The Effect And The Functional Mechanism Study Of MicroRNA-20a On Regulating The Differentiation Of Osteoclast

Background and Objective:Periodontal diseases(PD)are characterized as a peripheral infection involving multiple species of Gram negative organisms.Porphyromonas gingivalis is among the bacteria that have been implicated in the association with the pathogenesis of periodontal disease.It has been suggested that lipopolysaccharide(LPS)and other pathogenic factors from these organisms may stimulate production of tissue-destructive factors by host cells.Thererfore,Persistent bacterial infection of the periodontal tissues leads to increased inflammation and in severe cases culminates in the destruction of the alveolar bone and tooth loss.Bone resorption in periodontal diseases is regulated by actions of osteoclast.Osteoclasts are derived from monocyte-macrophage precursors that arise from multipotent haematopoietic stem cells.It have been demonstrated that Once differentiated to macrophages,THP-1 human acute monocytic leukemia cell line(THP-1 cells)can be differentiated to osteoclasts.Many cytokines and exogenous hormones have been identified to be involved in osteoclastogenesis through transcription factors that positively or negatively modulate osteoclast proliferation,survival,differentiation and function.M-CSF and RANKL,both produced by osteoblast or activated T cells,are significant cytokines for osteoc lasto genes is.RANKL can induce the expression of nuclear factor of activated T-cells cytoplasmic(NFATcl).Osteoclast-specific markers,such as tartrate-resistant acid phosphatase(TRAP)and cathepsin K,have multiple sites recognized by NFATcl,a key transcription factor for osteoclastogenesis.MicroRNAs(miRNAs)are small non-coding RNAs of?22 nucleotides known to play important roles in post-tanscrptional regulation in various cellular processes.They contribute to every step of osteogenesis from embryonic bone development to maintenance of adult tissue,by regulating the growth,differentiation and functional activity of osteoblasts,osteoclasts and osteocytes.MicroRNA-20a is a member of the miR-17-92 cluster,which is one of the most extensively studied families.Some members of this family(miR17-5p,miR-20a and miR-106a)have been found to be involved in monocytic differentiation and maturation.MiR-17-92 genes cluster not only participates in skeleton formation,but maintains the skeleton in a healthy state as well.Various miRNAs have been reported to regulate osteoblastic differentiation,proliferation and bone formation.It is found that miR-20a negatively regulates the expression of Bambi and Criml,which are inhibitors of the BMP pathway.Further studies show that miR-20a can enhance the expression of BMP2,BMP4,Runx2,Osx/Sp7,Ocn and Opn to increase bone formation.Recently,the function of miRNAs in osteoclastic differentiation and activity is beginning to be disclosed.However,the expression and function of miR-20a in osteoclasts remain elusive.MiR-20a is widely expressed in different tissues and cells.It targets different genes to regulate the occurrence of cancer,immune,differentiation of skeletal muscle cells,inhibition of endothelial cell migration,impairment of angiogenesis.Among them,PPARy is one of its main targets of miR-20.Previous studies have shown that the upregulation of miR-20a expression leads to an increase in the protein expression levels of the adipogenic PPARy marker gene in rats,as compared with the controls.In bone tissue,miR-20a has been identified to target PPAR ? in osteoblasts.The tumor suppressor gene PTEN is located on chromosome 10q23.Pten is another main targets in breast cancer,ovarian cancer,hepatic cancer and gastric cancer.Recent studies have demonstrated that PTEN plays an essential role in regulating signaling pathways involved in cell growth,adhesion,migration,invasion,and apoptosis in a number of human tissues.In this paper,our experiments are dinvided into three parts.The first part involve that the expression of miR-20a have an effect on proliferation,cycle and apoptosis of the THP-1 cells.The second part contain exploring role of miR-20a in the differentiation of osteoclasts,the last part exploring role of miR-20a in LPS-induced osteoclast differe-ntiation.Methods:1.Effect of miR-20a on THP-1 cells by cell proliferation assay,Cell cycle analysis,Cell apoptosis assayA human monocyte cell line THP-1 cells(Santa Cruz,sc-7274)was routinely expanded in RPMI 1640 medium(Sigma-Aldrich,St.Louis,MO)supplemented with 10%fetal bovine serum.The monocytes were then treated with 10 ng/ml of phorbol-12-myristate-13-acetate(PMA)(Sigma,79346)to induce the maturation into macrophages.To induce miR-20a expression,the miR-20a mimics,miR-20a inhibitor and miR-controls(10 nM)were both from GenePharma(Shanghai,China).For transfection,THP-1 cell-derived macrophages were seeded into 96-well plates(1-104 cells/well)and transfected with the above oligonucleotides for 6h using the Lipofectamin 2000(Invitrogen)according to the manufacturer's instructions.Then,cells were further incubated for additional 24h,The transfected effectiveness was assessed by qRT-PCR.THP-1 cell-derived macrophages were prepared above,Cells were seeded in 6-well plates and cultured after treated with miRNA-20a mimics,miRNA-20a inhibitor,and miR-control.Cell proliferation was detected using a CCK8 kit(Dojindo,EW615,Japan)following the manufacturer's instruction.The plates were then read on a micro plate reader using a test wave length of 450 nm.For survival rate assay,the number of viable cells were counted using Trypan blue.The cells were rinsed in PBS and fixed in ice-cold ethanol overnight(at 4?).The cells were then rinsed in PBS and incubated at room temperature in a 1-mL staining solution(20 mg/mL propidium iodide(PI)and 10 U/mL RNaseA)for 30 min.The cells were examined by fluorescence-activated cell sorting(FACS)using a flow cytometer(Accuri C6,BD),and the cell cycle populations were determined using the ModFit software.After 48h treatment,single cell suspensions were prepared and centrifuged for 5 min at 1000×g.After discarding the supernatant,cells were resuspended with PBS and centrifuged for another 5 min at 1000×g,followed with the removal of supernatant.Cells were washed in cold PBS,incubated with Annexin V-FITC and PI for 15 min in the dark.Then cell apoptosis was analyzed by flow cytometry(within 1 h).2.The expression levels of Trap and NFATCl and PPAR? in osteoclast differentiationTHP-1 cells were routinely expanded in RPMI 1640 medium supplemented with10%fetal bovine serum.Logarithmically,growing cells(105 cells/mL)were seeded into a 6-well plate(2X105 cells/well).The cells were then treated with 200 ng/ml of phorbol-12-myristate-13-acetate(PMA)(Sigma,MO)and 25 ng/ml M-CSF and 30 ng/mL RANKL(receptor activator of nuclear factor kappa-B ligand)for osteoclast differentiation as described previously with medium changes every 3-4 days.After 5 days,cells were stained and TRAP-positive multinuclear cells(MNCs)including more than 3 nuclei were counted as osteoclasts under microscopic examinatio.THP-lcells were treated with 200 ng/ml of PMA for 3 days to induce macrophage differentiation.Then,PMA treated cells were transfected with miRNA-20a mimics and miR-control for 6 h using Lipofectamin-2000(Invitrogen)according to the manufac-urer's instruction.Then,cells were further incubated in 25 ng/ml M-CSF and 30 ng/mL RANKL for 3 days and were collected for RT-PCR and Western blot analysis.3.The expression levels of Trap and NFATC1 and PPARy in osteoclast differentiation during LPS infectionTHP-1 cells were routinely expanded in RPMI 1640 medium supplemented with 10%fetal bovine serum.Logarithmically,growing cells(105 cells/mL)were seeded into a 6-well plate(2×105 cells/well).The cells were then treated with 200 ng/ml of phorbol-12-myristate-13-acetate(PMA)(Sigma,MO)and 25 ng/ml M-CSF and 30 ng/mL RANKL(receptor activator of nuclear factor kappa-B ligand)for osteoclast differentiation as described previously with medium changes every 3-4 days.After 5 days,cells were stained and TRAP-positive multinuclear cells(MNCs)including more than 3 nuclei were counted as osteoclasts under microscopic examination.THP-lcells were treated with 200 ng/ml of PMA for 3 days to induce macrophage differentiation.Then,PMA treated cells were transfected with miRNA-20a mimics and miR-control for 6 h using Lipofectamin-2000(Invitrogen)according to the manufacturer's instruction.Then,cells were further incubated in 25 ng/ml M-CSF and 30 ng/mL RANKL for 3 days,LPS was added for 24h,then cellswere collected for RT-PCR and Western blot analysis.Results:1.MiR-20a inhibits THP-1 cells growth and induces Gl/S arrestTo study the role of miR-20a in THP-1 cell proliferation,CCK8 assay was used.Results demonstrated that both of miR-20a mimic and inhibitor inhibited the proliferation of THP-1 cells compared with cells transfected with blank control.This was also observed on the first,second and third day after transfection with miR-20a mimics.This phenomenon was very obviously on the second day.We examined the effects of miR-20a on cell cycle and apotosis at second day after transfection.Our results shows that S stage during cell cycle are significant between THP-1 cells transfected with miR-20a mimics and with blank control,while G1-S stage are different between cells transfected with miR-20a inhibitor and blank control.We conclude that miR-20a maybe inhibited cell growth by inducing Gl/S arrest during cell cycle.Flow cytometry was used to determine whether miR-20a inhibited cell proliferation resulted from a promoted apoptosis checkpoint.Overexpression of miR-20a after transfection inhibited apoptosis in early and late stage and promoted survival cell rate at second day.compared with cells transfected with blank controLWhile lower expression of miR-20a showed an apoptosis-inducing tread.Therefore,we conclude that effect of miR-20a on cell proliferation and cell apoptosis follow two different signaling mechanism.2.Over-expression of miR-20a negatively regulates osteoclastogenesis and miR-20a directly targets PPAR,yThe complex osteoclast differentiation process has been widely accepted to be associated with periodontal diseases.To define the function of miR-20a during osteoclastogenesis,we treated THP-1 cells with miR-20a mimic in the course of osteoclastogenesis.The expression levels of Trap and NFATcl,both the master regulators of osteoclastogenesis,were notably decreased when miR-20a mimic was introduced into THP-1cells compared with cells transfected with blank or negative control Together,these results suggested that miR-20a may act as a negative regulator of RANKL-dependent osteoclastogenesis.To test whether miR-20a targets PPARy,We then examined the effect of miR-20a on the PPARy mRNA and protein levels in RANKL-induced osteoclastogenesis.The results demonstrated that miR-20a leads to a decrease in PPARy mRNA and protein level in RANKL-induced THP-1 cells compared with cells transfected with blank or negetive control.The results demonstrated that miR-20a leads to a decrease in Pten mRNA level in RANKL-induced THP-1 cells compared with cells transfected with blank or negative control.However,there is no detectable change in Pten protein level These results showed that PPARy may be the target of miR-20a in osteoclhst differentiation.2.Over-expression of miR-20a positively regulates osteoclastogenesis and miR-20a directly targets PPARy during LPS infectionThe complex osteoclast differentiation process has been widely accepted to be associated with periodontal diseases.To define the function of miR-20a during osteoclastogenesis,we treated THP-1 cells with miR-20a mimic in the course of osteoclastogenesis.The expression levels of TRAP and NFATc1,both the master regulators of osteoclastogenesis,were notably increased when miR-20a mimic was introduced into THP-1cells compared with cells transfected with blank or negetive control.Together,these results suggested that miR-20a may act as a positive regulator of RANKL-dependent osteoclastogenes is.To test whether miR-20a targets PPAR?,We then examined the effect of miR-20a on the PPARy mRNA and protein levels in RANKL-induced osteoclastogenesis.The results demonstrated that miR-20a leads to a increase in PPARy protein and mRNA levels in RANKL-induced THP-1 cells compared with cells transfected with blank or negetive control.The results demonstrated that miR-20a leads to a increase in Pten mRNA level in RANKL-induced THP-1 cells compared with cells transfected with blank or negative control.However,there is no detectable change in Pten protein level These results showed that PPARy may be the target of miR-20a in osteoclast differentiation.Conclusion:1?MiR-20a inhibits THP-1 cells growth,induce G1/S arrest and inhibite THP-1 cells apoptosis2.MiR-20a may act as a negative regulator of RANKL-dependent osteoclastogenesis and PPAR? may be the target of miR-20a in osteoclast differentiation3.MiR-20a may act as a positive regulator of RANKL-dependent osteoclastogenesis and PPARy may be the target of miR-20a in LPS-induced osteoclast differentiation...