Study of the role of microRNAs on biological effects of TGF-beta1 on human dental pulp fibroblasts

Abstract

Dental pulp is persistently affected by environmental insults along human life. The repair ability of the dental pulp is limited and the mechanisms involved in this biological event are not completely understood. MicroRNAs are molecules responsible for the tight regulation of protein synthesis. The aim of this project is to investigate the role of microRNAs in dental pulp repair. Primary culture of human dental pulp fibroblasts will be established through an explant technique. The fibroblasts will be treated with TGF-b1 for 6, 24 and 72 h for analysis and validation for the expression of the microRNAs miR-144 and let-7 family. Based on these results, the expressed miRNAs will be silenced utilizing Locked Nucleic Acids and then the cells will be treated with TGF-b1 determined at the abovementioned assays. The expression of the interaction candidate targets of the microRNAs will be evaluated by means of RT-PCR and protein detection will be performed through Flow Cytometry (Fibronectin, integrin b8, integrin b3, integrin ±3, TGFBRI), Western Blot (collagen 1A1, 1A2, collagen 3, fibrillin 1, SMAD4) and ELISA (thrombospondin-1 and p-SMAD2/3). To detect myofibroblasts and odontoblast-like differentiation, immunofluorescence for staing of ±-SMA and DMP-1 will be performed. Additionally, silenced and control cells for cell growth and cell cycle. To evaluate miRNAs expression during dental pulp repair, Balb/c mice will be treated with a TGFBRI inhibitor (SB-431542) and subjected to dental pulp exposure followed by pulp capping with mineral trioxide aggregate. After 1, 2, 3 and 4 weeks, specimens will be collected for microRNAs detection by means of in situ hybridization. Based on the results obtained with in vitro experiments and by in situ hybridization, in vivo silencing of the miRNAs will be performed. Balb/c mice will be treated with miRNA LNA inhibitors and subjected to dental pulp exposure as described. Four weeks later, the animals will be euthanized and dental pulp repair will be evaluated through hematoxilin/eosin stained sections.