反义寡核苷酸处理伊马替尼耐药慢粒细胞后Bcl-x剪接异构体的变化
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摘要
目的探讨反义寡核苷酸Vivo-Morpholinos(vMO)处理对伊马替尼(Imatinib,IM)耐药慢性粒细胞白血病(chronic myeloid leukemia,CML)细胞中Bcl-x基因选择性剪接的影响。方法将IM(1μM)组、荧光标记的vMO、IM联合vMO组(IM+vMO)、空白对照组分别与IM耐药CML细胞株K562/G01细胞进行共培养48h,通过RT-PCR和Western Blot分别检测K562/G01细胞胞内Bcl-x剪接异构体Bcl-xL/Bcl-xS mRNA和蛋白比值变化。结果分别与IM组和vMO组处理K562/G01细胞相比,IM+vMO组的Bcl-xL mRNA和蛋白表达量显著降低,同时Bcl-xS mRNA和蛋白表达量明显升高,而对照组中Bcl-x剪接异构体变化不明显。结论 K562/G01细胞在vMO处理后Bcl-xL/Bcl-xS mRNA和蛋白比值显著下调。
Objective To explore the effect of alternative splicing isoforms of Bcl-x in IM resistant CML cells treated with Antisense oligonucleotides. Methods IM group(1μM),Fluorescent labeled vMO group,IM+vMO group and untreated group(blank control) were co-cultured with K562/G01 cells for 48 hours. The ratio of Bcl-xL/Bcl-xS m RNA and protein in K562/G01 cells were detected by RT-PCR and western blot,respectively. Results Compared with IM or vMO group alone,the expression of BclxL was significantly decreased and the expression of Bcl-xS was significantly increased in IM+vMO group,but the changes of Bclx alternative splicing isoform was not obvious in control group. Conclusion The ratio of Bcl-xL/Bcl-xS mRNA and protein decreased significantly after treatment with vMO.
Objective To explore the effect of alternative splicing isoforms of Bcl-x in IM resistant CML cells treated with Antisense oligonucleotides. Methods IM group(1μM),Fluorescent labeled vMO group,IM+vMO group and untreated group(blank control) were co-cultured with K562/G01 cells for 48 hours. The ratio of Bcl-xL/Bcl-xS m RNA and protein in K562/G01 cells were detected by RT-PCR and western blot,respectively. Results Compared with IM or vMO group alone,the expression of BclxL was significantly decreased and the expression of Bcl-xS was significantly increased in IM+vMO group,but the changes of Bclx alternative splicing isoform was not obvious in control group. Conclusion The ratio of Bcl-xL/Bcl-xS mRNA and protein decreased significantly after treatment with vMO.
引文
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[2]Paschalis A,Sharp A,Welti JC,et al.Alternative splicing in prostate cancer[J].Nat Rev Clin Oncol,2018,15(11):663-675.
[3]张静,李书琪,姜钰环,等.PKM剪接体在伊马替尼敏感及耐药慢粒细胞中的差异表达[J].实验与检验医学,2017,35(2):277-279.
[4]Li Z,Li Q,Han L,et al.Pro-apoptotic effects of splice-switching oligonucleotides targeting Bcl-x pre-mRNA in human glioma cell lines[J].Oncol Rep,2016,35(2):1013-1019.
[5]Bielli P,Busa R,Di Stasi SM,et al.The transcription factor FBI-1inhibits SAM68-mediated BCL-X alternative splicing and apoptosis[J].EMBO Rep,2014,15(4):419-427.
[6]Niklaus NJ,Humbert M,Tschan MP.Cisplatin sensitivity in breast cancer cells is associated with particular DMTF1 splice variant expression[J].Biochem Biophys Res Commun,2018,503(4):2800-2806.
[7]Mueller AA,van Velthoven CT,Fukumoto KD,et al.Intronic polyadenylation of PDGFRαin resident stem cells attenuates musclefibrosis[J].Nature,2016,540(7632):276-279.
[8]刘静,张静,黄波,等.选择性剪接机制及其在白血病诊疗中的应用[J].中华检验医学杂志,2015,38(11):730-732.
[9]Harb JG,Neviani P,Chyla BJ,et al.Bcl-xL anti-apoptotic network is dispensable for development and maintenance of CML but is required for disease progression where it represents a new therapeutic target[J].Leukemia,2013,27(10):1996-2005.
[10]Holm F,Hellqvist E,Mason CN,et al.Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal[J].Proc Natl Acad Sci USA,2015,112(50):15444-15449.
[11]Wang F,Wei ZL,Sun XR,et al.Apoptosis Inducing Factor Is Involved in Stretch-Induced Apoptosis of Myoblast via a Caspase-9Independent Pathway[J].J Cell Biochem,2017,118(4):829-838.
[12]Oh ST,Lee S,Hua C,et al.Decursin induces apoptosis in glioblastoma cells,but not in glial cells via a mitochondria-related caspase pathway[J].Korean J Physiol Pharmacol,2019,23(1):29-35.