B-Cell Lymphoma In Eu-Mir-17~92 Transgenic Mice

MicroRNAs (miRNAs) are small, 18–24 nucleotide long, non-coding RNAs that regulate gene expression by binding to the 3` untranslated region (UTR) of the target mRNAs, resulting in mRNA degradation or translational repression. The polycistronic miRNA cluster miR-17~92 consists of six miRNAs (miR-17, miR-18a, miR-19a, miR-19b, miR-20a and miR-92a) and is frequently overexpressed in various solid and lymphoid malignancies. Loss-of-function of this cluster revealed its role in heart and lung development (Ventura et al., 2008, Cell), whereas gain-of-function study associated it to acceleration of myc induced B cell lymphomas, lymphoproliferative disorders and autoimmunity (Xiao et al., 2008 Nat Immunol; He et al., 2005 Nature). Some of the key targets of this miRNA include the well known tumor suppressor gene, Pten and the proapoptotic gene, Bim. In order to further understand the mechanisms of miR-17~92 induced B-cell malignancies, we generated a B-cell specific miR-17~92 transgenic mouse model.

Overexpression of miR-17~92 cluster in mice was driven by the Em enhancer and immunoglobulin heavy chain promoter. The transgenic construct contained Green Fluorescent Protein (GFP) tag in order to track the miRNA. Northern Blot analysis for miRNA expression identified three positive founder lines. The animals were maintained and bred at the OSU vivarium under the IACUC approved protocol and guidelines. The mice were followed for two years for survival, phenotype characteristics and disease. Phenotyping involved flow cytometry to study the distribution of various hematopoietic cell populations in the spleen and Hematoxylin and Eosin (H&E) staining to analyze the pathology and histology of various tissues from transgenic and wild type mice. Clonality of the lymphomas was assessed by using JH4 probe for immunoglobulin heavy chain and TCR-a for T-cell receptor rearrangement, using Southern blot on EcoRI digested genomic DNA from total splenocytes. For gene expression analysis, total RNA was isolated from FACs sorted GFP+ malignant and MACS sorted normal CD19+ B cells using Trizol from transgenic and wild type mice, respectively. RNA was hybridized to Mouse430_2 genome arrays and data was analyzed using BRB-Array tools.

Northern Blot analysis of miR-17~92 in total splenocytes and bone marrow cells of transgenic mice showed increased expression for all six members of the cluster with highest expression in the spleen. Approximately 40% of the transgenic mice developed high grade B-cell lymphoma by nine to twelve months of age. The diseased mice had ascites, enlarged lymph nodes, and spleen, and the malignant cells at these sites were GFP positive. Histological analysis showed these mice have lymphomas infiltrating multiple organs (lungs, kidney, brain) at nine to twelve months of age. Preliminary analysis of spleenic lymphocytes showed significantly expanded CD5+ CD19+ B-cell populations. Southern Blot analysis showed these cells to be oligoclonal B cells, while no T cell clonality was observed. The results showed 782 genes to be up- and 1019 genes to be downregulated in transgenic B-cells versus wild type CD19+ B-cells. Ingenuity pathway analyses showed that majority of the downregulated genes were associated with hematological system development and function, and inflammatory response pathways, while the upregulated genes were represented by cell cycle pathway. Some of the upregulated genes in miR-17~92 over-expressing malignant B-cells include ZAP-70, LEF1, TGFBR3, were previously reported in B-CLL gene signature (Jelinek et al., 2003 MCR; Klein et al., 2001 JEM). Among other overexpressed genes, cJUN, cFOS and ATF3 were upregulated by 5-, 67- and 18-fold, respectively. These results were also confirmed by western blots. Jun:Fos and Jun:ATF complexes represent two classes of AP-1 transcription factor family. This suggests that AP-1 dependent transcription may be the key survival factor in the miR-17~92 driven lymphomas. Currently, we are exploring the negative regulators of the oncogenic cJun signaling pathway which may be miR-17~92 targets.

This model provides a useful tool to study role of this miRNA and its targets in lymphomagenesis. In addition, this model can be used for therapeutic targeting of miR-17~92 driven malignancies, both lymphomas and solid tumors using miR-inhibitors or antimiRs.

No relevant conflicts of interest to declare.