慢病毒介導的雙干擾RNA同時沉默IGF1R和EGFR蛋白治療肝癌的實驗研究_《中國普外基礎與臨床雜志》

作者：

牛堅 ¹ , 劉斌 ¹ , 潘晴 ² , 于彬 ³ , 李向農 ¹

1.徐州醫學院附屬醫院普外科（江蘇徐州221002）;;
2.上海交通大學附屬新華醫院消化科(上海200092);;
3.上海交通大學腫瘤研究所癌基因及相關基因國家重點實驗室（上海200032）;

關鍵詞：

RNA干擾人胰島素樣生長因子1類受體表皮樣生長因子受體慢病毒載體構建肝癌

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目的　研究雙靶向IGF1R和EGFR基因的小干擾RNA（siRNA）慢病毒對肝癌細胞SMMC7721中IGF1R和EGFR表達的影響及其對細胞生長的作用。
方法　針對已經篩選確定的IGF1R和EGFR基因干擾有效序列，合成靶序列的Oligo DNA，退火形成雙鏈DNA，與pLVTHM載體連接產生pLVTHM-IGF1R。RT-PCR合成含H1啟動子EGFR-siRNA表達框,克隆入pLVTHM-IGF1R中形成pLVTHM-IGF1R-EGFR-siRNA。用pLVTHM-IGF1R-EGFR-siRNA、psPAX2和pMD2G 3質粒共轉染包裝細胞293T細胞，包裝產生慢病毒LVTHM-IGF1R-EGFR-siRNA，大量擴增，氯化銫梯度離心純化，測病毒滴度。用LVTHM-IGF1R-EGFR-siRNA感染SMMC7721細胞(感染組),以未經處理的SMMC7721細胞（細胞對照組）及空載體質粒LVTHM（空載體對照組）作為對照。RT-PCR及Western blot法檢測細胞中IGF1R和EGFR的表達，Cell Counting Kit-8法檢測細胞生長，TUNEL法檢測細胞凋亡。
結果　成功構建慢病毒LVTHM-IGF1R-EGFR-siRNA；病毒滴度為4.58×109 pfu/ml； LVTHM-IGF1R-EGFR-siRNA明顯抑制肝癌細胞中IGF1R和EGFR的mRNA和蛋白的表達，分別為細胞對照組及空載體對照組的5%、4%和4%、3%以及10%、11%和8%、9%（P＜0.05），同時能抑制肝癌細胞生長（P＜0.05），促進肝癌細胞凋亡（P＜0.05）。
結論　靶向IGF1R和EGFR基因siRNA慢病毒可同時有效封閉肝癌細胞中的IGF1R及EGFR，降低肝癌細胞的增殖能力，為以IGF1R和EGFR基因為靶點的肝癌生物治療奠定了理論基礎。

引用本文： 牛堅,劉斌,潘晴,于彬,李向農. 慢病毒介導的雙干擾RNA同時沉默IGF1R和EGFR蛋白治療肝癌的實驗研究. 中國普外基礎與臨床雜志, 2009, 16(9): 734-739. doi: 復制

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1. Mendelsohn J, Baselga J. The EGF receptor family as targets for cancer therapy [Ｊ]. Oncogene, 2000; 19(56): 6550-6565.
2. Dalle S, Imamura T, Rose DW, et al. Insulin induces heterologous desensitization of G-protein-coupled receptor and insulin-like growth factor Ⅰ signaling by downregulating beta-arrestin-1 [Ｊ]. Mol Cell Biol, 2002; 22(17): 6272-6285.
3. Peretz S, Kim C, Rockwell S, et al. IGF1 receptor expression protects against microenvironmental stress found in the solid tumor [Ｊ]. Radiat Res, 2002; 158(2): 174-180.
4. Blum G, Gazit A, Levitzki A. Development of new insulin-like growth factor-1 receptor kinase inhibitors using catechol mimics [Ｊ]. J Biol Chem, 2003; 278(42): 40442-40454.
5. Kari C, Chan TO, Rocha de Quadros M, et al. Targeting the epidermal growth factor receptor in cancer: apoptosis takes center stage [Ｊ]. Cancer Res, 2003; 63(1): 1-5.
6. Gilmore AP, Valentijn AJ, Wang P, et al. Activation of BAD by therapeutic inhibition of epidermal growth factor receptor and transactivation by insulin-like growth factor receptor [Ｊ]. J Biol Chem, 2002; 277(31): 27643-27650.
7. Scotlandi K, Manara MC, Nicoletti G, et al. Antitumor activity of the insulin-like growth factor-Ⅰ receptor kinase inhibitor NVP-AEW541 in musculoskeletal tumors [Ｊ]. Cancer Res, 2005; 65(9): 3868-3876.
8. Shepherd FA, Rodrigues Pereira J, Ciuleanu T, et al. Erlotinib in previously treated non-small-cell lung cancer [Ｊ]. N Engl J Med, 2005; 353(2): 123-132.
9. Tsao MS, Sakurada A, Cutz JC, et al. Erlotinib in lung can-cer-molecular and clinical predictors of outcome [Ｊ]. N Engl J Med, 2005; 353(2): 133-144.
10. 牛堅, 錢海鑫, 黃健, 等. RNA干涉胰島素樣生長因子1類受體的研究 [Ｊ]. 中華實驗外科雜志， 2006； 23(7): 872-873.
11. 劉蘇健, 申慶民, 劉宏, 等. 靶向PIK3cb雙位點shRNA干擾抑制血管移植術后再狹窄 [Ｊ]. 中國普外基礎與臨床雜志， 2008； 15(6): 396-401.
12. 何滿西, 李耿, 姚有貴, 等. 胰腺癌細胞survivin表達RNAi抑制載體的構建及其抑制作用的研究 [Ｊ]. 中國普外基礎與臨床雜志， 2008； 15(6): 829-834.
13. Pan M, Wei QJ, Cao F, et al. Inhibition of cell proliferation by siRNA targeting hPRLR in breast cancer MCF-7 cell line [Ｊ]. JNMU, 2007; 21（6）: 372-376.
14. Morris KV, Rossi JJ. Lentiviral-mediated delivery of siRNAs for antiviral therapy [Ｊ]. Gene Ther, 2006; 13(6): 553-558.
15. Cockrell AS, Kafri T. Gene delivery by lentivirus vectors [Ｊ]. Mol Biotechnol, 2007; 36(3): 184-204.
16. Sumimoto H, Kawakami Y. Lentiviral vector-mediated RNAi and its use for cancer research [Ｊ]. Future Oncol, 2007; 3(6): 655-664.
17. 牛堅, 李向農, 韓澤廣. 運用siRNA建立穩定低表達IGF1R基因的肝癌細胞株的實驗研究 [Ｊ]. 中國普外基礎與臨床雜志， 2007； 14(6): 679-684.
18. Sachdev D, Li SL, Hartell JS, et al. A chimeric humanized single-chain antibody against the type Ⅰ insulin-like growth factor (IGF) receptor renders breast cancer cells refractory to the mitogenic effects of IGF-Ⅰ [Ｊ]. Cancer Res, 2003; 63(3): 627-635.
19. Andrews DW, Resnicoff M, Flanders AE, et al. Results of a pilot study involving the use of an antisense oligodeoxynucleotide directed against the insulin-like growth factor type Ⅰ receptor in malignant astrocytomas [Ｊ]. J Clin Oncol, 2001; 19(8): 2189-2200.
20. Zhao J, Pettigrew GJ, Thomas J, et al. Lentiviral vectors for delivery of genes into neonatal and adult ventricular cardiac myocytes in vitro and in vivo [Ｊ]. Basic Res Cardiol, 2002; 97(5): 348-358.
21. Bauer G, Dao MA, Case SS, et al. In vivo biosafety model to assess the risk of adverse events from retroviral and lentiviral vectors [Ｊ]. Mol Ther, 2008; 16(7): 1308-1315.
22. Kock N, Kasmieh R, Weissleder R, et al. Tumor therapy mediated by lentiviral expression of shBcl-2 and S-TRAIL [Ｊ]. Neoplasia, 2007; 9(5): 435-442.
23. Haynes JR, Dokken L, Wiley JA, et al. Influenza-pseudotyped Gag virus-like particle vaccines provide broad protection against highly pathogenic avian influenza challenge [Ｊ]. Vaccine, 2009; 27(4): 530-541.
24. Lillico SG, Sherman A, McGrew MJ, et al. Oviduct-specific expression of two therapeutic proteins in transgenic hens [Ｊ]. Proc Natl Acad Sci USA, 2007; 104(6): 1771-1776.
25. Baba K, Goto-Koshino Y, Mizukoshi F, et al. Inhibition of the replication of feline immunodeficiency virus by lentiviral vector-mediated RNA interference in feline cell lines [Ｊ]. J Vet Med Sci, 2008; 70(8): 777-783.

《中國普外基礎與臨床雜志》

慢病毒介導的雙干擾RNA同時沉默IGF1R和EGFR蛋白治療肝癌的實驗研究

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《中國普外基礎與臨床雜志》

慢病毒介導的雙干擾RNA同時沉默IGF1R和EGFR蛋白治療肝癌的實驗研究

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