Dicer-dependent Pathway Contribute To The Osteogenesis Mediated By Regulation Of Runx2

Osteogenesis is controlled by intricate interactions of numerous biological processes and molecular functions,as well as by miRNAs.Either positively or negatively through multiple signaling pathways,various miRNAs have been reported to regulate bone homeostasis and osteoblasts differentiation.Numerous biological processes,including proliferation,apoptosis,differentiation,organogenesis and tumorigenesis are regulated by miRNAs.Likewise,various miRNAs have been found to play critical roles in bone homeostasis by regulating their targeting gene associated with osteogenesis.Primary miRNA(pri-miRNA)stem-loop structures encoded in genes is cut into the precursor miRNA(pre-miRNA)approximately 60-70 nucleotides in length by the ribonuclease ? enzyme Drosha in nucleus.After translocated into the cytoplasm by Exportin-5,the pre-miRNAs are further processed by Dicer,a second RNase ? ribonuclease,into the mature miRNA and loading onto RISC complexes.Therefore Dicer is required for generation of functional miRNAs biogenesis.Hence,it's necessary to investigate the extent to which function of miRNAs are obligatory for osteogenic control of bone metabolism by examining the transcriptional regulation of Dicer and the role of Dicer in osteoblast differentiation and mineralization.ObjectivesTo determine the global influence of miRNAs on regulation of osteogenesis of pre-osteoblast cells,the transcriptional regulation of Dicer and the role of Dicer in osteoblast differentiation and mineralization were investigated.1.To assess expression changes in osteogenic marker genes during the differentiation from pre-osteoblast to mature osteoblast cells.2.To explore the possibility mechanism of Runx2 upregulation on Dicer.3.To determine the role of Dicer in osteoblast differentiation and mineralization.4.To explored the role of PTEN and miR-21a-5p in the Akt/GSK3 signaling pathway of Dicer knockdown cells during osteogenesis,Methods1.A time course of mRNA and miRNA expression of MC3T3-E1 cells inducted to osteogenesis from 0 to 13 day were performed.Cells were cultured in osteoinductive medium for 2,4 and 6 days respectively and then detected by western-blot and Immunofluorescence scanned by laser confocal microscopy.2.MC3T3-E1 cells were transiently transfected with p CMV-Runx2 plasmid or siRunx2 prior to osteogenic induction.Hek293 T cells were co-transfected with p CMV-Runx2 plasmid of gradual increase dose from 100 ng to 400 ng and p GL3-Dicer promoter constructs and MC3T3-E1 cells were co-transfected with p GL3-Dicer promoter plasmid mixed with siRunx2 or pCMV-Runx2.Dual-luciferase system was used to measure the promoter activity.Chromatin immune complexes obtained from osteoinductive medium cultured MC3T3-E1 cells upon 3 days were analyzed by q PCR using Dicer promoter gene specific primers for the Runx2-binding region.3.MC3T3-E1 cells were transfected with siRNA targeting Dicer and cultured in complete medium or osteoinductive medium separately.The osteogenic phenotype of Dicer knockdown cells was assessed by detecting the enzymatic activity and calcification of ALP as well as the expression levels of several osteoblastic genes.MC3T3-E1 cells in Dicer knockdown or control were both cultured in osteoinductive medium and assessed at 24 h intervals from day 0,when the expression of these genes was initiated,until day 5 of induction.We examined potential functional protein expression by western-blot in cells transfected with sidicer and cultured in osteoinductive medium for 2,4,6 days.4.MC3T3-E1 cells were treated with si Dicer or VO-OHpic trihydrate and followed by osteogenic induction.Expression of Dicer,Runx2 and PTEN were also observed on the mRNA and protein levels by Realtime-PCR and western-blot.Protein levels of Dicer,?-catenin,total and phosphorylated Akt,PTEN,total and phosphorylated GSK3? on Ser9 were detected by western blot analysis.Results1.Immunofluorescence showed the expression of Dicer and Runx2 in MC3T3-E1 cells during osteogenesis was more than control cells,and Dicer mainly located in cytoplasm while Runx2 more expressed in nucleus.q PCR showed Runx2 increased as cells proceeded from proliferation to day 3 and decreased upon day 5 and a similar trend was observed in Dicer but decreased more earlier on day 3.ALP mRNA increased steadily from day 0 to day 7 and began to decline gently.Osterix significantly increased during the initial stage of differentiation upon day 5 and rapidly declined on day 7.An initial slight increase was found in the OPN and OCN,while the OCN mRNA levels reached steady state,the OPN mRNA expression declined during late mineralization on Day 9.Pre-miRNA21 remained at relatively steady level,a trend was observed for the increase in mature miR21a-5p,suggested posttranscriptional regulation of the miRNA.The expression profile displayed expression patterns consistent with the progression of osteogenesis and suggested a relationship between Runx2,Dicer and miR21a-5p to regulate the osteoblast differentiation at multiple stages.Western-blot,showing that osteoinductive medium significantly induced Dicer,?-catenin and Runx2 protein production in a time-dependent manner,but reduced PTEN expression.2.The gradated dose of Runx2 in the transfected MC3T3-E1 cells were confirmed and the Dicer mRNA expression correspondingly rose 0.84,2.45 and 3.27 fold respectively,suggesting the endogenous Dicer mRNA and protein levels increasing in a Runx2 dose dependent manner.A similar dose dependent was observed in OSX.Over-expression Runx2 also significantly increased OPN and OCN mRNA expression,whereas had no effect on expression of the gene encoding ALP.siRunx2 have been shown to efficiently knockdown Runx2 mRNA and protein levels.Moreover,MC3T3-E1 transfected with siRunx2 showed significantly lower levels of endogenous Dicer and down-regulation of other osteogenic marker gene was seen to be affected by Runx2 activity.Osteogenic induction significantly increased Runx2 binding to the Dicer promoter region,while less binding on the promoter in Chromatin immunoprecipitation obtained with a control Ig G or from cells in non-induced(-).The same anti-Runx2 antibody binding was detected in promoter region of osteocalcin,a widely known gene transcription regulated by Runx2,for positive control.Dual luciferase report gene assay in HEK-293 cells demonstrated a dose dependent increase in Dicer expression,with increasing concentrations of transfected p CMV-Runx2 plasmid.Over-expression of Runx2 in MC3T3-E1 promoted the transcription of Dicer,while siRunx2 inhibited Dicer promoter luciferase reporter gene activity.In contrast,no significant change in gene promoter activity was observed in cells transfected with empty vector and si NC.These results indicated that Runx2 could occupy and directly regulate the endogenous Dicer promoter.3.As the growth curves shown,knockdown of Dicer promoted the proliferation of MC3T3-E1.Dicer knockdown significantly inhibited the induced ALP activities of cell lysis and cultural supernatants.Staining for ALP calcification and Alizarin red for mineral deposition revealed the Dicer knockdown significantly inhibited osteoblast maturation while Runx2 overexpression promoted ALP expression at day7 and mineralization at day 14.q PCR showed that Dicer knockdown could decreased the expression of osteogenic genes,such as Runx2,ALP,OSX,OCN and OPN,whereas expressed significantly greater amounts of PTEN.Decreased protein levels of ?-catenin and Runx2 in response to Dicer knockdown and a tendency towards of higher PTEN protein were also observed.4.ALP staining demonstrated that Dicer Knockdown resulted in significant reduction of ALP production,while restoration of miR-21a-5p or inhibition of PTEN with VO-OHpic trihydrate in Dicer knockdown cells significantly rescued osteoblast mineralization.Similar tendencies were also observed on the mRNA and protein levels in MC3T3-E1 cells treated the same as above determined by Realtime-PCR and western-blot,suggesting miR-21a-5p targeting PTEN may play a role in lineage commitment,although it is likely that other miRNAs as well as osteogenic regulators also contribute to control of osteogenesis.Western blot analysis revealed that Dicer knockdown increased the expression of PTEN and inhibited the phosphorylation of Akt and GSK-3? on ser9.Abatement was also observed in ?-catenin in Dicer knockdown cells compared with control cells during the differentiation.In turn,we also verified that inhibitor of PTEN VO-OHpic trihydrate can promote the phosphorylation of Akt and GSK-3? on ser9 and then increased the accumulation of ?-catenin in Dicer knockdown cells.These results demonstrated that miR-21a-5p and PTEN may participate in osteogenesis through Akt/GSK3? signaling to regulate the stabilization of ?-catenin,which would act as a transcriptional co-factor to activate osteogenesis.Conclusion:In the present study,Dicer,Runx2 and mature miR-21a-5p expressions were induced during early stage of osteogenesis and Runx2 binding in the Dicer promoter directly was identified.Further,knockdown of Dicer in osteoblast gave rise to loss of the mineralization and down-regulation of differentiation markers whereas inhibition of PTEN diminished this effect,which may be associated with related signaling.These results imply a molecular mechanism underlying lineage commitment by a Runx2/Dicer/miRNAs regulatory network during osteogenic differentiation.