Identification of microRNAs implicated in the late differentiation stages of normal B cells suggests a central role for miRNA targets ZEB1 and TP53

// Giorgio Malpeli 1, 2 , Stefano Barbi 2 , Simonetta Zupo 3 , Gabriele Tosadori 4 , Giovanni Scardoni 4 , Anna Bertolaso 2 , Silvia Sartoris 5 , Stefano Ugel 5 , Caterina Vicentini 2, 10 , Matteo Fassan 6 , Annalisa Adamo 7 , Mauro Krampera 7 , Maria Teresa Scupoli 8 , Carlo Maria Croce 9 , Aldo Scarpa 2, 10 1 Department of Surgical Sciences, Dentistry, Gynecology and Pediatrics, Section of Surgery, University of Verona, Verona, Italy 2 Department of Diagnostics and Public Health, Section of Pathological Anatomy, University of Verona, Verona, Italy 3 Laboratory of Molecular Diagnostics, IRCCS-AOU San Martino-IST, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy 4 Center for BioMedical Computing (CBMC), University of Verona, Verona, Italy 5 Department of Medicine, Section of Immunology, University of Verona, Verona, Italy 6 Department of Medicine, Surgical Pathology and Cytopathology Unit, University of Padua, Padua, Italy 7 Department of Medicine, Section of Hematology, Stem Cell Research Laboratory, University of Verona, Italy 8 Department of Medicine, Section of Hematology, University of Verona, Italy 9 Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA 10 Applied Research on Cancer-Network (ARC-NET), University of Verona, Verona, Italy Correspondence to: Giorgio Malpeli, email: giorgio.malpeli@univr.it Keywords: B cell development, follicle, germinal centre, microRNAs, network analysis Received: June 17, 2016      Accepted: December 12, 2016      Published: January 17, 2017 ABSTRACT In the late B cell differentiation stages, miRNAs expression modifications promoting or inhibiting key pathways are only partially defined. We isolated 29 CD19 + human B cell samples at different stages of differentiation: B cells from peripheral blood; naive, germinal center (GC) and subepithelial (SE) B cells from tonsils. SE cells were further split in activated and resting B cell. The miRNA expression profile of these B cells was assessed by microarray analysis and selected miRNAs were validated by quantitative RT-PCR and in situ hybridization on normal tonsils. The comparison of all samples showed changes in 107 miRNAs in total. Among 48 miRNAs differentially expressed in naive, GC and SE cells, we identified 8 miRNAs: mir-323, mir-138, mir-9*, mir-211, mir-149, mir-373, mir-135a and mir-184 , strictly specific to follicular cells that had never been implicated before in late stages of B cell development. Moreover, we unveiled 34 miRNAs able to discriminate between CD5 − activated B cells and resting B cells. The miRNAs profile of CD5 − resting B cells showed a higher similarity to naive CD5 + than CD5 − activated B cells. Finally, network analysis on shortest paths connecting gene targets suggested ZEB1 and TP53 as key miRNA targets during the follicular differentiation pathway. These data confirm and extend our knowledge on the miRNAs-related regulatory pathways involved in the late B cell maturation.