Mechanoresponsive MiR-138-5p Targets MACF1 to Inhibit Bone Formation

Mechanical stimuli play an essential role in maintaining bone remodeling and skeletal integrity.Meanwhile,bone can respond to the changes of mechanical condition to adjust its mass and architecture.Clinical studies discover that bedridden patients showed osteoporotic T-scores and low bone mineral density,and long-term immobilized patients presented reduced markers of bone formation.However,as bone formation mediated by osteoblast differentiation is a complex process,the underlying molecular mechanism of mechanical stimuli regulating bone formation is still unclear.Recent evidences show that microRNAs(miRNAs)are involved in mechanical stimuli regulating bone formation or osteoblast differentiation.Nevertheless,no direct evidence identifies mechanoresponsive miRNA in both human and animal bones,and clarifies its mechanoresponsive role under different mechanical conditions(e.g.mechanical unloading,reloading,loading).In the current study,we screened for differentially expressed miRNAs in bone specimens of bedridden patients with fractures,then identified that the expression of miR-138-5p,but not the other miRNAs,altered withbedridden time and was negatively correlated with the expression of the bone formation marker genes Alp(alkaline phosphatase).Moreover,miR-138-5p was up-regulated with reduced bone formation during unloading and down-regulated with increased bone formation during reloading in hind4imb unloaded mice.In addition,miR-138-5p was verified to be responsive to different mechanical unloading condition and cyclic mechanical stretch condition in primary osteogenic cells,respectively.Further in vitro data suggested that mechanoresponsive miR-138-5p directly targeted microtubule actin crosslinking factor 1(MACF1)to inhibit osteoblast differentiation.In vivo,we constructed an osteoblastic miR-138-5p transgenic mice model(TG138)with the Runx2promoter,and found that overexpression miR-138-5p supressed bone formation.Moreover,osteoblast-targeted inhibition of miR-138-5p sensitized bone anabolic response to mechanical loading in TG138 mice.Predominantly,the osteoblast-targeted inhibition of miR-138-5p could counteract bone formation reduction induced by hind limb unloading.Taken together,the mechanoresponsive miR-138-5p inhibited bone anabolic response for developing a novel bone anabolic sensitization strategy.

Mechanical Sensitive Molecule MACF1 Promotes Osteoblast Differentiation

The decreased osteoblast differentiation associated with reduced bone formation is one main cause of microgravityinduced bone loss.Our previous studies have demonstrated that microtubule actin crosslinking factor 1(MACF1)is downregulated in association with the decreased osteoblast differentiation and bone formation under simulated microgravity conditions.These findings suggest that MACF1 is sensitive to mechanical condition and may be critical for osteoblast differentiation and bone formation.To verify this hypothesis,current study investigates the role and mechanism of MACF1 in regulatingosteoblast differentiation by adopting MACF1 knockdown(MACF1-KD)osteoblasts.The results showed that MACF1 knockdown suppressed mineralized nodules formation,alkaline phosphatase(ALP)activity,osteogenic gene expression andβ-catenin signaling transduction.Moreover,we used RNA sequencing(RNA-seq)and chromatin immunoprecipitation sequencing(ChIP-seq)to investigate further mechanism.Interestingly,we found that MACF1 sequesterd repressors of osteoblast differentiation in cytoplasm.In conclusion,MACF1 is sensitive to mechanical condition and plays key role in activatingβ-catenin signaling transduction and sequestering repressors of osteoblast differentiation,which further promotes osteoblast differentiation.

Bioengineered miR-27b-3p and miR-328-3p modulate drug metabolism and disposition via the regulation of target ADME gene expression

Drug-metabolizing enzymes, transporters, and nuclear receptors are essential for the absorption, distribution, metabolism, and excretion(ADME) of drugs and xenobiotics. MicroRNAs participate in the regulation of ADME gene expression via imperfect complementary Watson–Crick base pairings with target transcripts. We have previously reported that Cytochrome P450 3A4(CYP3A4) and ATP-binding cassette sub-family G member 2(ABCG2) are regulated by miR-27b-3p and miR-328-3p,respectively. Here we employed our newly established RNA bioengineering technology to produce bioengineered RNA agents(BERA), namely BERA/miR-27b-3p and BERA/miR-328-3p, via fermentation. When introduced into human cells, BERA/miR-27b-3p and BERA/miR-328-3p were selectively processed to target miRNAs and thus knock down CYP3A4 and ABCG2 mRNA and their protein levels,respectively, as compared to cells treated with vehicle or control RNA. Consequently, BERA/miR-27b-3p led to a lower midazolam 10-hydroxylase activity, indicating the reduction of CYP3A4 activity. Likewise,BERA/miR-328-3p treatment elevated the intracellular accumulation of anticancer drug mitoxantrone, a classic substrate of ABCG2, hence sensitized the cells to chemotherapy. The results indicate that biologic miRNA agents made by RNA biotechnology may be applied to research on miRNA functions in the regulation of drug metabolism and disposition that could provide insights into the development of more effective therapies.

MACF1 deficiency suppresses tooth mineralization through IGF1 mediated crosstalk between odontoblasts and ameloblasts

Tooth mineralization is a ubiquitous and tightly regulated process involving complicated interactions between dental epithelium and mesenchyme.Key molecules in tooth mineralization remain poorly identified.Microtubule actin cross-linking factor 1(MACF1)is a spectraplakin protein that plays pivotal roles in the brain,muscle,lung,and bone developmental process.^(1-3) To study the specific functions of MACF1 in bone formation,we established Macf1 conditional knockout mice using the Cre-LoxP system driven by Osxterix promoter(Osx-Cre;Macf1^(f/f)).^(2) Not surprisingly,Osx-Cre;Macf1^(f/f) mice displayed the phenotypes of delayed ossification and decreased bone mass.Moreover,the OsxCre;Macf1^(f/f) mice unexpectedly showed a white and opaque appearance of incisors,contrary to the normal yellowbrown and transparent incisors.Since Osxterix is expressed in dental mesenchyme during tooth development,the abnormal tooth appearance might imply a new function of MACF1 in odontoblasts,or even ameloblasts.Therefore,the present study aimed to investigate the role of MACF1 during tooth development.

Global gene expression profiling of blast lung injury of goats exposed to shock wave

Purpose:Blast lung injury(BLI)is the most common damage resulted from explosion-derived shock wave in military,terrorism and industrial accidents.However,the molecular mechanisms underlying BLI induced by shock wave are still unclear.Methods:In this study,a goat BLI model was established by a fuel air explosive power.The key genes involved in were identified.The goats of the experimental group were fixed on the edge of the explosion cloud,while the goats of the control group were 3 km far away from the explosive environment.After successful modeling for 24 h,all the goats were sacrificed and the lung tissue was harvested for histopathological observation and RNA sequencing.Gene ontology(GO)and kyoto encyclopedia of genes and genomes(KEGG)analysis were performed to identify the main enriched biological functions of differentially expressed genes(DEGs).Quantitative real-time polymerase chain reaction(qRT-PCR)was used to verify the consistency of gene expression.Results:Of the sampled goat lungs,895 genes were identified to be significantly differentially expressed,and they were involved in 52 significantly enriched GO categories.KEGG analysis revealed that DEGs were highly enriched in 26 pathways,such as cytokine-cytokine receptor interaction,antifolate resistance,arachidonic acid metabolism,amoebiasis and bile secretion,JAK-STAT,and IL-17 signaling pathway.Furthermore,15 key DEGs involved in the biological processes of BLI were confirmed by qRTPCR,and the results were consistent with RNA sequencing.Conclusion:Gene expression profiling provide a better understanding of the molecular mechanisms of BLI,which will help to set strategy for treating lung injury and preventing secondary lung injury induced by shock wave.

Microtubule actin crosslinking factor 1 functions as a novel therapeutic target in lung metastasis of osteosarcoma

Lung metastasis is the primary cause of death in osteosarcoma(OS)patients.1 A better understanding of the molecular mechanisms underlying OS tumorigenesis and metastasis is urgently needed to identify therapeutic targets.Microtubule actin crosslinking factor 1(MACF1),which belongs to the spectraplakin family of cytoskeletal crosslinking proteins,2 is critical for cell migration and polarization due to its regulation of the cytoskeleton.Recently,MACF1 was indicated to be involved in the metastatic invasion of some human cancers,3 but the function of MACF1 in OS is still unclear.

An integrated genome-wide analysis identifies HUR/ELAVL1 as a positive regulator of osteogenesis through enhancing theβ-catenin signaling activity

Osteoporosis is a prevalent multifactorial bone disease with a strong genetic contribution.The heritability of traits that contribute to osteoporosis(bone mass,bone mineral density(BMD),bone size,bone loss and fractures)ranges from 50 to 85%,suggesting that a comprehensive understanding of the genetic basis may help identify new therapeutic targets.1 However,the genetic characteristics remain obscure,and the existing drug targets are associated with various challenges.Numerous studies have demonstrated that high-throughput sequencing data analysis is fruitful for identifying novel targets of human diseases.2 We therefore integrated GWAS and transcriptome analyses through Multi-marker Analysis of GenoMic Annotation(MAGMA)and weighted gene co-expression network analysis3(WGCNA)to identify new network modules and potential therapeutic genes for osteoporosis.As an illustration,the flow chart presenting the process of the present study was shown in Figure S1.