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Published on: September 1, 2019
Bcr-abl silencing by specific small-interference RNA expression vector as a potential treatment for chronic myeloid
Ali Zaree Mahmodabady1, Hamid Reza Javadi, Mehdi Kamali
1Dept. of Biochemistry, Baqiyatallah University, Tehran, Iran.
Background:
RNA interference (RNAi) is the mechanism of gene silencing-mediated messenger RNA degradation by small interference RNA (siRNA), which becomes a powerful tool for in vivo research, especially in the areas of cancer. In this research, the potential use of an expression vector as a specific siRNA producing tool for silencing of Bcr-abl in K562 cell line has been investigated.
Methods:
siRNA specific for Bcr-abl as short hairpin RNA (shRNA) was designed and cloned in expression vector (pRNAH1.1/Neo). K562 cells were cultured in RPMI media and transfected with shRNA expressing vector using lipofectamin 2000. Successful transfection was confirmed by significant increase of enhanced green fluorescent protein (EGFP) levels in K562-treated cells with expression vector (pEGFP-C1). In vitro studies in human K562 cell line entailed modulation of endogenous Bcr-abl mRNA levels which induced apoptosis. Effects of siRNA treatment on K562 cells were measured by ELISA.
Results:
Successful expression of siRNA was confirmed by significant reduction of Bcr-abl mRNA levels in K562 cells treated with expression vector (pRNAH1.1/Neo). siRNA directed against Bcr-abl effectively induced apoptosis and reduced viability in human K562 cell lines.
Conclusion:
Expression vector of siRNA can be used in vitro to target specific RNA and to reduce the levels of the specific gene product in the targeted cells. Results of this work suggest that RNAi has potential application for the treatment of a variety of diseases, including those involving abnormal gene expression and viral contamination.
Insights
This study shows that an expression vector can produce small interfering RNA (siRNA) to silence the Bcr-abl gene in K562 cells, inducing apoptosis. This RNA interference (RNAi) approach holds promise for treating diseases with abnormal gene expression.
Area of Science:
- Molecular Biology
- Gene Silencing
- Cancer Research
Background:
- RNA interference (RNAi) is a gene silencing mechanism utilizing small interfering RNA (siRNA) for messenger RNA degradation.
- RNAi is a valuable tool in in vivo research, particularly in oncology.
- This study investigates using an expression vector to produce siRNA for Bcr-abl gene silencing in K562 cells.
Purpose of the Study:
- To evaluate the efficacy of an expression vector-based siRNA system for targeting Bcr-abl.
- To assess the impact of Bcr-abl gene silencing on K562 cell viability and apoptosis.
- To explore the potential of RNAi as a therapeutic strategy for Bcr-abl-related conditions.
Main Methods:
- Designed and cloned Bcr-abl-specific short hairpin RNA (shRNA) into an expression vector (pRNAH1.1/Neo).
- Transfected K562 cells with the shRNA-expressing vector using lipofectamine 2000.
- Confirmed successful transfection via enhanced green fluorescent protein (EGFP) expression and measured Bcr-abl mRNA modulation and apoptosis via ELISA.
Main Results:
- Confirmed successful siRNA expression by a significant reduction in Bcr-abl mRNA levels in treated K562 cells.
- Demonstrated that siRNA targeting Bcr-abl effectively induced apoptosis in K562 cells.
- Observed a reduction in K562 cell viability following siRNA treatment.
Conclusions:
- Expression vectors can effectively produce siRNA in vitro to target and reduce specific gene products.
- RNA interference presents a promising therapeutic avenue for diseases characterized by abnormal gene expression.
- This approach may also be applicable to managing viral infections involving specific gene targets.
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