| ObjectiveTNF-?,one of the highly expressed inflammatory factors?is actively involved in a variety of inflammation-related bone diseases by manipulating multiple signaling pathways.As a secreted inhibitor of the canonical Wnt signaling pathway,sclerostin(Sost)also plays a critical role in the regulation of bone metabolism,especially in an inflammatory microenvironment.Unfortunately,the molecular mechanisms underlying the post-transcriptional regulation of Sost are still largely unknown.In this study,to investigate whether TNF-? participates in the post-transcriptional regulation of the murine Sost gene,MC3T3-E1 murine pre-osteoblast-like cells and primary calvarial osteoblasts were treated with TNF-?.Moreover,the 3'UTR of the Sost gene was subcloned and ligated to pMIR-REPORT,creating a new luciferase-3 ' UTR construct named as pMIR-REPORT-SOST UTR.In addition,emerging lines of evidence indicate that TNF-? regulates the expressions of many miRNAs during various physiological and pathological processes.Moreover,it has been reported that miR-204-5p and miR-218 can bind to the 3'UTR of the Sost gene and post-transcriptionally inhibit its expression.To further investigate the molecular mechanisms underlying the post-transcriptional regulation of the Sost gene by TNF-?,MC3T3-E1 cells and primary calvarial osteoblasts were treated with TNF-? and the expressions and functions of miR-204-5p and miR-218 were evaluated.The possibly involved signaling pathways were also investigatedMethodsPart ?:(1)MC3T3-E1 cells and primary calvarial osteoblasts were treated with TNF-a at different doses for different time periods.Real-time RT-PCR and western blot were used to measure the mRNA and protein levels of Sost,respectively.(2)3'UTR of the murine Sost gene was amplified and subcloned into the Sacl restriction site of pMIR-REPORT miRNA expression reporter.The resulted plasmid was named as pMIR-REPORT-SOST UTR.(3)Calvarial osteoblasts,MC3T3-E1 cells and NIH3T3 murine fibroblasts were transfected with pMIR-REPORT-SOST UTR or pMIR-REPORT.Cells transfected with pMIR-REPORT-SOST UTR were named as the pMIR-REPORT-SOST UTR group,while those transfected with pMIR-REPORT named as the pMIR-REPORT group.The luciferase levels were then determined.Part ?:(1)MC3T3-E1 cells were transfected with pMIR-REPORT-SOST UTR or pMIR-REPORT.Cells transfected with pMIR-REPORT-SOST UTR were named as the pMIR-REPORT-SOST UTR group,while those transfected with pMIR-REPORT named as the pMIR-REPORT group.Seventy-two hours after the transfection,the luciferase activities were measured.In addition,the cells were were treated with TNF-a at different doses for different time periods before the measurement of the luciferase activity.(2)MC3T3-E1 cells and primary calvarial osteoblasts were treated with TNF-a at different doses for different time periods.RT-qPCR was used to determine the expression levels of miR-204-5p and miR-218.(3)Overexpression of miR-204-5p was achieved in HEK293 cells and MC3T3-E1 cells via transfection with miR-204-5p mimics(named as the miR-204-5p group).Cells transfected with the negative control,mimic NC,served as controls(named as the mimic NC group).Twenty-four hours after the transfection,the cells were treated with TNF-a at the doses of Ong/ml or 15ng/ml for 48 hours.RT-qPCR were performed to measure the level of miR-204-5p.Cells were named as the mimic NC group,the miR-204-5p group,the mimic NC+TNF-a group and the miR-204-5p+TNF-a group accordingly.(4)MC3T3-E1 cells were co-transfected with pMIR-REPORT-SOST UTR and miR-204-5p mimics or mimic NC,and were named as the miR-204-5p group and the mimic NC group,respectively.Twenty-four hours after the transfection,the cells were treated with TNF-a at the doses of Ong/ml or 15ng/ml for 48 hours and the luciferase activities were monitored.(5)MC3T3-E1 cells and primary calvarial osteoblasts were treated with TNF-a and/or BAY 11-7082,an inhibitor of NF-?B signaling pathway.The cells were named as the Control group,the TNF-a group,the TNF-a+BAY 11-7082 group and the BAY 11-7082 group.Forty-eight hours after the treatment,mRNA levels and protein levels of the Sost gene,as well as the expression levels of miR-204-5p,were determined using RT-qPCR and western blot.MC3T3-E1 cells were also transfected with pMIR-REPORT-SOST UTR.Twenty-four hours after the transfection,the cells were treated with TNF-a and/or BAY 11-7082 for 48 hours.The cells were named as the Control group,the TNF-a group,the TNF-a+BAY 11-7082 group and the BAY 11-7082 group,and the luciferase activities were measured.ResultsPart ?:(1)TNF-a treatment time-and dose-dependently down-regulated the mRNA levels while up-regulating the protein levels of the Sost gene in MC3T3-E1 cells and the primary calvarial osteoblasts.(2)pMIR-REPORT-SOST UTR was successfully constructed.(3)In both MC3T3-E1 cells and the primary calvarial osteoblasts,luciferase levels were significantly lower in the pMIR-REPORT-SOST UTR group when compared with the pMIR-REPORT group.Interestingly in NIH3T3 cells,no statistically significant difference was detected between the pMIR-REPORT-SOST UTR group and the pMIR-REPORT group.Part?:(1)In MC3T3-E1 cells treated with TNF-?,luciferase levels were still lower in the pMIR-REPORT-SOST UTR group than in the pMIR-REPORT group.However,TNF-a treatment resulted in time-and dose-dependent increases in the luciferase levels in the pMIR-REPORT-SOST UTR group when compared with the pMIR-REPORT group.When the dose of TNF-? was at or above 15ng/ml or the time period of TNF-a treatment reached 48 hours,no statistically significant difference in luciferase levels was detected between the pMIR-REPORT-SOST UTR group and the pMIR-REPORT group.(2)In MC3T3-E1 cells and the primary calvarial osteoblasts,the expressions of miR-204-5p were significantly decreased by TNF-a treatment.In contrast,no statistically significant difference in the expressions of miR-218 was detected between TNF-a treated cells and the control cells.(3)In HEK293 cells and MC3T3-E1 cells,expression levels of miR-204-5p were dramatically elevated in the mimic204-5p group when compared with the mimic NC group.In addition,the expression levels of miR-204-5p were also dramatically elevated in the miR-204-5p+TNF-a group when compared with the mimic NC+TNF-a group.However,no statistically significant difference was detected in miR-204-5p levels between the miR-204-5p group and the miR-204-5p+TNF-a group,indicating that the effect of endogenous miR-204-5p was negligible in cells overexpressing miR-204-5p.(4)In MC3T3-E1 cells co-transfected with pMIR-REPORT-SOST UTR and miR-204-5p mimics or mimic NC,the luciferase level in miR-204-5p group was 30%lower than that in mimic NC group.When cells were treated with TNF-a after the co-transfection,the luciferase level in the miR-204-5p group was decreased by 28%when compared with that in the mimic NC group.The intact inhibitory effect of miR-204-5p on luciferase expression after TNF-a treatment suggested that the binding and inhibitory function of miR-204-5p on the 3 'UTR of Sost gene was not disturbed by TNF-a treatment.(5)Although showing no effect on the inhibition of TNF-a in Sost mRNA expression,BAY 11-7082 treatment completely reversed the positive effect of TNF-a on Sost protein levels.In addition,the inhibitory effect of TNF-a on the expression of miR-204-5p was also partly reversed.In MC3T3-E1 cells transfected with pMIR-REPORT-SOST UTR,the luciferase level was significantly higher in the TNF-a group when compared with the control group.However,this positive effect of TNF-a on luciferase levels was completely reversed by BAY 11-7082 treatment.ConclusionsPart?:TNF-a actively participated in the post-transcriptional regulation of the Sost gene.Through transfecting pMIR-REPORT-SOST UTR into different cell lines,we found that the post-transcriptional regulation of the Sost gene via its 3'UTR is cell-specific.Part?:Although showing no regulatory effects on the expression of miR-218,TNF-a post-transcriptionally increased the protein level of Sost by down-regulating the expression level of miR-204-5p.However in MC3T3-E1 cells,TNF-a showed no effect on the binding and inhibitory function of miR-204-5p on the 3;UTR of Sost.The post-transcriptional regulation of Sost by TNF-a was,at least partly,mediated by the NF-?B signaling pathway. |
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