| BackgroundIn the process of space flight,astronauts are affected by various environmental factors including weightlessness.With the rapid development of China’s Space Station,the impact of long-term weightlessness on astronauts has become increasingly prominent.Weightlessness can lead to cardiovascular dysfunction,muscle atrophy and decreased immune function.One of the most special is the impact on the bone system,that is,the occurrence of osteopenia induced by weightlessness.With the extension of exposure time,the severity of bone loss is increasing.Therefore,bone loss caused by weightlessness becomes one of the most important limiting factors of the long-term manned space missions.The reduced osteogenesis is the main characteristics of bone loss caused by weightlessness,and the decrease of osteoblast number and activity are the main reasons.Non-coding RNAs,such as mi RNAs and lnc RNAs affect the regulation of osteoblast proliferation and other functions.However,it has not yet been studied how mi RNAs and lnc RNAs interact and regulate osteoblast function under simulated weightlessness.ObjectiveBased on the previous sequencing results of simulated weightlessness sensitive mi RNAs and lnc RNAs,the research aimed at detecting the change characteristics,functions and mechanisms of key mi RNA and lnc RNA in osteoblasts under simulated weightlessness environment,and intended to observe the effect of in vivo intervention on the expression of key mi RNA and lnc RNA against bone loss caused by simulated weightlessness.Methods1.After 21 days of tail suspension,six-month-old male C57BL/6j mice were anesthetized,bilateral femurs and tibiae were removed from the mice.Micro-CT was used to detect bone loss in HU mice.MC3T3-E1 osteoblasts were cultured in vitro and divided into control group and simulated weightlessness group.We utilized q RT-PCR to examine mi RNAs in the femurs of HU mice and osteoblast under clinoration.MC3T3-E1 cells were cultured in vitro,transfected with mimic-139,inhibitor-139 and their corresponding negative controls,and the regulatory effects of mi R-139-3p on osteoblast differentiation,mineralization,proliferation and apoptosis were examined.2.Bioinformatics analysis and dual luciferase reporter gene system to detect whether mi R-139-3p could bind to ELK1.We utilized q RT-PCR,Western blotting and cellular immunofluorescence to confirm if mi R-139-3p could inhibit ELK1 expression at the post-transcriptional level.Mimic-139 and ELK1 overexpression plasmids p EX-ELK1 were double-transfected into MC3T3-E1 cells to verify whether mi R-139-3p regulated osteoblast differentiation and apoptosis through ELK1.3.We utilized bioinformatics analysis and q RT-PCR analysis to find lnc RNA that could bind to mi R-139-3p and significantly reduced in the femurs of HU mice and MC3T3-E1 cells under clinoration.RACE and Northern blotting were used to confirm the full-length sequence of lnc RNA ODSM,and its protein coding ability was predicted by CPC and CPAT software.The subcellular localization of lnc RNA ODSM was detected by software prediction,RNA FISH and nucleoplasmic separation PCR experiments.q RT-PCR,anti-Ago2 RIP and dual-luciferase reporter gene system were utilized to examine the interaction and regulatory relationship between mi R-139-3p and lnc RNA ODSM.After transfection of p EX-ODSM,si RNA ODSM and NC into MC3T3-E1 cells,the regulatory effects of lnc RNA ODSM on osteoblast differentiation and apoptosis were examined.After double transfection of p EX-ODSM and mimic-139 into MC3T3-E1 cells,the expression of ELK1 in osteoblasts was confirmed by dual luciferase reporter gene system,Western blotting and cellular immunofluorescence assay.4.MC3T3-E1 cells transfected with inhibitor-139 and inhibitor-NC,and then cultured for 48 h under clinoration to test whether inhibiting the expression of mi R-139-3p under simulated weightlessness could regulate osteoblast differentiation and apoptosis.p EX-ODSM and mimic-139 were double-transfected into osteoblast and cultured under clinoration for 48 h.Then,we examined whether lnc RNA ODSM could regulate ELK1 protein expression,osteoblast differentiation and apoptosis level through mi R-139-3p under simulated weightlessness.5.Targeted inhibition of mi R-139-3p or supplementation of lnc RNA ODSM in the bone formation area of HU mice using an osteogenic targeting delivery system.The expression levels of mi R-139-3p and lnc RNA ODSM were examined by q RT-PCR,the protein levels of ELK1 and Bglap were detected by immunohistochemical staining of bone tissue,and the apoptotic ratio of bone tissue was detected by TUNEL staining;H&E staining,calcein double-label,micro CT and three-point bending test were used to detect the protective effect of targeting inhibition of mi R-139-3p or supplementation of lnc RNA ODSM on bone loss caused by simulated weightlessness.Results1.After 21 days of HU,significant bone loss occurred in the femur of six-month-old male C57BL/6j mice.Mi R-139-3p was upregulated in the femurs of HU mice and MC3T3-E1 cells under clinoration.In MC3T3-E1 cells,mi R-139-3p inhibited the m RNA and protein expression levels of Runx2,Bglap and Col1a1.Mi R-139-3p inhibited the m RNA expression and protein activity of ALP.Mi R-139-3p inhibited the formation of mineralized nodules and promoted apoptosis.However,mi R-139-3p had no effect on PCNA expression and proliferation activity.2.Mi R-139-3p directly targeted ELK1 in MC3T3-E1 cells.Mi R-139-3p inhibited ELK1 expression at the post-transcriptional level,and could regulate osteoblast differentiation and apoptosis through ELK1.3.The expression of lnc RNA ODSM was significantly reduced in the femurs of HU mice and MC3T3-E1 cells under clinoration.Lnc RNA ODSM was a full length of1974 nt lnc RNA without ability to encode proteins,and mainly localized in the cytoplasm.In MC3T3-E1 cells,mi R-139-3p and lnc RNA ODSM could interact with and repress each other’s expression.Lnc RNA ODSM could regulate ELK1 expression by interacting with mi R-139-3p,and could promote osteoblast differentiation and inhibit apoptosis in MC3T3-E1 cells.4.Simulated weightlessness could suppress osteogenic differentiation in osteoblasts.Under simulated weightlessness,reducing the expression of mi R-139-3p could promote the m RNA and protein expression levels of Runx2,Bglap and Col1a1.Moreover,inhibitor-139 promoted the m RNA expression and protein activity of ALP.Knocking down endogenous mi R-139-3p expression partially alleviated the differentiation inhibited by simulated weightlessness.Furthermore,the apoptosis of MC3T3-E1 cells increased in simulated weightlessness environment.Knocking down mi R-139-3p expression partly decreased apoptosis caused by simulated weightlessness.In addition,lnc RNA ODSM-mediated expression of ELK1 and regulation of osteoblast differentiation and apoptosis partially depended on mi R-139-3p under simulated weightlessness environment.5.Targeted inhibition of mi R-139-3p or supplementation of lnc RNA ODSM in the bone formation area of HU mice using an osteogenic targeting delivery system resulted in increased expression of ELK1 and Bglap,decreased apoptotic ratio of bone tissue,increased bone mass and bone formation rate,and improved bone micro-structure and biomechanical properties.Conclusion1.The expression of mi R-139-3p is significantly increased in bone tissue of HU mice and in osteoblasts under simulated weightlessness induced by clinorotation.Mi R-139-3p can inhibit osteoblast differentiation and mineralization,meanwhile promote osteoblast apoptosis.2.In osteoblasts,ELK1 is the target gene of mi R-139-3p,and mi R-139-3p can regulate osteoblast differentiation and apoptosis through ELK1.Lnc RNA ODSM can interact with mi R-139-3p and regulate ELK1 expression through mi R-139-3p.3.Inhibiting the expression of mi R-139-3p in osteoblasts under simulated weightlessness can improve osteoblast differentiation and reduce osteoblast apoptosis.Lnc RNA ODSM can regulate ELK1 expression by combining with mi R-139-3p,and regulate the differentiation and apoptosis of osteoblasts under simulated weightlessness.4.Targeted inhibition of mi R-139-3p or supplementation of lnc RNA ODSM in osteoblasts in vivo can significantly alleviate bone loss in HU mice.This study finds that the expression of mi R-139-3p is significantly increased under simulated weightlessness,and the lnc RNA ODSM/mi R-139-3p/ELK1 pathway can regulate osteoblast differentiation and apoptosis.Targeted inhibition of mi R-139-3p or supplementations of lnc RNA ODSM in osteoblasts in vivo significantly alleviate bone loss in HU mice.Our study further explores the interaction mechanism of key mi RNAs and lnc RNAs in simulated weightlessness environment and provides new molecular targets for the prevention and treatment of weightlessness-induced bone loss. |
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