Abstract
Conventional approaches for studying and molecular typing of tumors include PCR, blotting, omics, immunocytochemistry, and immunohistochemistry. The last two methods are the most used, as they enable detecting both tumor protein markers and their localizations within the cells. In this study, we have investigated a possibility of using RNA aptamers, in particular, 2′-F-pyrimidyl-RNA aptamer ME07 (48 nucleotides long), specific to the receptor of epidermal growth factor (EGFR, ErbB1, Her1), as an alternative to monoclonal antibodies for aptacytochemistry and aptahistochemistry for human glioblastoma multiforme (GBM). A specificity of binding of FAM-ME07 to the receptor on the tumor cells has been demonstrated by flow cytometry; an apparent dissociation constant for the complex of aptamer – EGFR on the cell has been determined; a number of EGFR molecules has been semi-quantitatively estimated for the tumor cell lines having different amount of EGFR: A431 (106 copies per cell), U87 (104 copies per cell), MCF7 (103 copies per cell), and ROZH, primary GBM cell culture derived from patient (104 copies per cell). According to fluorescence microscopy, FAM-ME07 interacts directly with the receptors on A431 cells, followed by its internalization into the cytoplasm and translocation to the nucleolus; this finding opens a possibility of ME07 application as an escort aptamer for a delivery of therapeutic agents into tumor cells. FAM-ME07 efficiently stains sections of GBM clinical specimens, which enables an identification of EGFR-positive clones within a heterogeneous tumor; and providing a potential for further studying animal models of GBM.
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Abbreviations
- 2′-F-Py:
-
2′-fluoropyrimidine
- A431:
-
human epidermoid carcinoma cell line
- aK D :
-
apparent dissociation constant
- EGFR:
-
receptor of epidermal growth factor
- FAM:
-
5(6)-carboxyfluorescein
- GBM:
-
human glioblastoma multiforme
- MCF7:
-
human luminal breast adenocarcinoma cell line
- MoRE:
-
molecular recognition element
- U87:
-
human glioblastoma cell line
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Acknowledgments
The authors are grateful to A. A. Bisyaeva, S. F. Drozd, and M. V. Ryzhova for permanent help during this work.
Funding
This work was supported by the Ministry of Education and Science of the Russian Federation [project no. 075-15-2020-809 (13.1902.21.0030)] and by the Russian Foundation for Basic Research (project no. 18-29-01047 mk).
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The authors declare no conflict of interest in financial or in any other area. All procedures used in the studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Written consent was obtained from all participants in the study.
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Kopylov, A.M., Fab, L.V., Antipova, O. et al. RNA Aptamers for Theranostics of Glioblastoma of Human Brain. Biochemistry Moscow 86, 1012–1024 (2021). https://doi.org/10.1134/S0006297921080113
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DOI: https://doi.org/10.1134/S0006297921080113