Regulation Of MACF1 And Its Related NcRNAs To Osteoblast Function And Its Mechanism

Osteoblast is one of the most essential cells regulating bone formation in bone tissue.Inhibition of osteoblast proliferation and differentiation will cause significant decrease in bone formation and bone metabolism disorder,and eventually result in osteoporosis.The functions of osteoblasts were regulated by multiple factors including internal epigenetics and nonepigenetics factors,and also including external mechanical stimulations.While the mechanisms in which factors osteoblast response to mechanical stimulations and active internal signaling pathways is still unclear.Microtubule Actin Crosslinking Factor 1(MACF1)is an important cytoskeletal protein connecting microtube and actin.MACF1 regulates cellular skeletal dynamics,and thus regulating mechanical stimulation response,cell signal transduction,cell migration,and diseases.However,the regulation mechanisms of MACF1 to osteoblast proliferation and differentiation are still unknown.Therefore,this study clarified the regulation mechanisms of MACF1 to osteoblast proliferation and differentiation under mechanical unloading condition.The study also investigated the effects of MACF1 related non-coding RNAs on osteoblast differentiation.The study had provided experimental and theoretical basis for investigating MACF1 functions and the mechanism of osteoblast proliferation and differentiation under mechanical stimulation,and also provided potential targets for prevention and treatment of osteoporosis.Research Methods:By adopting random position machining and hind limb unloading mechanical unloading model,the expression levels of MACF1 to mechanical unloading condition in MC3T3-E1 preosteoblast,C57BL/6 mice femur tissue and femur bone derived MSCs was investigated.The effects of MACF1 to osteoblast alkaline phosphatase activities,cellular mineral node numbers,osteoblast differentiation marker genes and osteoblast proliferation rates were analyzed using MACF1 overexpression/knockdown MC3T3-E1 preosteoblast model.Mechanical unloading condition was also combined with MACF1 overexpression/knockdown osteoblast model to investigate the effects of MACF1 on ?-catenin expression levels and activities under mechanical unloading condition,and further clarify the effects of ?-catenin on osteoblast proliferation.LncRNA,miRNA and mRNA expression profile microarray were established on MACF1 knockdown preosteoblast.MACF1 related osteogenic non-coding RNAs were screened through bioinformatics analysis.By adopting CHIP-seq and JASPAR databaseanalysis,regulation of MACF1 to screened non-coding RNAs was forecasted.Expression levels of the screened non-coding RNAs Lnc-DIF and miR-489-3p in the femur tissues of aging and hind limb unloading osteoporosis mice model were investigated.Through the transfection of Lnc-DIF-siRNA or miR-489-3p mimic/inhibitor,expression levels of osteoblast differentiation marker genes,alkaline phosphatase activities,and mineral node numbers in MC3T3-E1 preosteoblast were analyzed.The effects of Lnc-DIF-siRNA to miR-489-3p were also clarified.In preosteoblast,the distribution of Lnc-DIF was analyzed by fluorescence in situ hybridization,and the bonding of Lnc-DIF to miR-489-3p was investigated by luciferase reporter assay.Bone-targeting delivery system was used to transfect Lnc-DIF-siRNA to ovariectomized osteoporosis mice model,femur microstructures were analyzed by microCT.AK016739-siRNA in vitro transfection were employed in MC3T3-E1 preosteoblast to investigate alkaline phosphatase activities,mineral node numbers,and the expression levels of osteoblast differentiation marker genes and osteogenic transcription factors.Super Depth Digital Microscope scanning,calcein labeling,immunohistochemical staining and RT-PCR were applied to detect the effects of AK016739-siRNA transfection to skull thicknesses,bone formation rates,osteogenic protein levels,and osteogenic transcription factors expression levels in ovariectomized osteoporosis mice model.Research Results:(1)MACF1 plays important role in the response of osteoblast to mechanical stimulation,and regulates osteoblast function.Both MACF1 mRNA and protein expression were decreased by random positioning machining mechanical unloading treatment.MACF1 expression were also decreased in mice femur and bone-derived MSCs after 28 d HLU exposure.Overexpression/knockdown MACF1 in osteoblasts resulted in increment/reduction of alkaline phosphatase activities,mineral node numbers and osteoblast differentiation marker gene expression levels.Osteoblast proliferation rates were also promoted by MACF1.The effects of mechanical unloading treatment to osteoblast proliferation were changed by MACF1 expression levels,inhibition effects of mechanical unloading to osteoblast proliferation were declined when MACF1 were knocked down.Moreover,in MACF1 overexpression/knockdown osteoblasts,expressions and activities of ?-catenin were promoted/inhibited respectively.The inhibition effects of mechanical unloading to osteoblast proliferation were declined when MACF1 were knocked down.Interference of ?-catenin expression by siRNA suppressed osteoblast proliferation,while lithium chloride treatment rescued such effects.Taken together,mechanical unloadingcondition inhibited MACF1 expression in osteoblasts.MACF1 played an essential role in response of osteoblast to mechanical unloading condition.MACF1 promoted osteoblast proliferation and differentiation.Moreover,MACF1 promoted osteoblast proliferation through its regulation to ?-catenin signaling pathway.(2)Screening of MACF1 related non-coding RNA Lnc-DIF and miR-489-3p,and their functions on osteoblast differentiation and bone formation.Bioinformatics analysis of lncRNA,miRNA,mRNA expression profile microarray on MACF1 knockdown osteoblasts results showed that: miR-489-3p expression was significantly decreased in in MACF1 knockdown osteoblast and presented the highest correlation with osteogenic genes;Lnc-DIF expression was significantly increased in MACF1 knockdown osteoblast,and multiple miR-489-3p bonding sites were predicted in Lnc-DIF sequence.Through CHIP-seq and JASPAR database forecast,binding sites of transcription factors that bonded with MACF1 were found in Lnc-DIF promoter region,indicated the regulation of MACF1 to Lnc-DIF expression.Thus,we assumed that MACF1 may regulate Lnc-DIF expression,and through which affected the regulation effects of miR-489-3p to osteogenic genes.Lnc-DIF expression was increased in the femur tissues of aging and hind limb unloading osteoporosis mice model.Lnc-DIF-siRNA transfection in MC3T3-E1 preosteoblast upregulated osteoblast differentiation marker genes expression,and enhanced alkaline phosphatase activities and mineral node numbers in preosteoblasts.In preosteoblast,Lnc-DIF mainly distributed in cytoplasm,as proved by fluorescence in situ hybridization.Lnc-DIF-siRNA transfection increased miR-489-3p levels;luciferase reporter assay also proved Lnc-DIF bond with miR-489-3p.Moreover,Lnc-DIF-siRNA transfection through bone-targeting delivery system by tail intravenous injection would rescue the trabecular microstructures of ovariectomized osteoporosis mice.miR-489-3p expression was decreased in aging osteoporosis patients bone tissues and in of aging and hind limb unloading osteoporosis mice femur tissues.Transfection of miR-489-3p mimic/inhibitor would promote/inhibit osteoblast differentiation respectively,which confirming the involvement of miR-489-3p in osteogenesis.In summary,Lnc-DIF inhibited osteoblast differentiation and bone formation through binding with miR-489-3p.MACF1 regulates osteoblast differentiation via Lnc-DIF-miR-489-3p axis.By inhibiting Lnc-DIF transcription,MACF1 promotes miR-489-3p function and increase bone formation.(3)Screening of MACF1 related long non-coding RNA AK016739 and its functions on osteoblast differentiation and bone formation.Microarray and bioinformatics analysis results showed that,in all long non-coding RNAs,AK016739 presented the highest correlation between osteogenic genes.CHIP-seq and JASPAR database forecast indicted the regulation of MACF1 to AK016739 promoter.Transfection of AK016739-siRNA in MC3T3-E1 preosteoblast enhanced cellular alkaline phosphatase activities and mineral node numbers,and also increased the expression of osteoblast differentiation marker genes and osteogenic transcription factors.Transfection of AK016739-siRNA in vivo rescued the decrease of the skull thicknesses,bone formation rates,osteogenic protein levels,and osteogenic transcription factors expression levels in ovariectomized osteoporosis mice model.In conclusion,MACF1 inhibited AK016739 transcription via transcription factors,and AK016739 inhibited osteoblast differentiation and bone formation.ConclusionTo sum up,MACF1 played an important role in the response of mechanical unloading condition of osteoblast.It promoted osteoblast proliferation and differentiation.MACF1 promoted osteoblast proliferation via ?-catenin and promoted osteoblast differentiation through its downstream non-coding RNA: Lnc-DIF-miR-489-3p axis and AK016739.The study expounded the effects and mechanisms of mechano-responsive MACF1 to osteoblast proliferation and differentiation.And we discovered novel epigenetic signaling pathways regulating osteoblast differentiation.The study had provided theoretical basis for investigating the response of osteoblast to mechanical unloading condition and its effects on bone formation.Study have also provided potential targets for prevention and treatment of bone metabolism and related diseases.