Peptide-15 Changes miRNA Expression in Osteoblast-Like Cells Annalisa Palmieri, PhD,* Furio Pezzetti, PhD,† Giorgio Brunelli, MD,‡ Marcella Martinelli, PhD,§ Lorenzo Lo Muzio, MD, Antonio Scarano, DDS,¶ Marco Degidi, MD,# Adriano Piattelli, MD,** and Francesco Carinci, MD†† S everal research projects have been involved in the identification of factors that could help in the re- generation of lost tissue. 1 One avenue of research has been the identification of the specific cell-binding domain of type I collagen. 1 Type I collagen represents about one third of the body proteins. 2 Collagen, moreover, is a major determi- nant of the architecture and tensile strength of the tissues, and modulates cell proliferation, migration, differentia- tion, and specific gene expression. 2 Peptide-15 is an analog of the cell- binding domain of collagen. 2 P-15 com- petes for cell surface sites for attachment of collagen and, when immobilized on surfaces, it promotes adhesion of cells. 3 P-15 has been shown to facilitate phys- iological processes in a way similar to collagen, to facilitate the exchange of mechanical signals, and to promote cell differentiation. 4–6 Like other bone aug- mentation materials, P-15 associated with anorganic-derived bone matrix, has been shown to be helpful in the treat- ment of periodontal defects, and sinus- lifting procedures. 7–12 Because the mechanism by which P-15 stimulates osteoblast activity to promote bone formation is poorly un- derstood, any information relative to bone biology could be helpful to reach a better comprehension of the clinical effect induced by P-15. From this point of view microRNAs (miRNAs) are a new field of research. miRNAs represent a class of small, functional, noncoding RNAs of 19 to 23 nucleotides (nt) cleaved from 60 to 110 nt hairpin precursors. 13,14 Hundreds of miRNAs have been identified in plants and animals. The miRNAs are involved in various biological processes, includ- ing cell proliferation and cell death during development, stress resistance, and fat metabolism, through the regu- lation of gene expression 15 in posttran- scriptional RNA silencing pathways. The RNA interference and the miRNA pathway regulate gene expression by inducing degradation or translational repression of target mRNAs. These pathways are generally initiated by various forms of double-stranded RNA, which are processed by Dicer, an RNase III family endonuclease, to 21 to 22 nt-long RNA molecules that serve as sequence-specific guides for silencing. 16,17 miRNAs are transcribed as long primary transcripts, which are processed by a nuclear RNase III Drosha- containing complex into short hairpin *Post Doctoral Fellow, Institute of Histology, University of Bologna and Center of Molecular Genetics, CARISBO Foundation, Bologna, Italy. †Associate Professor, Institute of Histology, University of Bologna and Center of Molecular Genetics, CARISBO Foundation, Bologna, Italy. ‡Senior Lecturer, Department of DMCCC, Section of Maxillofacial Surgery, University of Ferrara, Ferrara, Italy. §Assistant Professor, Institute of Histology, University of Bologna and Center of Molecular Genetics, CARISBO Foundation, Bologna, Italy. Full Professor, Dental Clinic, University of Foggia, Foggia, Italy. ¶Assistant Professor, Dental Clinic, University of Chieti, Chieti, Italy. #Senior Lecturer, Dental Clinic, University of Bologna, Bologna, Italy. **Full Professor, Dental Clinic, University of Chieti, Chieti, Italy. ††Associate Professor, Department of DMCCC, Section of Maxillofacial Surgery, University of Ferrara, Ferrara, Italy. ISSN 1056-6163/08/01701-100 Implant Dentistry Volume 17 • Number 1 Copyright © 2008 by Lippincott Williams & Wilkins DOI: 10.1097/ID.0b013e318166d182 Purpose: Peptide-15 (P-15) is an analog of the cell-binding domain of collagen. P-15 has been shown to fa- cilitate physiological process in a way similar to collagen, to serve as an- chorage for cells, and to promote the binding, migration, and differentiation of cells. However, how P-15 alters os- teoblast activity to promote bone for- mation is poorly understood. We therefore attempted to address this question by using microarray tech- niques to investigate the microRNA (miRNA) expression in osteoblasts ex- posed to P-15. Materials: The miRNA oligonu- cleotide microarray provides a novel method to carry out genome-wide miRNA profiling in human samples. By using miRNA microarrays contain- ing 329 probe designed from human miRNA sequence, we identified in osteoblast-like cells line (MG-63) cul- tured with P-15 several miRNA whose expression is significantly modified. Results: We identified 11 up- regulated miRNA (i.e., mir-337, mir- 15b, mir-377, mir-100, mir-148a, mir-125a, mir-199a, mir-221, mir-let- 7d, mir-92, mir-23b) and six down- regulated miRNA (i.e., mir-422a, mir-19a, mir-224, mir-145, mir-22, mir-29a). Conclusion: The data reported are, to our knowledge, the first on translation regulation in osteoblasts exposed to P-15. They can be relevant to better understand the molecular mechanism of bone regeneration and can serve as a model for comparing other materials with similar clinical effects. (Implant Dent 2008;17:100 – 108) Key Words: P-15, bone morphoge- netic protein, miRNA, microarray, gene expression, gene profiling 100 PEPTIDE-15 CHANGES MIRNA EXPRESSION IN OSTEOBLAST-LIKE CELLS