Abstract
Objectives
Synthetic biology is primarily an emerging research field that consists of designing new synthetic gene circuits dedicated to targeted functions and therapies such as cancer therapy. In this study, a genetic logic NOT-IF gate is used to reduce the multidrug resistance and facilitate the malignant cancer therapy. MCF7 cancer cells were cultured in RPMI-1640 medium and transfected with lentiviral vectors including MDR1 gene and the corresponding shRNA against MDR1 with controllable promoters. Transcript levels and protein levels of MDR1 gene were quantified.
Results
Our results showed that when doxycycline (DOX) and sodium butyrate were present and IPTG was absent, these led to a 74,354-fold increase in MDR1 gene expression. Upon IPTG treatment, the MDR1 gene expression was not detected due to the lack of the inducer. In addition, following IPTG induction in the presence of DOX and sodium butyrate and expressing shRNA, there was a 75% reduction in MDR1 gene expression compared to those cells treated only with sodium butyrate and DOX.
Conclusions
We successfully designed and implemented the genetic logic NOT-IF gate at the transcriptional level using the inducible expression of both MDR1 drug resistance pump and its specific shRNA in MCF7 cancer cells, using the third generation lentiviral vectors.
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References
Amos M (2012) A reconfigurable NAND/NOR genetic logic gate. BMC Syst Biol 6:126. https://doi.org/10.1186/1752-0509-6-126
Amos M, Goñi-Moreno A (2018) Cellular computing and synthetic biology. In: Stepney S, Rasmussen S, Amos M (eds) Computational matter. Natural computing series. Springer, Cham, pp 93–110
Ausländer S, Ausländer D, Müller M, Wieland M, Fussenegger M (2012) Programmable single-cell mammalian biocomputers. Nature 487:123–127. https://doi.org/10.1038/nature11149
Ausländer S, Ausländer D, Fussenegger M (2017) Synthetic biology the synthesis of biology. Angew Chem Int Ed Engl 56:6396–6419. https://doi.org/10.1002/anie.201609229
Canton B, Labno A, Endy D (2008) Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26:787–793. https://doi.org/10.1038/nbt1413
Chownk M, Thakur K, Purohit A, Vashisht A, Kumar S (2019) Applications and future perspectives of synthetic biology systems. In: Singh SP, Pandey A, Du G, Kumar S (eds) Current developments in biotechnology and bioengineering. Elsevier, Amsterdam, pp 393–412. https://doi.org/10.1016/B978-0-444-64085-7.00016-2
Coley HM (2009) Mechanisms and consequences of chemotherapy resistance in breast cancer. Eur J Cancer. https://doi.org/10.1016/S1359-6349(09)70003-5
Frezza BM, Cockroft SL, Ghadiri MR (2007) Modular multi-level circuits from immobilized DNA-based logic gates. J Am Chem Soc 129:14875–14879. https://doi.org/10.1021/ja0710149
Kramer BP, Fischer C, Fussenegger M (2004) BioLogic gates enable logical transcription control in mammalian cells. Biotechnol Bioeng 87:478–484. https://doi.org/10.1002/bit.20142
Liu Y, Zeng Y, Liu L, Zhuang C, Fu X, Huang W, Cai Z (2014) Synthesizing AND gate genetic circuits based on CRISPR-Cas9 for identification of bladder cancer cells. Nat Commun 5:5393. https://doi.org/10.1038/ncomms6393
Liu Y, Beyer A, Aebersold R (2016) On the dependency of cellular protein levels on mRNA abundance. Cell 165:535–550. https://doi.org/10.1016/j.cell.2016.03.014
Madec M, Gendrault Y, Lallement C, Haiech J (2010) Design methodology and modeling of synthetic biosystems. Int J Microelectron Comput Sci 1:147–155
Manzoni R, Urrios A, Velazquez-Garcia S, de Nadal E, Posas F (2016) Synthetic biology: insights into biological computation. Integr Biol 8:518–532. https://doi.org/10.1039/C5IB00274E
Moon TS, Lou C, Tamsir A, Stanton BC, Voigt CA (2012) Genetic programs constructed from layered logic gates in single cells. Nature 491:249–253. https://doi.org/10.1038/nature11516
Pasotti L, Quattrocelli M, Galli D, De Angelis MGC, Magni P (2011) Multiplexing and demultiplexing logic functions for computing signal processing tasks in synthetic biology. Biotechnol J 6:784–795. https://doi.org/10.1002/biot.201100080
Pevsner J (2015) Bioinformatics and functional genomics, 3rd edn. Wiley, Chichester
Rinaudo K, Bleris L, Maddamsetti R, Subramanian S, Weiss R, Benenson Y (2007) A universal RNAi-based logic evaluator that operates in mammalian cells. Nat Biotechnol 25:795–801. https://doi.org/10.1038/nbt1307
Ruder WC, Lu T, Collins JJ (2011) Synthetic biology moving into the clinic. Science 333:1248–1252. https://doi.org/10.1126/science.1206843
Sabolić I, Breljak D, Herak-Kramberger CM, Ljubojević M, Thévenod F (2008) Expression of multidrug resistance P-glycoprotein Mdr1 (Abcb1) in rat kidney proximal tubules is up-regulated by nephrotoxic metals. Metal Ions Biol Med 10:315–321
Wang L et al (2015) SynBioLGDB: a resource for experimentally validated logic gates in synthetic biology. Sci Rep 5:1–5. https://doi.org/10.1038/srep08090
Weiss R, Basu S, Hooshangi S, Kalmbach A, Karig D, Mehreja R, Netravali I (2003) Genetic circuit building blocks for cellular computation, communications, and signal processing. Nat Comput 2:47–84. https://doi.org/10.1023/A:1023307812034
Xie Z, Wroblewska L, Prochazka L, Weiss R, Benenson Y (2011) Multi-input RNAi-based logic circuit for identification of specific cancer cells. Science 333:1307–1311. https://doi.org/10.1126/science.1205527
Yu Y, Lowy MM, Elble RC (2016) Tet-On lentiviral transductants lose inducibility when silenced for extended intervals in mammary epithelial cells. Metab Eng Commun 3:64–67. https://doi.org/10.1016/j.meteno.2016.03.001
Zhang H, Wang J, Cai K, Jiang L, Zhou D, Yang C, Chen J, Chen D, Dou J (2012) Downregulation of gene MDR1 by shRNA to reverse multidrug-resistance of ovarian cancer A2780 cells. J Cancer Res Ther 8:226–231. https://doi.org/10.4103/0973-1482.98975
Acknowledgements
The authors would like to thank the officials and staff of Shahrekord University of Medical Sciences, and all those who assisted us in carrying out this study.
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ME coordinated the study and revised the final manuscript. MEB, SAM and RH designed the vectors, transfection strategies and participated in the intellectual manuscript writing. NS was the student involving in the experiments as parts of her PhD thesis.
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Soheili, N., Eshghi, M., Emadi-Baygi, M. et al. Design and evaluation of biological gate circuits and their therapeutic applications in a model of multidrug resistant cancers. Biotechnol Lett 42, 1419–1429 (2020). https://doi.org/10.1007/s10529-020-02851-y
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DOI: https://doi.org/10.1007/s10529-020-02851-y