Studies On The Role Of MicroRNA-3064-3p In Cementoblast Differentiation

Part One Study on the microRNA profile during the mineralization of mouse cementoblastObjectiveTo establish the mineralization cell model of mouse cementoblast.To study the microRNA(miRNA)expression profile during the differentiation and mineralization of cementoblast.Methods:Mouse cementoblast cell line OCCM-30 was cultured in vitro.The cells were differentiated and mineralized in the presence of 50 mg/mL ascorbic acid,10 mmol/L sodium ?-glycerophosphate,and 10nmol/L dexamethasone.Quantitative Real-time Polymerase Chain Reaction(qRT-PCR)was performed to verify the mineralization by testing the expression level of Alkaline phosphatase(ALP)and osteocalcin(OCN).The cementoblast mineralization model was established.Lastly,the different miRNA expression profile during the differentiation and mineralization of cementoblast was screened by miRNA microarray and verified by qRT-PCR.MiR-3064-3p was chosen for further study for its significant change.Results1.After 21 days of culture of cementoblast,the expression level of mineralization markers ALP and OCN increased gradually,which proved that ascorbic acid,sodium ?-glycerophosphate and dexamethasone were able to induce the differentiation and mineralization of cementoblast.2.MiRNA microarray showed that during the differentiation and mineralization of cementoblast,the number of miRNAs with significant change was 336.3.The results of qRT-PCR were consistent with that of miRNA microarray,which showed the accuracy of miRNA microarray.ConclusionThe cementoblast mineralization model was successfully established.MiRNA microarray showed that there were 336 miRNAs that changed significantly during the differentiation and mineralization of cementoblast.The significantly changed miR-3064-3p was chosen to investigate its regulating effect on cementoblast mineralization.Part Two Study on the effect of miR-3064-3p in cementoblast mineralizationObjectiveTo investigate the function of miR-3064-3p in the mineralization of cementoblast.MethodsOCCM-30 was differentiated and mineralized in the presence of 50 mg/mL ascorbic acid,10 mmol/L sodium ?-glycerophosphate,and 10nmol/L dexamethasone.The miR-3064-3p expression profile was detected by qRT-PCR during this process.MiR-3064-3p agomir,miR-3064-3p antagomir,or negative control(NC)was individually transfected into OCCM-30 cells by Lipofectamine 2000.After mineralization induction,qRT-PCR was used to detect the expression level of ALP and OCN.ARS and ALPase activity assay were also performed to test the mineralization results,so as to verify the function of miR-3064-3p during cementoblast differentiation.Results1.The expression of miR-3064-3p in cementoblast was detected after mineralization induction and decreased progressively with time.2.Change expression of miR-3064-3p was obtained in cementoblast after transfected with miR-3064-3p agomir or antagomir compared with NC.Overexpression of miR-3064-3p inhibited the expression of mineralization markers and decreased the formation of mineralized nodules,while inhibition of miR-3064-3p enhanced the expression of mineralization markers and increased the formation of mineralized nodules.ConclusionThe expression of miR-3064-3p decreased during cementoblast mineralization.Overexpression of miR-3064-3p inhibited cementoblast mineralization while inhibition of miR-3064-3p promoted cementoblast mineralization.Part Three The roles and mechanisms of miR-3064-3p in cementoblast mineralizationObjectiveTo predict and verify whether miR-3064-3p regulate cementoblast mineralization by targeting DKK1.To elucidate the role of miR-3064-3p/DKK1 pathway in cementoblast mineralization.MethodsVarious miRNA target prediction software such as TargetScan and miRBase were used to predict DKK1(Dickkopf-related protein 1)as the target genes of miR-3064-3p.QRT-PCR and Western Blot were used to test the mRNA and protein level of DKK1 in cementoblast after transfected with miR-3064-3p agomir or antagomir,so as to verify the regulatory effect of miR-3064-3p on DKK1.To create wide type(WT)luciferase reporter vector pmirGLO-DKKl-WT,a segment of the DKK1 3' UTR including the putative target site of was amplified by PCR and cloned into the downstream of the stop codon in the pmirGLO Dual-Luciferase miRNA Target Expression Vector.The mutation site of DKK1 3' UTR was synthesized and constructed into pmirGLO to create mutant type(MUT)luciferase reporter vector pmirGLO-DKK1-MUT.These two reporter vectors were cotransfected with miR-3064-3p agomir or NC into 293e cell lines,and Dual-Luciferase Reporter Assay System was used to detect the luminescent signal.Recombinant mouse DKK1 was added into cementoblast with miR-3064-3p agomir transfected.By testing whether overexpression of DKK1 could rescue the antagomiry effect of miR-3064-3p on cementoblast differentiation,we verified whether the regulatory effect of miR-3064-3p on cementoblast mineralization was via the downregulation of DKK1 expression.Results1.Target prediction analysis tools identified Dickkopf-related protein 1(DKK1)as a direct target of miR-3064-3p.2.Compared with NC transfection,miR-3064-3p agomir was able to downregulate DKK1 expression level on both mRNA and protein levels,while miR-3064-3p antagomir had the contrary functions.3.Compared with NC,co-transfection of miR-3064-3p agomir with pmirGLO-DKK1-WT significantly suppressed the luciferase activity.Co-transfection of miR-3064-3p agomir with pmirGLO-DKK1-MUT abolished this repression,which confirmed DKK1 as the target gene of miR-3064-3p.4.Recombinant mouse DKK1 was able to promote the mineralization level of cementoblast,which performed as the rise of mineralization markers such as ALP and OCN.5.Recombinant mouse DKK1 was able to rescue the miR-3064-3p-mediated suppression of cementoblast differentiation,which showed that after co-transfection of DKK1 with miR-3064-3p agomir,the expression levels of mineralization markers had significant difference with miR-3064-3p agomir group,while had no difference with NC group.Conclusion1.MiR-3064-3p suppressed the expression of DKK1 and inhibited cementoblast mineralization.2.We constructed WT and MUT DKK1 3' UTR dual-luciferase reporter vector.3.Dual-luciferase reporter assay showed that DKK1 was one of the target genes of miR-3064-3p.MiR-3064-3p was able to regulate DKK1 directly.4.MiR-3064-3p regulated cementoblast mineralization by targeting DKK1.