RNAi在胰腺癌基因治疗中的交叉抑制性效果研究
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摘要
目的:构建表达短发卡RNA(shRNA)的质粒载体,观察其在K-ras基因突变类型为GAT和GTT的人胰腺癌细胞株PANC-1和CFPAC-1间的交叉抑制效果,为RNAi技术应用于治疗胰腺癌奠定一定的理论和实验基础。
方法:1、针对GAT和GTT两种K-ras基因突变类型,分别设计两条shRNA序列,并分别加载到质粒载体pGenesil-1上,构建针对人胰腺癌细胞株PANC-1和CFPAC-1的K-ras基因的质粒pGenesil-1GAT和pGenesil-1GTT。2、应用质粒pGenesil-1GAT和pGenesil-1GTT瞬时转染K-ras突变类型为GAT的人胰腺癌细胞株PCNA-1 ,同时将质粒pGenesil-1GAT和pGenesil-1GTT瞬时转染具有K-ras突变类型为GTT的人胰腺癌细胞株CFPAC-1。每种细胞分4组,分别为特异性抑制组,交叉性抑制组,空白质粒组和对照组,其中特异性抑制组转染同细胞K-ras基因突变类型相同的质粒,交叉性抑制组转染与其K-ras基因突变类型不同的质粒,空白对照组转染空白质粒pGenesil-1KB,对照组以1×PBS转染作为对照。用逆转录-聚合酶链反应(RT-PCR)和免疫印迹法(Western-blot)方法检测转染后细胞K-ras基因的表达情况,并采用CCK-8(ceIl counring kit-8)活细胞计数法,绘制转染前后细胞的生长曲线,以检测质粒pGenesil-1GAT对胰腺癌细胞PANC-1的K-ras基因的抑制效果和质粒pGenesil-1GTT对PANC-1细胞的交叉抑制效果,以及质粒pGenesil-1GTT对胰腺癌细胞CFPAC-1的K-ras基因的抑制效果和质粒pGenesil-1GAT对CFPAC-1细胞的交叉抑制效果。
结果:1、通过酶切鉴定和送检基因测序证实插入shRNA序列正确,成功构建了质粒pGenesil-1GAT和pGenesil-1GTT。2、将质粒pGenesil-1GAT和pGenesil-1GTT瞬时转染到PANC-1细胞后,PANC-1特异性抑制组(转染pGenesil-1GAT的细胞)细胞的K-ras基因mRNA和蛋白表达水平明显下降,与交叉抑制组(转染pGenesil-1GTT的细胞)相比结果有显著性差异(p<0.05),与转染空白质粒组(转染pGenesil-1KB的细胞)相比结果有显著性差异(p<0.05),与对照组相比结果亦有显著性差异(p<0.05),细胞生长明显受限,对数生长期后移,平台期水平低,细胞生长曲线明显向右下移动,而PANC-1细胞交叉抑制组(转染pGenesil-1GTT的细胞)的K-ras基因mRNA和蛋白表达水平下降不明显,与转染空白质粒组(转染pGenesil-1KB的细胞)相比结果无显著性差异(p>0.05),与未转染的空白对照组最相比结果亦无显著性差异(p>0.05)。细胞生长未见明显影响。3、将质粒pGenesil-1GAT和pGenesil-1GTT瞬时转染到CFPAC-1细胞后,特异性抑制组(转染pGenesil-1GTT的细胞)K-ras基因mRNA和蛋白表达水平明显下降,与交叉抑制组(转染pGenesil-1GAT的细胞)相比结果有显著性差异(p<0.05),与空白质粒组(转染pGenesil-1KB的细胞)相比结果有显著性差异(p<0.05),与对照组相比结果亦有显著性差异(p<0.05),细胞生长亦明显受限。而交叉抑制组(转染pGenesil-1GAT的细胞)K-ras基因mRNA和蛋白表达水平下降不明显,与空白质粒组(转染pGenesil-1KB的细胞)相比结果无显著性差异(p>0.05),与对照组相比结果亦无显著性差异(p>0.05),细胞生长未受影响。
结论:1、针对胰腺癌细胞K-ras基因突变位点设计的shRNA能够有效地加载到pGenesil-1质粒载体上,并且重构的质粒载体都可以高效的转染人胰腺癌细胞株PANC-1和CFPAC-1。2、突变特异性的shRNA可以特异性的抑制相应突变类型的K-ras基因的表达,从而达到抑制具有相应K-ras基因突变类型的人胰腺癌细胞生长的目的,说明K-ras基因在肿瘤细胞增殖方面起到非常重要的作用,因此应用RNA干扰技术治疗胰腺癌是可行的。3、针对K-ras突变类型GAT和GTT所设计的两条shRNA序列,无相互交叉抑制现象的出现,这说明质粒载体介导的RNAi特异性非常强,一般不会出现碱基错配现象,这为RNAi应用于胰腺癌的基因治疗提供了更加全面的数据。
Objective: To construct plasmids vector expressing short hairpin RNA(shRNA) to understand the possibility of cross-inhibition of K-ras gene with different kinds of mutation by vector-based RNAi in human pancreatic cancer cells PANC-1 and CFPAC-1. And to establish certain theoretic groundwork for the use of RNAi technique in the treatment of pancreatic cancer.
Methods: To silence the expression of two kinds of K-ras gene mutation, GAT and GTT, we synthesized 2 shRNA sequences and colon these two sequences into pGenesil-1,to construct 2 recombinant K-ras shRNA plasmids, pGenesil-1GAT and pGenesil-1GTT. These two kinds of plasmids were transfected into the human pancreatic cancer cells PANC-1, with K-ras mutation of GAT, and the human pancreatic cancer cells CFPAC-1, with K-ras mutation of GTT. Each kind of cells was divided into 4 groups (special interference group, cross interference group, empty plasmid transfected group and control group). In the special interference group, cells were transfected with the recombinant shRNA plasmid, which was designed for the same mutation of K-ras gene as the cells. In the cross interference group, cells were transfected with the recombinant shRNA plasmid, which was not designed for the same mutation of K-ras gene as the cells. In the empty plasmid transfected group, the cells were transfected with the empty plasmid pGenesil-1KB, and the cells were transfected with the PBS in the control group. RT-PCR and Western blot were used to identify K-ras mRNA and protein expression. And the cell proliferation was analyzed by employing CCK-8 methods. In this way, we detected he interferential effect of the plasmid pGenesil-1GAT in PANC-1 and the plasmid pGenesil-1GTT in CFPAC-1, and the cross-interferential effect of the plasmid pGenesil-1GTT in PANC-1 and the plasmid pGenesil-1GAT in CFPAC-1.
Results: 1. Plasmids expressing specific shRNA targeting K-ras gene were constructed successfully,and were certified through agarose gel electrophoresis and gene sequencing. 2. The recombinant plasmids were efficiently transfected into all human pancreatic cancer cells. K-ras mRNA and protein expression were significantly inhibited by mutation-specific shRNA in PANC-1 cells, transfected with pGenesil-1GAT (special interference group), and showed a reduced proliferation after the transfection compared with the empty plasmid group and control group (p<0.05), while the mRNA and protein expression of the PANC-1 cells, transfacted with pGenesil-1GTT (cross interference group), were not inhibited, and the cells proliferated as well as empty plasmid transfected group and control group(p>0.05). Similarly, in CFPAC-1 cells, which was transfected with pGenesil-1GTT(special interference group), the K-ras mRNA and protein expression were reduced sharply and the proliferation of the cells decreased compared with the empty plasmid group and control group(P<0.05), while the K-ras gene of CFPAC-1 cells, transfected with pGenesil-1GAT (cross interference group), was not blocked, and the cells proliferation did not decrease compared with the empty plasmid group and control group(p>0.05).
Conclusions: 1. The shRNA sequence targeting K-ras can be inserted into the pGenesil-1 plasmid, and the recombinant plasmids expressing specific shRNA can be transfected into the human pancreatic cancer cell line PANC-1 and CFPAC-1 successfully. 2. Mutation-specific shRNA can specifically inhibit the relevant mutant K-ras genes, and the proliferation of the cells was decreases. So K-ras gene is very important for the cancer cells proliferation, especially in the pancreatic cancer cells. Therefore, it is feasible to treat pancreatic cancer by using RNAi targeting the mutated K-ras gene. 3. There is Even though there was only one nucleic acid different between the 2 K-ras gene mutation, there is no effective cross-silence between each other in present research. This indicated that the plasmid-based RNA interference significantly inhibit the expression of the target gene. And there is no cross-silence in RNAi, which provides more data for the application of RNAi in the treatment of pancreatic cancer.
方法:1、针对GAT和GTT两种K-ras基因突变类型,分别设计两条shRNA序列,并分别加载到质粒载体pGenesil-1上,构建针对人胰腺癌细胞株PANC-1和CFPAC-1的K-ras基因的质粒pGenesil-1GAT和pGenesil-1GTT。2、应用质粒pGenesil-1GAT和pGenesil-1GTT瞬时转染K-ras突变类型为GAT的人胰腺癌细胞株PCNA-1 ,同时将质粒pGenesil-1GAT和pGenesil-1GTT瞬时转染具有K-ras突变类型为GTT的人胰腺癌细胞株CFPAC-1。每种细胞分4组,分别为特异性抑制组,交叉性抑制组,空白质粒组和对照组,其中特异性抑制组转染同细胞K-ras基因突变类型相同的质粒,交叉性抑制组转染与其K-ras基因突变类型不同的质粒,空白对照组转染空白质粒pGenesil-1KB,对照组以1×PBS转染作为对照。用逆转录-聚合酶链反应(RT-PCR)和免疫印迹法(Western-blot)方法检测转染后细胞K-ras基因的表达情况,并采用CCK-8(ceIl counring kit-8)活细胞计数法,绘制转染前后细胞的生长曲线,以检测质粒pGenesil-1GAT对胰腺癌细胞PANC-1的K-ras基因的抑制效果和质粒pGenesil-1GTT对PANC-1细胞的交叉抑制效果,以及质粒pGenesil-1GTT对胰腺癌细胞CFPAC-1的K-ras基因的抑制效果和质粒pGenesil-1GAT对CFPAC-1细胞的交叉抑制效果。
结果:1、通过酶切鉴定和送检基因测序证实插入shRNA序列正确,成功构建了质粒pGenesil-1GAT和pGenesil-1GTT。2、将质粒pGenesil-1GAT和pGenesil-1GTT瞬时转染到PANC-1细胞后,PANC-1特异性抑制组(转染pGenesil-1GAT的细胞)细胞的K-ras基因mRNA和蛋白表达水平明显下降,与交叉抑制组(转染pGenesil-1GTT的细胞)相比结果有显著性差异(p<0.05),与转染空白质粒组(转染pGenesil-1KB的细胞)相比结果有显著性差异(p<0.05),与对照组相比结果亦有显著性差异(p<0.05),细胞生长明显受限,对数生长期后移,平台期水平低,细胞生长曲线明显向右下移动,而PANC-1细胞交叉抑制组(转染pGenesil-1GTT的细胞)的K-ras基因mRNA和蛋白表达水平下降不明显,与转染空白质粒组(转染pGenesil-1KB的细胞)相比结果无显著性差异(p>0.05),与未转染的空白对照组最相比结果亦无显著性差异(p>0.05)。细胞生长未见明显影响。3、将质粒pGenesil-1GAT和pGenesil-1GTT瞬时转染到CFPAC-1细胞后,特异性抑制组(转染pGenesil-1GTT的细胞)K-ras基因mRNA和蛋白表达水平明显下降,与交叉抑制组(转染pGenesil-1GAT的细胞)相比结果有显著性差异(p<0.05),与空白质粒组(转染pGenesil-1KB的细胞)相比结果有显著性差异(p<0.05),与对照组相比结果亦有显著性差异(p<0.05),细胞生长亦明显受限。而交叉抑制组(转染pGenesil-1GAT的细胞)K-ras基因mRNA和蛋白表达水平下降不明显,与空白质粒组(转染pGenesil-1KB的细胞)相比结果无显著性差异(p>0.05),与对照组相比结果亦无显著性差异(p>0.05),细胞生长未受影响。
结论:1、针对胰腺癌细胞K-ras基因突变位点设计的shRNA能够有效地加载到pGenesil-1质粒载体上,并且重构的质粒载体都可以高效的转染人胰腺癌细胞株PANC-1和CFPAC-1。2、突变特异性的shRNA可以特异性的抑制相应突变类型的K-ras基因的表达,从而达到抑制具有相应K-ras基因突变类型的人胰腺癌细胞生长的目的,说明K-ras基因在肿瘤细胞增殖方面起到非常重要的作用,因此应用RNA干扰技术治疗胰腺癌是可行的。3、针对K-ras突变类型GAT和GTT所设计的两条shRNA序列,无相互交叉抑制现象的出现,这说明质粒载体介导的RNAi特异性非常强,一般不会出现碱基错配现象,这为RNAi应用于胰腺癌的基因治疗提供了更加全面的数据。
Objective: To construct plasmids vector expressing short hairpin RNA(shRNA) to understand the possibility of cross-inhibition of K-ras gene with different kinds of mutation by vector-based RNAi in human pancreatic cancer cells PANC-1 and CFPAC-1. And to establish certain theoretic groundwork for the use of RNAi technique in the treatment of pancreatic cancer.
Methods: To silence the expression of two kinds of K-ras gene mutation, GAT and GTT, we synthesized 2 shRNA sequences and colon these two sequences into pGenesil-1,to construct 2 recombinant K-ras shRNA plasmids, pGenesil-1GAT and pGenesil-1GTT. These two kinds of plasmids were transfected into the human pancreatic cancer cells PANC-1, with K-ras mutation of GAT, and the human pancreatic cancer cells CFPAC-1, with K-ras mutation of GTT. Each kind of cells was divided into 4 groups (special interference group, cross interference group, empty plasmid transfected group and control group). In the special interference group, cells were transfected with the recombinant shRNA plasmid, which was designed for the same mutation of K-ras gene as the cells. In the cross interference group, cells were transfected with the recombinant shRNA plasmid, which was not designed for the same mutation of K-ras gene as the cells. In the empty plasmid transfected group, the cells were transfected with the empty plasmid pGenesil-1KB, and the cells were transfected with the PBS in the control group. RT-PCR and Western blot were used to identify K-ras mRNA and protein expression. And the cell proliferation was analyzed by employing CCK-8 methods. In this way, we detected he interferential effect of the plasmid pGenesil-1GAT in PANC-1 and the plasmid pGenesil-1GTT in CFPAC-1, and the cross-interferential effect of the plasmid pGenesil-1GTT in PANC-1 and the plasmid pGenesil-1GAT in CFPAC-1.
Results: 1. Plasmids expressing specific shRNA targeting K-ras gene were constructed successfully,and were certified through agarose gel electrophoresis and gene sequencing. 2. The recombinant plasmids were efficiently transfected into all human pancreatic cancer cells. K-ras mRNA and protein expression were significantly inhibited by mutation-specific shRNA in PANC-1 cells, transfected with pGenesil-1GAT (special interference group), and showed a reduced proliferation after the transfection compared with the empty plasmid group and control group (p<0.05), while the mRNA and protein expression of the PANC-1 cells, transfacted with pGenesil-1GTT (cross interference group), were not inhibited, and the cells proliferated as well as empty plasmid transfected group and control group(p>0.05). Similarly, in CFPAC-1 cells, which was transfected with pGenesil-1GTT(special interference group), the K-ras mRNA and protein expression were reduced sharply and the proliferation of the cells decreased compared with the empty plasmid group and control group(P<0.05), while the K-ras gene of CFPAC-1 cells, transfected with pGenesil-1GAT (cross interference group), was not blocked, and the cells proliferation did not decrease compared with the empty plasmid group and control group(p>0.05).
Conclusions: 1. The shRNA sequence targeting K-ras can be inserted into the pGenesil-1 plasmid, and the recombinant plasmids expressing specific shRNA can be transfected into the human pancreatic cancer cell line PANC-1 and CFPAC-1 successfully. 2. Mutation-specific shRNA can specifically inhibit the relevant mutant K-ras genes, and the proliferation of the cells was decreases. So K-ras gene is very important for the cancer cells proliferation, especially in the pancreatic cancer cells. Therefore, it is feasible to treat pancreatic cancer by using RNAi targeting the mutated K-ras gene. 3. There is Even though there was only one nucleic acid different between the 2 K-ras gene mutation, there is no effective cross-silence between each other in present research. This indicated that the plasmid-based RNA interference significantly inhibit the expression of the target gene. And there is no cross-silence in RNAi, which provides more data for the application of RNAi in the treatment of pancreatic cancer.
引文
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11 Hruban RH, Van-mansfela AD, Offerhans GJ, et al. K-ras oncogene activation in adenocarcinoma of the human pancreacreas: A study of 82 carcinomas using a combination of matant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide hybridization. Am J Pathal, 1993, 143(2):545
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13 Smit VT, Boot AT, smits Am, et al. KRAS codon 12 mutations occur very frequently in pancreatic adenocarcinoma. Nucleic Acids Res, 1988, 16(16):7773~7782
14 Howe JR, Conlon KC. The molecular genetics of pancreatic cancer. Surg Oncol, 1997, 6(1):1~18
15 Fleming JB, Shen GL, Holloway SE, et al. Molecular consequences of silencing mutant K-RAS in pancreaticcancer cells: justification for K-RAS-directed therapy. Mol Cancer Res, 2005, 3(7):413~423
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32 Boden D, Pusch O, Lee F, et al. Human immunodeficiency virus type 1 escape from RNA interference. J Virol, 2003, 2003, 77(21):11531~11535.
33 Brummelkamp TR, Nijman SM, Dirac AM, et al. Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-κB. Nature, 2003, 424(6950):797~801
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23 Wilda M, Fuchs U, Wossmann W, et al. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference(RNAi). Oncogene, 2002, 21(37):5716
24 Crans-Vargas HN, Landaw EM, Bhatia S, et al. Expression of cyclic adenosine monophosphate response-element binding protein in acute leukemia. B lood, 2002, 99 (7):2617~2619
25 Yasuharu Hayashida, Kazufumi Honda. E-cadherin regulates the association of β-catenin and actinin-4. Proc Amer Asso Cancer Res, 2005, 46:5632
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27 Salvi A, Arici B, De Petro G, et al. Small interfering RNA urokinase silencing inhibits invasion and migration of human hepatocellular carcinoma cells. Mol Cancer Ther, 2004, 3(6):671~678
28 Ryo A, Uemura H, Ishiguro H, et al. Stable suppression of tumorigenicity by Pin1-targeted RNA interference in prostatecancer. Clin Cancer Res, 2005, 11(20):7523~7531
29 Zhang D, Li F, Weidner D, et al. Physical and functional interaction between myeloid cell leukemia 1 protein (MCL1) and Fortilin. The potential role of MCL1 as a fortilin chaperone. J Biol Chem, 2002, 277(40):37430~37438
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32 Boden D, Pusch O, Lee F, et al. Human immunodeficiency virus type 1 escape from RNA interference. J Virol, 2003, 2003, 77(21):11531~11535.
33 Brummelkamp TR, Nijman SM, Dirac AM, et al. Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-κB. Nature, 2003, 424(6950):797~801