| Part.I Expression and function of miR-361-3p during cementoblasts differentiationObjectiveTo identify remarkable changes in miRNA expression patterns during cementoblasts differentiation and determine the influences of miR-361-3p overexpression or silence on osteogenic differentiation of cementoblasts.Materials and methods1.OCCM-30 cells,the mouse cementoblast cell line,were cultured in mineralization medium containing 0.01 ?M dexamethasone,50 ?g/ml vitamin C,and 10 m M Na?-glycerophosphate.2.miRNA microarray profiling was performed to screen the differentially expressed miRNAs during cementoblasts differentiation.Data were analyzed.3.q RT-PCR was used to confirm expression of miR-361-3p during cementoblasts differentiation.m RNA expression levels of osteogenic marker genes(Osx,Bsp,Bglap)were also determined.4.Overexpressing or silencing miR-361-3p in OCCM-30 cells by lentivirus packaging system.The effects of cell transfections were validated by q RT-PCR.5.q RT-PCR,Western blot,ALP staining,ALP activity assay and alizarin red staining(ARS)were performed to detect the effects of miR-361-3p overexpression or silence on osteogenic differentiation ability of OCCM-30 cells.Results1.336 differentially expressed miRNAs during cementoblast differentiation were identified by miRNA microarray.Among the differentially expressed miRNAs,miR-361-3p was significantly down-regulated.2.q RT-PCR confirmed that the expression level of miR-361-3p has been on declining during the mineralization of OCCM-30 cells,consistent with miRNA microarray data,along with significant increases in the expression levels of osteogenic marker genes(Osx,Bsp,Bglap).3.Compared with the control group,the expression level of miR-361-3p in overexpression group increased more 20 times.The expression levels of putative target genes Nfat5 and Ogt in the silenced group increased significantly,suggested that miR-361-3p-sponge functioned well in silencing miR-361-3p.4.Results of q RT-PCR and Western blot revealed that the forced expression of miR-361-3p resulted in significantly inhibited m RNA and protein expressions of Osx,Bsp,and Bglap,while miR-361-3p silence enhanced m RNA and protein expressions of Osx,Bsp,and Bglap.5.The forced expression of miR-361-3p resulted in significantly inhibited osteogenic differentiation of cementoblasts,illustrated as enhanced ALP staining and ALP activity and ARS staining.Correspondingly,the downregulated miR-361-3p by sponge exhibited the opposite effects.miR-361-3p overexpression also inhibited calcium deposition,as assessed by ARS at day 14.One the other hand,miR-361-3p silence yielded the opposite effect on ALP staining,ALP activity and ARS.Conlusion1.Expression level of miR-361-3p decreased during cementoblasts osteogenic differentiation;2.Overexpressing miR-361-3p inhibited osteogenic differentiation of cementoblasts,while silencing miR-361-3p yielded the opposite effects.Part.II miR-361-3p negatively targets Nfat5 during cementoblast differentiationObjectiveTo identify target gene of miR-361-3p in cementoblast and investigate the function of target gene in cementoblasts.Materials and methods1.Bioinformatics analysis: DIANA Tools(containing micro CLIP,micro T-CDS,Tarbase,MR-micro T and mir Path)combined with NCBI,MGI and Ensembl databases were used to perform bioinformatics analysis.q RT-PCR and Western blot were performed to confirm expression levels of selected candidated target genes2.Dual luciferase assay: The wild-type Nfat5-3'UTR containing one of the miR-361-3p targeting sites and nonsense mutated 3'UTR sequence were synthesized and cloned into pmir GLO plasmid.We performed dual luciferase assay by co-transfecting OCCM-30 cells with reporter plasmids containing wild-type / mutant Nfat5 3'UTR and i R-361-precursor / miR-361-3p-sponge / miR-CTL.After 48 h of infection,luciferase activity was measured.3.q RT-PCR and Western blot were used to detect expression level of Nfat5 during cementoblast differentiation.4.Immunofluorescent staining and immunohistochemical staining were used to observe the expression and subcellular location of NFAT5 in cementoblast in vitro and in vivo.5.Stably knocked down Nfat5 in OCCM-30 cells.q RT-PCR,Western blot,ALP staining,ALP activity assay and alizarin red staining(ARS)were performed to detect the effects of Nfat5 knockdown on osteogenic differentiation ability of OCCM-30 cells.6.Nfat5 knockdown OCCM-30 cells were treated with mineralization medium for 4 days,then Western blot was performed to detect multiple major signaling pathways which have been implicated in certain aspects of differentiation programs,including Erk1/2,JNK,p38,PI3K-Akt,NF-?B and Wnt/?-catenin pathways.Results1.8 candidated target genes of miR-361-3p were selected,including Nfat5,Mat?,Ogt,Atg12,Upf2,Smg1,Dcun1d1 and Tspyl2.Results of q RT-PCR and Western blot showed that among the candidate target genes,Nfat5 not only decreased in miR-361-3p overexpressed cells but also increased dramatically in miR-361-3p knockdown cells.2.Overexpressing miR-361-3p attenuated the luciferase activity of wild type Nfat5-3'UTR reporter,but not that of the mutant reporter.Conversely,silencing miR-361-3p in OCCM-30 cells considerably enhanced the luciferase activity of type Nfat5-3'UTR reporter,but not that of the mutant.The result of dual luciferase assay verified that Nfat5 is the direct target gene of miR-361-3p in cementoblast.3.Both results of q RT-PCR and Western blot revealed that Nfat5 was up-regulated during in vitro cementoblast differentiation,presenting an inverse correlation with the expression pattern of miR-361-3p.4.NFAT5 expressed in cementoblasts both in vitro and in vivo,localized mainly in the nucleus and also localized in cytoplasm.5.Downregulation of Nfat5 inhibited the m RNA and protein expression levels of Osx,Bsp,and Bglap.Compared with control group,Nfat5 knockdown resulted in inhibited ALP staining,ALP activity and ARS staining of OCCM-30 cells treated with mineralization medium.6.Western blot showed that downregulation of Nfat5 during cementoblast differentiation resulted in the activation of various signaling pathways,including Erk1/2,JNK,p38,PI3K-Akt and NF-?B pathways,and blocking of the Wnt/?-catenin pathway.Conlusion1.Nfat5 is a direct target gene of miR-361-3p in cementoblast.2.Knockdown of Nfat5 mimicked the inhibitory effect of overexpressing miR-361-3p in cementoblast.3.Multiple signaling pathways,including Erk1/2,JNK,p38,PI3K-Akt,and NF-?B pathways were notably activated,and Wnt/?-catenin pathway was blocked by downregulation of Nfat5 or forced expression of miR-361-3p in cementoblast differentiation.Part.III Multiple signaling pathways are involved in miR-361-3p/Nfat5 axis during cementoblast differentiationObjectiveTo explore the mechanism of miR-361-3p/Nfat5 axis regulating cementoblast differentiation.Materials and methods1.miR-361-3p overexpressed or silenced OCCM-30 cells were treated with mineralization medium for 4 days.Western blot was used to detect signaling pathways,including Erk1/2,JNK,p38,PI3K-Akt,NF-?B and Wnt/?-catenin pathways.2.Dual luciferase assay: miR-361-3p overexpressed or silenced or the control OCCM-30 cells were co-transfected with the reporter plasmid TOPflash/FOPflash and the internal control plasmid p RL-TK.After 48 h of infection,luciferase activity was measured.3.Complementary approach of signaling pathways: specific chemical inhibitors of Erk1/2,JNK,p38,PI3K-Akt and NF-?B signaling pathways and Licl were added to miR-361-3p overexpressed OCCM-30 cells respectively during mineralization induction.Mi R-361-3p overexpressed or control OCCM-30 cells added the same dosage of DMSO during mineralization induction were used as control.m RNA changes of Osx,Bsp and Bglap were detected by q RT-PCR at day 4.Results1.Upregulation of miR-361-3p could also activate Erk1/2,JNK,p38,PI3K-Akt,and NF-?B pathways and block the Wnt/?-catenin pathway during cementoblast differentiation,consistent with the function of sh-Nfat5.On the other hand,downregulation of miR-361-3p by sponge yielded the opposite effects.2.Dual luciferase assay confirmed that overexpressing miR-361-3p in OCCM-30 cells impeded the activity of Wnt/?-catenin pathway,while silencing miR-361-3p in OCCM-30 cells enhanced the activity of Wnt/?-catenin pathway.3.Compared with miR-361-3p overexpressed OCCM-30 cells merely added DMSO during mineralization induction,blocking Erk1/2 pathway could significantly increase Osx expression;blocking JNK pathway could increase Bglap and Bsp expression while decrease Osx expression;blocking p38 pathway exacerbated the inhibition of miR-361-3p on Osx,Bglap and Bsp expression;blocking PI3K-Akt pathway raised Osx and Bglap expression obviously,also raised the Bsp expression;Both blocking NF-?B and activating Wnt/?-catenin pathway enhanced the inhibition function of miR-361-3p on Osx and Bglap expression,while blocking NF-?B and activating Wnt/?-catenin pathway had the opposite effects on Bsp expression.ConlusionmiR-361-3p regulated cementoblast differentiation via or partially via Erk1/2 and PI3K-Akt.Overall,our study elucidated that JNK,p38,NF-?B,and Wnt/?-catenin pathways act as balancing players in miR-361-3p/Nfat5/signaling axis during cementoblast differentiation. |
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