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

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Abstract

Introduction

Alternative splicing of hTERT pre-mRNA is an important step in the regulation of telomerase activity, but the regulation mechanisms and functions remain unclear.

Methods

RT-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.

Results

hTERT 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.

Conclusions

Our findings provided new targets and important clues for the gene therapy of gliomas by regulating the alternative splicing pattern of hTERT pre-mRNA.

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Funding

This work was supported by the Chinese National Natural Science Foundation (Grant Nos. 81671054, 81771135, 91649102, 31771520).

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Authors and Affiliations

  1. Department of Neurology, General Hospital, Tianjin Medical University, Tianjin, 300052, China

    Fei Wang, Yajing Cheng & Chi Zhang

  2. Department of Clinical Laboratory, General Hospital, Tianjin Medical University, Tianjin, 300052, China

    Guangming Chang

  3. Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Immune Microenvironment and Disease, Ministry of Education, Tianjin Medical University, 22nd Qixiangtai Road, Tianjin, 300070, China

    Xin Geng

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  1. Fei Wang
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  2. Yajing Cheng
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  3. Chi Zhang
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Contributions

XG, FW: Conception and design, FW, XG, YC, CZ, GC: Development of methodology, FW,YC, CZ GC: Analysis and interpretation of data, FW, YC, CZ, XG: Writing, review and revision of the manuscript.

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Correspondence to Xin Geng.

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Wang, F., Cheng, Y., Zhang, C. et al. A novel antisense oligonucleotide anchored on the intronic splicing enhancer of hTERT pre-mRNA inhibits telomerase activity and induces apoptosis in glioma cells. J Neurooncol 143, 57–68 (2019). https://doi.org/10.1007/s11060-019-03150-x

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