Abstract
To investigate the effects of small interference RNA (siRNA) targeting BCR/ABL mRNA on proliferation and apoptosis in the K562 human chronic myeloid leukemia (CML) cell line and to provide a theoretical rationale and experimental evidence for its potential clinical application for anti-CML treatment. The gene sequence for BCR/ABL mRNA was found from the GeneBank. The target gene site on the BCR/ABL mRNA were selected according to Max-Planck-Institute (MPI) and rational siRNA design rules, the secondary structure of the candidate targeted mRNA was predicted, the relevant thermodynamic parameters were analyzed, and the targeted gene sequences were compared with BLAST to eliminate any sequences with significant homology. Inhibition of proliferation was evaluated by MTT assay and colony-formation inhibiting test. Apoptosis was determined by flow cytometry (FCM) and the morphology of apoptotic cells was identified by Giemsa-Wright staining. Western blotting was used to analyze the expression of BCR/ABL fusion protein in K562 cells after siRNA treatment. The mRNA local secondary structure calculated by RNA structure software, and the optimal design of specific siRNA were contributed by bioinformatics rules. Five sequences of BCR/ABL siRNAs were designed and synthesized in vitro. Three sequences, siRNA1384, siRNA1276 and siRNA1786, which showed the most effective inhibition of K562 cell growth, were identified among the five candidate siRNAs, with a cell proliferative inhibitory rate nearly 50% after exposure to 12.5 nmol/L~50 nmol/L siRNA1384 for 24,48 and 72 hours. The 50% inhibitory concentrations (IC50) of siRNA1384, siRNA1276 and siRNA1786 for 24 hours were 46.6 nmol/L, 59.3 nmol/L and 62.6 nmol/L, respectively, and 65.668 nmol/L, 76.6 nmol/L, 74.4 nmol/L for 72 hours. The colony-formation inhibiting test also indicated that, compared with control, cell growth of siRNA treated group was inhibited. FCM results showed that the rate of cell apoptosis increased 24 hours after transfecting siRNA. The results of annexinV/PI staining indicated that the rate of apoptosis imcreased (1.53%, 15.3%, 64.5%, 57.5% and 21.5%) following treamtne with siRNAs (siRNA34, siRNA372, siRNA1384, siRNA1276 and siRNA1786). Morphological analysis showed td typical morphologic changes of apoptosis such as shrunken, fragmentation nucleus as well as "apoptotic bodies" after K562 cell exposure to siRNA. Western blot analysis showed that BCR/ABL protein was reduced sharply after a single dose of 50 nmol/L siRNA transfection. Proliferation of K562 cells was remarkbly inhibited by siRNAs (siRNA1384, siRNA1276 and siRNA1786) in a concentration-dependent manner in vitro, with effective induction of apoptosis at a concentration of 50 nmol/L. One anti-leukemia mechanism in K562 cells appeared that BCR/ABL targeted protein was highly down-regulated. The siRNAs (siRNA1384, siRNA1276 and siRNA1786) may prove valuable in the treatment of CML.
Highlights
Small interfering RNA, sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 base pairs in length. small interference RNA (siRNA) plays many roles, but it is most notable in the RNA interference (RNAi) pathway, where it interferes with the expression of specific genes with complementary nucleotide sequence (Seyed-Gogani et al, 2013). small interfering RNAs (siRNAs) acts in RNAi-related pathways, e.g., as an antiviral mechanism or in shaping the chromatin structure of a genome (Shehata et al, 2010)
The target gene site on the BCR/ABL mRNA were selected according to Max-Planck-Institute (MPI) and rational siRNA design rules, the secondary structure of the candidate targeted mRNA was predicted, the relevant thermodynamic parameters were analyzed, and the targeted gene sequences were compared with BLAST to eliminate any sequences with significant homology
A siRNA labeled by fluorescein at 3’ end of antisense strand was used to determine efficiency of cellular transfection
Summary
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 base pairs in length. siRNA plays many roles, but it is most notable in the RNA interference (RNAi) pathway, where it interferes with the expression of specific genes with complementary nucleotide sequence (Seyed-Gogani et al, 2013). siRNA acts in RNAi-related pathways, e.g., as an antiviral mechanism or in shaping the chromatin structure of a genome (Shehata et al, 2010). There is intense interest in the use of combinations of apoptosis inducing agents, for example tumor necrosis factor-related apoptosis-inducing ligand, with siRNAs that silence genes which inhibit apoptosis such as Bcl-2, FLICE-like inhibitory protein or inhibition of apoptosis proteins Another approach that has been used to augment the effects of radiation and chemotherapy in cancer cells is RNAi-mediated silencing of DNA repair factors. Short RNAs targeting the pivotal proteins, ataxia telangiectasiamutated protein (ATM), ataxia telangiectasia-related (ATR) protein and DNA-dependent protein kinase catalytic subunit (DNA-PKCs), in DNA damage signaling and repair pathways, have been used to enhance radiation and chemotherapy-mediated cell killing in human cancer cells (Hirose et al, 2010) These findings imply that short RNA molecules can potentially be developed for use as radio-or chemo-sensitizing agents (Inayoshi et al, 2010). The siRNAs (siRNA1384, siRNA1276 and siRNA1786) may prove valuable in the treatment of CML
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