Volume 23, Issue 2 (2020)
mjms 2020, 23(2): 67-74 |
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Shafaati M, Jamalidoust M, Kargar M, Arefian E, Kafilzadeh F. Luciferase Assay for Demonstrate the Competence of Selective MicroRNA of let-7b in Suppressing HCV Replication. mjms 2020; 23 (2) :67-74
URL: http://mjms.modares.ac.ir/article-30-41254-en.html
URL: http://mjms.modares.ac.ir/article-30-41254-en.html
1- Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran
2- Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran ,mjamalidoust@gmail.com
3- Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran
2- Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran ,
3- Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran
Abstract: (2339 Views)
Aims: The development of new antiviral agents is an appropriate approach to eradicate hepatitis C infection. Due to the lack of suitable animal models, there is always a barrier to the proper evaluation of antiviral compounds in vivo. The growing attention to microRNAs is a new strength in antiviral therapy. The aim of the present study was to use luciferase assay to confirm the specific interaction between miRNA and genomic RNA of hepatitis C virus (HCV) genotype 1b to suppress the replication of the virus.
Materials & Methods: The NS5B genomic fragments of the HCV genome were sub-cloned into the psiCHECK-2-TM vector as MRE. The relative expression of lentivirus vectors expressing miRNAs in Huh7.5 cells was assessed through fluorescence microscopy and real-time PCR. The lentivirus expressing let-7b was transduced to Huh7.5 cells. The NS5B-psiCHECK-2-TM (MRE) was transfected to the Huh7.5 cell. The relative expression of luciferase was measured using a luciferase dual assay kit.
Findings: With the use of lentiviruses expressing let-7b, high and permanent expression of let-7b was created in the target cell. On the other hand, the specific attachment of the responsive sequence (NS5B) to the microRNA of let-7b was shown by decreasing luciferase light.
Materials & Methods: The NS5B genomic fragments of the HCV genome were sub-cloned into the psiCHECK-2-TM vector as MRE. The relative expression of lentivirus vectors expressing miRNAs in Huh7.5 cells was assessed through fluorescence microscopy and real-time PCR. The lentivirus expressing let-7b was transduced to Huh7.5 cells. The NS5B-psiCHECK-2-TM (MRE) was transfected to the Huh7.5 cell. The relative expression of luciferase was measured using a luciferase dual assay kit.
Findings: With the use of lentiviruses expressing let-7b, high and permanent expression of let-7b was created in the target cell. On the other hand, the specific attachment of the responsive sequence (NS5B) to the microRNA of let-7b was shown by decreasing luciferase light.
Conclusion: Lentiviral vectors are used to maintain high and stable expression of microRNAs in cells. The use of luciferase assay is one of the most appropriate methods to confirm the interaction between miRNA-mRNA that can be used for other viral genes with different microRNAs.
References
1. Binkowski B, Fan F, Wood K. Engineered luciferases for molecular sensing in living cells. Curr Opin Biotechnol. 2009;20(1):14-8. [Link] [DOI:10.1016/j.copbio.2009.02.013]
2. Wang L, Fu Q, Dong Y, Zhou Y, Jia Sh, Du J, et al. Bioluminescence imaging of Hepatitis C virus NS3/4A serine protease activity in cells and living animals. Antivir Res. 2010;87(1):50-6. [Link] [DOI:10.1016/j.antiviral.2010.04.010]
3. Moradpour D, Penin F, Rice CM. Replication of hepatitis C virus. Nat Rev Microbiol. 2007;5(6):453-63. [Link] [DOI:10.1038/nrmicro1645]
4. Murakami Y, Aly HH, Tajima A, Inoue I, Shimotohno K. Regulation of the hepatitis C virus genome replication by miR-199a. J Hepatol. 2009;50(3):453-60. [Link] [DOI:10.1016/j.jhep.2008.06.010]
5. Pockros PJ. New direct-acting antivirals in the development for hepatitis C virus infection. Ther Adv Gastroenterol. 2010;3(3):191-202. [Link] [DOI:10.1177/1756283X10363055]
6. Mortazavi M, Hosseinkhani S. Design of thermostable luciferases through arginine saturation in solvent-exposed loops. Protein Eng Des Sel. 2011;24(12):893-903. [Link] [DOI:10.1093/protein/gzr051]
7. Kozomara A, Griffiths-Jones S. miRBase: Integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res. 2010;39(suppl-1):D152-7. [Link] [DOI:10.1093/nar/gkq1027]
8. Lanford RE, Hildebrandt-Eriksen ES, Petri A, Persson R, Lindow M, Munk ME, et al. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science. 2010;327(5962):198-201. [Link] [DOI:10.1126/science.1178178]
9. Deiters A. Small molecule modifiers of the microRNA and RNA interference pathway. AAPS J. 2010;12(1):51-60. [Link] [DOI:10.1208/s12248-009-9159-3]
10. Li L, Xu J, Yang D, Tan X, Wang H. Computational approaches for microRNA studies: A review. Mamm Genome. 2010;21(1-2):1-12. [Link] [DOI:10.1007/s00335-009-9241-2]
11. Shrivastava Sh, Steele R, Ray R, Ray RB. MicroRNAs: Role in hepatitis C virus pathogenesis. Genes Dis. 2015;2(1):35-45. [Link] [DOI:10.1016/j.gendis.2015.01.001]
12. Connelly CM, Thomas M, Deiters A. High-throughput luciferase reporter assay for small-molecule inhibitors of microRNA function. J Biomol Screen. 2012;17(6):822-8. [Link] [DOI:10.1177/1087057112439606]
13. vita.mbc.nctu.edu.tw [Internet]. Hsinchu: National Chiao Tung University; 2006 [cited 2017 July 9]. Available from: http://vita.mbc.nctu.edu.tw [Link]
14. Cheng JC, Yeh YJ, Tseng CP, Hsu SD, Chang YL, Sakamoto N, et al. Let-7b is a novel regulator of hepatitis C virus replication. Cell Mol Life Sci. 2012;69(15):2621-33. [Link] [DOI:10.1007/s00018-012-0940-6]
15. Rougvie AE. Control of developmental timing in animals. Nat Rev Genet. 2001;2(9):690-701. [Link] [DOI:10.1038/35088566]
16. Shafaati M, Jamalidoust M, Ziyaeyan M, Kargar M, Arefian E. Liver micro-RNAs and their role in different HCV genotype infections. Pathobiol Res. 2018;20(4):1-19. [Persian] [Link]
17. Alipour BS, Hosseinkhani S, Ardestani SK, Moradi A. The effective role of positive charge saturation in bioluminescence color and thermostability of firefly luciferase. Photochem Photobiol Sci. 2009;8(6):847-55. [Link] [DOI:10.1039/b901938c]
18. Du J, Zhao F, Zhou Y, Yan H, Duan XG, Liang SQ, et al. Bioluminescence imaging allows monitoring hepatitis C virus core protein inhibitors in mice. PLoS One. 2010;5(11):e14043. [Link] [DOI:10.1371/journal.pone.0014043]
19. Gupta P, Cairns MJ, Saksena NK. Regulation of gene expression by microRNA in HCV infection and HCV-mediated hepatocellular carcinoma. Virol J. 2014;11(1):64. [Link] [DOI:10.1186/1743-422X-11-64]
20. Fukuhara T, Kambara H, Shiokawa M, Ono Ch, Katoh H, Morita E, et al. Expression of microRNA miR-122 facilitates an efficient replication in nonhepatic cells upon infection with hepatitis C virus. J Virol. 2012;86(15):7918-33. [Link] [DOI:10.1128/JVI.00567-12]
21. Wang H, Gao H, Duan Sh, Song X. Inhibition of microRNA-199a-5p reduces the replication of HCV via regulating the pro-survival pathway. Virus Res. 2015;208:7-12. [Link] [DOI:10.1016/j.virusres.2015.05.002]
22. Houdebine LM. Design of vectors for optimizing transgene expression. In: Pinkert CA, editor. Transgenic animal technology. 3rd Edition. Amsterdam: Elsevier; 2014. pp. 489-511. [Link] [DOI:10.1016/B978-0-12-410490-7.00017-7]
23. Chan CH, Yu TH, Wong KB. Stabilizing salt-bridge enhances protein thermostability by reducing the heat capacity change of unfolding. PloS One. 2011;6(6):e21624. [Link] [DOI:10.1371/journal.pone.0021624]
24. Wilson JA, Zhang C, Huys A, Richardson CD. Human Ago2 is required for efficient microRNA 122 regulation of hepatitis C virus RNA accumulation and translation. J Virol. 2011;85(5):2342-50. [Link] [DOI:10.1128/JVI.02046-10]
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