Sains Malaysiana 42(8)(2013): 1131–1137


Targeted RNAi of the Mitogen-activated Protein Kinase Pathway Genes in

Acute Myeloid Leukemia Cells

(RNAi Sasar Gen Tapak Jalan Protein Kinase Diaktifkan-Mitogen dalam Mieloid Leukemia Akut)



M.R. Mohd Hafiz1*, M.Z. Mazatulikhma2, F.A. Mohd Faiz1 & M.S. Mohamed Saifulaman1


1Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor



2Institute of Science, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia


Diserahkan: 2 April 2012/Diterima: 16 Disember 2012



In this study, RNA interference (RNAi) was carried out as an experimental technique to knockdown three mitogen-activated protein kinase (MAPK) pathway genes, raf-1, mekk1 and mlk3 in acute myeloid leukemia (AML) cells. Conventionally, RNAi knockdown experiments target a single gene for functional studies or therapeutic purposes. We wanted to explore the potential differences or similarities between targeting single targets or multiple target genes in a single application. We achieved knockdown of gene expression levels of between 40 and 60% for the RNAi experiments, with better knockdown observed in single target gene experiments in comparison with the multiple target gene experiment. Microarray analysis indicated that the transfection process had most likely induced the immune response from the cells in every RNAi treatment. This might indicate that when the MAPK signaling pathway is partially blocked, in tandem with the immune response, the cells will begin signaling for apoptosis leading to cellular death of the leukemic cells.


Keywords: Acute myeloid leukemia; immune response; MAPK pathway; RNA interference



Dalam kajian ini, penggangguan RNA (RNAi) digunakan sebagai teknik uji kaji untuk menurunkan tiga gen protein kinase diaktifkan-mitogen (MAPK) iaitu gen raf1, mekk1 dan mlk3 di dalam sel mieloid leukemia akut (AML). Kebiasaannya, eksperimen RNAi dijalankan untuk menyasar satu gen sahaja demi mengkaji fungsi atau peranan terapi. Kami telah mengkaji potensi perbezaan atau persamaan antara menyasar satu atau lebih gen dalam satu aplikasi. Kami berjaya mencapai penurunan pengekspresan gen daripada 40% hingga 60% dan RNAi kelihatan lebih berkesan melalui penyasaran satu gen. Analisis mikroatur menunjukkan bahawa proses transfeksi kemungkinan tinggi telah mengaruh tindak balas imun dalam setiap perlakuan RNAi yang telah dilakukan. Ini mungkin memberi petunjuk bahawa apabila pengisyaratan tapak jalan MAPK dihalang separa, disertakan pengaruhan tindak balas imun, tapak laluan apoptosis akan dimulakan dan mengakibatkan kematian sel kepada sel-sel leukemia.


Kata kunci: Mieloid leukemia akut; penggangguan RNA; tapak jalan MAPK; tindak balas imun


Alejandro, E.U. & Johnson, J.D. 2008. Inhibition of Raf-1 alters multiple downstream pathways to induce pancreatic β-cell apoptosis. Journal of Biochemistry 238(4): 2401-2417.

Brancho, D., Ventura, J.J., Jaeschke, A., Doran, B., Flavell, R.A. & Davis, R.J. 2005. Role of MLK3 in the regulation of mitogen-activated protein kinase signaling cascades. Molecular & Cellular Biology 25(9): 3670-3681.

Chadee, D.N. & Kyriakis, J.M. 2004. A novel role for Mixed Lineage Kinase 3 (MLK3) in B-Raf / Raf-1 activation and cell proliferation. Cell Cycle 3(10): e73-e75.

Chen, J., Miller, E.M. & Gallo, K.A. 2010. MLK3 is critical in breast cancer cell migration and promotes a malignant phenotype in mammary epithelial cells. Oncogene 29: 4399-4411.

Cheng, J.C., Moore, T.B. & Sakamoto, K.M. 2003. RNA interference and human disease. Molecular Genetic & Metabolism 80(1-2): 121-128.

Elbashir, S.M., Lendeckel, W. & Tuschl, T. 2001. RNA interference is mediated by 21-22 nucleotide RNAs. Genes & Development 15(2): 188-200.

English, J.M., Pearson, G., Hockenberry, T., Shivakumars, L., White, M.A. & Cobb, M.H. 1999. Contribution of the ERK5/ MEK5 pathway to Ras/Raf signaling and growth control. The Journal of Biological Chemistry 274(44): 31588-31592.

Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E. & Mello, C.C. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391(6669): 806-811.

Fuchs, S.Y., Adler, V., Pincus, M.R. & Ronai, Z. 1998. MEKK1/ JNK signaling stabilizes and activates p53. Proceedings of the National Academy of Science 95: 10541-10546.

Gallagher, E.D., Xu, S., Moomaw, C., Slaughter, C.A. & Cobb, M.H. 2002. Binding of JNK/SAPK to MEKK1 is regulated by phosphorylation. The Journal of Biological Chemistry 277(48): 45785-45792.

Gantier, M.P., Tong, S., Behlke, M.A., Irving, A.T., Lappas, M., Nilsson, U.W., Latz, E., McMillan, N.A.J. & Williams, B.R.G. 2010. Rational design of immunostimulatory siRNAs. Molecular Therapy 18(4): 785-795.

Giuliano, Jr. J.S., Lahni, P.M., Wong, H.R. & Wheeler, D.S. 2011. Extracellular heat shock proteins: Alarmins for the host immune system. The Open Inflammation Journal 4(Suppl 1-M6): 49-60.

Gregory, T.K., Wald, D., Chen, Y., Vermaat, J.M., Xiong, Y. & Tse, W. 2009. Molecular prognostic markers for adult acute  myeloid leukemia with normal cytogenetics. Journal of Hematology and Oncology 2: 23.

Haferlach, T. 2008. Molecular genetic pathways as therapeutic targets in acute myeloid leukaemia. Hematology 2008: 400-411.

Hirano, T., Shino, Y., Saito, T., Komoda, F., Okutomi, Y., Takeda, A., Ishihara, T., Yamaguchi, T., Saisho, H. & Shirasawa, H. 2002. Dominant negative MEKK1 inhibits survival of pancreatic cancer cells. Oncogene 21: 5923-5928.

Hood, E. 2004. RNAi: What’s all the noise about gene silencing?. Environmental Health Perspectives 112(4): A224-A229.

Huang, D.W., Sherman, B.T. & Lempicki, R.A. 2009. Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nature Protocols 4(1): 44-57.

Kim, D.H. & Rossi, J.J. 2007. Strategies for silencing human disease using RNA interference. Nature Reviews Genetic 8: 173-184.

Kim, H., Kojima, K., Swindle, C.S., Cotta, C.V., Huo, Y., Reddy, V. & Klug, C.A. 2008. FLT3-ITD cooperates with inv (16) to promote progression to acute myeloid leukaemia. Blood 111(3): 1567-1574.

Kim, K.Y., Kim, B.C., Xu, Z. & Kim, S.J. 2004. Mixed Lineage Kinase 3 (MLK3)-activated p38 MAP kinase mediates transforming growth factor – β – induced apoptosis hepatoma cells. The Journal of Biological Chemistry 279(28): 29478- 29484.

Kim, N.V. 2003. RNA interference in functional genomics and medicine. Journal Korean Medical Science 18: 309-318.

Kingsley, D.M. 1994. The TGF-β superfamily: New members, new receptors, and new genetic tests of function in different organisms. Genes & Development 8: 133-146.

Maekawa, T., Shinagawa, T., Sano, Y., Sakuma, T., Nomura, S., Nagasaki, K., Miki, Y., Saito-Ohara, F., Inazawa, J., Kohno, T., Yokota, J. & Ishii, S. 2007. Reduced levels of ATF-2 predispose mice to mammary tumors. Molecular & Cellular Biology 27(5): 1730-1744.

Minoo, P., Zlobec, I., Baker, K., Tornillo, L., Terraciano, L., Jass, J.R. & Lugli, A. 2007. Loss of raf-1 kinase inhibitor protein expression is associated with tumor progression and metastasis in colorectal cancer. American Journal of Clinical Pathology, 127: 820-827.

Sebolt-leopold, J.S., Dudley, D.T., Herrera, R. Becelaere, K.V., Wiland, A., Gowan, R.C., Tecle, H., Barrett, S.D., Bridges, A., Przybranowski, S., Leopold, W.R. & Saltiel, A.R. 1999. Blockade of the MAP kinase pathway supresses growth of colon tumors in vivo. Nature Medicine 5: 810-816.

Sondarva, G., Kundu, C.N., Mehrotra, S., Mishra, R., Rangasamy, V., Sathyanarayana, P., Ray, R.S., Rana, B. & Rana, A. 2010. TRAF2 – MLK3 interaction is essential for TNF – α – induced MLK3 activation. Cell Research 20: 89-98.

Su, F., Li, H., Yan, C., Jia, B., Zhang, J. & Chen, X. 2009. Depleting MEKK1 expression inhibits the ability of invasion and migration of human pancreatic cancer cells. Journal Cancer Research & Clinical Oncology 135: 1655-1663.

Sundstrom, C. & Nilsson, K. 1976. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). International Journal of Cancer 17(5): 565-577.

Tallman, M.S., Gililand, D.G. & Rowe, J.M. 2005. Drug therapy for acute myeloid leukemia. Blood 106: 1154-1163.

Tibbles, L.A., Ing, Y.L., Kiefer, F., Chan, J., Iscove, N., Woodgett, J.R. & Lassam, N.J. 1996. MLK-3 activates the SAPK/JNK and p38/RK pathways via SEK1 and MKK3/6. The EMBO Journal 15(24): 7026-7035.

Towatari, M., Iida, H., Tanimoto, M., Iwata, H., Hamaguchi, M. & Saito, H. 1997. Constitutive activation of mitogen-activated protein kinase pathway in acute leukemia cells. Leukemia 11: 479-484.

Weissinger, E.M., Eissner, G., Grammar, C., Fackler, S., Haefner, B., Yoon, L.S., Lu, K.S., Bazarov, A., Sedivy, J.M., Mischak, H. & Kolch, W. 1997. Inhibition of Raf-1 kinase by cyclic AMP agonists causes apoptosis in v-abl transformed cells. Molecular and Cellular Biology 17(6): 3229-3241.

Whelan, J. 2005. First clinical data on RNAi. Drug Discovery Today 10: 1014-1015.

Zamore, P.D., Tuschl, T., Sharp, P.A. & Bartel, D.P. 2000. RNAi: Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21-23 nucleotide intervals. Cell 101: 25-33.



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