作者投稿
专家审稿
编辑办公
邮件订阅
RSS
Effect of RNA Interference Plasmid on the Expression of Oncogene AKT2 in Pancreatic Cancer Cell Line Panc-1
-
摘要:
目的 探讨RNA干扰质粒抑制胰腺癌细胞系Panc-1原癌基因AKT2的表达对胰腺癌细胞生长和凋亡的影响, 并初步探讨其作用机制。 方法 选择胰腺癌细胞系Panc-1, 构建特异性抑制AKT2表达的RNA干扰质粒, 瞬时和稳定转染胰腺癌细胞, 采用MTT法及软琼脂克隆形成实验检测胰腺癌细胞生长能力, Heochst染色及Annexin V-FITC/PI染色法检测细胞凋亡情况, 通过Western blot方法检测凋亡蛋白caspase-3表达; 并进行裸鼠移植瘤体内转染实验。 结果 采用RNA干扰质粒沉默胰腺癌细胞系Panc-1原癌基因AKT2, 能够有效抑制胰腺癌细胞Panc-1体外生长能力、促进细胞凋亡, 诱导凋亡激酶caspase-3的表达; 动物体内实验结果显示, 干扰质粒能够有效抑制胰腺癌细胞系Panc-1在动物体内的成瘤能力。 结论 RNA干扰质粒抑制原癌基因AKT2表达, 可有效抑制胰腺癌细胞生长, 促进凋亡, 针对原癌基因AKT2的基因治疗对胰腺癌具有重要的潜在应用价值。 Abstract:Objective To explore the effect of RNA interference (RNAi) plasmid on proliferation, apoptosis of status of Panc-1 cells by silencing oncogene AKT2, and investigate its possible mechanism. Methods Pancreatic cancer cell line Panc-1 was applied to constructe the RNAi plasmid-targeting oncogene AKT2 and to transfect cells transiently and stably. The proliferation status was determined using CCK-8 method and soft agar colone formation test. The apotosis status of the cancer cells in vitro was determined using Heochst and Annexin V-FITC/PI methods. The protein level of AKT2 and caspase-3 kinase were detected using Western blotting. Finally, we evaluate the in vivo effect of the recombinant plamids on Panc-1 cells. Results By silencing the oncogenes AKT2, RNAi plasmid effectively down-regulate the mRNA and protein levels of of AKT2 in Panc-1 cells reduced cell proliferation and colony formation of Panc-1 cells, induced apoptosis in Panc-1 cells, increased the protein level of caspase-3 protein, and inhibited tumor growth in vivo. Conclusions RNAi can inhibit the expression of oncogene AKT2 and therefore effectively inhibit the growth of pancreatic cancer cells and promote their apoptosis. Gene therapy targeting AKT2 may be a promising target for pancreatic cancer. -
Key words:
- RNA interference /
- pancreatic cancer /
- Panc-1 cell line /
- gene therapy /
- protein kinase B
-
图 1 AKT2干扰质粒对胰腺癌细胞系Panc-1 AKT2基因和蛋白表达的影响
A. Panc-1细胞AKT2 mRNA水平变化, 与未处理组或阴性载体组比较, * P < 0. 05; B. Panc-1细胞AKT2蛋白水平变化
图 2 AKT2干扰质粒对胰腺癌细胞系Panc-1生长的影响
A.细胞生长曲线; B.第5天Panc-1细胞生长情况, 与未处理组或阴性载体组比较, * P < 0. 05
图 3 AKT2干扰质粒对胰腺癌细胞系Panc-1软琼脂克隆形成能力的影响
A. Panc-1细胞软琼脂克隆形成实验; B. MTT法软琼脂克隆染色计数, 与未处理组或阴性载体组比较, * P < 0. 05
图 5 Annexin V/PI法检测anti-AKT2干扰质粒对胰腺癌细胞系Panc-1调亡的影响
A.流式细胞术示加入Annexin V/PI后不同处理组胰腺癌细胞系Panc-1的凋亡; B.不同质粒转染胰腺癌细胞系Panc-1凋亡细胞统计分析, 与未处理组或阴性载体组比较, * P < 0. 05
-
[1] Davidson BL, McCray PB Jr. Current prospects for RNA in-terference-based therapies[J]. Nat Rev Genet, 2011, 12:329-340. doi: 10.1038/nrg2968 [2] Murthy SS, Tosolini A, Taguchi T, et al. Mapping of AKT3, encoding a member of the Akt/protein kinase B family, to human and rodent chromosomes by fluorescence in situ hybridization[J]. Cytogenet Cell Genet, 2000, 88:38-40. doi: 10.1159/000015481 [3] Testa JR, Bellacosa A. AKT plays a central role in tumorigenesis[J]. Proc Natl Acad Sci USA, 2001, 98:10983-10985. doi: 10.1073/pnas.211430998 [4] Ruggeri BA, Huang L, Wood M, et al. Amplification and overexpression of the AKT2 oncogene in a subset of human pancreatic ductal adenocarcinomas[J]. Mol Carcinog, 1998, 21:81-86. doi: 10.1002/(SICI)1098-2744(199802)21:2<81::AID-MC1>3.0.CO;2-R [5] Altomare DA, Tanno S, De Rienzo A, et al. Frequent activation of AKT2 kinase in human pancreatic carcinomas[J]. J Cell Biochem, 2002, 87:470-476. doi: 10.1002/jcb.10287 [6] Haller DG. New perspectives in the management of pancreas cancer[J]. Semin Oncol, 2003, 30:3-10. doi: 10.1016/S0093-7754(03)00119-2 [7] Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature, 1998, 391:806-811. doi: 10.1038/35888 [8] Hannon GJ, Rossi JJ. Unlocking the potential of the human genome with RNA interference[J]. Nature, 2004, 431:371-378. doi: 10.1038/nature02870 [9] Scherr M, Battmer K, Winkler T, et al. Specific inhibition of bcr-abl gene expression by small interfering RNA[J]. Blood, 2003, 101:1566-1569. doi: 10.1182/blood-2002-06-1685 [10] Michael MZ, O'Connor SM, van Holst Pellekaan NG, et al. Reduced accumulation of specific microRNAs in colorectal neoplasia[J]. Mol Cancer Res, 2003, 1:882-891. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=8333ff0709bc69cc65a1352ce570517f [11] Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference[J]. Cancer Cell, 2002, 2:243-247. doi: 10.1016/S1535-6108(02)00122-8 [12] Yamamoto S, Tomita Y, Hoshida Y, et al. Prognostic significance of activated Akt expression in pancreatic ductal adenocarcinoma[J]. Clin Cancer Res, 2004, 10:2846-2850. doi: 10.1158/1078-0432.CCR-02-1441 [13] Liu X, Shi Y, Giranda VL, et al. Inhibition of the phosphatidylinositol 3-kinase/Akt pathway sensitizes MDAMB468 human breast cancer cells to cerulenin-induced apoptosis[J]. Mol Cancer Ther, 2006, 5:494-501. doi: 10.1158/1535-7163.MCT-05-0049 [14] Shimamura H, Terada Y, Okado T, et al. The PI3-kinaseAkt pathway promotes masangial cell survival and inhibits apoptosis in vitro via NF-kappa B and Bad[J]. J Am Soc Nephrol, 2003, 14:1427-1434. doi: 10.1097/01.ASN.0000066140.99610.32 [15] Guo S, Sonenshein GE. Forkhead box transcription factor FOXO3a regulates estrogen receptor alpha expression and is repressed by the Her-2/neu/phosphatidylinositol 3-kinase/Akt signaling pathway[J]. Mol Cell Biol, 2004, 24:8681-8690. doi: 10.1128/MCB.24.19.8681-8690.2004 [16] Terraqni J, Graham JR, Adams KW, et al. Phosphatidylinositol 3-kinase signaling in proliferating cells maintains an anti-apoptotic transcriptional program mediated by inhibition of FOXO and non-canonical activation of NFkappaB transcription factors[J]. BMC Cell Biol, 2008, 9:6. doi: 10.1186/1471-2121-9-6 [17] Zhang X, Jin B, Huang C. The PI3K/Akt pathway and its downstream transcriptional factors as targets for chemoprevention[J]. Curr Cancer Drug Targets, 2007, 7:305-316. doi: 10.2174/156800907780809741 [18] Kang YC, Kim KM, Lee KS, et al. Serum bioactive lysophospholipids prevent TRAIL-induced apoptosis via PI3K/Akt-dependent cFLIP expression and Bad phosphorylation[J]. Cell Death Differ, 2004, 11:1287-1298. doi: 10.1038/sj.cdd.4401489 [19] Panka DJ, Mano T, Suhara T, et al. Phosphatidylinositol 3-kinase/Akt activity regulates c-FLIP expression in tumor cells[J]. J Biol Chem, 2001, 276:6893-6896. doi: 10.1074/jbc.C000569200 -
下载:
点击查看大图
图(7)
计量
- 文章访问数: 207
- HTML全文浏览量: 51
- PDF下载量: 8
- 被引次数: 0
