The Mir-17~92 Cluster Family Determines the Responsiveness of CLL Cells with Unmutated IGHV Genes to Toll-Like Receptor 9 Triggering

The clinical course of chronic lymphocytic leukemia (CLL) is highly variable and prognosis is strongly associated with the mutational status of the IGHV genes. Recently, it has been observed that CLL cells expressing unmutated (UM) IGHV genes can be more efficiently induced to proliferate by stimulation of Toll-like Receptor 9 (TLR9) with unmethylated CpG oligonucleotides (CpG) than CLL cells expressing mutated (M) IGHV genes. MicroRNA (miRNAs) are 18- to 22-nucleotide-long RNA molecules that regulate gene expression and play a key role in several biological process including oncogenesis. Although the recognized pathogenetic relevance of miRNAs in CLL, their involvement in regulating activation/proliferation processes of CLL cells has still to be elucidated.

Freshly-isolated negatively-selected CLL cells from 19 patients (9 UM and 10 M CLL) were stimulated with CpG or left unstimulated for 18 hours. MiRNA profiling and Gene expression profiling (GEP) were performed according to Agilent Technologies protocols. Bioinformatics analyses were performed integrating three different methods for supervised analysis (LIMMA algorithm, Agilent and Partek softwares).

The miRNA profile of CLL cells treated or not with CpG was separately evaluated in M and UM CLL. Consistent with the notion that M CLL cells are usually non-responsive to CpG stimulation, no miRNA was found to be differentially expressed between CpG-stimulated and unstimulated CLL cells belonging to this subgroup. In contrast, in UM CLL, as many as 28 miRNAs resulted differentially expressed, 24 up-regulated (miR-1260, miR-1274a, miR-1274b, miR-1280, miR-155, miR-155*, miR-17, miR-17*, miR-18a, miR-196a, miR-19b-1*, miR-20a, miR-20b, miR-221, miR-221*, miR-222, miR-29b-1*, miR-30b*, miR-30d*, miR-374b*, miR-720, miR-886-3p, miR-92a-1*, miR-939) and 4 down-regulated (miR-1226*, miR-125a-3p, miR-135a*, miR-150*) upon CpG stimulation. Data were confirmed by quantitative real time PCR. In order to identify the miRNAs actually involved in regulating activation/proliferation processes induced by TLR9 triggering, a concomitant GEP was performed comparing the same UM CLL cells exposed or not to CpG. Data analysis was carried out by taking advantage of the T-REX software that, by integrating four algorithms and six different target prediction programs, allows the identification of the regulated miRNAs on the basis of their repression activity on target mRNA. T-REX application selected four miRNAs whose mRNA targets resulted significantly down-regulated upon TLR9 triggering, namely miR-17, miR-20a, miR-20b and miR-93a. All these miRNAs belong to the miR-17~92 cluster family, known to be over-expressed in a variety of B-cell lymphomas, including diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma and mantle cell lymphoma. Notably, three of these miRNAs and four additional miRNAs also belonging to the miR-17~92 cluster family (e.g. miR-17*, miR-18a, mir-19b-1* and mir-92a-1*) turned out to be among the 24 up-regulated miRNAs in CpG-stimulated UM CLL cells. In-silico analyses performed with the “Onto-Express” software, found that several differentially expressed genes were included in Gene Ontology (GO) categories related to regulation of cell proliferation, G1/S transition, apoptosis and NFkB signalling, in keeping with the typical proliferative response induced by CpG stimulation in UM CLL cells. The down-regulated genes included in these categories comprised CDKN1B/P27, CCNG2, NCOA3, E2F5, MAPK4, TRIM8, ZBTB4 and TP53INP1, all known target of miR-17~92 cluster family. Notably, the gene for the negative cell cycle regulator CDKN1B/P27 is also targeted by miR-221 and miR-222, two miRNAs both up-regulated in UM CLL cells upon CpG stimulation. Finally, transcripts for the proto-oncogene MYC also resulted over-expressed upon CpG stimulation. This observation may be relevant given the capacity of MYC to directly and positively regulate expression of miRNAs belonging to the miR-17~92 cluster family.

Induction of the miR-17~92 family is a specific feature of UM CLL cells triggered through TLR9 and is associated with down-regulation of genes involved in cell cycle control and apoptosis regulation. MiRNAs belonging to the miR-17~92 family may represent promising novel targets for biological therapies of high risk CLL.

No relevant conflicts of interest to declare.