Construction and Identification of Lentiviral Vector-mediated Small Interfering RNA Targeting Human Vascular Endothelial Growth Factor A Gene_West China Medical Journal

Authors：

 JiangXiaofei ,  YeFen , ShiLi , ChenXi , ZhuWeimin , ZhangYeming

Department of Cardiology, Zhuhai People's Hospital, Zhuhai, Guangdong 519000, P. R. China;

Corresponding author：

YeFen, Email: ye2260@163.com

Keywords：

RNA interference; Vascular endothelial growth factor; Lentivirus; Human umbilical vein endothelial cells

DOI：

10.7507/1002-0179.20160001

Video：

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Objective To construct a lentiviral vector-mediated gene-targeted small interfering RNA (siRNA) vector to vascular endothelial growth factor (VEGF), and choose the RNAi with the highest silence efficiency to VEGFA gene. Methods Three kinds of VEGFA gene-targeted hairpin siRNA was designed (KD1, KD2, KD3), then two complementary oligo nucleotide strand were synthesized and inserted into pGCSIL-GFP vector. After annealing, the recombined vector pGCSIL-GFP-siVEGFA was gotten, which was digested by restrictive enzyme and sequenced, and was co-transfected with the pHelper 1.0 and pHelper 2.0 into 293T cells by Lipofectamine 2000. After that, the new vector was transfected into human umbilical vein endothelial cells (HUVECs), and the mRNA expression level of VEGFA gene in cells was detected by RT-PCR. Then we compared the mRNA expression level of VEGFA gene of the 3 groups. Results pGCSIL-GFP-siVEGFA was built successfully, and all the siRNA could silence the expression of VEGFA mRNA in the HUVECs, and the relative expressions of VEGFA mRNA to the control group were 0.614±0.043 (KD1), 0.334±0.030 (KD2), and 0.201±0.015 (KD3) respectively. Conclusion We've successfully constructed the siRNA vector for VEGFA mRNA, which can obviously suppress the expression of VEGFA mRNA.

Citation： JiangXiaofei, YeFen, ShiLi, ChenXi, ZhuWeimin, ZhangYeming. Construction and Identification of Lentiviral Vector-mediated Small Interfering RNA Targeting Human Vascular Endothelial Growth Factor A Gene. West China Medical Journal, 2016, 31(1): 1-6. doi: 10.7507/1002-0179.20160001 Copy

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2.	Sarkar N, Rück A, Källner G, et al. Effects of intramyocardial injection of phVEGF-A165 as sole therapy in patients with refractory coronary artery disease-12-month follow-up:angiogenic gene therapy[J]. J Intern Med, 2001, 250(5):373-381.
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5.	Heeschen C, Jang JJ, Weis M, et al. Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis[J]. Nat Med, 2001, 7(7):833-839.
6.	Vander ZR, Murohara T, Luo Z, et al. Vascular endothelial growth factor/vascular permeability factor augments nitric oxide release from quiescent rabbit and human vascular endothelium[J]. Circulation, 1997, 95(4):1030-1037.
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8.	Elbashir SM, Harborth J, Weber K, et al. Analysis of gene function in somatic mammalian cells using small interfering RNAs[J]. Methods, 2002, 26(2):199-213.
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12.	Nguyen QD, Rodrigues S, Rodrigue CM, et al. Inhibition of vascular endothelial growth factor (VEGF)-165 and semaphorin 3A-mediated cellular invasion and tumor growth by the VEGF signaling inhibitor ZD4190 in human colon cancer cells and xenografts[J]. Mol Cancer Ther, 2006, 5(8):2070-2077.
13.	Binion DG, Otterson MF, Rafiee P. Curcumin inhibits VEGF-mediated angiogenesis in human intestinal microvascular endothelial cells through COX-2 and MAPK inhibition[J]. Gut, 2008, 57(11):1509-1517.
14.	Rad FH, Le BH, Paturance S, et al. VEGF kinoid vaccine, a therapeutic approach against tumor angiogenesis and metastases[J]. Proc Natl Acad Sci USA, 2007, 104(8):2837-2842.
15.	Folkman J. Therapeutic angiogenesis in ischemic limbs[J]. Circulation, 1998, 97(12):1114-1123.
16.	Henry TD, Annex BH, McKendall GR, et al. The VIVA trial:vascular endothelial growth factor in ischemia for vascular angiogenesis[J]. Circulation, 2003, 18, 107(10):1359-1365.
17.	Hedman M, Hartikainen J, Syvanne M, et al. Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia:phase Ⅱ results of the Kuopio Angiogenesis Trial (KAT)[J]. Circulation, 2003, 107(21):2677-2683.
18.	Sarkar N, Rück A, Källner G, et al. Effects of intramyocardial injection of phVEGF-A165 as sole therapy in patients with refractory coronary artery disease-12-month follow-up:angiogenic gene therapy[J]. J Intern Med, 2001, 250(5):373-381.
19.	Henry TD, Rocha SK, Isner JM, et al. Results of intracoronary recombinant human vascular endothelial growth factor (rhVEGF) administration trial[J]. J Am Coll Cardiol, 1998, 31(Suppl A):65A.
20.	Laitinen M, Hartikainen J, Hiltunen MO, et al. Catheter-mediated vascular endothelial growth factor gene transfer to human coronary arteries after angioplasty[J]. Hum Gene Ther, 2000, 11(2):263-270.
21.	李丽, 张日, 岑建农. VEGF基因RNAi慢病毒载体的构建与鉴定[J].苏州大学学报:医学版, 2008, 28(1):20-22.

1. Carmeliet P. Angiogenesis in health and disease[J]. Nat Med, 2003, 9(6):653-660.
2. Sarkar N, Rück A, Källner G, et al. Effects of intramyocardial injection of phVEGF-A165 as sole therapy in patients with refractory coronary artery disease-12-month follow-up:angiogenic gene therapy[J]. J Intern Med, 2001, 250(5):373-381.
3. Vale PR, Losordo DW, Milliken CE et al. Left ventricular electromechanical mapping to assess efficacy of phVEGF (165) gene transfer for therapeutic angiogenesis in chronic myocardial ischemia[J]. Circulation, 2000, 102(9):965-974.
4. Celletti FL, Hilfiker PR, Ghafouri P, et al. Effect of human recombinant vascular endothelial growth factor165 on progression of atherosclerotic plaque[J]. J Am Coll Cardiol, 2001, 37(8):2126-2130.
5. Heeschen C, Jang JJ, Weis M, et al. Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis[J]. Nat Med, 2001, 7(7):833-839.
6. Vander ZR, Murohara T, Luo Z, et al. Vascular endothelial growth factor/vascular permeability factor augments nitric oxide release from quiescent rabbit and human vascular endothelium[J]. Circulation, 1997, 95(4):1030-1037.
7. Wang SC, Liu H, Ren LF, et al. Carcinoma growth and metastasis by blocking the expression of VEGF using RNA interference[J]. Neoplasia, 2008, 10(4):399-407.
8. Elbashir SM, Harborth J, Weber K, et al. Analysis of gene function in somatic mammalian cells using small interfering RNAs[J]. Methods, 2002, 26(2):199-213.
9. Xia XB, Xiong SQ, Song WT, et al. Inhibition of retinal neovascularization by siRNA targeting VEGF165[J]. Mol Vis, 2008, 14(5):1965-1973.
10. Jayson GC, Mulatero C, Ranson M, et al. Phase I investigation of recombinant anti-human vascular endothelial growth factor antibody in patients with advanced cancer[J]. Eur J Cancer, 2005, 41(4):555-563.
11. Fan X, Krieg S, Kuo CJ, et al. VEGF blockade inhibits angiogenesis and reepithelialization of endometrium[J]. FASEB J, 2008, 22(10):3571-3580.
12. Nguyen QD, Rodrigues S, Rodrigue CM, et al. Inhibition of vascular endothelial growth factor (VEGF)-165 and semaphorin 3A-mediated cellular invasion and tumor growth by the VEGF signaling inhibitor ZD4190 in human colon cancer cells and xenografts[J]. Mol Cancer Ther, 2006, 5(8):2070-2077.
13. Binion DG, Otterson MF, Rafiee P. Curcumin inhibits VEGF-mediated angiogenesis in human intestinal microvascular endothelial cells through COX-2 and MAPK inhibition[J]. Gut, 2008, 57(11):1509-1517.
14. Rad FH, Le BH, Paturance S, et al. VEGF kinoid vaccine, a therapeutic approach against tumor angiogenesis and metastases[J]. Proc Natl Acad Sci USA, 2007, 104(8):2837-2842.
15. Folkman J. Therapeutic angiogenesis in ischemic limbs[J]. Circulation, 1998, 97(12):1114-1123.
16. Henry TD, Annex BH, McKendall GR, et al. The VIVA trial:vascular endothelial growth factor in ischemia for vascular angiogenesis[J]. Circulation, 2003, 18, 107(10):1359-1365.
17. Hedman M, Hartikainen J, Syvanne M, et al. Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia:phase Ⅱ results of the Kuopio Angiogenesis Trial (KAT)[J]. Circulation, 2003, 107(21):2677-2683.
18. Sarkar N, Rück A, Källner G, et al. Effects of intramyocardial injection of phVEGF-A165 as sole therapy in patients with refractory coronary artery disease-12-month follow-up:angiogenic gene therapy[J]. J Intern Med, 2001, 250(5):373-381.
19. Henry TD, Rocha SK, Isner JM, et al. Results of intracoronary recombinant human vascular endothelial growth factor (rhVEGF) administration trial[J]. J Am Coll Cardiol, 1998, 31(Suppl A):65A.
20. Laitinen M, Hartikainen J, Hiltunen MO, et al. Catheter-mediated vascular endothelial growth factor gene transfer to human coronary arteries after angioplasty[J]. Hum Gene Ther, 2000, 11(2):263-270.
21. 李丽, 张日, 岑建农. VEGF基因RNAi慢病毒载体的构建与鉴定[J].苏州大学学报:医学版, 2008, 28(1):20-22.

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