Bcr-abl silencing by specific small-interference RNA expression vector as a potential treatment for chronic myeloid leukemia

Abstract

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.

Fig. 1

RNA interference in k562 cells. (A) Bcr-abl fusion sequence and schematic representation of b3a2-1, b3a2-2 siRNA and siControl that target scrambled sequence. TT indicates the deoxythymidine dimer as 3' overhang. The arrow marks the fusion between bcr (left) and abl (right) sequences of b3a2-bcr-abl. (B) The siRNA expression vector was tested in Ph + K562 cells

Fig. 2.

Transfection of K562 cells with b3a2-1 expression vector (pRNAH1.1/Neo). Transfection efficacy was analyzed using the GFP expression system (green color). (B) Transfection was investigated using electroporation, lipofectamin ^th2000 and CdCl₂ methods in K562 cells. The highest efficacy was about 50% in electroporation (n = 3

Fig. 3

Cell growth of k562 cells treated and untreated with specific anti-Bcr-abl siRNA expression vector. Viable cells were counted by trypan blue exclusion during suspension cultures after transfection with anti-Bcr-abl siRNA, siControl, vector control and GFP expression vector (n=3),  P 0.05.  P 0.01

. Typical RT-PCR (48 h following transfection) showing the level of mRNA relative to that of Bcr-abl and GAPDH in k562 cells treated and untreated with anti-Bcr-abl siRNA (lane 1), siControl (lane 2), vector control (lane 3) and normal cell (lane 4).

Fig. 5

(A) A time-dependently increase in the apoptotic cell population when treated with sib3a2 (n = 3). ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001; (B) eight-fold increase in apoptotic cell population was observed in specifically silenced Bcr-abl cells using b3a2-1 siRNA.