Construction, Screening, and Verification of The Silencing Effects for Human Interference Lentiviral Vector of Inhibitor of Differentiation-1_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

 DINGRui , LIXiao ,  DOUKe-feng

Department of Hepato-Biliary and Pancreato-Splenic Surgery, The First Affiliated Hospital, The Forth Military Medical University, Xi'an 710032, Shaanxi Province, China;

Corresponding author：

DOUKe-feng, Email: gdwkgwx@fmmu.edu.cn

Keywords：

Inhibitor of differentiation-1; Interference lentiviral vector; Gene silence; HepG2 cell line; Hepatocellular carcinoma

DOI：

10.7507/1007-9424.20140358

Video：

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Objective To construct the human small interfering RNA (siRNA) lentiviral vector who targeting inhibitor of differentiation-1 (Id1) gene, and to detect its efficiency of gene silence for the HepG2 cells. Methods The most effective RNA interference sequences was screened from 4 kinds of siRNA vectors targeting Id1 gene (included pCGSIL-GFP-Id1-1, pCGSIL-GFP-Id1-2, pCGSIL-GFP-Id1-3, and pCGSIL-GFP-Id1-4), who was transfected to 293T cells. The selected siRNA vector was used to build lentiviral vector (Id1-RNAi-LV) and then infected human HepG2 cells. Then the expression levels of Id1 mRNA and its protein were detected by the real time PCR and Western blot method respectively. Results Expression level of Id1 protein in pCGSIL-GFP-Id1-4 group was lower than those of pCGSIL-GFP-Id1-1 group, pCGSIL-GFP-Id1-2 group, and pCGSIL-GFP-Id1-3 group (P < 0.05), who had the best efficiency of gene silence. The Id1-siRNA lentiviral vector (Id1-RNAi-LV) was successfully constructed by using pCGSIL-GFP-Id1-4. The titer of lentiviral was 2.0×10⁹ TU/mL.results of real time-PCR and Western blot showed that, the expression levels of Id1 mRNA and its protein in HepG2 cells of Id1-RNAi-LV group were lower than those of blank control group and negative control group (P < 0.05). Conclusions The specific lentiviral can constantly down-regulate the expression of Id1 gene.

Citation： DINGRui, LIXiao, DOUKe-feng. Construction, Screening, and Verification of The Silencing Effects for Human Interference Lentiviral Vector of Inhibitor of Differentiation-1. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2014, 21(12): 1511-1517. doi: 10.7507/1007-9424.20140358 Copy

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9.	Schoppmann SF, Schindl M, Bayer G, et al.Overexpression of Id-1 is associated with poor clinical outcome in node negative breast cancer[J].Int J Cancer, 2003, 104(6):677-682.
10.	Schindl M, Oberhuber G, Obermair A, et al.Overexpression of Id-1 protein is a marker for unfavorable prognosis in early-stage cervical cancer[J].Cancer Res, 2001, 61(15):5703-5706.
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27.	Naldini L, Blömer U, Gallay P, et al.In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector[J].Science, 1996, 272(5259):263-267.

1. Kamangar F, Dores GM, Anderson WF.Patterns of cancer incidence, mortality, and prevalence across five continents:defining priorities to reduce cancer disparities in different geographic regions of the world[J].J Clin Oncol, 2006, 24(14):2137-2150.
2. Arbuthnot P, Kew M.Hepatitis B virus and hepatocellular carcinoma[J].Int J Exp Pathol, 2001, 82(2):77-100.
3. Murakami S.Hepatitis B virus X protein:structure, function and biology[J].Intervirology, 1999, 42(2-3):81-99.
4. Murakami S.Hepatitis B virus X protein:a multifunctional viral regulator[J].J Gastroenterol, 2001, 36(10):651-660.
5. Benezra R, Davis RL, Lockshon D, et al.The protein Id:a negative regulator of helix-loop-helix DNA binding proteins[J].Cell, 1990, 61(1):49-59.
6. Wong YC, Wang X, Ling MT.Id-1 expression and cell survival[J].Apoptosis, 2004, 9(3):279-289.
7. Schindl M, Schoppmann SF, Ströbel T, et al.Level of Id-1 protein expression correlates with poor differentiation, enhanced malignant potential, and more aggressive clinical behavior of epithelial ovarian tumors[J].Clin Cancer Res, 2003, 9(2):779-785.
8. Ouyang XS, Wang X, Lee DT, et al.Over expression of ID-1 in prostate cancer[J].J Urol, 2002, 167(6):2598-2602.
9. Schoppmann SF, Schindl M, Bayer G, et al.Overexpression of Id-1 is associated with poor clinical outcome in node negative breast cancer[J].Int J Cancer, 2003, 104(6):677-682.
10. Schindl M, Oberhuber G, Obermair A, et al.Overexpression of Id-1 protein is a marker for unfavorable prognosis in early-stage cervical cancer[J].Cancer Res, 2001, 61(15):5703-5706.
11. Lin CQ, Singh J, Murata K, et al.A role for Id-1 in the aggressive phenotype and steroid hormone response of human breast cancer cells[J].Cancer Res, 2000, 60(5):1332-1340.
12. Matsuda Y, Yamagiwa S, Takamura M, et al.Overexpressed Id-1 is associated with a high risk of hepatocellular carcinoma development in patients with cirrhosis without transcriptional repression of p16[J].Cancer, 2005, 104(5):1037-1044.
13. Damdinsuren B, Nagano H, Kondo M, et al.TGF-beta1-induced cell growth arrest and partial differentiation is related to the suppression of Id1 in human hepatoma cells[J].Oncol Rep, 2006, 15(2):401-408.
14. Fellmann C, Lowe SW.Stable RNA interference rules for silencing[J].Nat Cell Biol, 2014, 16(1):10-18.
15. Nickoloff BJ, Chaturvedi V, Bacon P, et al.Id-1 delays senescence but does not immortalize keratinocytes[J].J Biol Chem, 2000, 275(36):27501-27504.
16. Tang J, Gordon GM, Müller MG, et al.Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen induces expression of the helix-loop-helix protein Id-1 in human endothelial cells[J].J Virol, 2003, 77(10):5975-5984.
17. Alani RM, Hasskarl J, Grace M, et al.Immortalization of primary human keratinocytes by the helix-loop-helix protein, Id-1[J].Proc Natl Acad Sci U S A, 1999, 96(17):9637-9641.
18. Desprez PY, Lin CQ, Thomasset N, et al.A novel pathway for mammary epithelial cell invasion induced by the helix-loop-helix protein Id-1[J].Mol Cell Biol, 1998, 18(8):4577-4588.
19. Ling MT, Lau TC, Zhou C, et al.Overexpression of Id-1 in prostate cancer cells promotes angiogenesis through the activation of vascular endothelial growth factor (VEGF)[J].Carcinogenesis, 2005, 26(10):1668-1676.
20. Cheung HW, Ling MT, Tsao SW, et al.Id-1-induced Raf/MEK pathway activation is essential for its protective role against taxol-induced apoptosis in nasopharyngeal carcinoma cells[J].Carcino-genesis, 2004, 25(6):881-887.
21. Zhang X, Ling MT, Wang X, et al.Inactivation of Id-1 in prostate cancer cells:a potential therapeutic target in inducing chemosensitization to taxol through activation of JNK pathway[J].Int J Cancer, 2006, 118(8):2072-2081.
22. Lin JC, Chang SY, Hsieh DS, et al.The association of Id-1, MIF and GSTpi with acquired drug resistance in hormone indepen-dent prostate cancer cells[J].Oncol Rep, 2005, 13(5):983-988.
23. Lee TK, Man K, Ling MT, et al.Over-expression of Id-1 induces cell proliferation in hepatocellular carcinoma through inactivation of p16INK4a/RB pathway[J].Carcinogenesis, 2003, 24(11):1729-1736.
24. Kim VN.RNA interference in functional genomics and medicine[J].J Korean Med Sci, 2003, 18(3):309-318.
25. Stewart SA, Dykxhoorn DM, Palliser D, et al.Lentivirus-delivered stable gene silencing by RNAi in primary cells[J].RNA, 2003, 9(4):493-501.
26. Schambach A, Zychlinski D, Ehrnstroem B, et al.Biosafety features of lentiviral vectors[J].Hum Gene Ther, 2013, 24(2):132-142.
27. Naldini L, Blömer U, Gallay P, et al.In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector[J].Science, 1996, 272(5259):263-267.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Construction, Screening, and Verification of The Silencing Effects for Human Interference Lentiviral Vector of Inhibitor of Differentiation-1

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