Effects Of Btbd7-small Interfering RNA On The Biological Behavior Of Human Dental Pulp Cells

Backgroud and ObjectiveThe dental pulp which has the capacity to formthe dentin lays under a protective shell.Its blood supply comes mainly from the tiny vessels in the apical foramen,which has no effective collateral circulation and is stimulated by external inflammation.The local injury of pulp tissue is usually irreversible,which can lead to the necrosis of pulp tissue and further development to periapical tissue.In response to external factors,the dental pulpalso mayproduce a type of reactionary/reparativedentine.Human dental pulp cells(hDPCs)play an important role in dentin formation and regeneration throughout life.Odontoblasts,the main cell types in dental pulp cells,can produce dentin.During tooth morphogenesis,a series of reciprocal epithelial-Mesenchymal interactions lead to pdontoblast differentation and dentin mineralization.Many genes have been reported to play a role during the progress.Many factors may lead to gene up-or down-regulation or even to cause cell death-in the worstcase.Dspp isfound in dentin as one of the major non-collagenous protein,that is essential for odontoblast differentiation and dentin mineralization.However,a number of genes still undetected in human dental pulp cells and their funtion have not been identified.The BTB domainwhich is first found as a sequence motif is a protein-protein interaction motif in genes of a DNA virusthroughout eukaryotes.Recently,BTB/POZ domain-containing protein7(Btbd7)was identify as a dynamic regulator of epithelial tissue remodeling and formation of branched organs.Btbd7 also plays an inportant role in various cancers,including lung cancer,liver cancer and primary salivary adenoid cystic carcinoma by regulating E-cadherin.However,the expression of Btbd7in human dental pulp cells has not been identified.Our group found that Btbd7 gene exists in human dental pulp cells.In our study,we generated Btbd7 knockdown hDPCs by means of siRNA to identify the effects of Btbd7 on the biological behavior of hDPCs,in the terms of cell proliferation and the ralationship of Dspp.Materials and Methods1.Select Wistar rat,take off the neck to death,after anatomy of the upper and lower jaws,take the first molar tooth.Five serial sections were cut for HE staining,immunohistochemical staining and negative control(3 sections)respectively.We observed the expression of Btbd7 in dental pulp tissues.2.Cell culture We collected freshly extracted caries-free third molars from patients(18-25 years of age)at Department of Oral and Maxillofacial Surgery of Stomatology hospital of Shandong University(written informed consent from patients was obtained).The hDPCs were isolated by method of enzymatic digestion of Dental pulp tissues.Dental pulp cells grown out from the pulp were cultured.Cells that fromthird to fifth passages in good condition were used for further research.3.Cell ImmunohistochemistryHDPCs were plated onto coverslips.Primary rabbit anti-human polyclonal antibody Btbd7 was used for Immunohistochemical steps in hDPCs.The primary antibody was instead of phosphate buffer solution(PBS)for control groups.4.Small interfering RNA(siRNA)transfection We usedSmall interfering RNA for transient gene knockdown studies.Lipofectamine 2000 was used with siRNA-Btbd7 or siRNA-Ncontrol for transfection in hDPCs according to the manufacturer's instructions.Prior to transfection,hDPCs were cultured in serum-and antibiotic-free medium for 24 hours.The efficiency of gene knock down was evaluated by RT-PCR and Western blot after an incubation period of 48 hours.5.Reverse transcription-polymerase chain reaction was used to evaluate Btbd7mRNA and DsppmRNA in hDPCs after siRNA transfection for 48 hours.6.Western blot analysis was used to evaluate Btbd7 protein and Dspp protein in hDPCs after siRNA transfection for 48 hours.7.Cell proliferation assayThe Cell Counting Kit-8 assay was used to examine the effect of Btbd7 on the proliferation of hDPCs.The absorbance was measure at 450 nm using a microplate reader.8.Statistical analysisSPSS 17.0 software was used to analyze all data.They are presented as means±SD of three independent replicates.Values of P<0.05 were considered statistically significant.One-way analysis of variance was used to evaluateStatistical significance.Results1.Compared to normal negative control,The positive immunohistochemical staining of Btbd7was observed in Wistar rat dental pulp in vivo and in human dental pulp cells in vitro.It exists in the cytoplasm and nucleus in dental pulp cells.2.Establishment of Btbd7 knockdown cells and the expression of Dspp.Compared to the siRNA-Ncontrol group,the mRNA level and protein expression of Btbd7 were significantly lower.The results demonstrated that Btbd7knock-down was successful.The mRNA level and protein expression of Dspp in the siRNA-Btbd7 transfected group were also significantly lower than those in the siRNA-Ncontrol group.3.SiRNA-Btbd7 promotes the proliferation of hDPCs After CCK-8 assays were performed on human dental pulp cells which transfected with siRNA-Btbd7 and siRNA-Ncontrol at different time points(days 1 to 5).Compared with the siRNA-Ncontrol group,the result showed that siRNA-Btbd7 significantly promoted the proliferation of hDPCs.ConclusionOur group found that Btbd7 expression in Wistar rat dental pulp in vivo and in human dental pulp cells in vitro.The established Btbd7 knockdown cells showed lower dentin sialophosphoprotein(Dspp)expression level compared to siRNA-Ncontrol group.Human genetic studies have demonstrated that mutations in,or knockout of,the Dspp gene result in mineralization defects in dentin.In present study,lower Dspp expression indicating that Btbd7 may be play a role in dentin formation and regenerationby regulating the expression of Dspp.The results showedthat siRNA-Btbd7 significantly promoted the proliferation of hDPCs.This suggest that Btbd7 inhibit the proliferation of hDPCs.In conclusion,Our experiments has shown the involvement of Btbd7 in hDPCs.Although we found Btbd7 knockdown can inhibit Dspp expression and the proliferation of hDPCs,furthur reseach is still needed to identify other functions of Btbd7 in hDPCs.