| BackgroundOsteoporosis is a bone disease characterized by low bone mineral density(BMD),impaired micro-architecture,increased risk of fragility fractures,and the growing mortality rate of aged people.The disability and mortality rate are high,it can seriously affect the patient’s function and the quality of life,and cause the serious social and the economic burden.Osteoclasts and osteoblasts play an important role in the development and formation of bone.Osteoclasts and osteoblasts are closely related to the function of osteoporosis,and their functions are opposite.Excessive activation of osteoclasts can reduce the BMD and significantly contribute to the development of osteoporosis.Long non-coding RNAs(lncRNAs)are a class of Long transcripts that have no protein-coding function and play a key role in gene silencing and disease progression.In recent years,lncRNAs have attracted extensive attention as potential biological and pathological regulatory factors.LncRNA X chromosome inactivation specific transcription factor(Xist)is expressed only in the X inactivation center of the inactivated X chromosome and is essential for the initiation and spread of X chromosome inactivation.LncRNA Xist has been identified to promote the progression of osteoporosis by suppressing the osteogenic differentiation from bone marrow mesenchymal stem cells(BMSC).It has been reported that lncRNAs regulate gene expression by acting as competing endogenous RNAs(ceRNAs)of microRNAs,thus participating in many processes in various diseases.According to bioinformatics analysis by LncBase Experimental v.2 software(http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=lncbasev2%2Findex-experimental),there was a binding site of miR-590-3p on the sequence of lncRNA Xist.Furthermore,the concentration of miR-590-3p was decreased in the serum of osteoporosis patients.Nevertheless,whether lncRNA Xist can regulate the osteoclast differentiation in osteoporosis by acting as a ceRNA of miR-590-3p remains unknown.A previous study indicated that Transforming growth factor-beta-induced factor 2(Tgif2)was one of the regulators of osteoclast differentiation and its deficiency can suppress the osteoclast differentiation and bone loss in mice.In addition,Tgif2 can be regulated by miRNAs in various diseases.A recent study has demonstrated that miR-34a participates in the development of osteoporosis by targeting Tgif2 and inhibiting osteoclast differentiation.The bioinformatics analysis by microT-CDS software(http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=microT_CDS/index)showed there was a miR-590-3p binding site on the 3’-untranslated region(3’-UTR)of Tgif2.However,whether Tgif2 expression can be regulated by miR-590-3p in osteoclast differentiation has not been fully elucidated.Based on the above evidence and the bioinformatics analysis,we intended to investigate the mechanism by which lncRNA Xist competitively binds to miR-590-3p with Tgif2 to regulate osteoclast differentiation through the ceRNA mechanism.It will influence the mechanism of osteoporosis and provide the basis and contribution for the research,prevention and treatment of osteoporosis.Part one The expressions of lncRNA Xist,miR-590-3p,and Tgif2 in femoral bone marrow of OVX mice and osteoclastsObjective:To understand the expression levels of lncRNA Xist,miR-590-3p,and Tgif2 in the development of osteoporosis.Methods:For OVX mice,bilateral ovaries and the surrounding adipose tissues were excised to induce osteoporosis,while only surrounding adipose tissue was excised in Sham mice.Five weeks after surgery,the mice were sacrificed and the femurs were isolated for further detection.RAW264.7 cells were treated with 50 ng/mL mouse recombinant Receptor Activator of Nuclear Factor-κ B Ligand(RANKL)and 100 ng/mL mouse recombinant Colony Stimulating Factor 1,Macrophage(M-CSF)to induce osteoclast differentiation.1.Representative micro-CT scanning images(left)and the BS/TV(right)of mouse femurs.2.The expressions of lncRNA Xist and miR-590-3p,and Tgif2 mRNA level in femurs of mice were detected by qRT-PCR.3.Representative images of TRAP staining of osteoclasts to observe the differentiation of osteoclasts.4.The expressions of lncRNA Xist,miR-590-3p,and Tgif2 in Osteoclasts were detected by qRT-PCR.5.The protein level of Tgif2 in Osteoclasts was measured by western blot.Results:1.The micro-CT parameters bone surface/tissue volume(BS/TV)of OVX mice was lower than that of Sham mice(P<0.001).2.The increased lncRNA Xist expression and Tgif2 mRNA level and decreased expression of miR-590-3p were found in the femoral bone marrow of OVX mice(P<0.001).3.Compared with NC(negative control)group,the TRAP-positive cells were obviously observed in the Osteoclasts group.4.LncRNA Xist expression and the mRNA level of Tgif2 were increased(P<0.001),while the miR-590-3p expression was decreased(P<0.05)in the Osteoclasts group.5.The protein level of Tgif2 was increased in the Osteoclasts group.Conclusion:LncRNA Xist and Tgif2 were up-regulated and miR-590-3p was down-regulated both in femurs of OVX mice and Osteoclasts.Part two Interference with lncRNA Xist or miR-590-3p overexpression inhibited osteoclast differentiation and Tgif2 expressionObjective:To investigate the effects of lncRNA Xist,miR-590-3p,and Tgif2 on osteoclast differentiation.Methods:RAW264.7 cells were transfected with lncRNA Xist overexpression vector(oe-Xist),shRNA vector(sh-Xist),or negative controls(oe-NC and sh-NC)and then treated with 50 ng/mL RANKL and 100 ng/mL M-CSF for 7 days.1.The expression levels of lncRNA Xist,miR-590-3p,and Tgif2 in RAW264.7 cells were detected by qRT-PCR.2.The protein level of Tgif2 in RAW264.7 cells was detected by western blot.RAW264.7 cells were transfected with miR-590-3p overexpression vector(miR-590-3p-mimic),miR-590-3p inhibition vector(miR-590-3p-inhibitor),or negative controls(mimic-NC and inhibitor-NC)and then treated with 50 ng/mL RANKL arid 100 ng/mL M-CSF for 7 days.3.The expression levels of lncRNA Xist,miR-590-3p,and Tgif2 mRNA in RAW264.7 cells were detected by qRT-PCR.4.The protein level of Tgif2 in RAW264.7 cells was examined by western blot.RAW264.7 cells were transfected with sh-Xist or miR-590-3p-mimic and then treated with 50 ng/mL RANKL and 100 ng/mL M-CSF for 7 days.5.Representative images of TRAP staining of RAW264.7 cells to observe the differentiation of osteoclasts.Results:1.The overexpression of lncRNA Xist promoted Tgif2 mRNA level and protein level(P<0.001;P<0.01)and interference with lncRNA Xist inhibited Tgif2 mRNA level and protein level(P<0.01)in RAW264.7 cells,while neither IncRNA Xist overexpression nor interference had a significant effect on miR-590-3p expression.2.The overexpression of miR-590-3p inhibited the expressions of lncRNA Xist and Tgif2 in RAW264.7 cells(P<0.001),while interference with miR-590-3p facilitated the expressions of lncRNA Xist and Tgif2(P<0.001;P<0.01).3.Interference with lncRNA Xist or miR-590-3p overexpression suppressed the osteoclast differentiation.Conclusion:Interference with lncRNA Xist or miR-590-3p overexpression could inhibit Tgif2 expression and osteoclast differentiation.Part three miR-590-3p targeted lncRNA Xist and Tgif2Objective:To further explore the relationship between lncRNA Xist and miR-590-3p,as well as miR-590-3p and Tgif2.Methods:The wild type(WT)and mutated(mut)3’-UTR sequences of the Tgif2 sequence were cloned into pGL3 vectors carrying the luciferase gene.Then the recombinant pGL3 vectors were co-transfected into 293T cells or osteoclasts with miR-590-3p-mimic/inhibitor or negative controls.1.The relative luciferase activity in 293T cells was examined after overexpression of miR-590-3p.2.The relative luciferase activity in osteoclasts was detected.The wild type(WT)and mutated(mut)sequences of lncRNA Xist were cloned into pGL3 vectors carrying the luciferase gene.Then the recombinant pGL3 vectors were co-transfected into 293T cells or osteoclasts with miR-590-3p-mimic/inhibitor or negative controls.3.The relative luciferase activity in 293T cells was examined.4.The relative luciferase activity in osteoclasts was detected.Results:1.In 293T cells,overexpression of miR-590-3p reduced the luciferase activity in the pGL3-WT-Tgif2 group(P<0.05),while had no significant effect on the luciferase activity in the pGL3-mut-Tgif2 group.2.In osteoclasts,overexpression of miR-590-3p decreased the luciferase activity in the pGL3-WT-Tgif2 group(P<0.05),and interference with miR-590-3p increased the luciferase activity in the pGL3-WT-Tgif2 group(P<0.05),but had no significant effect on the luciferase activity of pGL3-mut-Tgif2.3.In 293T cells,overexpression of miR-590-3p reduced the luciferase activity in the pGL3-WT-Xist group(P<0.05),while had no significant effect on the luciferase activity in the pGL3-mut-Xist group.4.In osteoclasts,overexpression of miR-590-3p decreased the luciferase activity in the pGL3-WT-Xist group(P<0.05),and interference with miR-590-3p increased the luciferase activity in the pGL3-WT-Xist group(P<0.05),but had no significant effect on the luciferase activity of pGL3-mut-Tgif2.Conclusion:1.miR-590-3p can bind with Tgif2 and regulate the expression level of Tgif2.2.miR-590-3p can bind with lncRNA Xist and regulate the expression level of lncRNA Xist.Part four LncRNA Xist promoted osteoclast differentiation by competitively binding to miR-590-3pObjective:To verify whether lncRNA Xist regulates osteoclast differentiation by competitively binding miR-590-3p with Tgif2.Methods:RAW264.7 cells were transfected with miR-590-3p mimic or co-transfected with oe-Xist and miR-590-3p mimic,and then treated with 50 ng/mL mouse recombinant RANKL and 100 ng/mL mouse recombinant M-CSF for 7 days.1.The expressions of lncRNA Xist,miR-590-3p,and Tgif2 in RAW264.7 cells were detected by qRT-PCR.2.The protein level of Tgif2 in RAW264.7 cells was measured by western blot.3.Representative images of TRAP staining of RAW264.7 cells.Results:1.Overexpression of miR-590-3p inhibited the expression of lncRNA Xist and the mRNA level and protein level of Tgif2 in osteoclasts(P<0.001 or P<0.05),while the overexpression of lncRNA Xist reversed the effects(P<0.001).2.Overexpression of miR-590-3p inhibited the differentiation of osteoclasts,while the overexpression of lncRNA Xist abrogated this effect.Conclusion:LncRNA Xist promoted osteoclast differentiation by competitively binding to miR-590-3p with Tgif2 to up-regulate Tgif2 and then promote osteoclast differentiation through the ceRNA mechanism.Summary:1.LncRNA Xist and Tgif2 were up-regulated and miR-590-3p was down-regulated in femoral bone marrow of OVX mice and osteoclast.2.Interference with lncRNA Xist or overexpressing miR-590-3p inhibited osteoclast differentiation and Tgif2 expression.3.miR-590-3p targeted IncRNA Xist and Tgif2.4.LncRNA Xist promoted osteoclast differentiation by competitively binding to miR-590-3p with Tgif2 through the ceRNA mechanism. |
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