ALTERNATIVE SPLICING OF mRNA TRAIL REGULATES APOPTOSIS IN THE GLIOBLASTOMA MULTIFORME T-98G CELL LINE
Keywords:Pro-apoptotic, mRNA isoform, TRAIL, Glioblastoma multiforme
Objective: This is an in vitro experimental study designed to analyze the role of alternative splicing of mRNA in the apoptotic process of the cancer cells. Here we induced apoptosis in the glioblastoma multiforme (GBM) T-98G cell line to obtain a better understanding in the regulation of mRNA expression of the soluble Tumor Necrosis factor-related Apoptosis-Inducing Ligand (sTRAIL) gene.
Methods: Cells were induced to undergo apoptosis by treatment with rotenone at 10, 20 and 40 µM for 6 h. Dimethylsulphoxide (DMSO) was used to dissolve rotenone and as a negative control. The morphology of the GBM-T98G cells was viewed with an inverted microscope. DNA, RNA and protein extractions were performed to analyse apoptotic DNA fragmentation by a DNA laddering assay, a quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) for TRAIL mRNA expression and ELISA for caspase-9 protein expression. Electrophoresis was also performed on TRAIL complementary DNA (cDNA) produced from TRAIL qRT-PCR mRNA.
Results: Nucleosomal DNA degradation was confirmed by DNA laddering, whereas the TRAIL melting curve and the cDNA electrophoresis showed a shift in the balance of the TRAIL mRNA isoform to the pro-apoptotic mRNA isoform, in conjunction with a significant increase in expression of TRAIL mRNA and caspase-9 protein.
Conclusion: These findings indicate the regulation of apoptotic events at the level of TRAIL mRNA expression, as indicated by the shift in the balance of mRNA expression of the TRAIL isoform towards the pro-apoptotic isoform.
Davis, Mary. Glioblastoma: an overview of disease and treatment. Clin J Oncol Nursing 2016;20:S2–8.
Department of Medical Oncology, Centro Hospitalar de Sao Joao, Porto, Portugal, Catarina Fernandes, Andreia Costa, et al. Current standards of care in glioblastoma therapy. In: Glioblastoma. edited by department of neurosurgery, university hospitals leuven, Leuven, Belgium and Steven De Vleeschouwer, Codon Publications; 2017. p. 197–241.
Li Nianyu, Kathy Ragheb, Gretchen Lawler, Jennie Sturgis, Bartek Rajwa, J Andres Melendez, et al. Mitochondrial complex i inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production J Biol Chem 2003;278:8516–25.
Heinz, Sabrina, Alexius Freyberger, Bettina Lawrenz, Ludwig Schladt, Gabriele Schmuck, et al. Mechanistic Investigations of the mitochondrial complex i inhibitor rotenone in the context of pharmacological and safety evaluation. Scientific Reports 2017;7:454-65.
Wong, Rebecca SY. Apoptosis in cancer: from pathogenesis to treatment. J Exp Clin Cancer Res 2011;30:87.
Iglesias Guimarais, Victoria, Estel Gil Guinon, Gisela Gabernet, Merce Garcia-Belinchon, Maria Sanchez Osuna, et al. Apoptotic DNA degradation into oligonucleosomal fragments, but not apoptotic nuclear morphology, relies on a cytosolic pool of DFF40/CAD endonuclease. J Biol Chem 2012;287:7766–79.
Spano, Carlotta, Giulia Grisendi, Giulia Golinelli, Filippo Rossignoli, Malvina Prapa, et al. Soluble TRAIL armed human MSC as gene therapy for pancreatic cancer. Scientific Reports 2019;9:1788.
Allen, Joshua E, Wafik S El-Deiry. Regulation of the human TRAIL gene. Cancer Biol Ther 2012;13:1143–51.
Sheridan JP. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 1997;277:818–21.
Naimi, Adel, Ali Akbar Movassaghpour, Majid Farshdousti Hagh, Mehdi Talebi, Atefeh Entezari, et al. TNF-related apoptosis-inducing ligand (TRAIL) as the potential therapeutic target in hematological malignancies. Biomed Pharmacother 2018;98:566–76.
Wu, Gen Sheng. TRAIL as a target in anti-cancer therapy. Cancer Lett 2009;285:1–5.
Mizikar, Alisa. Encyclopedia of cancer (3rd Edition) 2013. 21 Edited by Manfred Schwab. Encyclopedia of Cancer (3rd Edition). Berlin: Springer; 2011.
Krieg A, T Krieg, M Wenzel, M Schmitt, U Ramp, B Fang, et al. TRAIL-β and TRAIL-γ: two novel splice variants of the human TNF-related apoptosis-inducing ligand (TRAIL) without apoptotic potential. Br J Cancer 2003;88:918–27.
Krohn, Randall I. The colorimetric detection and quantitation of total protein. In: Current Protocols in Cell Biology. Edited. Juan S. Bonifacino, Mary Dasso, Joe B. Harford, Jennifer Lippincott-Schwartz, and Kenneth M. Yamada, cba03hs52. Hoboken, NJ, USA: John Wiley and Sons, Inc; 2011.
Livak, Kenneth J, Thomas D Schmittgen. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 2001;25:402–8.
Le Kai-qin, Bellur S Prabhakar, Wan-jin Hong, Liang cheng Li. Alternative splicing as a biomarker and potential target for drug discovery. Acta Pharmacologica Sinica 2015;36:1212–8.
Huang, Ying, Xiang Yang, Tianrui Xu, Qinghong Kong, Yaping Zhang, et al. Overcoming resistance to TRAIL-Induced apoptosis in solid tumor cells by simultaneously targeting death receptors, c-FLIP and IAPs. Int J Oncol 2016;49:153–63.
Dubuisson, Agathe, Cecile Favreau, Eric Fourmaux, Sabrina Lareure, Rafael Rodrigues Saraiva, et al. Generation and characterization of novel anti-DR4 and anti-DR5 antibodies developed by genetic immunization. Cell Death Disease 2019;10:101.
Wang, Shulin, Wafik S El-Deiry. TRAIL and apoptosis induction by TNF-family death receptors. Oncogene 2003;22:8628–33.