In this study we investigated the effect of elevated expression of the miR-17−92 cluster, a miRNA cluster highly expressed in some human lymphomas, on lymphocytes and its possible contribution to lymphomagenesis, by genetically increasing miR-17−92 expression in lymphocytes and identifying two tumor suppressors, Pten
, as functionally important targets. We propose that the downregulation of Pten and Bim protein expression by miR-17−92 miRNAs contributes to the lymphoproliferative and autoimmune phenotype observed in miR-17−92
transgenic mice, and to lymphoma development in patients in which the miR-17−92 coding region is amplified in tumor cells. In TG/TG mice, elevated miR-17−92 expression was noted in all B and T cells, as it was driven by the expression of the hCD2-iCre
. As Pten and Bim play important roles in maintaining central and peripheral tolerance 6, 7, 11, 12
, downregulation of Pten and Bim protein expression could potentially cause the breakdown of both tolerance mechanisms. In vitro
activation assays showed that TG/TG CD4+
T cells proliferated more and survived better upon activation, suggesting the breakdown of peripheral T cell tolerance mechanisms. Whether peripheral B cell tolerance mechanisms and central tolerance mechanisms are intact in TG/TG mice remains to be tested in separate experiments before a firm conclusion can be reached. However, in a first approach, we examined the percentage of bone marrow Hardy Fraction E cells, and transitional and mature splenic B cells expressing Igλ proteins, whose selective loss during B cell maturation has been attributed to negative selection 38
. We did not find any significant difference between control and TG/TG mice (data not shown), suggesting that negative selection of autoreactive B cells during development is intact in TG/TG mice. We also examined the number of Foxp3+
T cells in the thymus and the spleen and did not find any significant difference between control and TG/TG mice (data not shown). Therefore, it is likely that elevated miR-17−92 expression leads to breakdown of T cell tolerance in the periphery, causing the accumulation of activated CD4+
T cells, which provide sufficient help to stimulate B cells to enter germinal center reactions, where they undergo class switch recombination and somatic hypermutation. Some of these cells may differentiate into plasma cells and produce autoantibodies that were detected in the serum of TG/TG mice. Deposition of IgG immune complexes caused tissue damage, as shown by histological analysis. Similar observations have been reported for sanroque
mice, which carry a point mutation in the roquin
gene. In sanroque
mice, accumulation of activated CD4+
T cells leads to B cell activation, germinal center reactions, and autoantibody production 39
. TG/TG mice may die of autoimmune disease before TG/TG lymphocytes accumulate sufficient oncogenic mutations for malignant transformation.
In lymphoma patients carrying amplifications of the miR-17−92 coding region in the tumor cells, this amplification likely occurs somatically in individual B or T cells during the course of their malignant transformation. Such an amplification will not give rise to systemic autoimmune disease similar to that observed in TG/TG mice. However, cells with elevated miR-17−92 expression have a lower threshold for activation, proliferate more and survive better upon antigen recognition. These features could lead to an expansion of the mutant cell population, which would increase the probability that such cells, rather than their un-mutated counterparts, acquire additional oncogenic mutations. Elevated miR-17−92 expression may also cooperate with pre-existing oncogenic mutations.
These proposed molecular mechanisms may also explain the accelerated lymphomagenesis observed upon retroviral expression of miR-17−92 in Eμ-Myc
transgenic mice 22
. Bim functions as a tumor suppressor in Eμ-Myc
transgenic mice, and Bim protein expression is increased in Eμ-Myc
transgenic B cells and mediates apoptosis. Loss of one allele of Bim accelerates cMyc-induced lymphoma development, almost as effectively as loss of both alleles of Bim 8
. Retroviral expression of miR-17−92 accelerated Myc-induced lymphoma development to the same degree as loss of one Bim allele22
. We measured Bim protein concentrations in lymphocytes of Bim+/−
mice by immunoblot and the 50% reduction we observed was close to that caused by elevated miR-17−92 expression in TG/TG mice. This suggests that down-regulation of Bim protein expression by miR-17−92 is a major mechanism underlying the accelerated cMyc-induced lymphomagenesis induced by miR-17−92.
How do miR-17−92 miRNAs control Pten and Bim protein expression? Both Pten and Bim 3’UTRs contain multiple binding sites for miR-17−92 miRNAs, and they are among the top ten targets when mRNAs containing at least one site for each of the distinct miRNA families in the miR-17−92 cluster are ranked by the total number of binding sites for these miRNAs. Based on the results from the reporter assays performed in NIH3T3 cells over-expressing miR-17−92 and in HeLa cells with individual miR-17−92 miRNAs sequestered by antisense LNA oligonucleotides, the first miR-17−5p site and the second miR-19 site in the P1 fragment cooperate to mediate the repression of the Pten protein levels. For the Bim 3’UTR, the two miR-92 sites seem to play dominant roles. These observations are interesting, in that they confirm the idea derived from previous bioinformatic analyses that co-expressed miRNAs may cooperate to suppress a common target gene bearing binding sites for these miRNAs 34
. They also suggest that not all miRNA binding sites are functional in a given cellular context. Recent studies have shown that the context or accessibility of miRNA binding sites, and the protein factors binding in close proximity of the sites, strongly influence the function of individual sites 40-42
. It is therefore possible that non-functional sites in NIH3T3 and/or HeLa cells could be functional in lymphocytes or other cells. Alternatively, the expression of other miRNAs with identical seed regions could confer functional redundancy in cells in which reporter assays were performed.
To what degree does downregulation of Pten and Bim protein contribute to the phenotype of TG/TG mice? hCD2-iCre
mice exhibited an abnormal, progressive accumulation of antigen-experienced CD4+
T cells and germinal center B cells, similar to the TG/TG mice. However, the massive lymphocyte population expansion seen in the latter mice did not occur in hCD2-iCre
mice. Therefore, additional miR-17−92 target genes must be taken into account to fully explain the phenotype of TG/TG mice. E2F family transcription factors have been implicated as functional targets of miRNAs in the miR-17−92 cluster 43,44
. E2F1 and E2F2 are important cell cycle regulators, and a reduction in their protein expression leads to the development of hematopoietic malignancy and autoimmunity 45-48
. It is thus conceivable that down-regulation of E2F1 and E2F2 protein synergizes with that of Pten and Bim in causing the severe disease in TG/TG mice. Because of the unavailability of suitable antibodies against murine E2F1 and E2F2 we were unable to directly test this hypothesis.
Taken together, our data indicate that TG/TG mice develop a dramatic lymphoproliferative and autoimmune disease. Pten and Bim are direct targets of miRNAs in the miR-17−92 cluster, and hCD2-iCre;Ptenfl/+;Bim+/− mice exhibit part of the phenotype displayed by TG/TG, demonstrating that miR-17−92-mediated down-regulation of Pten and Bim protein expression indeed contributes to the disease phenotype in TG/TG mice. Clearly, however, miR-17−92 over-expression affects additional pathways in the cells, whose de-regulation, together with that of Pten and Bim, are required for the massive population expansion seen in TG/TG mice. With respect to lymphomagenesis, our results suggest that miR-17−92 over-expression may well play a role in early phases of disease.