Construction and Identification of Lentiviral Vector of Expressing siRNA Targeting IGF1R, EGFR Gene and Its Inhibition of Liver Cancer Cell Growth_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

NIU Jian ¹ , LIU Bin ¹ , PAN Qing ² , YU Bin ³ , LI Xiangnong ¹

1.Department of General Surgery, Affiliated Hospital of Xuzhou Medical College， Xuzhou 221002, Jiangsu Province, China;;
2.Department of Digestion, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai 200092, China;;
3.Tumor Research Institute of Shanghai Jiaotong University and National Intensive Laboratory of Cancer or Relative Gene, Shanghai 200032, ChinaCorresponding Author: NIU Jian, E-mail: njnj_001@163.com;

Corresponding author：

Keywords：

RNA interference; Human insulin like growth factor receptor 1; Epidermal growth factor receptor; Lentivirus; Vector reconstruction; Liver cancer

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Objective 　To study the interferencing and anti-tumor effects of lentiviral vector of siRNA targeting IGF1R and EGFR gene of the liver cancer cell.
Methods 　The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and connected to the pLVTHM vector, named pLVTHM-IGF1R, into whom the EGFR-siRNA expression frame containing H1 promotor synthesized by RT-PCR was cloned to generate pLVTHM-IGF1R-EGFR-siRNA. The 293T cells were cotransfected by 3 plasmids of pLVTHM-IGF1R-EGFR-siRNA, psPAX2 and pMD2G to enclose LVTHM-IGF1R-EGFR-siRNA, which was amplified in large amount and purified by caesium chloride density gradient centrifugation for measurement of virus titer. SMMC7721 cells infected by LVTHM-IGF1R-EGFR-siRNA were infection group, the untreated SMMC7721 cells and blank vector plasmid LVTHM were two control groups (SMMC7721 cell group and blank vector group). The effect of LVTHM-IGF1R-EGFR-siRNA on IGF1R and EGFR expressions of SMMC7721 cells were detected by RT-PCR and Western blot. The antitumor potential of LVTHM-IGF1R-EGFR-siRNA to SMMC7721 cells was evaluated by Cell Counting Kit-8 assay for cell growth and TUNEL for apoptosis respectively.
Results 　LVTHM-IGF1R-EGFR-siRNA was constructed successfully. Functional pfu titers of LVTHM-IGF1R-EGFR-siRNA was 4.58×109 pfu/ml. Protein and mRNA expression of IGF1R and EGFR of infection group were less than those of blank vector group and SMMC7721 cell group （P＜0.05）, LVTHM-IGF1R-EGFR-siRNA was more effective to inhibit the proliferation and promote apoptosis of SMMC7721 cells （P＜0.05）.
Conclusion 　LVTHM-IGF1R-EGFR-siRNA expressing IGF1R-EGFR-siRNA can inhibit the expression of IGF1R and EGFR, and may be used for further investigation of gene therapy of liver cancer.

Citation： NIU Jian,LIU Bin,PAN Qing,YU Bin,LI Xiangnong. Construction and Identification of Lentiviral Vector of Expressing siRNA Targeting IGF1R, EGFR Gene and Its Inhibition of Liver Cancer Cell Growth. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2009, 16(9): 734-739. doi: Copy

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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. 牛坚, 钱海鑫, 黄健, 等. RNA干涉胰岛素样生长因子1类受体的研究 [Ｊ]. 中华实验外科杂志， 2006； 23(7): 872-873.
11. 刘苏健, 申庆民, 刘宏, 等. 靶向PIK3cb双位点shRNA干扰抑制血管移植术后再狭窄 [Ｊ]. 中国普外基础与临床杂志， 2008； 15(6): 396-401.
12. 何满西, 李耿, 姚有贵, 等. 胰腺癌细胞survivin表达RNAi抑制载体的构建及其抑制作用的研究 [Ｊ]. 中国普外基础与临床杂志， 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. 牛坚, 李向农, 韩泽广. 运用siRNA建立稳定低表达IGF1R基因的肝癌细胞株的实验研究 [Ｊ]. 中国普外基础与临床杂志， 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.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Construction and Identification of Lentiviral Vector of Expressing siRNA Targeting IGF1R, EGFR Gene and Its Inhibition of Liver Cancer Cell Growth

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