MiRNA-Mediated Knock-Down of Bcl-2 and Mcl-1 Increases Fludarabine-Sensitivity in CLL-CII Cells

Abstract

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.

Keywords: Bcl-2; CLL; Fludarabine; Mcl-1; MiRNA-15a.

Figure 1

The Effect of miRNA-15a on Sensitivity of the CLL-CII Cells to Fludarabine. Cells were treated with miRNA-15a (50 nM) and different concentrations of fludarabine for 24 h (A and B) and 48 h (C and D). Next, the cell survival rate was determined using MTT assay. Cell survival curves were plotted by Prism software. The data are expressed as mean ± SD (n=3). Results from three independent experiments were used to plot the combination index (CI) versus fractional effect (Fa) using the Chou and Talalay method and CalcuSyn software. Dashed lines represent CI=1.

Figure 2

Gene Expression Analysis in CLL-CII Cells Treated with miRNA-15a and Fludarabine. The cells were treated with miRNA-15a, fludarabine and combination of them for 24 and 48 h. Relative Bcl-2 (A) and Mcl-1(B) mRNA expression was measured using RT-qPCR and 2 ^{- (∆∆Ct)} method. The results are presented as mean±SD of the results of three experiments. *p<0.05 versus corresponding blank control or NC miRNA transfected cells

Figure 3

Proliferation Curve of CLL-CII Cells Treated with miRNA-15a and Fludarabine. Cell proliferation was measured using trypan blue exclusion assay over a period of 5 days. Results are expressed as mean ± SD (n=3). *p< 0.05 versus blank control or NC miRNA

Figure 4

Combination Effects of miRNA-15a and Fludarabine on Apoptosis of CLL Cells. Cells were treated with miRNA-15a (50 nM), negative control (NC) miRNA (50 nM) and fludarabine (IC50 doses of 24 and 48 h). Next, the apoptosis was quantified using ELISA apoptosis assay (A). Caspase-3 activity of CLL cells was detected according to caspase-3 activity assay Kit (B). The data are presented mean ± SD (n=3) of three independent experiments. *p<0.05 compared with control; #p<0.05 versus miRNA-15a or fludarabine monotreatment