| Objective:Fluid shear stress(FSS)plays a critical role in osteoblast proliferation and apoptosis.However,the roles of lnc RNAs and micro RNAs in fluid shear stress-regulated osteoblast proliferation and apoptosis and the possible mechanisms remain unclear.The aim of the present study was to investigate whether miR-140-5p and miR-34a regulate osteoblast proliferation and apoptosis under FSS and its molecular mechanisms.Methods:(1)MC3T3-E1 cells were exposed to FSS(12 dyn/cm~2),miR-140-5p expression levels were measured by q RT-PCR.(2)MC3T3-E1 cells were transfected with mimic-140-5p or inhibitor-140-5p.Cell proliferation capacity was detected by CCK-8 assay and Ed U labeling assay.The expression levels of PCNA,CDK4 and Cyclin D1 were identified through q RT-PCR and western blot.And we further studied whether overexpression of miR-140-5p could inhibit osteoblast proliferation induced by FSS.(3)Luciferase assay was used to validate that VEGFA was a target of miR-140-5p.q RT-PCR,western blot and immunofluorescence were used to detect the effect of overexpression or knockdown of miR-140-5p on VEGFA expression.Then we tested whether overexpression of miR-140-5p could inhibit the up-regulation of VEGFA protein expression induced by FSS.(4)pc DNA3.1-VEGFA and si RNA-VEGFA were used to overexpress and knockdown VEGFA expression in MC3T3-E1 cells,respectively.Then we investigated the effect of VEGFA on osteoblast proliferation.In addition,the co-transfection of mimic-140-5p with pc DNA3.1-VEGFA was performed to verify whether miR-140-5p could regulate osteoblast proliferation and ERK5 activation through VEGFA.(5)MC3T3-E1 cells were exposed to FSS(12 dyn/cm~2),miR-34a expression levels were measured by q RT-PCR.(6)MC3T3-E1 cells were transfected with mimic-34a or inhibitor-34a.Cell proliferation capacity was detected by CCK-8 assay and Ed U labeling assay.The expression levels of PCNA,CDK4 and Cyclin D1 were identified through q RT-PCR and western blot.The apoptosis rate was detected by flow cytometric analysis and Hoechest 33258 staining.The expression levels of Bax,Bcl-2 and cleaved caspase-3were identified by western blot.And we further studied whether overexpression of miR-34a could attenuate FSS-induced promotion of proliferation and suppression of apoptosis.(7)Luciferase assay was used to validate that FGFR1 was a target of miR-34a.q RT-PCR,western blot and immunofluorescence were used to detect the effect of overexpression or knockdown of miR-34a on FGFR1 expression.Then we tested whether overexpression of miR-34a could inhibit the up-regulation of FGFR1protein expression induced by FSS.(8)pc DNA3.1-FGFR1 and si RNA-FGFR1 were used to overexpress and knockdown FGFR1 expression in MC3T3-E1 cells,respectively.Then we investigated the effect of FGFR1 on osteoblast proliferation and apoptosis.In addition,the co-transfection of mimic-34a with pc DNA3.1-FGFR1 was performed to verify whether miR-34a could regulate the proliferation and apoptosis of osteoblasts through FGFR1.(9)MC3T3-E1 cells were exposed to FSS(12 dyn/cm~2),lnc RNA TUG1 expression levels were measured by q RT-PCR.RNA-FISH assay analysed the cellular location of lnc RNA TUG1 in MC3T3-E1 cells.The mutual regulation between lnc RNA TUG1 and miR-34a was verified by transfection of mimic-34a or si RNA-TUG1.Luciferase assay was used to validate whether lnc RNA TUG1 could directly bind to miR-34a.pc DNA3.1-TUG1 and si RNA-TUG1 were used to overexpress and knockdown lnc RNA TUG1 expression in MC3T3-E1 cells,respectively.Then we investigated the effect of lnc RNA TUG1 on osteoblast proliferation and apoptosis.Moreover,si RNA-TUG1 and inhibitor-34a were co-transfected into MC3T3-E1 cells before FSS exposure.The expression level of FGFR1 was detected by western blot.(10)q RT-PCR and western blot were used to detect the expression changes of lnc RNA TUG1,miR-34a and FGFR1,PCNA,CDK4and Cyclin D1,and Bax,Bcl-2 and cleaved caspase-3 in the femoral tissue after overexpression of lnc RNA TUG1 in ovariectomized(OVX)osteoporosis mice model.And the structure of mice femoral tissue was observed by micro-CT and HE staining.Results:(1)FSS down-regulated miR-140-5p expression levels in MC3T3-E1cells.(2)Up-regulation of miR-140-5p inhibited osteoblast proliferation and inhibited the m RNA and protein expression levels of PCNA,CDK4 and Cyclin D1.On the contrary,down-regulation of miR-140-5p promoted osteoblast proliferation and promoted the m RNA and protein expression levels of PCNA,CDK4 and Cyclin D1.In addition,Up-regulation of miR-140-5p inhibited FSS-induced proliferation of osteoblasts.(3)miR-140-5p could directly bind to the 3'UTR of VEGFA.miR-140-5p mainly influenced protein expression levels of VEGFA but had little effect on m RNA expression levels in MC3T3-E1 cells.Moreover,up-regulation of miR-140-5p inhibited the protein expression levels of VEGFA induced by FSS.(4)VEGFA promoted osteoblast proliferation.In addition,miR-140-5p regulated osteoblast proliferation and ERK5 activation through VEGFA.(5)FSS down-regulated miR-34a expression levels in MC3T3-E1 cells.(6)Up-regulation of miR-34a inhibited osteoblast proliferation and inhibited the m RNA and protein expression levels of PCNA,CDK4 and Cyclin D1.And up-regulation of miR-34a also promoted osteoblast apoptosis and promoted the protein expression levels of Bax and cleaved caspase-3,and inhibited Bcl-2 protein expression levels.On the contrary,down-regulation of miR-34a promoted osteoblast proliferation and promoted the m RNA and protein expression levels of PCNA,CDK4 and Cyclin D1.And down-regulation of miR-34a also inhibited osteoblast apoptosis and inhibited the protein expression levels of Bax and cleaved caspase-3,and promoted Bcl-2 protein expression levels.In addition,up-regulation of miR-34a attenuated FSS-induced promotion of proliferation and suppression of apoptosis of osteoblasts.(7)miR-34a could directly bind to the 3'UTR of FGFR1.miR-34a mainly influenced protein expression levels of FGFR1 but had little effect on m RNA expression levels in MC3T3-E1 cells.Moreover,up-regulation of miR-34a inhibited the protein expression levels of FGFR1 induced by FSS.(8)FGFR1 promoted proliferation and inhibited apoptosis of osteoblasts.In addition,miR-34a regulated the proliferation and apoptosis of osteoblasts through FGFR1.(9)FSS up-regulated lnc RNA TUG1expression levels in MC3T3-E1 cells.RNA FISH analysis further confirmed that lnc RNA TUG1 localized in the cytoplasm and nuclei of MC3T3-E1 cells.In addition,LncRNA TUG1 and miR-34a repressed each other.And lnc RNA TUG1 could directly bind to miR-34a.LncRNA TUG1 promoted proliferation and inhibited apoptosis of osteoblasts.In addition,transfection of inhibitor-34a partly reversed the si RNA-TUG1-induced reduction in FGFR1 protein expression level under FSS.(10)Transfection of pc DNA3.1-TUG1 in vivo could reverse the decrease of lnc RNA TUG1 expression,the increase of miR-34a expression,the decrease of FGFR1 m RNA and protein expression,the decrease of PCNA,CDK4 and Cyclin D1 m RNA and protein expression,the increase of Bax and cleaved caspase-3 protein expression,and the decrease of Bcl-2 protein expression in the ovariectomized osteoporosis mice model.The results of micro-CT and HE staining indicated that overexpression of lnc RNA TUG1 could alleviate the osteoporosis in ovariectomized mice.Conclusions:(1)FSS-induced down-regulation of miR-140-5p facilitates osteoblast proliferation via activating VEGFA/ERK5 signaling pathway.The mechanism is as follows:FSS down-regulates miR-140-5p expression levels in osteoblasts.VEGFA is a target of miR-140-5p.Down-regulation of miR-140-5p activates ERK5 by up-regulating VEGFA,thereby promoting osteoblast proliferation.(2)FSS-induced down-regulation of miR-34a facilitates proliferation and inhibits apoptosis of osteoblasts via targeting FGFR1.The mechanism is as follows:FSS down-regulates miR-34a expression levels in osteoblasts.FGFR1 is a target of miR-34a.Down-regulation of miR-34a promotes proliferation and inhibits apoptosis of osteoblast by up-regulating FGFR1.(3)FSS facilitates proliferation and inhibits apoptosis of osteoblasts via the lnc RNA TUG1/miR-34a/FGFR1 axis.The mechanism is as follows:FSS up-regulates lnc RNA TUG1 expression levels in osteoblasts.LncRNA TUG1 up-regulates FGFR1expression levels by sponging miR-34a,thereby promoting proliferation and inhibiting apoptosis of osteoblasts.Additionally,lnc RNA TUG1 alleviates osteoporosis in ovariectomized mice through miR-34a/FGFR1 axis.The above findings may provide a novel mechanism of FSS-regulated osteoblast proliferation and apoptosis,and offer a new avenue to further investigate osteogenesis induced by mechanical loading,which may provide potential therapeutic targets for osteoporosis. |
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