Study Of Tooth Eruption Via RUNX2-MiR-31-SATB2 Loop In Dental Follicle Cells

Cleidocranial dysplasia(CCD) is an autosomal dominant inherited skeletal disorder due to mutations causing haploinsufficiency of Runt related transcription factor 2(RUNX2) on chromosome 6p21. The clinical features of CCD are characterized by skeletal dysplasia, delayed tooth eruption and impacted teeth. Studies have shown that RUNX2 haploinsufficiency may lead to disturbed homeostasis of osteogenesis and osteoclastogenesis mediated by dental follicle cells in CCD patients. RUNX2 inactivates micro RNA-31(mi R-31) and mi R-31 also inhibits SATB2 which in turn induces RUNX2 activation, thus establishes a physiological regulatory loop. However, whether such loop participates in tooth eruption regulated by dental follicle cells is still unknown. Our research aims to study the biological characterization of dental follicle cells of CCD patient and investigate the influence of RUNX2-mi R-31-SATB2 in dental follicle cells to osteoclastogenesis signalings and tooth eruption. Part Ⅰ Isolation,culture and biological characterization of dental follicle cells of CCD patient Objective: To isolate and culture dental follicle cells of CCD patient(DFCs-CCD) and normal control(DFCs) and to study the invitro biologic characters of DFCs-CCD including proliferation ability, clony-forming efficiency, osteogenesis and osteoclastogenesis abilities. Methods: DFCs were isolated and cultured by dental sac adherent culture and were indentified by immunofluorescence of Vimentin and Cytokeratin. Crystal violet staining with cells cultured for 12 days for clony-forming efficiency; MTT assays and Brdu cell proliferation for cell proliferationo ability; Western blot and alizarin red staining for osteogenesis ability and real-time PCR for osteoclastogenesis ability. Results:(1) No obvious morphological difference was found between DFCs and DFCs-CCD. Both DFCs and DFCs-CCD exhibited positive expression of Vimentin and negative expression of Cytoketatin.(2) DFCs-CCD exhibited stronger colony formation ability and Brd U positive rate. The optical density value(OD value) of DFCs-CCD was higher than DFCs at the same time in MTT assays.(3) DFCs-CCD presented weaker expression of Runx2, Osrerix, Ocn and alizarin red staining compared to DFCs.(4) DFCs-CCD also expressed stronger levels of OPG and RANK(p<0.05), but no statistical difference of RANKL(p>0.05). Conclusions: DFCs-CCD exhibited stronger cell proliferation and clony-forming abilities, weaker osteogenesis and disorganized osteoclastogenesis abilities compared to DFCs. Part Ⅱ RUNX2 –mi R-31-SATB2 loop of dental follicle cells in tooth eruption Objective: To analysis the influence of RUNX2-mi R-31-SATB2 loop to invasion and osteoclastogenesis activation abilities of dental follicle cells and to study the mechanism of tooth eruption. Methods:(1) Western blot for RUNX2 and SATB2 expression and real-time PCR for mi R-31 expression.(2) Transwell matrigel invasion assay for cell invasive ability, western blot and gelatin zymography for MMP9 and MMP2 expression and activity.(3) Real-time PCR for osteoclastogenesis factors expression and dental follicle cells-bone marrow cells co-culture for osteoclast inductive ability.(4) Mi R-31 knockdown in DFCs-CCD for detecting the changes of RUNX2, SATB2 and osteoclast inductive ability and mi R-31 overexpression in DFCs for reversely verifying the changes of RUNX2, SATB2 and osteoclast inductive ability. Results:(1) DFCs-CCD exhibited lower expression of RUNX2, SATB2 and higher mi R-31 than DFCs.(2) The invasive ability, MMP9 and MMP2 expression of DFCs-CCD were weaker than DFCs.(3) The expression of osteoclastogenesis factors and osteoclast inductive ability of DFCs-CCD were also weaker than DFCs.(4) Knockdown of mi R-31 in DFCs-CCD could rescue the situation above. Otherwise, overexpression mi R-31 in DFCs could mimic DFCs-CCD phenotypes. Conclusions: Mutation of RUNX2 caused its decreased expression, which lead to increased mi R-31 expression and downregulated SATB2. RUNX2-mi R-31-SATB2 loop may participate in delayed tooth eruption of CCD patients. Manipulation of the RUNX2-mi R-31-SATB2 loop may be a potential way to facilitate tooth eruption in CCD patients.Part Ⅲ invivo study of RUNX2-mi R-31-SATB2 loop Objective: To study the influence of RUNX2-mi R-31-SATB2 loop in tooth eruption by in vivo RUNX2 interference with neonatal mice. Methods: The si RUNX2 nucleotides were intraperitoneally and locally injected into neonatal C57BL/6J mice(postnatal day 1). Immunofluorescence for RUNX2 expression and TRAP staining for active osteoclasts around dental follicles of mandible and extraction the protein and RNA of dental follicles for detection the expression of RUNX2, SATB2 and mi R-31 on day 8. Micro CT for tooth eruption on day 14. Results:(1) Immunofluorescence and western blot results showed reduced RUNX2, SATB2 expression and increased mi R-31 expression in dental follicles from si RUNX2-injected mice compared to control animals.(2) Osteoclasts around dental follicles were significant decreased in si RUNX2 group.(3) Micro-CT results showed delayed tooth eruption in si RUNX2 animals. Conclusions: In vivo interference of RUNX2 could mimic CCD patients’ tooth eruption. RUNX2-mi R-31-SATB2 loop may be the significant mechanism of dental follicle cells mediated tooth ertption.