Introduction: microRNAs are involved in posttranscriptional regulation of mRNAs. Acute leukemia cells show a number of differentially expressed miRNA genes compared to normal cells. Some of the dysregulated miRNAs are highly specific for hematopoetic cells, others are involved in leukemogenesis. miR181a is involved in T-cell receptor signalling and expression correlates with morphology in myeloid leukemia cells. Here we determined the miR181a expression in acute lymphoblastic leukemia (ALL) cells and investigated by LNA-miR-181a specific knockdown whether the expression of its potential targets CD4 and high mobility group Box 1 (HMGB1) as well as cell survival are influenced. Methods: B- and T-ALL cell lines with different cytogenetics and phenotypes (REH, Jurkat, MOLT-4) were analysed. Expression of the miRNA was determined using stem-loop RT-PCR. Expression of HMGB1 was detected by Western Blot as well as quantitative RT-PCR. Expression of surface markers was determined by flow cytometry. For the expression of CD4 the relative mean fluorescence intensity (rMFI) was calculated (MFI sample/MFI mock). For knock-down experiments cells were transfected using electroporation and LNA - modified oligonucleotides (Exiqon A/S) complementary to the sequence of the hsa-miR181a, an unspecific oligonucleotide and water as mock control. After 15, 24, 48 and 72h samples were taken to define cell proliferation, expression of miRNA181a and its target genes. Results: miRNA181a was expressed in all analysed ALL cell lines. Expression levels were higher than in CD34+ cells. Transfection with both, unmodified and LNA-modified oligonucleotides, led to transfection rates of >70% (range 62% - 85%) and a strong knock down of miRNA 181a. In samples transfected with miRNA181a specific oligonucleotides a 19.5 - 59 fold reduction of miRNA181a expression compared to the mock controls was observed over time. The expression of CD4 increased in T-cell ALL MOLT-4 cells after transfection with the LNA-mi181a specific oligonucleotid compared to the unspecific control. The most pronounced effect on CD4 expression was seen after 24h (rMFI 0.92 (control) vs. 1.23 (specific); p=0.003). HMGB1 mRNA expression did not differ between specific transfected cells and controls. However, the protein expression of HMGB1 was reduced in REH cells after 15h and 24h following miRNA181a specific knock down. miRNA181a knock down did not influence proliferation in any analysed ALL cell line. Conclusion: ALL suspension cells can be successfully transfected with miRNA181a specific LNA-oligonucleotides leading to a significant reduction of miRNA expression. miRNA181a knockdown increases the surface expression of CD4 and seems to reduce the protein expression of HMGB1. miRNA181a knockdown does not influence cell proliferation.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2534.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

American Association for Cancer Research
2009