BCR/ABL mRNA Targeting Small Interfering RNA Effects on Proliferation and Apoptosis in Chronic Myeloid Leukemia

  • Zhu, Xi-Shan (Clinical Research Center, Affiliated Hospital of Guangdong Medical College) ;
  • Lin, Zi-Ying (Clinical Research Center, Affiliated Hospital of Guangdong Medical College) ;
  • Du, Jing (Department of Urology, Weifang Traditional Chinese Medicine Hospital) ;
  • Cao, Guang-Xin (Department of Vascular Surgery,Weifang People's Hospital) ;
  • Liu, Gang (Clinical Research Center, Affiliated Hospital of Guangdong Medical College)
  • Published : 2014.06.30


Background: 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. Materials and Methods: 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. Results: 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 12.5nmol/L~50nmol/L siRNA1384 for 24,48 and 72 hours. The 50% inhibitory concentrations ($IC_{50}$) of siRNA1384, siRNA1276 and siRNA1786 for 24hours 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 50nmol/L siRNA transfection. Conclusions: 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.


  1. Zhang H, Zhu L, He H, et al (2014). NF-kappa B mediated Up-regulation of CCCTC-binding factor in pediatric acute lymphoblastic leukemia. Mol Cancer, 13, 5. [Epub ahead of print]
  2. Xu X, Li B, Huang P, et al (2013). Citrate induces apoptosis of the acute monocytic leukemia U937 cell line through regulation of HIF-$1\alpha$ signaling. Mol Med Rep, 8, 1379-84.
  3. Xiao X, Yang J, Li R, et al (2013). Deregulation of mitochondrial ATPsyn-$\beta$ in acute myeloid leukemia cells and with increased drug resistance. PLoS One, 8, 83610.
  4. Zhang J, Wang H, Wang L, et al (2014). Adenovirusmediated delivery of the human IFN-$\gamma$ gene potentiates the cytotoxicity of daunorubicin against leukemic cells through downregulation of the $\alpha4\beta1$ integrin/ILK/apoptosis pathway. Oncol Lett, 7, 361-368.
  5. Zhang H, Chang G, Wang J, et al (2013). CUEDC2 sensitizes chronic myeloid leukemic cells to imatinib treatment. Leuk Res, 37, 1583-91.
  6. Zhao X, Liu J, Peng M, Liu J, Chen F (2013). BMP4 is involved in the chemoresistance of myeloid leukemia cells through regulating autophagy-apoptosis balance. Cancer Invest, 31, 555-62.
  7. Suresh S, McCallum L, Crawford LJ, et al (2013). The matricellular protein CCN3 regulates NOTCH1 signalling in chronic myeloid leukaemia. J Pathol, 231, 378-87.
  8. Shehata M, Schnabl S, Demirtas D, et al (2010). Reconstitution of PTEN activity by CK2 inhibitors and interference with the PI3-K/Akt cascade counteract the antiapoptotic effect of human stromal cells in chronic lymphocytic leukemia. Blood, 116, 2513-21.
  9. Snyder M, Huang XY, Zhang JJ (2010). Stat3 directly controls the expression of Tbx5, Nkx2.5, and GATA4 and is essential for cardiomyocyte differentiation of P19CL6 cells. J Biol Chem, 285, 23639-46.
  10. Song Y, Ding N, Kanazawa T, Yamashita U, Yoshida Y (2013). Cucurbitacin D is a new inflammasome activator in macrophages. Int Immunopharmacol, 17, 1044-50.
  11. Tao Q, Cui-Qing F, Ning Z, Yan S, Mei-Hong C (2013). Knockdown of proteasome subunit $\alpha7$ with small interfering RNA inhibits cell proliferation of k562 cell line. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 35, 601-6.
  12. Timblin GA, Schlissel MS (2013). Ebf1 and c-Myb repress rag transcription downstream of Stat5 during early B cell development. J Immunol, 191, 4676-87.
  13. Wang L, Luo J, Nian Q, et al (2013). Ribosomal protein S14 silencing inhibits growth of acute myeloid leukemia transformed from myelodysplastic syndromes via activating p53. Hematology. [Epub ahead of print]
  14. Wang Y, Zhang J, Wang Q, et al (2013). Bryostatin 5 induces apoptosis in acute monocytic leukemia cells by activating PUMA and caspases. Eur J Pharmacol, 718, 340-9.
  15. Woyach JA, Bojnik E, Ruppert AS, et al (2013). Bruton's tyrosine kinase (BTK) function is important to the development and expansion of chronic lymphocytic leukemia (CLL). Blood. [Epub ahead of print]
  16. Misumi S, Inoue M, Dochi T, et al (2010). Uncoating of human immunodeficiency virus type 1 requires prolyl isomerase Pin1. J Biol Chem, 285, 25185-95.
  17. Li XJ, Luo XQ, Han BW, et al (2013). MicroRNA-100/99a, deregulated in acute lymphoblastic leukaemia, suppress proliferation and promote apoptosis by regulating the FKBP51 and IGF1R/mTOR signalling pathways. Br J Cancer, 109, 2189-98.
  18. Li G, Chang H, Zhai YP, Xu W (2013). Targeted silencing of inhibitors of apoptosis proteins with siRNAs: a potential anti-cancer strategy for hepatocellular carcinoma. Asian Pac J Cancer Prev, 14, 4943-52.
  19. Markman JL, Rekechenetskiy A, Holler E, Ljubimova JY (2013). Nanomedicine therapeutic approaches to overcome cancer drug resistance. Adv Drug Deliv Rev, 65, 1866-79.
  20. Okuhashi Y, Itoh M, Nara N, Tohda S (2013). NOTCH knockdown affects the proliferation and mTOR signaling of leukemia cells. Anticancer Res, 33, 4293-8.
  21. Omedes Pujol M, Coleman DJ, Allen CD, Heidenreich O, Fulton DA (2013). Determination of key structure-activity relationships in siRNA delivery with a mixed micelle system. J Control Release, 172, 939-45.
  22. Qiao SK, Ren HY, Shi YJ, Liu W (2013). Silencing HCCR2 expression inhibits the proliferation of leukemia cells by inducing apoptosis and promoting cell cycle arrest. Int J Mol Med, 32, 1373-9.
  23. Ramakrishnan V, Painuly U, Kimlinger T, et al (2014). Inhibitor of apoptosis proteins (IAPs) as therapeutic targets in multiple myeloma (MM). Leukemia.
  24. Seyed-Gogani N, Rahmati M, Zarghami N, et al (2013). Nucleostemin depletion induces post-g1 arrest apoptosis in chronic myelogenous leukemia k562 cells. Adv Pharm Bull, 4, 55-60.
  25. Kamata M, Liu S, Liang M, Nagaoka Y, Chen IS (2010). Generation of human induced pluripotent stem cells bearing an anti-HIV transgene by a lentiviral vector carrying an internal murine leukemia virus promoter. Hum Gene Ther, 21, 1555-67.
  26. Humphries LA, Godbersen JC, Danilova OV, et al (2013). Pro-apoptotic TP53 homolog TAp63 is repressed via epigenetic silencing and B-cell receptor signalling in chronic lymphocytic leukaemia, Br J Haematol, 163, 590-602.
  27. Inayoshi Y, Okino Y, Miyake K, et al (2010). Transcription factor YY1 interacts with retroviral integrases and facilitates integration of moloney murine leukemia virus cDNA into the host chromosomes. J Virol, 84, 8250-61.
  28. Isakson P, Bjoras M, Boe SO, Simonsen A (2010). Autophagy contributes to therapy-induced degradation of the PML/RARA oncoprotein. Blood, 116, 2324-31.
  29. Kanduri M, Sander B, Ntoufa S, et al (2013). A key role for EZH2 in epigenetic silencing of HOX genes in mantle cell lymphoma. Epigenetics, 18, 1280-8.
  30. Karami H, Baradaran B, Esfahani A, et al (2013). siRNAmediated silencing of survivin inhibits proliferation and enhances etoposide chemosensitivity in acute myeloid leukemia cells. Asian Pac J Cancer Prev, 14, 7719-24.
  31. Koide N, Naiki Y, Odkhuu E, et al (2013). Involvement of oncogenic protein $\beta$-catenin in LPS-induced cytotoxicity in mouse mononuclear leukemia RAW 264.7 Cells. Oncol Res, 21, 59-65.
  32. Lee SH, Lee JH, Lee JH, Kim DK (2010). Involvement of MITF-A, an alternative isoform of mi transcription factor, on the expression of tryptase gene in human mast cells. Exp Mol Med, 42, 366-75
  33. Lennon JC, Bright SA, Carroll E, et al (2014). The novel pyrrolo-1, 5-benzoxazepine, PBOX-6, synergistically enhances the apoptotic effects of carboplatin in drug sensitive and multidrug resistant neuroblastoma cells. Biochem Pharmacol, 87, 611-24.
  34. Almalik A, Day PJ, Tirelli N (2013). HA-coated chitosan nanoparticles for CD44-mediated nucleic acid delivery. Macromol Biosci, 13, 1671-80.
  35. Choi ES, Chung T, Kim JS, et al (2013). Mithramycin A induces apoptosis by regulating the mTOR/Mcl-1/tBid pathway in androgen-independent prostate cancer cells. J Clin Biochem Nutr, 53, 89-93.
  36. Gao P, Bauvy C, Souquere S, et al (2010). The Bcl-2 homology domain 3 mimetic gossypol induces both Beclin 1-dependent and Beclin 1-independent cytoprotective autophagy in cancer cells. J Biol Chem, 285, 25570-81.
  37. Hands KJ, Cuchet-Lourenco D, Everett RD, Hay RT (2014). PML isoforms in response to arsenic: high-resolution analysis of PML body structure and degradation. J Cell Sci, 127, 365-75.
  38. Hirose K, Inukai T, Kikuchi J, et al (2010). Aberrant induction of LMO2 by the E2A-HLF chimeric transcription factor and its implication in leukemogenesis of B-precursor ALL with t (17;19). Blood, 116, 962-70.
  39. Hou KK, Pan H, Ratner L, Schlesinger PH, Wickline SA (2013). Mechanisms of nanoparticle-mediated siRNA transfection by melittin-derived peptides. ACS Nano, 7, 8605-15.
  40. Hsiao PC, Hsieh YH, Chow JM, et al (2013). Hispolon induces apoptosis through JNK1/2-mediated activation of a caspase-8, -9, and -3-dependent pathway in acute myeloid leukemia (AML) cells and inhibits AML xenograft tumor growth in vivo. J Agric Food Chem, 61, 10063-73.

Cited by

  1. Parameters Involved in Autophosphorylation in Chronic Myeloid Leukemia: a Systems Biology Approach vol.16, pp.13, 2015,
  2. Systemic delivery of CRISPR/Cas9 with PEG-PLGA nanoparticles for chronic myeloid leukemia targeted therapy vol.6, pp.6, 2018,