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MiRNA-Mediated Knock-Down of Bcl-2 and Mcl-1 Increases Fludarabine-Sensitivity in CLL-CII Cells
Nooshin Ashofteh1, Razieh Amini1, Neda Molaee2
1Department of Molecular Medicine and Biotechnology, Faculty of Medicine, Arak University of Medical Sciences, Arak, Iran.
Background:
Over-expression of anti-apoptotic proteins such as Bcl-2 and Mcl-1 is associated with resistance to chemotherapeutic agents such as fludarabine. Moreover, an inverse relationship between miRNA-15a levels with Bcl-2 and Mcl-1 expression has been observed in CLL patients. In this study, the effect of miRNA-15a on apoptosis and sensitivity of the CLL cells to fludarabine was investigated.
Methods:
After treatments, the Mcl-1 and Bcl-2 expression levels were quantified by RT-qPCR. Trypan blue assay was used to explore the effects of miRNA-15a and fludarabine on cell proliferation. The cytotoxicity was measured using MTT assay and combination index analysis. Cell death was determined using cell death detection ELISA assay and caspase-3 activity assay Kits.
Results:
Results showed that miRNA-15a clearly decreased the mRNA levels of Bcl-2 and Mcl-1 in a time dependent manner, which led to CLL-II cell proliferation inhibition and enhancement of apoptosis (p < 0.05, relative to control). Transfection of the miRNA-15a synergistically reduced the cell survival rate and lowered the IC50 value of fludarabine. Furthermore, miRNA-15a significantly enhanced the apoptotic effect of fludarabine.
Conclusions:
Our data propose that suppression of Bcl-2 and Mcl-1 by miRNA-15a can effectively inhibit the cell proliferation and sensitize CLL cells to fludarabine. Therefore, miRNA-15a can be considered as a potential therapeutic target in CLL resistant patients.
Insights
MicroRNA-15a suppresses Bcl-2 and Mcl-1, inhibiting chronic lymphocytic leukemia (CLL) cell proliferation and enhancing fludarabine sensitivity. This suggests miRNA-15a as a potential therapeutic target for resistant CLL patients.
Area of Science:
- Molecular Biology
- Oncology
- Gene Regulation
Background:
- Over-expression of anti-apoptotic proteins Bcl-2 and Mcl-1 is linked to fludarabine resistance in chronic lymphocytic leukemia (CLL).
- Inverse correlation observed between miRNA-15a levels and Bcl-2/Mcl-1 expression in CLL patients.
Purpose of the Study:
- Investigate the impact of miRNA-15a on apoptosis in CLL cells.
- Determine the effect of miRNA-15a on CLL cell sensitivity to fludarabine.
Main Methods:
- Quantification of Mcl-1 and Bcl-2 mRNA levels using RT-qPCR.
- Assessment of cell proliferation via trypan blue assay.
- Measurement of cytotoxicity and apoptosis using MTT assay, combination index analysis, ELISA, and caspase-3 activity assays.
Main Results:
- miRNA-15a significantly decreased Bcl-2 and Mcl-1 mRNA levels, inhibiting CLL-II cell proliferation and promoting apoptosis (p < 0.05).
- miRNA-15a transfection synergistically reduced cell survival and lowered fludarabine's IC50 value.
- miRNA-15a markedly enhanced the apoptotic effect of fludarabine.
Conclusions:
- Suppression of Bcl-2 and Mcl-1 by miRNA-15a effectively inhibits CLL cell proliferation.
- miRNA-15a sensitizes CLL cells to fludarabine, indicating its potential as a therapeutic target for resistant CLL.
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