282270 CEBPA Mutant Regulates miR181a Expression in AML Cells: A Single Cell Study by Nanochannel Electroporation

Thursday, November 1, 2012: 9:42 AM
Somerset East (Westin )
Xi Zhao1, Yun Wu2, Xinmei Wang2, Daniel Gallego-Perez2, Pouyan E. Boukany2, Xiaomeng Huang2, Sebastian Schwind3, Guido I. Marcucci3 and L. James Lee1, (1)William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, (2)Nanoscale Science and Engineering Center, The Ohio State University, Columbus, OH, (3)Division of Hematology, Department of Internal Medicine, Comprehensive Cancer Center, The Ohio State University, Columbus, OH

One of the major disadvantages of conventional electroporation is the compromise between viability and efficiency, especially with large plasmids. With certain combination of gene and cell type, the transfection could be so poor that it no longer remains a meaningful tool for the study of gene expression. We have previously reported a nanochannel electroporation (NEP) technology which provides high transfection efficiency and uniform dosage with minimal cell damage, thus high viability, for individual cells1.

Clinically, mutation of CCAAT/enhancer-binding protein alpha (CEBPα) gene is observed in some population of acute myeloid leukemia (AML) patients2, of which certain sub-groups show significant advantage of disease progression. Although the full and detailed effects of such mutation are yet to be established, we have previously found correlations between favorable clinical outcome, certain sub-type of mutation and increase of miR181a expression level in patient samples3. However, verification of such correlation in vitro in AML cell lines that do not express endogenous CEBPα was unsuccessful due to the low expression and viability after conventional electroporation.

Here we applied our NEP platform to transfect wild type and mutated CEBPα gene into individual AML KG1a cells as well as observing miR181a expression level afterwards. The cell viability and gene expression were verified by EthD-1/Calcein staining kit and the use of CEBPα-GFP encoded plasmid. At different time points after transfection, we analyzed the expression of miR181a by injecting a locked nucleic acid (LNA) molecular beacon (MB) targeting mature miR181a into the cells, and retrieving the transfected cells for single cell qRT-PCR. Our results shows that while the wild type CEBPα gene, of which the mRNA can be translated to two polypeptides with different length (p42 and p30, respectively), can increase the expression of miR181, the N-terminal mutant of this gene, which exclusively express p30, results in a statistically larger upregulation of miR181. In contrast, the C-terminal mutant, which may inhibits the function of CEBPα, has no significant effect on the expression of miR181a.

Our NEP technology demonstrated here for the study of gene expression/regulation successfully combines precise transfection with target detection, and shows great compatibility with small cell population. In the case of rare cell analysis, this platform will be the perfect tool.

[1] Boukany PE, Morss A, Liao WC, et al., Nanochannel electroporation delivers precise amounts of biomolecules into living cells, Nature Nanotech, 2011;6, 747-754

[2] Leroy H, Roumier C, Huyghe P, et al., CEBPA point mutations in hematological malignancies, Leukemia, 2005;19, 329-334

[3] Marcucci G, Maharry K, Radmacher MD, et al., Prognostic significance of, and gene and microRNA expression signatures associated with, CEBPA mutations in cytogenetically normal acute myeloid leukemia with high-risk molecular features: a cancer and leukemia group B study, J Clin Oncol. 2008;26, 5078-5087

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