ISSN 2410-7751 (Друкована версія)
ISSN2410-776X (Електронна версія)
Biotechnologia Acta Т. 15, No. 2, 2022
P. 68-69, Bibliography 7, Engl.
UDC: 577.112.7:616
https://doi.org/10.15407/biotech15.02.068
O. Rudnytska, Y. Kulish, D. Trufanova, O. Minchenko
Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, Kyiv
Aim. In this study we investigate the impact of low doses of graphene oxide on the expression of key regulatory genes which control cell proliferation as well as microRNAs in normal human astrocytes.
Methods. The expression level of genes related to cell proliferation was studied by real-time qPCR in normal human astrocytes line NHA/TS (Cambrex Bio Science, Walkersville, MD, USA) using SYBRGreen Mix and specific for each mRNA forward and reverse primers. These astrocytes were treated with graphene oxide (1 and 4 ng/ml of medium) for 24 hrs. Graphene oxide (2 mg/ml, dispersion in water) was received from Sigma-Aldrich Chemie GmbH, Germany. Total RNA was extracted using TRIZOL reagent. For reverse transcription of mRNAs we used Thermo Scientific Verso cDNA Synthesis Kit (Germany). The values of mRNA expressions were normalized to the level of ACTB mRNA and represented as percent of control (100 %). For polyadenylation and reverse transcription of miRNAs we used Mir-X miRNA First-Strand Synthesis Kit (Takara, Japan). The expression level of microRNAs was studied by real-time qPCR using SYBRGreen Mix and specific for each miRNA forward primers and universal reverse primer. For normalization of microRNA expressions the level of U6 RNA expression was used.
Results. It was shown that the expression level of TOB1, HSPA5, EDEM1, MYBL1, and MYBL2 significantly increased in normal human astrocytes line NHA/TS, which were treated with graphene oxide (1 and 4 ng/ml of medium) for 24 hrs. Up-regulation of these genes expression was dose-dependent: bigger dose of graphene oxide (4 ng/ml of medium) introduced more significant changes in the expression of all these genes. Furthermore, bioinformatics analysis of 3?-untranslated regions of mRNA allowed identifying binding sites of microRNA: miR-19a for MYBL1, miR-143 for MYBL2 and miR-182 for TOB1. It was also shown that the expression of all these microRNA significantly down-regulated by graphene oxide, supporting the idea of both post-transcriptional and transcriptional regulation of MYBL1, MYBL2 and TOB1 gene expressions.
Conclusions. Graphene oxide significantly disturbs genome stability by up-regulation of the expression of key regulatory genes and down-regulation of microRNA.
Key words. Graphene oxide, proliferation, gene expression, microRNA, normal human astrocytes
© Palladin Institute of Biochemistry of National Academy of Sciences of Ukraine, 2022
References
1. Avitabile E., Bedognetti D., Ciofani G., Bianco A., Delogu L.G. How can nanotechnology help the fight against breast cancer? Nanoscale, 2018, 10 (25), 11719?11731. https://doi.org/10.1039/C8NR02796J
2. Backes C., Behera R.K., Bianco A., Casiraghi C., Doan H., Criado A., Galembeck F., Goldie S., Gravagnuolo A.M., Hou H.L., Kamali A.R., Kostarelos K., Kumar V., Lee W.H., Martsinovich N., Palermo V., Palma M., Pang J., Prato M, Samori P., Silvestri A., Singh S., Strano M., Wetzl C. Biomedical applications: general discussion. Faraday Discuss., 2021, 227, 245?258. https://doi.org/10.1039/D1FD90003J
3. Dash B.S., Jose G., Lu Y.J., Chen J.P. Functionalized Reduced Graphene Oxide as a Versatile Tool for Cancer Therapy. Int. J. Mol. Sci., 2021, 22, 2989. https://doi.org/10.3390/ijms22062989
4. Fadeel B., Bussy C., Merino S., V?zquez E., Flahaut E., Mouchet F. et al. Safety Assessment of Graphene-Based Materials: Focus on Human Health and the Environment. ACS Nano, 2018, 12, 10582?10620. https://doi.org/10.1021/acsnano.8b04758
5. Jia P.P., Sun T., Junaid M., Yang L., Ma Y.B., Cui Z.S., Wei D.P., Shi H.F., Pei D.S. Nanotoxicity of different sizes of graphene (G) and graphene oxide (GO) in vitro and in vivo. Environ. Pollut., 2019, 247, 595?606. https://doi.org/10.1016/j.envpol.2019.01.072
6. Li J., Zeng H., Zeng Z., Zeng Y., Xie T. Promising Graphene-Based Nanomaterials and Their Biomedical Applications and Potential Risks: A Comprehensive Review. ACS Biomater. Sci. Eng., 2021, 7, 5363?5396 https://doi.org/10.1021/acsbiomaterials.1c00875
7. Rudnytska O.V., Khita O.O., Minchenko D.O., Tsymbal D.O., Yefimova Y.V., Sliusar M.Y., Minchenko O.H. The low doses of SWCNTs exhibit a genotoxic effect on the normal human astrocytes by disrupting the functional integrity of the genome. Curr. Res. Toxicol., 2021, 2, 64?71. https://doi.org/10.1016/j.crtox.2021.02.001