Francesco Carinci

MicroRNA expression profiling of oral carcinoma identifies new markers of tumor progression.

Oral squamous cell carcinoma, the most frequently occurring malignant head and neck tumour, generally exhibits poor prognosis and metastases are the main cause of death. The discovery of reliable prognostic indicators of tumour progression could greatly improve clinical practice. MicroRNAs are involved in the regulation of basic cellular processes such as cell proliferation, differentiation, and apoptosis. Since miRNAs have been shown to be abnormally expressed in different tumours their importance as potential cancer prognostic indicators is increasing. To define the role of miRNA in OSCC tumours we investigated the expression profile of 15 OSCC (8 without metastasis and 7 with lymph node metastasis) using microarray analysis. Thirteen miRNA were significantly overexpressed (miR-489, miR-129, miR-23a, miR-214, miR-23b, miR-92, miR-25, miR-210, miR-212, miR-515, miR-146b, miR-21, miR-338) and 6 miRNA were underexpressed (miR-520h, miR-197, miR-378, miR-135b, miR-224, miR-34a) in oral tumours. Underexpression of mir-155, let-7i, mir-146a was found to characterize progression to metastastatic tumours. Further investigations will elucidate whether differentially expressed miRNAs will help to better classify OSCCs, thus improving diagnoses and patient care.

Comparison between genetic portraits of osteoblasts derived from primary cultures and osteoblasts obtained from human pulpar stem cells.

Harvesting bone for autologous grafting is a daily problem encountered by craniofacial and oral surgeons. Stem cells derived from human dental pulp are able to differentiate in osteoblasts and are a potential source of autologous bone produced in vitro. However, as stem cells are characterized by self-renewing and commitment in several cellular subtypes (ie, pluripotential differentiation), some concerns may arise as regards their potential uncontrolled proliferation. To screen the behavior of osteoblasts derived from human pulpar stem cells (ODHPSCs), we used microarray techniques to identify genes that are differently regulated in ODHPSC in comparison to normal osteoblasts (NOs). Osteoblasts derived from human pulpar stem cells were obtained from human dental pulp, and cells were selected using a cytometer. The cell profile was c-kit+/CD34+/STRO-1+/CD45−. These cells were capable of differentiation of osteoblasts in vitro. By using DNA microarrays containing 19,200 genes, we identified in ODHPSC some genes whose expression was significantly up- and downregulated compared to NO. The differentially expressed genes have different functional activities: (a) cell differentiation, (b) developmental maturation, (c) cell adhesion, and (d) production of cytoskeleton elements. Thus, some molecular differences exist between NO and ODHPSC, although the previously considered histologic parameters show a normal phenotype.

Study of the PVRL1 Gene in Italian Nonsyndromic Cleft Lip Patients with or without Cleft Palate

Nonsyndromic cleft lip with or without cleft palate (CL/P) is a complex genetic trait and little is known about its aetiology. Recent investigations on rare clefting syndromes provided interesting clues about genes involved in face development. The PVRL1 gene encodes nectin1, a cell‐to‐cell adhesion molecule. Mutations in its sequence have been shown to cause the rare autosomal recessive syndrome CL/P‐ectodermal dysplasia syndrome (CLPED1), while heterozygosity for the mutation W185X seemed to increase the risk of non syndromic CL/P in a population from northern Venezuela. In the present study, we screened 143 Italian CL/P patients for mutations in PVRL1. Three rare sequence variants in exon 3 that create amino‐acid changes were detected in a total of 7 patients. Two of these mutations were not found in a panel of 292 unaffected controls, while the third was found in two controls. This study describes new mutations that may represent genetic risk factors for CL/P. Even though a study to look at the effects of the mutations on nectin1 function was not feasible, supporting evidence was reported, thus confirming the involvement of PVRL1 in the aetiology of non‐syndromic CL/P malformation.

Peptide-15 changes miRNA expression in osteoblast-like cells.

Purpose:Peptide-15 (P-15) is an analog of the cell-binding domain of collagen. P-15 has been shown to facilitate physiological process in a way similar to collagen, to serve as anchorage for cells, and to promote the binding, migration, and differentiation of cells. However, how P-15 alters osteoblast activity to promote bone formation is poorly understood. We therefore attempted to address this question by using microarray techniques to investigate the microRNA (miRNA) expression in osteoblasts exposed to P-15. Materials:The miRNA oligonucleotide microarray provides a novel method to carry out genome-wide miRNA profiling in human samples. By using miRNA microarrays containing 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.

Genes causing clefting syndromes as candidates for non‐syndromic cleft lip with or without cleft palate: a family‐based association study

Clefts of the orofacial region are among the most common congenital defects, caused by abnormal facial development during gestation. Non-syndromic cleft lip with or without cleft palate (NSCLP) is a complex trait most probably caused by multiple interacting loci, with possible additional environmental factors. As facial clefts form part of more than 300 syndromes, one strategy for identifying the genetic causes of NSCLP could be to study candidate genes responsible for clefting syndromes. Three genes were selected for this investigation: TP63, which codes for the tumour protein p63 and causes Ectrodactyly-Ectodermal dysplasia-orofacial Cleft syndrome; JAG2, a downstream gene of TP63; and MID1, which is responsible for Opitz syndrome. A linkage disequilibrium investigation was performed with intragenic single nucleotide polymorphisms on each of these genes in a sample study of 239 patients/parents trios. Evidence which suggests that JAG2 and MID1 may play a role in NSCLP was obtained.

Cleft lip with or without cleft palate: implication of the heavy chain of non-muscle myosin IIA

Non-syndromic cleft lip with or without palate (CL/P) is one of the most common malformations among live births, but most of the genetic components and environmental factors involved remain to be identified. Among the different causes, MYH9, the gene encoding for the heavy chain of non-muscle myosin IIA, was considered a potential candidate, because it was found to be abundantly and specifically expressed in epithelial cells of palatal shelves before fusion. After fusion, its expression level was shown to decrease and to become limited to epithelial triangles before disappearing, as fusion is completed. To determine whether MYH9 plays a role in CL/P aetiology, a family-based association analysis was performed in 218 case/parent triads using single-nucleotide polymorphism (SNP) markers. Pairwise and multilocus haplotype analyses identified linkage disequilibrium between polymorphism alleles at the MYH9 locus and the disease. The strongest deviation from a null hypothesis of random sharing was obtained with two adjacent SNPs, rs3752462 and rs2009930 (global p value  = 0.001), indicating that MYH9 might be a predisposing factor for CL/P, although its pathogenetic role needs to be investigated more accurately.

PerioGlas regulates osteoblast RNA interfering.

PURPOSE PerioGlas (PG) is an alloplastic material that has been used for grafting periodontal osseous defects since the 1990s. In animal models, it has been proven that PG achieves histologically good repairs of surgically created defects. In clinical trials, PG is effective as an adjunct to conventional surgery in the treatment of intrabony defects; however, how PG alters osteoblast activity to promote bone formation is poorly understood. We therefore attempted to address this question by using microRNA (miRNA) microarray techniques to investigate the translation process in osteoblasts exposed to PG. MATERIALS AND METHODS By using miRNA microarrays containing 329 probes designed from human miRNA sequences, we identified several miRNA whose expression was significantly modified in osteoblast-like cell lines (MG-63) cultured with PG. RESULTS There were ten up-regulated miRNA (mir-337, mir-377, mir-9, mir-516, mir-515-3p, mir-496, mir-200b, mir-489, mir-25, mir-423) and two down-regulated miRNA (mir-26a, mir-30d). CONCLUSION PG acts on miRNAs, which in turn regulate several messengers. Among them there are mRNAs related to bone formation and skeletal and cartilage development. The vast majority of detected genes are down-regulated, and some are homeobox genes like NOG, EN1, and CHRD. Other down-regulated genes are receptors (like GHRHR) and extracellular matrix proteins (like COMP). Although the exact mechanism of PG action on osteoblasts is still incompletely understood, these data demonstrate that PG has not only an osteoconductive effect, but also regulates bone formation.

Zirconium oxide regulates RNA interfering of osteoblast-like cells.

Zirconium oxide (ZO) has outstanding mechanical properties, high biocompatibility and high resistance to scratching. Since dental implants are made with ZO and the genetic effects of ZO on osteoblasts are incompletely understood, we used microRNA microarray techniques to investigate the translation process in osteoblasts exposed to ZO. By using miRNA microarrays containing 329 probes designed from Human miRNA sequences, we identified in osteoblast-like cells line (MG-63) cultured on ZO disks several miRNA whose expression was significantly modified. The most notable regulated genes acting on osteoblasts are: NOG, SHOX, IGF1, BMP1 and FGFR1. The data reported below represent the first study on translation regulation in osteoblasts exposed to zirconium and one in which the effect of ZO on bone formation has been detected.

Medpor ® regulates osteoblast's microRNAs

Porous polyethylene (PP or Medpor) is an alloplastic material worldwide used for craniofacial reconstruction. Although several clinical studies are available, there is a lack as regard the genetic effects. Because PP is always fixed on bone and the mechanism by which PP acts on osteoblasts is unknown, we therefore attempted to address this question by using microRNA microarray techniques to investigate the translation regulation in osteoblasts exposed to PP. The miRNA oligonucleotide microarray provides a novel method to carry out genome-wide microRNA profiling in human samples. By using miRNA microarrays containing 329 probe designed from Human miRNA sequence, we identified in osteoblast-like cells line (MG-63) cultured with Medpor (Porex Corporation, Fairburn, Georgia, USA) several miRNA which expression is significantly modified. We identified 16 up-regulated miRNA (i.e. mir-337, mir-515-3p, mir-377, mir-153, mir-367, mir-152, let-7b, mir-92, mir-155, mir-424, mir-148b, mir-368, mir-18b, mir-520d, mir-20b, mir-128a) and 2 down-regulated miRNA (i.e. mir-143, mir-32). The data reported are, to our knowledge, the first study on translation regulation in osteoblasts exposed to PP. They can be relevant to better understand the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

Short-period Effects of Zirconia and Titanium on Osteoblast MicroRNAs

BACKGROUND MicroRNAs (miRNAs) are a class of small, functional, noncoding RNAs of 19 to 23 nucleotides which induce degradation of specific messenger RNAs (mRNAs), thus controlling the translational process (ie, synthesis of proteins from mRNAs). In addition, mRNAs regulate the promoter of specific miRNAs activating an autoregulatory feedback loop. PURPOSE Titanium and zirconium dioxide ceramics (ZDCs) are used to make dental implants. Because the molecular mechanism by which ZDC and Ti act on osteoblasts is incompletely understood, we attempted to get more information by comparing the effect of ZDC and Ti on osteoblast miRNAs. MATERIALS AND METHODS By using miRNA microarray technique, we identified in osteoblast-like cell line (MG63) grown on grade 3 Ti and ZDC disks several miRNAs whose expression was modified. We collected mRNAs after 24 hours of cell culturing to better understand molecular events related to early bone healing around inserted implants. An mRNA microarray technique was then performed as a control. RESULTS There were six up- and four down-regulated miRNAs. Because every miRNA regulates hundreds of genes, we focused only on those related to bone formation. Among them, the most notable are BMP4 and 7, which are both up-regulated in osteoblasts cultured on Ti disks. CONCLUSION The detected miRNAs differentially expressed in osteoblast-like cells grown on ZDC versus Ti act on a limited number of miRNAs and bone-related genes. The most notable are BMP4 and 7, which are more expressed in osteoblasts exposed to Ti surface. Consequently, we suggest that Ti surfaces could provide some advantages to immediate load implantology.