Analysis Alternative Splicing Pattern Of ADAR2Pre-mRNA In Human Glioma Cell Lines, And Apoptosis-promoting Effect Of Splice-switching Oligonucleotides Targeting Bcl-x Pre-mrna On Human Glioma Cell Lines

Part I Analysis alternative splicing pattern of ADAR2pre-mRNA inhuman glioma cell linesObjective：To analysis the differences of alternative splicing pattern of ADAR2between glioma cell lines U87, U251, A172and normal human astrocyte HA1800.Methods：A-to-I editing level at the Q/R-Site of GluR-2was analysis by RT-PCRand sequencing. The expression of ADAR2mRNA in the human glioma cell linesand normal human astrocyte were examined using real-time PCR. Alternativesplicing sites in the ADAR2pre-mRNA in human glioma cell lines and normalhuman astrocyte were analyzed by RT-PCR and sequencing blast. Five specificprimer pairs were designed to amplify different regions of the ADAR2cDNA, whichcollectively contained all the alternative splicing sites. The PCR products wereanalyzed with agarose gel electrophoresis and then cloned and sequenced. Real-timePCR was performed to detect the expression level of each alternatively splicingvariant using specific primer which was confirmed to amplify only the targetedtemplate and not other alternatively spliced variant fragments. Results：We verifythat the Q/R-Site of GluR-2is underedited in glioma cell lines. However, wedemonstrate with reverse transcription–real-time quantitative PCR that theexpression of ADAR2mRNA is not significantly altered in glioma cell lines. Threealternative splicing sites are utilized in both glioma cell lines and normal humanastrocytes: the first, located between exons–1and1, causes the inclusion of exon1a;the second causes the removal of exon2, which encodes two double-strandedRNA-binding domains; and the third, located between exons4and6, causes theinclusion of alternative exon5a, introducing a120nucleotide coding Alu-repeatsequence in frame. However, the expression ratio of two kinds of transcripts (withand without exon5a) is altered in the glioma cells. Transcripts with exon5a, which generate an ADAR2isoform with~50%reduced activity, are predominantlyexpressed in the glioma cell lines, whereas transcripts without exon5a arepredominantly expressed in normal human astrocytes. Conclusion：There wereevident differences of splicing at the site of exon5a between glioma cell lines andnormal human astrocyte. The different alternatively splicing pattern at exon5a mayattribute to the decreased activity of ADAR2. Part II Apoptosis-promoting effect of splice-switchingoligonucleotides targeting Bcl-x pre-mRNA on human glioma celllinesObjective：To analyze the alternative splicing pattern of Bcl-x pre-mRNA in gliomacell lines U87, U251, A172and normal human astrocyte HA1800, and investigatethe proliferation inhibitory and apoptosis-promoting and effect of Splice-switchingoligonucleotides targeting Bcl-x pre-mRNA (Bcl-x SSO) on human glioma cell lineU251.Methods： Two alternatice splicing variants of Bcl-x pre-mRNA, Bcl-xL(anti-apoptosis) and Bcl-xS (pro-apoptosis) were detected in glioma cell lines U87,U251, A172and normal human astrocyte HA1800by reversetranscription-polymerase chain reaction (RT-PCR) and verified by sequencing. Theexpression levels of Bcl-xL and Bcl-xS mRNA were analyzed by quantitativereal-time PCR. The expression levels of Bcl-xL and Bcl-xS protein were examinedby western blot. The Bcl-x SSO was designed to bind to the5’-splice site of exon IIin Bcl-x pre-mRNA. An oligonucleotide targeted to aberrantly splice human β-globin intron was used as control SSO. SSOs were modified by2’-O-methoxyethyl (MOE) and phosphorothioate (PS). The SSOs were deliveredwith lipofectamine into glioma cell line U251by cationic liposome. The proliferationinhibitory rate of U251was assessed by MTT. Flow cytometry (FCM) was used forthe detection of apoptosis rate. Modulation from Bcl-xL to Bcl-xS was analyzed byRT-PCR and western blot. Results: Compared to normal human astrocyte HA1800,the expression level of Bcl-xL mRNA was increased in glioma cell lines (P＜0.01);however, the expression level of Bcl-xS mRNA was decreased in glioma cell lines.The results were consistent at the protein level (P＜0.01). We show that Bcl-x SSOresulted in proliferation inhibitory and induced apoptosis in dose-dependent mannerin glioma cell line U251, while control SSO had no evident effect. The expression ofBcl-xL mRNA and protein was increased while the expression level of Bcl-xSmRNA was decreased in glioma cell line U251treated with Bcl-x SSO (P＜0.01).Conclusion: Alternative splicing of Bcl-X pre-mRNA is aberrant in glioma cell lines.The aberrantion is manifested in transformation from Bcl-XS to Bcl-XL, which isprobably related to pathogenesis of gliomas and may be a new target for therapy. Ourfindings demonstrate that Bcl-x SSO can induce apoptosis in glioma cell line U251.The mechanism is redirection of Bcl-x splicing from Bcl-xl to Bcl-xs. Bcl-x SSOsmay be potential anti-cancer drugs used in gliomas therapy.