Changes of Protein Expression after CRISPR/Cas9 Knockout of miRNA-142 in Cell Lines Derived from Diffuse Large B-Cell Lymphoma.

BACKGROUND: As microRNA-142 (miR-142) is the only human microRNA gene where mutations have consistently been found in about 20% of all cases of diffuse large B-cell lymphoma (DLBCL), we wanted to determine the impact of miR-142 inactivation on protein expression of DLBCL cell lines. METHODS: miR-142 was deleted by CRISPR/Cas9 knockout in cell lines from DLBCL. RESULTS: By proteome analyses, miR-142 knockout resulted in a consistent up-regulation of 52 but also down-regulation of 41 proteins in GC-DLBCL lines BJAB and SUDHL4. Various mitochondrial ribosomal proteins were up-regulated in line with their pro-tumorigenic properties, while proteins necessary for MHC-I presentation were down-regulated in accordance with the finding that miR-142 knockout mice have a defective immune response. CFL2, CLIC4, STAU1, and TWF1 are known targets of miR-142, and we could additionally confirm AKT1S1, CCNB1, LIMA1, and TFRC as new targets of miR-142-3p or -5p. CONCLUSIONS: Seed-sequence mutants of miR-142 confirmed potential targets and novel targets of miRNAs can be identified in miRNA knockout cell lines. Due to the complex contribution of miRNAs within cellular regulatory networks, in particular when miRNAs highly present in RISC complexes are replaced by other miRNAs, primary effects on gene expression may be covered by secondary layers of regulation.

Biochemical characterisation of the proteins encoded by the DiGeorge critical region 6 (DGCR6) genes.

The DiGeorge critical region 6 (DGCR6) gene exists in two highly homologous copies (DGCR6 and DGCR6L) on chromosome 22q11 and is deleted in patients with velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS). The DGCR6 mRNA levels are increased in metastatic mammary tumour cells and regulate the expression of neighbouring genes at the 22q11 region. Newly developed monoclonal antibodies detected predominantly nuclear phosphoproteins of approximately 25 kDa, with low expression levels in the cytoplasm. Both proteins have half-lives of about 2.5 h. Exogenously expressed DGCR6 and DGCR6L migrated with slightly different mobility in SDS-gels in accordance with two immunoreactive bands observed for the endogenous proteins. DGCR6 is found at low levels in primary human fibroblasts or peripheral blood mononuclear cells, while tumour cells, B-cells transformed by EBV as well as activated normal human T cells, contain elevated levels of the proteins. The proteins are differentially expressed in mammalian tissues, with high protein levels in heart, liver and skeletal muscle. These observations are important as some patients with DGCR6 syndrome exhibit a T-cell deficiency and/or cardiac malformations. As the DGCR6 protein(s) influence gene expression in trans, we analysed the influence of DGCR6/DGCR6L on the Epstein-Barr virus-encoded oncoproteins EBNA2 and EBNA3c in the activation of the viral LMP1 promoter, as well as LMP1-mediated activation of NFkB, but found no effect in either setting.