The Function Of HTERT Pre-mRNA Alternative Splicing In Modulation Of Telomerase In Glioma

Objectives:Telomere and telomerase are closely related to cell senescence,apoptosis and cancerization.Human telomerase reverse transcriptase(hTERT),which serves as a catalytic subunit,is the key factor in regulating telomerase activity.The alternative splicing of mRNA plays a vital role in regulating telomerase activity.In this study,we made use of the combination between antisense oligonucleotides(AOs)and ESEs of hTERT pre-RNA so as to regulate hTERT splicing patterns,repress full-length hTERT mRNA,inhibit telomerase activity,thus affect proliferation,apoptosis and invasion of glioma cells.Methods:Part 1:Detect hTERT ASVs of six glioma cell lines and glioma tissue specimens by RT-PCR,detect telomerase activity according to TRAP,the correlation between ASVs and telomerase activity has also been taken into account.Part 2:Antisense oligonucleotides will be designed and synthesized on the basis of ESE sequences to transfect U251 glioma cells,in order to investigate the effect on structure as well as telomerase activity,telomere length of glioma cells produced by different chemical modification.Part 3:Using MTT assay,plate-clone formation,PI staining cell cycle detection,SCGE assay,DNA Ladder,Annexin V/PI staining apoptosis detection,2DMatrigel assay,3 DMatrigel assay,Transwell assay and so on,the methods were used to analyze proliferation,apoptosis and change of invasive ability after chronic treatment of U251 with AOs.Using western blot,to detect change of expression of tumor cells malignant phenotype related protein.We also observed the formation of neurospheres after chronic treatment of U87 stem cells with antisense oligonucleotide and detected telomerase activity and telomere length of U87 stem cells by TRAP and TRF method.Results:Part 1:hTERT ASVs expressed in both glioma cells and tissues.A correlation existed between telomerase activity and amount of expression of full-length hTERT mRNA,but the total hTERT mRNA had nothing to do with telomerase activity.Part 2:The designed 2'-O-methyl thio-phosphate oligonucleotides could regulate splicing of hTERT pre-mRNA to induce an exon skipping leading to a deletion of exon 7 and exon 8,so that telomerase activity of U251 was repressed.Part 3:After long term treatment with AOs,U251 telomerase activity decreased,telomere length shortens,and the proliferating activity is inhibited,cell apoptosis event increased significantly,invasive ability markedly decreased,expression of tumor cells malignant phenotype related protein was significantly lower than before.The neurospheres after chronic treatment of U87 stem cells with antisense oligonucleotide was poorly formatted,meanwhile telomerase activity decreased,telomere length shortened.Conclusion:1.Only proteins produced by transcription of full-length hTERT mRNA have promoting effect on telomerase activity.The alternative splicing of hTERT pre-mRNA,namely the regulation at posttranscriptional level plays a crucial role in regulating telomerase activity.2.The complementary AOs of ESEs is able to regulate hTERT pre-mRNA splicing patterns result in a reduction of full-length hTERT mRNA expression.3.By changing the splicing patterns of hTERT pre-mRNA,AOs expressly down-regulate full-length hTERT mRNA,at the same time ? hTERT mRNA is relatively up-regulated,as a result telomerase activity is repressed and telomere length shortens,consequently for the cell,the proliferating activity is inhibited,apoptosis event increases significantly,invasive ability markedly decreases.AOs also have an effect on the formation of neurospheres of glioma stem cells.4.This study could provide a new approach and reference basis for the new generation development of telomerase inhibitors.It could provide new targets and important clues for glioma gene therapy and might become one of the future effective strategies for glioma treatment.