MicroRNA-Mediated Down-Regulation of the Tumor Suppressor Gene PRDM1/Blimp-1 in Diffuse Large B-Cell Lymphomas.

PR (PRDI-BF1-RIZ-homology) domain zinc finger protein 1 (PRDM1) is a master regulator in plasma cell differentiation recently identified as a tumor suppressor target for inactivation in diffuse large B-cell lymphomas (DLBCL) of the activated B-cell (ABC) type, implying interference of B-cell terminal differentiation as a pathogenetic mechanism in DLBCL. Besides deleterious gene mutations, it has been suggested that PRDM1 may also be inactivated in DLBCL by an epigenetic mechanism. In this study, we examined the hypothesis of microRNA (miRNA)-mediated down-regulation of PRDM1 in DLBCL. 10 DLBCL cell lines and 25 clinical DLBCL samples were analyzed for PRDM1α (PRDM1 functional isoform) RNA and protein expression by quantitative real-time reverse transcriptase-PCR, Western blotting and immunohistochemistry. The clinical samples included 5 ABC-DLBCLs with PRDM1 gene deletions and inactivating mutations (Group I), 12 ABC-DLBCLs without PRDM1 mutations (Group II) and 8 germinal center B-cell (GCB) type (Group III). The myeloma cell line U266, which expresses relatively abundant PRDM1α mRNA and protein, was used as a reference standard (arbitrarily set as 1). These expression studies identify desynchrony in PRDM1α mRNA and protein expression. PRDM1α is weakly expressed or undetectable in DLBCL cell lines regardless of levels of PRDM1α transcripts. For the primary DLBCL cases, the mean levels of PRDM1α mRNA in groups I, II and III were: 2.21+0.53 (p<0.05 vs. II & III), 0.84+0.19, and 0.43+0.24, respectively. However, immunohistochemistry demonstrated that in all three DLBCL groups, including Group II which has relatively high PRDM1α mRNA and harbors no PRDM1 mutations, an average of only <5% (range: 0 to 10%) of the neoplastic B cells weakly expressed PRDM1. These results suggest epigenetic down-regulation of PRDM1 protein expression in some DLBCLs. Several lines of evidence support a role for miRNA let-7 in mediating translation repression of PRDM1 in DLBCLs: (1) let-7a levels in DLBCL cell lines and primary cases, as determined by quantitative modified Invader assays, are higher (∼2 to 30 fold) than in U266; (2) The lowest (let-7a)/(PRDM1α mRNA) ratio is found in those ABC-DLBCLs harboring PRDM1 mutations; (3) Enforced expression of let-7a caused binding site-dependent reduction in reporter gene activities of at least 50%. This reduction is due to translation repression; (4) Enforced let-7a expression reduces PRDM1α levels by ∼ 50% in U266 cell lines, suggesting functional in vivo interaction of let-7a with PRDM1 mRNA. In conclusion, PRDM1 protein levels correlate poorly with PRDM1 mRNA expression in DLBCLs. Our studies suggest miRNA-mediated down-regulation as a mechanism of lowering PRDM1 activity in DLBCL, apart from genetic mutations and transcription repression. In ABC-DLBCLs without PRDM1 gene mutations, PRDM1 inactivation is likely mediated at least in part via translation repression of PRDM1 transcripts by high levels of let-7. Those ABC-DLBCLs with PRDM1 gene mutations might have “escaped” let-7-mediated down-regulation, for example, via higher levels of induction of PRDM1 transcripts or some other mechanisms. let-7 may be considered a potential target for therapeutic inhibition to restore terminal differentiation in DLBCL cells.