ALTERNATIVE SPLICING OF MRNA TRAIL REGULATES APOPTOSIS IN THE GLIOBLASTOMA MULTIFORME T-98G CELL LINE
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.
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