Study On Osteoclast Differentiation And Role Of Autophagy In Keratocystic Odontogenic Tumour

Part 1. The adaptor protein p62 mediates RANKL-induced autophagy and osteoclastogenesisObjective:Aim of this study was to investigate the potential role of p62, a characterized adaptor protein for autophagy, in RANKL-induced osteoclastogenesis.Methods:Real-time quantitative PCR and western blot analyses were used to evaluate the expression levels of autophagy-related markers during RANKL-induced osteoclastogenesis in mouse macrophage-like RAW264.7 cells. Meanwhile, the potential relationship between p62/LC3 localization and F-actin ring formation was tested using double-labeling immunofluorescence. Then, the expression of p62 in RAW264.7 cells was knocked down using small-interfering RNA (siRNA), followed by detecting its influence on RANKL-induced autophagy activation, osteoclast differentiation, and F-actin ring formation.Results:The data showed that several key autophagy-related markers including p62 were significantly increased during RANKL-induced osteoclast differentiation. Western blot analyses indicated that p62 was significantly down-regulated during the initial stages of RANKL-induced osteoclastogenesis and then gradually increased over time in osteoclast culture. This down-regulation of p62 protein was associated with the increase in the LC3-Ⅱ/LC3-I ratio, which indicates the autophagic degradation of p62.The expression and localization of p62 showed negative correlation with LC3 accumulation and F-actin ring formation, as demonstrated by immunofluorescence analyses. Importantly, knockdown of p62 obviously attenuated RANKL-induced expression of autophagy-and osteoclastogenesis-related genes, formation of TRAP-positive multinuclear cells, as well as formation of F-actin ring.Conclusions:The study indicated that p62 may play essential roles in RANKL-induced autophagy and osteoclastogenesis.Part 2. Effect of tunneling nanotubes-mediated intercellular communication on osteoclasts differentiationObjective:To evaluate the role of tunneling nanotubes (TNTs)-mediated intercellular communication between endothelial progenitor cells (EPCs) and osteoclast precursors (RAW264.7 cells), and the effect of TNTs on osteoclasts differentiation.Methods:EPCs and RAW264.7 cells were stained with different fluorescent dyes before direct co-cultured. Then the existence of TNTs was detected by scanning electron microscopic and immunofluorescence analysis. The direct co-cultured cells were sorted by flow cytometric analysis, and the ability of co-cultured RAW264.7 cells was determined by transwell assay and osteoclastogenesis assay. The material transfer between these two kinds of cells was further detected by immunofluorescence analysis. The role of TNTs between them was also investigated using TNTs inhibitor.Results:The scanning electron microscopic and immunofluorescence analysis showed the existence of TNT-like structures between EPCs and RAW264.7 cells, we also observed components transferred via TNTs from EPCs to RAW264.7 cells. The results indicated that TNTs played pivotal roles in intercellular communication between co-cultured EPCs and RAW264.7 cells by using TNTs inhibitor. The study demonstrated that the ability of RAW264.7 cells forming mature osteoclasts was inhibited after direct co-cultured with EPCs. Moreover, we also found that the expression of MIF was up-regulated in RAW264.7 cells after co-cultured with EPCs. Using the MIF inhibitor ISO-1 increased the formation of TRAP positive multinuclear osteoclasts and up-regulated the expression of osteoclastogenesis-associated genes in the co-cultured RAW264.7 cells.Conclusions:Between direct co-cultured EPCs and RAW264.7 cells, TNTs mediated MIF transferation from EPCs to RAW264.7 cells, which could inhibit the migration ability and differentiation potential of osteoclast precursors.Part 3. Role of autophagy in keratocystic odontogenic tumoursObjective:To evaluate the activation status of autophagy in keratocystic odontogenic tumor (KCOT), and investigate its possible association with the growth potential.Methods:The expression levels of some key autophagy-related proteins in clinical samples of KCOT and radicular cyst (RC) were detected and compared by immunohistochemical and real-time quantitative polymerase chain reaction (qPCR) analysis, respectively. The correlation between these tested autophagy-related proteins, and their correlation with the cell anti-apoptotic (Bcl-2) or proliferative (Ki-67) markers in KCOT was explored using Spearman rank correlation test and clustering analysis.Results:The results showed that both the expression of mRNA and immunoreactivity of the autophagy-related proteins tested were considerably increased in samples of KCOT compared with those in samples of radicular cysts. Our study also detected the expression of Bcl-2 significantly increased in KCOT samples. The Spearman rank correlation analyses showed that the immunostainings of tested autophagy-related proteins (Beclin 1, Atg7, LC3, p62) in KCOT samples were closely correlated with each other, and the immunostainings of these autophagy-related proteins were also revealed to closely correlate with the immunostainings of Bcl-2 and Ki-67 in KCOT. More importantly, double-labelling immunofluorescence analyses also revealed the partially synchronous distribution for LC3 and Ki-67 in KCOT samples compared with those in samples of radicular cysts.Conclusions:The study implicated the increased expression of autophagy-related genes in KCOT, and meanwhile showed its obvious association with epithelial proliferation markers Ki-67 in KCOT.