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

SIMPLE SUMMARY: The gene of the human tumor suppressive microRNA-142 (miR-142) carries mutations in about 20% of cases of diffuse large B-cell lymphoma (DLBCL). Because microRNAs post-transcriptionally regulate the protein expression of their cognate messenger RNA (mRNAs) targets, we determined the effect of miR-142 knockout on protein expression in two cell lines derived from DLBCL. We found a significant up-regulation of 52 proteins but also a down-regulation of 41 proteins upon miR-142 deletion. Knockout of a miRNA may be used to identify novel targets, and seed-sequence mutants of a miRNA unable to bind to their targets can be used to confirm potential novel targets. With this approach, we identify AKT1S1, CCNB1, LIMA1 and TFRC as novel targets of miR-142. As miR-142 is highly present in the miRNA processing RISC complexes, the deletion of this miRNA might result in its replacement by other miRNAs, thus introducing an additional layer of complexity regarding gene regulation. ABSTRACT: 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.