A novel antisense oligonucleotide anchored on the intronic splicing enhancer of hTERT pre-mRNA inhibits telomerase activity and induces apoptosis in glioma cells

Abstract

IntroductionAlternative splicing of hTERT pre-mRNA is an important step in the regulation of telomerase activity, but the regulation mechanisms and functions remain unclear.MethodsRT-PCR analysis was used to detect hTERT splicing in glioma cell lines and brain tissues. TRAP assay was used to detect the telomerase activity. Then, we designed and synthesized 2′-O-methyl-RNA phosphorothioate AONs and transfected them into glioma cells to detect the changes in telomerase activity. MTT assay, plate colony formation assay, western blotting and Annexin V/PI assay were used to detect cell proliferation and apoptosis. At last, bioinformatics analyses were used to predict the expression and function of splicing protein SRSF2 in gliomas.ResultshTERT splicing occurs both in glioma cell lines and glioma patients’ tissues. The telomerase activity was related to the expression level of the full-length hTERT, rather than the total hTERT transcript level. AON-Ex726 was complementary to the sequence of the intronic splicing enhancer (ISE) in intron six, and significantly altered the splicing pattern of hTERT pre-mRNA, reducing the expression level of the full-length hTERT mRNA and increasing the expression level of the -β hTERT mRNA. After transfection with AON-Ex726, the level of apoptosis was increased, while telomerase activity and cell proliferation were significantly decreased. By bioinformatic predictions, we found the AON-Ex726 anchoring sequence in ISE overlaps the binding site of SRSF2 protein, which is up-regulated during the development of gliomas.ConclusionsOur findings provided new targets and important clues for the gene therapy of gliomas by regulating the alternative splicing pattern of hTERT pre-mRNA.