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Construction and verification of NF-κB luciferase reporter gene system

Zhilan Guo1,2,3 Luyang Che4 Jingzhe Li Zhenxiao Sun1 Changzhen Liu

(1.College of Chinese Pharmacy, Beijing University of Chinese Medicine , Beijing, China 100102)
(2.Beijing Key Laboratory of Research of Chinese Medicine on Prevention and Treatment for Major Diseases, Experimental Research Center, China Academy of Chinese Medical Sciences, Beijing, China 100102)
(3.Beijing Key Laboratory of Research of Chinese Medicine on Prevention and Treatment for Major Diseases,Experimental Research Center,China Academy of Chinese Medical Sciences, Beijing ,China 100700)
(4.Department of Orthopedics, Chinese People's Liberation Army General Hospital, Beijing, China 100853)

【Abstract】To quantify the transcriptional activity of NF-κB and to screen drugs related to the regulation of NF-κB activation, we constructed a recombinant plasmid through deleting the original CMV promoter of retrovirus vector pQCXIP and inserting the NF-κB enhancer and NanoLuc luciferase sequence into the vector. Then, using the recombinant plasmid we constructed a cell line in which the expression of NanoLuc luciferase (NLuc) was regulated by NF-κB. The inserted sequences were verified by restriction endonuclease digestion and sequencing. Tumor necrosis factor-α( TNF-α), an NF-κB activator, acted on the constructed NLuc cell line and leaded to the specific luciferase reaction. The luciferase reaction showed a fine time and dose dependence to the TNF-α stimulation, indicating the successful construction of the NF-κB regulated NLuc-expressing cell line. Besides, the NF-κB inhibitor, triptolide, reduced the expression of NLuc in a dose-dependent way. The constructed reporter system in this study could be applied in the quantification of the NF-κB transcriptional activity and in the NF-κB regulation-related drug screening.

【Keywords】 NF-κB; NanoLuc luciferase; reporter gene system; NF-κB; NanoLuc luciferase; reporter gene system;

【DOI】

【Funds】 National Natural Science Foundation of China (Nos. 31170829, 81171762, 81550017, 81473418) National Natural Science Foundation of China(Nos.31170829,81171762,81550017,81473418) Research Project of CACMS (No. zz2015015) Research Project of CACMS(No.zz2015015)

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    References

    [1]Su JD, Wu LF. Relationship between nuclear factor-kappa B and cell apoptosis. World Chin J Digestol, 2007, 15(12): 1411–1416(in Chinese).

    [2]Gilmore TD. Introduction to NF-κB: players, pathways, perspectives. Oncogene, 2006, 25(51): 6680–6684.

    [3]Razani B, Reichardt AD, Cheng GH. Non-canonical NF-κB signaling activation and regulation: principles and perspectives. Immunol Rev, 2011, 244(1): 44–54.

    [4]Yang Y, Li JJ, Wang X, et al. Progresses on reporter gene and its application. Life Sci Res, 2011, 15(3): 277–282(in Chinese).

    [5]Gilmore TD. The Re1/NF-κB/IκB signal transduction pathway and cancer. Cancer Treat Res, 2003, 115: 241–265.

    [6]Laveti D, Kumar M, Hemalatha R, et al. Anti-inflammatory treatments for chronic diseases: a review. Inflamm Allergy Drug Targets, 2013, 12(5): 349–361.

    [7]Granic I, Dolga AM, Nijholt IM, et al. Inflammation and NF-κB in Alzheimer's disease and diabetes. J Alzheimers Dis, 2009, 16(4): 809–821.

    [8]Rico-Rosillo G, Vega-Robledo GB. The involvement of NF-κB transcription factor in asthma. Rev Alerg Méx, 2011, 58(2): 107–111.

    [9]Zubair A, Frieri M. Role of nuclear factor-κB in breast and colorectal cancer. Curr Allergy Asthma Rep, 2013, 13(1): 44–49.

    [10]Wong ET, Tergaonkar V. Roles of NF-κB in health and disease: mechanisms and therapeutic potential. Clin Sci, 2009, 116(6): 451–465.

    [11]Okamoto T. NF-κB and rheumatic diseases. Endocr Metab Immune Disord Drug Targets, 2006, 6(4): 359–372.

    [12]Hall MP, Unch J, Binkowski BF, et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol, 2012, 7(11): 1848–1857.

    [13]Park MH, Hong JT. Roles of NF-κB in cancer and inflammatory diseases and their therapeutic approaches. Cells, 2016, 5(2): 15.

    [14]Wang W, Nag SA, Zhang RW. Targeting the NF-κB signaling pathways for breast cancer prevention and therapy. Curr Med Chem, 2015, 22(2): 264–289.

    [15]Matsumoto G, Namekawa JI, Muta M, et al. Targeting of nuclear factorκB pathways by dehydroxymethylepoxyquinomicin, a novel inhibitor of breast carcinomas: antitumor and antiangiogenic potential in vivo. Clin Cancer Res, 2005, 11(3): 1287–1293.

    [16]Nishimura D, Ishikawa H, Matsumoto K, et al. DHMEQ, a novel NF-κB inhibitor, induces apoptosis and cell-cycle arrest in human hepatoma cells. Int J Oncol, 2006, 29(3): 713–719.

    [17]Poma P, Notarbartolo M, Labbozzetta M, et al. Antitumor effects of the novel NF-κB inhibitor dehydroxymethyl-epoxyquinomicin on human hepatic cancer cells: analysis of synergy with cisplatin and of possible correlation with inhibition of pro-survival genes and IL-6 production. Int J Oncol, 2006, 28(4): 923–930.

    [18]Watanabe M, Dewan MZ, Okamura T, et al. A novel NF-κB inhibitor DHMEQ selectively targets constitutive NF-κB activity and induces apoptosis of multiple myeloma cells in vitro and in vivo. Int J Cancer, 2005, 114(1): 32–38.

    [19]Pushkarev VV, Starenki DV, Pushkarev VM, et al. Inhibitor of the transcription factor NF-κB, DHMEQ, enhances the effect of paclitaxel on cells of anaplastic thyroid carcinoma in vitro and in vivo. Ukr Biochem J, 2015, 87(3): 63–74.

    [20]Togano T, Nakashima M, Watanabe M, et al. Synergistic effect of 5-azacytidine and NF-κB inhibitor DHMEQ on apoptosis induction in myeloid leukemia cells. Oncol Res, 2012, 20(12): 571–577.

    [21]Kozakai N, Kikuchi E, Hasegawa M, et al. Enhancement of radiosensitivity by a unique novel NF-κB inhibitor, DHMEQ, in prostate cancer. Br J Cancer, 2012, 107(4): 652–657.

    [22]Gupta SC, Sundaram C, Reuter S, et al. Inhibiting NF-κB activation by small molecules as a therapeutic strategy. Biochim Biophys Acta, 2010, 1799(10-12): 775–787.

    [23]Esser N, Paquot N, Scheen AJ. Anti-inflammatory agents to treat or prevent type 2 diabetes, metabolic syndrome and cardiovascular disease. Expert Opin Investig Drugs, 2015, 24(3): 283–307.

    [24]Jiang GC, Dandekar S. Targeting NF-κB signaling with protein kinase C agonists as an emerging strategy for combating HIV latency. AIDS Res Hum Retroviruses, 2015, 31(1): 4–12.

    [25]Orlando RA, Gonzales AM, Hunsaker LA, et al. Inhibition of nuclear factorκB activation and cyclooxygenase-2 expression by aqueous extracts of hispanic medicinal herbs. J Med Food, 2010, 13(4): 888–895.

    [26]Ajit D, Simonyi A, Li RT, et al. Phytochemicals and botanical extracts regulate NF-κB and Nrf2/ARE reporter activities in DI TNC1astrocytes. Neurochem Int, 2016, 97: 49–56.

This Article

ISSN:1000-3061

CN: 11-1998/Q

Vol 32, No. 10, Pages 1465-1473

October 2016

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Abstract

  • 1 Materials and methods
  • 2 Results and analysis
  • 3 Discussion
  • References