Production of Lentiviral Vector Expressing MicroRNA-148b

Document Type : Original Article (s)

Authors

1 PhD Candidate, Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

2 Professor, Biotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

3 Assistant Professor, Medical Genetics Research Center AND Department of Medical Genetics, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

Abstract

Background: Micro (mi)RNAs are non-coding endogenous RNAs which regulate gene expression by hybridization to specific binding sites in target mRNA sequences. Since several miRNAs are involved in proliferation and differentiation, miRNA-based therapies could be promising approach in regenerative medicine. Among different vehicles, lentiviral vector system is suitable for miRNA delivery. Besides, it is shown that miRNA-148b is involved in osteogenic differentiation. In this study, designing and cloning of miR-148b to lentiviral vector were investigated.Methods: We introduced miRNA-148b-3p/-5p into lentiviral vector through cloning producers. The sequences of lentiviral vectors carrying miRNA-148b were checked via analytical digestion as well as Sanger DNA sequencing. In the following, produced lentiviral vectors were used for mesenchymal stem cells transduction.Findings: Designed miR-148b-3p/-5p successfully cloned to the shuttle. Correctness and absence of any unintended mutations of lentiviral shuttle carrying miRNA-148b3p/-5p were confirmed followed by lentiviral production. Expression of enhanced green fluorescent protein (eGFP) demonstrated high efficiency of transfection as well as transduction.Conclusion: Viral vectors constructed in this study could be used for investigation of osteogenesis.

Keywords


  1. References
  2. Broderick JA, Zamore PD. MicroRNA therapeutics. Gene Ther 2011; 18(12): 1104-10.
  3. Collino F, Bruno S, Deregibus MC, Tetta C, Camussi G. MicroRNAs and mesenchymal stem cells. Vitam Horm 2011; 87: 291-320.
  4. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005; 120(1): 15-20.
  5. Taipaleenmaki H, Bjerre HL, Chen L, Kauppinen S, Kassem M. Mechanisms in endocrinology: micro-RNAs: Targets for enhancing osteoblast differentiation and bone formation. Eur J Endocrinol 2012; 166(3): 359-71.
  6. Hassan MQ, Gordon JA, Beloti MM, Croce CM, van Wijnen AJ, Stein JL, et al. A network connecting Runx2, SATB2, and the miR-23a~27a~24-2 cluster regulates the osteoblast differentiation program. Proc Natl Acad Sci USA 2010; 107(46): 19879-84.
  7. Dong S, Yang B, Guo H, Kang F. MicroRNAs regulate osteogenesis and chondrogenesis. Biochem Biophys Res Commun 2012; 418(4): 587-91.
  8. Kim KM, Lim SK. Role of miRNAs in bone and their potential as therapeutic targets. Curr Opin Pharmacol 2014; 16: 133-41.
  9. Lian JB, Stein GS, van Wijnen AJ, Stein JL, Hassan MQ, Gaur T, et al. MicroRNA control of bone formation and homeostasis. Nat Rev Endocrinol 2012; 8(4): 212-27.
  10. Kapinas K, Delany AM. MicroRNA biogenesis and regulation of bone remodeling. Arthritis Res Ther 2011; 13(3): 220.
  11. Zhang Y, Wang Z, Gemeinhart RA. Progress in microRNA delivery. J Control Release 2013; 172(3): 962-74.
  12. Singer O, Verma IM. Applications of lentiviral vectors for shRNA delivery and transgenesis. Curr Gene Ther 2008; 8(6): 483-8.
  13. Segura MM, Mangion M, Gaillet B, Garnier A. New developments in lentiviral vector design, production and purification. Expert Opin Biol Ther 2013; 13(7): 987-1011.
  14. Schoolmeesters A, Eklund T, Leake D, Vermeulen A, Smith Q, Force AS, et al. Functional profiling reveals critical role for miRNA in differentiation of human mesenchymal stem cells. PLoS One 2009; 4(5): e5605.
  15. Zavar-Reza J, Khaleghi N, Hatami A, Heidari M, Mansuri-Majumerd R, Shekhha MH, et al. Cloning and expression of truncated protein of epidermal growth factor-1 (EGFR-1) in Pichia pastoris yeast host. J Isfahan Med Sch 2016; 33(364): 2232-8. [In Persian].
  16. Song H, Yang PC. Construction of shRNA lentiviral vector. N Am J Med Sci 2010; 2(12): 598-601.
  17. Manjunath N, Wu H, Subramanya S, Shankar P. Lentiviral delivery of short hairpin RNAs. Adv Drug Deliv Rev 2009; 61(9): 732-45.
  18. Chen Y, Song YX, Wang ZN. The microRNA-148/152 family: multi-faceted players. Mol Cancer 2013; 12: 43.
  19. Song YX, Yue ZY, Wang ZN, Xu YY, Luo Y, Xu HM, et al. MicroRNA-148b is frequently down-regulated in gastric cancer and acts as a tumor suppressor by inhibiting cell proliferation. Mol Cancer 2011; 10: 1.
  20. Weilner S, Grillari-Voglauer R, Redl H, Grillari J, Nau T. The role of microRNAs in cellular senescence and age-related conditions of cartilage and bone. Acta Orthop 2015; 86(1): 92-9.