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Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells
Published on: April 26, 2017
Modification of alternative splicing of Bcl-x pre-mRNA in bladder cancer cells
Zhaohui Zhu1, Shi'an Xing, Ping Cheng
1Department of Urology, Union Hospital, Tongji Medical College, Huazhong Science and Technology University, Wuhan, China.
Abstract:
To modify the splicing pattern of Bcl-x and compare the effect of this approach with that of the antisense gene therapy in BIU-87 cell line of bladder cancer, by using 5'-Bcl-x AS to target downstream alternative 5'-Bcl-x splice site to shift splicing from Bcl-xL to Bcl-xS and 3'-Bcl-x AS antisense to the 3'-splice site of exon III in Bcl-x pre-mRNA to down regulation of Bcl-xL expression, the inhibitory effects on cancer cells by modification of alternative splicing and antisense gene therapy were observed and compared by microscopy, MTT Assay, RT-PCR, FACS, Western bloting and clone formation. The growth of cells BIU-87 was inhibited in a dose- and time-dependent manner. Its inhibitory effect began 12 h after the exposure, reaching a maximum value after 72h. The number of cells decreased in S phase and the number increased in G1 phase. The ability to form foci was reduced and the antisense gene therapy was approximately half as efficient as modification of alternative splicing in inducing apoptosis. It is concluded that modification of splicing pattern of Bcl-x pre-mRNA in bladder cancer cell BIU-87 is better than antisense gene therapy in terms of tumor inhibition.
Insights
Modifying Bcl-x splicing patterns in bladder cancer cells showed superior tumor inhibition compared to antisense gene therapy. This approach effectively reduced cancer cell growth and induced apoptosis.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- The Bcl-x gene plays a crucial role in apoptosis, with alternative splicing producing pro-survival (Bcl-xL) and pro-apoptotic (Bcl-xS) isoforms.
- Dysregulation of Bcl-x splicing is implicated in various cancers, including bladder cancer, making it a potential therapeutic target.
Purpose of the Study:
- To investigate the efficacy of modifying Bcl-x splicing patterns versus antisense gene therapy in inhibiting bladder cancer cell growth.
- To compare the therapeutic effects of shifting Bcl-x splicing towards Bcl-xS and downregulating Bcl-xL expression.
Main Methods:
- Utilized 5'-Bcl-x AS and 3'-Bcl-x AS antisense oligonucleotides to modulate Bcl-x pre-mRNA splicing in BIU-87 bladder cancer cells.
- Assessed cellular effects using microscopy, MTT assay, RT-PCR, FACS analysis, Western blotting, and clone formation assays.
Main Results:
- Both splicing modification and antisense gene therapy inhibited BIU-87 cell growth in a dose- and time-dependent manner.
- Splicing modification was more effective than antisense therapy in inhibiting cell growth, reducing foci formation, and inducing apoptosis.
- Cell cycle analysis revealed a decrease in S phase and an increase in G1 phase following treatment.
Conclusions:
- Modification of Bcl-x pre-mRNA splicing is a more effective strategy than antisense gene therapy for inhibiting bladder cancer progression.
- Targeting alternative splicing of Bcl-x offers a promising therapeutic avenue for bladder cancer treatment.
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