Abstract LB-289: Interaction between miR-155 and Quaking in the innate immune response and leukemia

Abstract

Abstract Rationale: miR-155 is a pro-inflammatory microRNA upregulated in human and mouse macrophages exposed to lipopolysaccharide (LPS) that is required to mount an effective immune response. High levels of miR-155 are observed in different solid tumors as well as leukemias, including chronic lymphocytic leukemia (CLL). Quaking (QKI) is a tumor-suppressor gene encoding a conserved RNA-binding protein. In silico analyses predict that the QKI transcript is a target of miR-155, and we hypothesized that miR-155 might carry out its pro-inflammatory and oncogenic signals at least in part by targeting QKI. Methods: Mouse RAW-264.7 macrophages were stimulated with LPS or mock PBS three times over a period of 6 days. qRT-PCR was used to monitor the expression of QKI, miR-155 and Tnf (tumor necrosis factor alpha), and Quaking protein (Qki) expression was analyzed by Western blot. Expression of miR-155 and QKI in Burkitt's lymphoma cell lines and CLL cell lines was measured by qRT-PCR; QKI expression was also determined by Western blot. Cell lines were transfected with miR-155 or miR-control, or 155-I, and Western blot analysis of QKI was performed after 48 hours. To study the in vivo effects of the cooperation between miR-155 and QKI, we analyzed Qki expression in splenic B cells from wild type C57B1/6 mice, miR-155−/- mice, and Eμ-miR-155 transgenic mice (which develop aggressive CLL), with and without LPS challenge. Finally, expression of Qki was determined in samples from patients with B-cell CLL. Results: After 8 hours of LPS challenge there was a 2-fold decrease in QKI expression, while miR-155 and Tnf both increased approximately 10-fold (p<0.05). QKI returned to pre-treatment levels at 2 days, while miR-155remained high for 3 days. LPS re-stimulation at 3 days (mimicking chronic inflammation) reduced QKI expression 2.3-fold (p<0.05) over a 48 hour period in parallel with a renewed up-regulation of miR-155, and Western blotting confirmed the above observed changes for Qki. CLL-derived cell lines showed significantly higher expression of miR-155, and lower expression of QKI, compared with Burkitt's lymphoma cell lines (p<0.05). Transfection of miR-155 led to decreased Qki expression in Burkitt's lymphoma lines, while 155-I transfection of MEC2 CLL cells caused Qki upregulation at 48 hours. Splenic B cells from leukemic Eμ-miR-155 mice had decreased Qki levels compared with C57B1/6 mice and miR-155−/−mice, and LPS challenge of cultured B cells resulted in a pronounced downregulation of Qki expression only in B cells coming from Eμ-miR-155 mice. Finally, reduced expression of Qki was observed in B cells coming from patients with CLL compared with healthy patients. Conclusions: The inverse relationship between miR-155 and Qki is specific, and may represent a regulatory mechanism for the immune response mounted by macrophages and B cells. This relationship also sheds light on the pro-inflammatory and oncogenic properties of miR-155 in leukemia. Citation Format: Timothy K. Richmond, Esmerina Tili, Melissa Brown, Marcela Chiabai, Dario Palmieri, Ri Cui, Tyler Sheetz, Carlo M. Croce. Interaction between miR-155 and Quaking in the innate immune response and leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-289. doi:10.1158/1538-7445.AM2015-LB-289