miR155 DOWNREGULATES EXPRESION of HGAL and INCREASES LYMPHOMA CELL MOTILITY

Abstract 464

HGAL is a germinal center (GC)-specific gene that inhibits lymphocyte and lymphoma cell motility and whose expression predicts improved survival of patients with diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). Its specific expression only in the GC B-cells and GC-derived lymphomas suggest the existence of a tight regulatory process. We have previously reported that IL-4 and IL-13 can induce HGAL RNA and protein expression (Lossos et al, Blood. 2003; Natkunam et al, Blood. 2007) and at this meeting we also report that HGAL transcription is regulated by BLIMP1 protein (Cubedo et al). However, recent observations suggest that the fine-tuned control of gene expression, necessary for the immune cell differentiation-specific gene expression, is frequently accomplished by miRNAs. Therefore we have searched three prediction algorithms, PicTar (http://pictar.mdc-berlin.de/), miRanda (http://cbio.mskcc.org/ mirnaviewer/), and TargetScan (http://www.targetscan.org/) to identify miRNAs potentially regulating HGAL expression. We have focused on miR155, predicted to target HGAL in the TargetScan algorithm, since miR155 is overexpressed in aggressive activated B-cell like (ABC) type of DLBCL, characterized by low expression of HGAL. Furthermore, miR155 plays an important role in the regulation of GC reaction (Thai et al, Science. 2007) and its overexpression in mice B-lymphocytes leads to preleukemic B cell proliferation followed by full blown B-cell malignancy (Costinean et al, Proc Natl Acad Sci USA. 2006). To test HGAL regulation by the miR155, precursor of hsa-miR-155 was transfected into VAL and Raji lymphoma cells that express endogenous HGAL protein. Western blotting of whole-cell lysates showed a decrease of native HGAL in both cell lines compared with control miRNA transfection. Transfection of hsa-miR155 precursor into 3 lymphoma cell lines did not result in uniform decreases in HGAL mRNA expression levels, suggesting main regulation at the protein translation level. Transfection efficacy in each experiment was confirmed by measurements of miR155 by TaqMan MicroRNA Assay. To confirm direct effect on HGAL expression, we fused 2614 bp 3'-UTR sequences of HGAL containing two putative binding sites (M1 positions 2285–2291 and M2 positions 1844–1849) to a luciferase reporter gene. Cotransfection of the hsa-miR155 precursor with the luciferase reporter significantly repressed luciferase activity compared to a nontargeting control. To identify the binding site and to further demonstrate the specificity of the interaction, we generated luciferase reporter constructs with mutations in the M1 and M2 binding sites. Mutagenesis of M2 but not M1 site reversed the inhibitory effect of miR155, thus establishing M2 as the binding site. Since HGAL inhibits motility and chemotaxis of B lymphocytes and B cell lymphoma cells, we next examined effect of mir155 on SDF1 and IL-6-induced chemotaxis and spontaneous motility of VAL and Raji cells. Decrease in HGAL protein levels by hsa-miR155 precursor led to increased cell chemotaxis, cell motility, and cell velocity and was associated with decreased actin polymerization (F-actin content). The-miR155 effect on chemotaxis was rescued by transfection of HGAL-encoding vector. Since HGAL exerts its motility inhibitory effects by activation of RhoA, we next evaluated effect of miR155 on levels of activated GTP-bound RhoA. Overexpression of miR155 resulted in decreased levels of GTP-bound RhoA in both Raji and Val cell lines. Our findings demonstrate that miR155 may not only lead to oncogenic transformation of B lymphocytes, but also increases lymphoma cell motility by down regulation of HGAL expression. This effect may contribute to lymphoma cell dissemination and aggressiveness, characteristic of ABC-like DLBCL typically expressing high levels of miR155 and lacking HGAL expression.