Mir-155 Contributed to Chronic Lymphocytic Leukemia Survival by Modulation of BCR-Signalling

Abstract

Abstract 620The microRNA miR-155 is encoded within the B-cell integration cluster bic, which is a frequent integration site for avian leucosis virus. This microRNA is over-expressed in certain B cell lymphomas, including chronic lymphocytic leukemia (CLL), suggesting miR-155 contributes to lymphoma development. Consistent with this are observations that mice made transgenic for miR-155 under a B-cell-specific promoter develop pre-B cell lymphomas. One of the target genes regulated by miR-155 is SHIP-1, a phosphatase that plays a critical role in down-modulating B-cell receptor (BCR) signaling, which appears to provide a growth and/or survival stimulus for CLL B cells. To test for this, we examined for relationships between the CLL-cell expression of miR-155, SHIP-1, magnitude of BCR-signaling, and time from diagnosis to initial therapy (TFS) in 33 pts with CLL. The relative level of miR-155 was determined by real-time PCR. SHIP-1 protein was determined by flow cytometry and immunoblot analyses. We noted variations in expression levels of miR-155 among patient samples studied. CLL cells that expressed levels of miR-155 higher than the median level of miR-155 for all patients studied had significantly lower expression levels of SHIP-1 protein than CLL cells that expressed levels of miR-155 lower than the median level of miR-155 had (7.0 ± 0.2 vs. 8.3 ± 0.47, mean ± SEM, P<0.05). CLL B cells were stimulated with anti-μ or control Ig and then examined for relative protein phosphorylation and calcium influx. CLL cases were segregated into groups with high-BCR signaling versus low BCR-signaling based on their responses to anti-μ stimulation. We found that CLL cells with high-BCR signaling expressed significantly higher levels of miR-155 (1.54 ± 0.22) than did CLL cells with low-BCR signaling (0.90 ± 0.13, p<0.05). CLL cells with high BCR-signaling had significantly lower amounts of SHIP-1 protein (7.1 ± 0.39) than did CLL cells with low BCR-signaling (9.12 ± 0.7, p<0.05). Moreover, Kaplan-Meier survival analysis revealed an association between higher expression levels or miR-155 and shorter survival times from diagnosis to initial therapy (TFS) (P<0.05). CLL patients with greater miR-155 expression had a median TFS of 25.9 months that was significantly shorter than the median TFS of 112.8 months for patients with low miR-155 expression. To further investigate its function role, CLL cells were transfected with miR155 mimic or a miR-155 inhibitor and examined for SHIP-1 expression, BCR signaling, and cell survival, with or without stromal cells. Introduction of miR-155 to CLL cells that had low expression levels of miR-155 significantly reduced SHIP-1 protein expression, enhanced BCR signaling and improved cell survival relative to that of mock transfect CLL cells or CLL cells tranfected with micro RNA. Conversely, transfection of CLL cells that had high expression levels of miR-155 with miR-155 inhibitor resulted in significantly increased SHIP-1 expression, reduced BCR signaling, and poor survival than mock transfected CLL cells. Moreover, transfection of miR-155 inhibitor significantly reduced the capacity of stromal cells to enhance CLL cell survival. We found that the viability of cells transfected with miR-155 inhibitor was significantly lower than that noted for cells transfected with control miRs when cocultured with stromal cells. These results demonstrate that expression of miR-155 can modulate CLL expression of SHIP-1, CLL cell BCR signaling, and survival. Disclosures:No relevant conflicts of interest to declare.