title: Pro-apoptotic effects of splice-switching oligonucleotides targeting Bcl-x pre-mRNA in human glioma cell lines.
abstract: Alternative splicing is a near-ubiquitous phenomenon with important roles in human diseases, including cancers. Splice-switching oligonucleotides (SSOs) have emerged as a class of antisense therapeutics that modulate alternative splicing by hybridizing to the pre-mRNA splice site. The Bcl-x gene is alternatively spliced to express anti‑apoptotic Bcl-xL and pro-apoptotic Bcl-xS. Bcl-xL expression is upregulated in many cancers and is considered a general mechanism by which cancer cells evade apoptosis. By redirecting Bcl-x pre-mRNA splicing from Bcl-xL to Bcl-xS, SSO exerted pro-apoptotic and chemosensitizing effects in various cancer cell lines. In this study, we investigated the effects of SSO targeting Bcl-x pre-mRNA in human glioma cell lines. First, we performed reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to determine the mRNA and protein expression levels of Bcl-xL in glioma cell lines (U87 and U251) and a normal human astrocyte cell line (HA1800). Then, the Bcl-x SSO was designed to bind to the downstream 5' alternative splice site of exon 2 in Bcl-x pre-mRNA and was modified using 2'-O-methoxyethyl-phosphorothioate. An oligonucleotide targeting aberrantly spliced human β-globin intron was used as a negative control. The SSOs were delivered with a cationic lipid into glioma and astrocyte cell lines. The antitumor effects of the SSOs were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and flow cytometry, and the switch in production from Bcl-xL to Bcl-xS was analyzed by RT-PCR and western blotting. Bcl-xL mRNA and protein were highly expressed in both glioma cell lines. The Bcl-x SSO modified Bcl-x pre-mRNA splicing and had pro-apoptotic effects on the glioma cell lines. By contrast, the lipid alone and the control SSO did not affect Bcl-xL expression or induce apoptosis. Our study demonstrated the antitumor activity of an SSO that targets Bcl-x pre-mRNA splicing in glioma cell lines. Bcl-x SSO may be a potential strategy for treating gliomas.
authors.affiliation: Department of Neurosurgery, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130031, P.R. China.
authors.forename: Zhaohui
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Neurosurgery, Heilongjiang Provincial Hospital, Harbin, Heilongjiang 150036, P.R. China.
authors.forename: Qingwei
authors.initials: Q
authors.lastname: Li
authors.affiliation: Department of Neurosurgery, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130031, P.R. China.
authors.forename: Liang
authors.initials: L
authors.lastname: Han
authors.affiliation: Department of Cell Biology, College of Life Science, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, P.R. China.
authors.forename: Nan
authors.initials: N
authors.lastname: Tian
authors.affiliation: Department of Neurosurgery, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130031, P.R. China.
authors.forename: Qianlei
authors.initials: Q
authors.lastname: Liang
authors.affiliation: Department of Neurosurgery, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130031, P.R. China.
authors.forename: Yanzhe
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Neurosurgery, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130031, P.R. China.
authors.forename: Xingli
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Department of Neurosurgery, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130031, P.R. China.
authors.forename: Chao
authors.initials: C
authors.lastname: Du
authors.affiliation: Department of Neurosurgery, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130031, P.R. China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Tian
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D017398:Alternative Splicing; D017209:Apoptosis / Q000235:genetics*; D001253:Astrocytes / Q000166:cytology; D001932:Brain Neoplasms / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D005091:Exons / Q000235:genetics; D005910:Glioma / Q000473:pathology; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D054735:Phosphorothioate Oligonucleotides / Q000494:pharmacology*; D012322:RNA Precursors / Q000037:antagonists & inhibitors*; D012334:RNA, Neoplasm / Q000037:antagonists & inhibitors*; D051020:bcl-X Protein / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics
chemical_list: D020121:5' Untranslated Regions; C494811:BCL2L1 protein, human; D016376:Oligonucleotides, Antisense; D054735:Phosphorothioate Oligonucleotides; D012322:RNA Precursors; D012334:RNA, Neoplasm; D051020:bcl-X Protein