| Background Weightlessness induced bone loss is one of the key scientific issues in aerospace medicine.The main cause of weightlessness induced bone loss is the imbalance of bone metabolism,due to the weakened bone formation ability and the enhanced degree of bone resorption by bone cells under weightlessness.Previous studies have shown that the reduced osteogenic differentiation ability of mesenchymal stem cells plays an important regulatory role in weightlessness induced bone loss.Long non-coding RNAs play an important role in gene transcription,post-transcriptional modification and protein function regulation,and are involved in the regulation of various physiological and pathological changes in the body.The role of long non-coding RNAs in bone loss has attracted increasing attention.Lnc RNA HOTAIR(HOX transcript antisense RNA)is a long non-coding RNA transcribed from the reverse sequence between Hox C11 and Hox C12 HOXC gene families on human chromosome 12.Studies have confirmed that HOTAIR is involved in the occurrence and development of various tumors by regulating chromatin remodeling,protein stabilization and mi RNA sponges.HOTAIR knockout mice show abnormal bone development.However,whether HOTAIR is involved in bone metabolism and the occurrence of weightless bone loss needs to be further investigated.ObjectiveBased on the previous work,we found that HOTAIR direct bound with mi R-214,a key molecule in the regulation of bone metabolism.In this study,we used different bone loss models to analyze the expression level of HOTAIR in the process of bone loss and reveal the role and mechanism of HOTAIR in the osteogenic differentiation of mesenchymal stem cells,investigate the effect of HOTAIR on bone development and bone formation in vivo by using genetically modified mice,and then explored the role of HOTAIR as a target against bone loss.Methods1.By using cell rotation model,mouse tail suspension model,rhesus monkey bed rest,OVX mouse,aging mouse and clinical bone tissue samples,Flow cytometry and q-PCR was used to analyze the expression levels of HOTAIR in osteoblast,BMSCs and the bone tissue of the process of bone loss.2.The expression level of HOTAIR during the osteogenic differentiation of BMSCs were analyzed by q-PCR,and the localization of HOTAIR in BMSCs and osteoblasts was analyzed by fluorescence in situ hybridization.The effect of HOTAIR on the osteogenic differentiation of BMSCs and osteoblasts’ function was studied by knock down or overexpression the level of HOTAIR.In order to verify HOTAIR’s function in vivo,we constructed the BMSCs and osteoblasts specific overexpressing HOTAIR transgenic mice,the effect of HOTAIR overexpression on bone development in mice was analyzed by alizarin red/alcian blue staining,and micro-CT was used to analyze bone mass in transgenic mice at different ages,and then the effect of HOTAIR on bone development and bone formation was analyze by bone tissue immunohistochemistry and Analysis of gene expression.3.In BMSCs,biotin-labeled HOTAIR probes and EZH2 antibody were used to analyze the interaction between HOTAIR and EZH2,then CHIP-PCR was used to analyze the H3K27me3 modification on the promoter region of Runx2 and Sp7 by HOTAIR.In osteoblasts,the interaction between HOTAIR and mi R-214 was analyzed by RIP and other techniques,luciferase reporter assay and other techniques were used to verify the molecular mechanism that HOTAIR promotes ATF4 protein level and regulate osteoblast function by competitively binding to mi R-214.4.We analyzed the RNA-binding proteins that interact with HOTAIR in BMSCs by mass spectrometry,then screened the RNA-binding proteins related to HOTAIR translocation,and further verified the interaction between HOTAIR and Hu R by RIP,FISH and other technologies in BMSCs.To analyze the effect of Hu R on the localization of HOTAIR in BMSCs and osteoblasts under osteogenic differentiation,specific si RNA,FISH and other methods were used to define the role of Hu R in the regulation of osteogenic differentiation of BMSCs by HOTAIR.5.The Prx1-HOTAIR and Ocn-HOTAIR transgenic mice were subjected to hindlimb suspension for 28-days.We performed micro-CT,tissue sections immunohistochemistry,serum biochemistry,and gene expression analysis to compare the changes of bone phenotype between wild-type and transgenic mice after hindlimb suspension,and to clarify the effect of mesenchymal stem cells and osteoblast-specific overexpressing HOTAIR transgenic mice on bone loss caused by hindlimb suspension.Results1.The level of HOTAIR in MC3T3-E1 cells was significantly reduced after 48-hours rotation.The level of HOTAIR in bone tissue was also significantly reduced after 28-days hindlimb suspension,the level of HOTAIR in osteoblast was significantly reduced,but didn’t change significantly in BMSCs after 28-days tail suspension.After 42-days of bed rest,the expression of HOTAIR was significantly reduced in macaques bone tissue.The level of HOTAIR in bone tissue of OVX mice was significantly lower than that of the sham group,the level of HOTAIR in osteoblasts was significantly decreased,while was significantly increased in BMSCs of OVX mice.The level of HOTAIR in bone tissue samples of osteoporosis was significantly lower than that in non-osteoporotic samples.The level of HOTAIR in osteoblasts of aging mice was significantly decreased,while was significantly increased in BMSCs.2.In the early stage of BMSCs osteogenic differentiation,the level of HOTAIR didn’t change significantly,but increased significantly in the later stage of differentiation.HOTAIR was mainly located in the nucleus in BMSCs,but in osteoblast differentiated BMSCs and osteoblasts,HOTAIR was mainly located in the cytoplasm.Knockdown of HOTAIR in BMSCs promoted the osteogenic differentiation of BMSCs,and knockdown of HOTAIR in osteoblasts inhibited the activity and function of osteoblast.In Prx1-HOTAIR transgenic mice,the mineralization capacity of calvaria,ribs and limb bones of suckling mice was reduced,and bone development was delayed.In 1 month-old mice,the bone formation of Prx1-HOTAIR transgenic mice was reduced,and the bone mass was significantly decreased,while in 6 months-old mice,the bone mass of the transgenic mice was not significantly changed between that of the wild-type mice.Compared to wild-type mice,the skeletal development of Ocn-HOTAIR transgenic mice was normal,and there was no significant difference in bone mass in 1 month-old mice,however,in 6 month-old adult mice,the bone volume fraction(BV/TV),bone mineral density(BMD),trabecular bone number(Tb.N)were significantly increased,the trabecular bone space(Tb.Sp)and bone structure model index(SMI)were significantly decreased in Ocn-HOTAIR transgenic mice.the function of osteoblasts in transgenic mice was enhanced,resulting a significant increase in bone mass of mice.3.In mesenchymal stem cells,we found that HOTAIR bound to EZH2,and inhibited the transcription levels of Runx2 and Sp7 genes by enhancing the H3K27me3 modification in the promoter regions of Runx2 and Sp7 genes.In osteoblasts,HOTAIR directly interacted with mi R-214,and significantly increased the protein level of ATF4,and HOTAIR significantly promote the activity of ATF4 3’UTR-Luc and OSE1-Luc reporter genes.4.In mesenchymal stem cells,Hu R directly bound to HOTAIR,and FISH staining showed that HOTAIR and Hu R co-localized in BMSCs.Knockdown of Hu R inhibited the translocation of HOTAIR from the nucleus to the cytoplasm in BMSCs.Overexpression of Hu R in BMSCs alleviated the inhibitory effect of HOTAIR on osteogenic differentiation,and knockdown of Hu R in osteoblasts reduced the promotion effect of HOTAIR on osteoblast function.In the process of BMSCs osteogenic differentiation,the expression of Hu R gradually increased,knockdown of Hu R significantly increased the protein level of H3K27me3,then strengthened the binding ability of H3K27me3 to the promoter regions of Runx2 and Sp7 genes,which in turn leaded to reduce the m RNA levels of Runx2 and Sp7,and their downstream genes including Alp,Bglap and Collagen1α1,which ultimately inhibited the osteogenic differentiation.5.After 28-days hindlimb suspension,Ocn-HOTAIR transgenic mice significantly alleviated the decrease of the bone volume fraction(BV/TV%),trabecular bone number(Tb.N),trabecular bone thickness(Tb.Th)and bone mineral density(BMD),and the increase of trabecular bone space(Tb.Sp)and structure model index(SMI),while the Prx1-HOTAIR transgenic mice didn’t alleviate effect on the reduction of bone mass caused by hindlimb suspension.Conclusion1.In this study,we find that the expression level of HOTAIR is closely related to the occurrence of bone loss,and HOTAIR shows different expression patterns in mesenchymal stem cells and osteoblasts in bone model.2.HOTAIR shows nucleoplasmic translocation and plays a different role in the process of BMSCs osteoblast differentiation.HOTAIR is mainly located in the nucleus of BMSCs,in which,HOTAIR interacts with EZH2 to promote the modification of H3K27me3 on Runx2 and Sp7 genes promoter,and inhibits their transcription,resulting in inhibiting osteogenic differentiation.In the process of BMSCs osteogenic differentiation,HOTAIR translocates from nucleus to cytoplasm.HOTAIR in osteoblasts is mainly located in the cytoplasm and promotes the function of osteoblasts.Cytoplasmic HOTAIR increases the level of ATF4 protein by competitively binding mi R-214 with ATF4 m RNA,thus promoting the function of osteoblasts.3.BMSCs and osteoblast specific overexpressing HOTAIR show different bone phenotypes.BMSCs specific overexpressing HOTAIR shows developmental delay,decrease bone mass and decreased osteogenic function in early age of mice.While the osteoblast specific overexpressing HOTAIR shows the increase osteoblast function and bone mass.4.HuR interacts with HOTAIR in mesenchymal stem cells and osteoblasts,and mediates HOTAIR nucleoplasmic translocation.5.HOTAIR acts as a spatio-temporal specific target against to bone loss.Osteoblast specific HOTAIR transgenic mice can significantly alleviate unloading induced bone loss,while mesenchymal stem cell specific HOTAIR transgenic mice don’t have that effect. |
