Oncogenic NK-Like Homeodomain Proteins Inhibit Etoposide-Induced Apoptosis in T-ALL Cell Lines by Activation of the Polycistronic miR-17-92 Transcript.

Homeobox genes of the NK-like familiy, including TLX1, TLX3 and NKX2-5, are ectopically activated in T-cell acute lymphoblastic leukemia (T-ALL) cells mostly via chromosomal aberrations. The pathologic function of these closely related genes is still unclear. Here we analyzed their effect on the C13ORF25 gene, containing the miR-17-92 cluster. Micro RNAs (miRNAs) are a class of small non-coding RNAs which are part of an evolutionarily highly conserved intracellular mechanism, regulating gene expression by hybridization to complementary sequences usually located in the 3′untranslated region of coding transcripts. The primary transcripts (pri-mRNA) are processed to short mature miRNAs, mediating either inhibition of mRNA translation or mRNA cleavage. Aberrant expression of specific miRNAs is involved in oncogenesis as recently described for several human malignancies. The miR-17-92 polycistron encodes miRNAs which decrease E2F1 protein expression. Transcription of both E2F1 and miR-17-92 is induced by MYC, itself a target of E2F1, generating a highly regulated interactive network. Depending on the cellular context, E2F1 performs conflicting tasks by triggering proliferation or inducing apoptosis. We investigated the expression of the miR-17-92 cluster in T-ALL cell lines. Real-time RT-PCR analysis of both miR-17-92 pri-mRNA and mature miRNAs revealed different expression levels in these cells, suggesting a possible implication of the NK-like homeodomain proteins in the regulation of the miR-17-92 cluster in T-ALL. HELA cells transfected with TLX1 or NKX2-5 expression constructs showed elevated miR-17-92 pri-mRNA expression, demonstrating an activating effect. Lentiviral-mediated overexpression of NKX2-5 in the T-ALL cell line MOLT-4 consistently showed increased miR-17-92 pri-mRNA levels and decreased E2F1 protein amounts. For functional analysis of these downstream targets, another T-ALL cell line (PEER) was lentivirally transduced with expression constructs for either miR-17-92 or E2F1, resulting in reduced or elevated E2F1 protein levels, respectively. Overexpression of miR-17-92 or E2F1 did not significantly influenced the cell proliferation. However, induction of apoptosis by treating these cells with etoposide, an inhibitor of topoisomerase II, indicated that overexpression of miR-17-92 and E2F1 resulted in enhanced and reduced cell viability, respectively, as analyzed by MTT assay. In summary, these data indicate an activatory effect of oncogenic NK-like homeodomain proteins on miR-17-92 expression, reducing E2F1 protein levels and thereby enhancing survival of leukemic T-cells.