17):4682-9.
4. Thomas GR, Forbes JR, et al, The Wilson disease gene:spectrum of mutations and their consequences, Nat Ge net,1995,9:210-217.
5. Semba S, Moriya T, Kimura W, et al. Phosphorylated AKT/PKB controls cell growth and apoptosis in intraductal papillary-mucinous tumor and inva sive ductal adenocarcinoma of the pancreas. Pancreas.2003; 26:250-257.
6. Wheeler AP, Ridley AJ. Why three Rho Proteins? RhoA, RhoB, RhoC and cell motility. Exp Ce Ⅱ Res,2004,30(1):43-49.
7. Sotiropoulos A, Gineitis D, Copeland J, Treisman R. Signal-regulated activation of serum response factor is mediated by changesin actin dynamics. Cell.1999; 98:159-169.
8. Yu Y, Xu F, Peng H, Fang X, Zhao S, Li Y, Cuevas B, Kuo W L, Gray, J W, Siciliano M, Mills G B, and Bast R C Jr NOEY2 (ARHI), an imprinted putative tumor suppressor gene in ovarian and breast carcinomas. Proc. Natl. Acad. Sci. USA 1999,96,214-219.
9. Bourne H R, Sanders D A, and McCormick F. The GTPase superfamily:Conserved structure and molecular mechanism. Nature.1991,349,117-127.
10. Luo R Z,Peng H Q,Xu F J, Bao J J,Pang Y,Pershad R,Issa J P J,Liao W S L, Bast RC Jr, and Yu Y H. Genomic structure and promoter characterization of an imprinted tumor suppressor gene ARHI. Biochim Biophys Acta.2001,1519,216-222.
11. Fujii S, Luo RZ, Yuan J, Kadota M, Oshimura M, Dent SR, Kondo Y, Issa JP, Bast RC Jr, and Yu Y. Reactivation of the silenced and imprinted alleles of ARHI is associated with increased histone H3 acetylation and decreased histone H3 lysine 9 methylation. Hum Mol Genet.2003,12,1791-1800.
12. Yuan J, Luo RZ, Fujii S, Wang L, Hu W, Andreeff M, Pan Y, Kadota M, Oshimura M, Sahin, AA, Issa JP, Bast RC Jr, and Yu Y. Aberrant methylation and silencing of ARHI,an imprinted tumor suppressor gene in which the function is lost in breast cancers. Cancer Res.2003,63,4174-4180.
13. Lu ZH, Chen J, Gu LJ, Luo YF, and Gu CF. ARHI mRNA and protein expression in pancreatic cancers. Zhong guo Yi Xue Ke Xue Yuan Xue Bao 2001.23,324-327.
14.杨红,陈原稼,徐峰极等.新的候选抑癌基因NOEY2在胰腺肿瘤中的表达.胰腺病学.2002,2(1)：28-30.
15. Bao J J, Le X F, Wang R Y, Yuan J, Wang L, Atkinson E N, LaPushin R, Andreeff M, Fang B, Yu Y, and Bast R C Jr. Reexpression of the tumor suppressor gene ARHI induces apoptosis in ovarian and breast cancer cells through a caspase-independent calpain-dependent pathway. Cancer Res.2002,62,7264-7272.
16. 胡益群等.ARHI基因在胰腺肿瘤中的抑癌作用及其可能细胞内信号传导的机制初探.2005.博士论文
17.路新卿等.ARHI基因对胰腺癌细胞体个增殖、凋亡和侵袭转移的影响及其相关机制研究.2009.博士论文
18.鲁嘉等,ARHI基因在胰腺癌中的抑癌作用及其对EGF-Ras-Raf-MAPK/ERK信号转导通路的影响.博士论文,2007
19. C.J. Der, T.G Krontiris, GM. Cooper, Transforming genes of human bladder and lung carcinoma cell lines are homologous to the ras genes of Harvey and Kirsten sarcoma viruses, Proc. Natl. Acad. Sci. U. S. A.79(1982) 3637-3640.
20. 苏震东.骆明德.K-ras基因突变在胰腺癌中的作用研究进展.期刊论文.国外医学(消化系疾病分册)2005(1)
21. Histopher A, ScoRe E. Molecular genetics of pancreatic carcinoma. In:Reher HA. Pancretie cancer pathogenesis, diagnosis and treatmen.1999:3—15.
22. Torrisani J, Bournet B, Cordelier P, Buscail L. New molecular targets in pancreatic cancer. Bull Cancer.2008 May;95(5):503-12.
23. 王文耀.K-ras基因在胰腺癌诊断中的应用(文献综述).国外医学外科学分册.2001,28-2
24. Yoshida T, Ohnami S, Aoki K. Development of gene therapy to target pancreatic cancer[J]. Cancer Sci,2004,95(4):283—289.
25. Jason B. Fleming, Guo-Liang Shen, Shane E. Holloway, Mishel Davis, and Rolf A. Brekken. Molecular Consequences of Silencing Mutant K-ras in Pancreatic Cancer Cells: Justification for K-ras-Directed Therapy
26. Morioka CY, Costa FP, Lima EMR, Saito S, Watanabe A, Huang CC, Arai H, Sakamoto T. Canantisense oligonucleotides specific to mutated K-ras gene inhibit the tumor growth, invasiveness, and MMP-2 and MMP-9 expression in hamster pancreatic cancer model in vitro and in vivo? EJC suppl 2007; 5:73
27. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002;296:550-553.
28. Caldas C, Kern SE. K-ras mutation and pancreatic adenocarcinoma. Int J Pancreatol 1995;18:1-6.