SLOWING THE GROWTH OF AN AGGRESSIVE GLIOBLASTOMA CELL LINE BY ENHANCER RNA SILENCING DEPENDS ON THE KNOCKDOWN METHOD

Abstract

Glioblastoma is the most frequent and aggressive brain tumor, with a low survival rate. One of the challenges in treating glioblastoma is the resistance of cells to temozolomide, the main chemical agent used to fight the disease. The STAT3 gene is known to play a significant role in the growth and metastasis of tumor cells. Inhibition of STAT3 or suppression of its expression reduces the resistance of glioblastoma cells to temozolomide. Enhancer RNAs (eRNAs) are non-coding RNAs that are transcribed from enhancer regions. eRNAs can affect the expression of key genes of various biological processes, including oncogenes. Previously, we conducted a search for an eRNAs that may potentially affect glioblastoma cell resistance to temozolomide. Knockdown of TMZR1-eRNA (ENSG00000289579) in DBTRG-05MG cell line resulted in decreased STAT3 gene expression and increased cell sensitivity to temozolomide. The effect of enhancer RNA knockdown on STAT3 gene promoter activity has been shown both alone and together with the enhancer from which this eRNA is transcribed. The current study was designed to investigate the potency of TMZR1-eRNA in a glioblastoma cell line U-251. Compared to the previously studied cell line DBTRG-05MG, the U-251 cell line is more aggressive, has a higher proliferation rate and was derived from patient tissue at a later stage of the disease. Knockdown of TMZR1-eRNA by small interfering RNAs resulted in a decrease in STAT3 gene expression. No effect on cell growth was observed both when temozolomide was added and in control conditions of culture medium with DMSO. To study the effect of constitutive suppression of eRNAs, U-251 cell cultures expressing short hairpin RNA (shRNA) targeting TMZR1-eRNA and control vector were transduced. A significant increase in STAT3 mRNA expression was observed in the obtained cell lines relative to the control. At the same time, viability analysis demonstrated a significant slowdown in the growth rate of the cell line expressing short hairpin RNAs to TMZR1-eRNA compared to the control. A significant effect was observed both when temozolomide was added and in culture medium with DMSO.

1. Singh N., Miner A., Hennis L., Mittal S. Mechanisms of temozolomide resistance in glioblastoma - a comprehensive review. Cancer Drug Resist. 2021, 4(1):17-43.

2. Fu W., Hou X., Dong L., Hou W. Roles of STAT3 in the pathogenesis and treatment of glioblastoma. Front Cell Dev Biol. 2023, 11:1098482.

3. Stasevich E.M., Murashko M.M., Zinevich L.S., Demin D.E., Schwartz A.M. The Role of Non-Coding RNAs in the Regulation of the Proto-Oncogene MYC in Different Types of Cancer. Biomedicines. 2021, 9(8):921.

4. Murashko M.M., Stasevich E.M., Schwartz A.M., Kuprash D.V., Uvarova A.N., Demin D.E. The Role of RNA in DNA Breaks, Repair and Chromosomal Rearrangements. Biomolecules. 2021, 11(4):550.

5. Stasevich E.M., Simonova A.V., Bogomolova E.A., Murashko M.M., Uvarova A.N., Zheremyan E.A., Korneev K.V., Schwartz A.M., Kuprash D.V., Demin D.E. Cut from the same cloth: RNAs transcribed from regulatory elements. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms, 2024, 195049.

6. Stasevich E.M, Uvarova A.N, Murashko M.M., Khabusheva E.R., Sheetikov S.A., Prassolov V.S., Kuprash D.V., Demin D.E., Schwartz A.M. Enhancer RNA AL928768.3 from the IGH Locus Regulates MYC Expression and Controls the Proliferation and Chemoresistance of Burkitt Lymphoma Cells with IGH/MYC Translocation. Int J Mol Sci. 2022, 23(9):4624.

7. Stasevich E.M., Simonova A.V. and Poteryakhina A.V., Bogomolova E. A., Uvarova A.N., Zheremyan E.A., Korneev K.V., Schwartz A.M. Kuprash D.V., Demin D.E. Enhancer Rna from Stat3 Locus Affects Temozolomide Chemoresistance of Glioblastoma Cells. 2024.

8. Fishilevich S., Nudel R., Rappaport N., Hadar R., Plaschkes I., Iny Stein T., Rosen N., Kohn A., Twik M., Safran M., Lancet D., Cohen D. GeneHancer: genome-wide integration of enhancers and target genes in GeneCards. Database (Oxford). 2017, 2017:bax028.

9. Kruse C.A., Mitchell D.H., Kleinschmidt-DeMasters B.K., Franklin W.A., Morse H.G., Spector E.B., Lillehei K.O. Characterization of a continuous human glioma cell line DBTRG-05MG: growth kinetics, karyotype, receptor expression, and tumor suppressor gene analyses. In Vitro Cell Dev Biol. 1992, 28A(9-10):609-14.

10. Kałuzińska-Kołat Ż., Kołat D., Kośla K., Płuciennik E., Bednarek A.K. Molecular landscapes of glioblastoma cell lines revealed a group of patients that do not benefit from WWOX tumor suppressor expression. Front Neurosci. 2023, 17:1260409.

11. Chumakov S.P., Kravchenko J.E., Prassolov V.S., Frolova E.I., Chumakov P.M. Efficient downregulation of multiple mRNA targets with a single shRNA-expressing lentiviral vector. Plasmid. 2010, 63(3):143-9.

12. Bravo-Estupiñan D.M., Aguilar-Guerrero K., Quirós S., Acón MS, Marín-Müller C., Ibáñez-Hernández M., Mora-Rodríguez R.A. Gene dosage compensation: Origins, criteria to identify compensated genes, and mechanisms including sensor loops as an emerging systems-level property in cancer. Cancer Med. 2023, 12(24):22130-22155.

13. Tolomeo M., Cascio A. The Multifaced Role of STAT3 in Cancer and Its Implication for Anticancer Therapy. Int J Mol Sci. 2021, 22(2):603.