Mechanical Force-induced SNHG8 And Histone Methylation Mediated Oxidative Changes Regulate Osteogenic Differentiation Of Periodontal Ligament Stem Cells

Background and objectivesAs an indispensable stimulus in physiological processes,the mechanical force has a wide range of biological functions in regulating gene expression or organelle generation.The mechanical force-guided adaptive remodeling of alveolar bone is the basis for achieving tooth movement and maintaining the position to avoid relapse.The osteogenic differentiation regulatory factors and mechanisms of multilineage stem cells osteogenic differentiation is the research emphasis of periodontal bone regeneration and remodeling.During tooth movement,periodontal ligament stem cells(PDLSCs),as the initial effector to respond to orthodontic force,change their basic physiological states such as morphology,cytoskeleton,and metabolism to guide the physiological activities including cell proliferation,programmed cell death,osteogenesis,and osteoclast differentiation.PDLSCs play a considerable role in alveolar bone reconstruction and guide the remodeling of fibrous tissue around the tooth roots and alveolar bone tissue to realize the movement of tooth position.Specifically,the mechanical force generated by the orthodontic appliances transmits to the periodontal ligament.The periodontal ligament on the pressure side is compressed,which leads to a reduction of blood flow,absorption of collagen fibers and matrix,then active osteoclastogenesis on the compression surface of alveolar bone.The periodontal ligament on the tension side is stretched,which leads to the widening of the periodontal ligament and active osteoblastogenesis to form new bone.This process involves multiple signal transduction pathways,protein interactions,and chemical molecular regulation.Our previous studies have found that there are multiple coding genes and pathways related to osteogenic differentiation regulated by mechanical stimuli in PDLSCs.It was also found that non-coding genes(such as miR-21)can mediate osteogenic differentiation and bone remodeling.Although existing research has confirmed the regulatory relationship between miRNAs and lncRNAs,it is still unclear whether compared to miRNAs with extremely short sequences,lncRNAs with longer sequences could also regulate the osteogenic differentiation of PDLSCs under mechanical force.Given the regulation of lncRNAs in the nucleus tend to achieve alternating effects through epigenetics,as well as the close relationship between stem cell behavior and epigenetics,this study explored the mechanism of the osteogenesis regulation of force-sensitive lncRNAs from the perspective of epigenetics.Research has shown that epigenetic pathways could regulate osteogenesis by alternating metabolic characteristics,such as the level of glycolysis.Metabolic factors,including the preference for glucose oxidation mode,also play a crucial role in regulating the fate of stem cells and their physiological activities.In particular,mitochondrial dysfunction and the increase of ROS levels caused by oxidative stress can significantly inhibit the osteogenic differentiation of stem cells.Previous studies have shown that mechanical stimulation can achieve specific metabolic regulation by changing crucial metabolic enzymes or regulating mitochondrial biogenesis.However,few studies have been conducted on the alteration of the oxidative mode induced by physical factors such as mechanical force in PDLSCs,and the impact of changing the oxidative profile on osteogenic differentiation.In this study,the results of chromatin immunoprecipitation sequencing(ChIP-seq)of PDLSCs under mechanical force showed significant differences in metabolic processes,such as changes in oxidation patterns.Therefore,this study investigated the regulation of mechanical force-sensitive lncRNA on osteogenic differentiation,identified the epigenetic impact of force-sensitive lncRNA,and determined the changes in histone methylation-mediated oxidation mode under mechanical force and its regulation on cell osteogenic differentiation.This study has significant implications for the study of PDLSCs differentiation and tissue regeneration under mechanical stimulation.Materials and methods1.Screening of mechanically-sensitive lncRNA and characterization of osteogenic differentiation functionFirstly,the primary PDLSCs were identified by flow cytometry,multi-lineage differentiation potential tests,CCK-8 assay,and wounding healing assay.Then we used FX6000 Tension System to apply mechanical force to PDLSCs for 0,6,12,and 24 hours.The high-throughput sequencing was used to analyze the differently expressed lncRNAs,twelve of them with significant changes and high expression levels were verified by quantitative reverse transcription polymerase chain reaction(qRT-PCR).The selected mechanical-sensitive lncRNA SNHG8 was then knocked down by lentivirus.The qRT-PCR,western blot,alkaline phosphatase staining,alizarin red staining,construction of rat tooth movement model,and ectopic osteogenesis in nude mice were used to verify the effect of SNHG8 on osteogenic differentiation.2.The epigenetic mechanism of mechanically-sensitive lncRNA on regulating osteogenic differentiationFirstly,the intracellular localization of SNHG8 was determined by fluorescence in situ hybridization(FISH)and the isolation of nuclear and cytoplasm.The interaction between SNHG8 and PRC2 subunits was verified by qRT-PCR and western blot combined with lentivirus transfection.To verify the changes of EZH2-related histone H3K27me3 and H3K4me3 and their regulatory effects on lncRNAs,western blot was used to detect the expression of H3K4me3 and H3K27me3 in PDLSCs under mechanical force while ChIP-seq was used to evaluate the overall changes of H3K4me3 and H3K27me3 under mechanical force and the enrichment of H3K4me3 at the promoters.The targeted biological processes and pathways of H3K4me3 and H3K27me3 were also analyzed.3.The alteration of oxidation mode and osteogenic differentiation of PDLSCs under mechanical forceFirstly,the mechanical force-induced alteration in the expression levels of the representative factors of aerobic and anaerobic oxidation,COX4 and LDH,were determined by qRT-PCR,western blot,immunofluorescence staining,and immunohistochemical staining.Then we detected the alteration of representative metabolites,ROS,and lactic acid.To clarify the mechanism of oxidation profile changes,the mitochondrial membrane potential(MMP)was detected by Tetramethylrhodamine staining,and the morphological changes of mitochondria in PDLSCs were analyzed by transmission electron microscopy.The mitochondrial fission was determined by western blot and immunofluorescence staining.The total level and phosphorylation activation of MFF,the representative factor related to mitophagy,were detected by western blot.To determine the regulatory effect of histone methylation on oxidation mode under mechanical force,H3K4me3 enrichment at the promoters of related genes was analyzed.To confirm the regulatory effect of mitophagy on the oxidation mode of PDLSCs under mechanical force,cyclosporin A was used to inhibit mitophagy.Then the MMP status,mitochondrial morphology,expression of LDH and COX4,and metabolite levels were remeasured after mechanical force with the inhibition of mitophagy.The effects of mitophagy on osteogenic differentiation were verified by qRT-PCR and western blot detection of ALP and RUNX2 in PDLSCs under mechanical force after cyclosporine A inhibited mitophagy.Results1.Mechanically sensitive lncRNA SNHG8 could negatively regulate the osteogenic differentiation of PDLSCsThe expression of SNHG8 in PDLSCs decreased steadily after force loading.The transcriptional and protein level of ALP and RUNX2 in SNHG8 knockdown PDLSCs augmented after the force loading.Alkaline phosphatase staining and alizarin red S staining showed that low SNHG8 levels could lead to the increase of alkaline phosphatase levels and mineralized tuberculosis in PDLSCs after osteogenesis induction to improve the ability of osteogenic differentiation.The above results showed that the down-regulation of SNHG8 significantly promoted the osteogenic differentiation of PDLSCs under mechanical stimulation or osteogenic induction.In vivo tooth movement experiments showed that the level of the homologous gene of SNHG8 in the periodontal tissue steadily declined from day 3 to day 14 during tooth movement and returned to baseline by day 21.The above results suggested the expression of SNHG8 decreased in the early stage of tooth movement in vivo,which is consistent with the in vitro results.The ectopic osteogenesis experiment in vivo also confirmed that infiltrating cells were found in the central areas of the scaffolds in the sh-SNHG8 group,with more collagen fibers and new bone formation.2.Mechanical force regulated SNHG8 expression and further metabolic changes through the epigenetic pathwayFISH assay and the isolation of nuclear and cytoplasmic showed that SNHG8 was mainly located in the nucleus.Therefore,the relationship between the expression levels of key subunits such as EZH2 of the PRC2 complex closely related to nuclear localization lncRNA and the intracellular levels of SNHG8 was examined.A significant decrease in EZH2 expression was observed in the PDLSCs of the sh-SNHG8 group,while the SNHG8 level in the sh-EZH2 group also decreased significantly,which confirmed the positive correlation between SNHG8 and EZH2.Results of the western blot showed that the level of H3K27me3,which is directly related to EZH2,decreased after the force loading,while the level of H3K4me3,which plays an antagonism with H3K27me3,rises significantly.The ChIP-seq of H3K27me3 and H3K4me3 in PDLSCs after mechanical stimuli showed that the enrichment of H3K4me3 in the promoter of SNHG8 and its preceding area decreased significantly after 24 hours of stretching,suggesting that mechanical force affects the expression of SNHG8 through epigenetic regulation of promoter.In addition,the results of ChIP-seq also revealed the changes in differentially expressed genes targeted metabolic processes such as oxidation patterns associated with the binding of H3K27me3 and H3K4me3 histones.3.Mechanical force enhances anaerobic oxidation and osteogenic differentiation by inducing mitophagy in PDLSCsThe transcription and protein level of COX4,the marker of aerobic oxidation,was significantly attenuated,while LDH,the key enzyme of anaerobic oxidation,was significantly elevated in PDLSCs under mechanical force.ALP and RUNX2,the pivotal genes of osteogenic differentiation,were also augmented after mechanical force loading.As for metabolites,ROS,the inevitable byproduct of aerobic oxidation,decreased after mechanical stimulation,while lactic acid,the representative product of anaerobic oxidation increased significantly.As regards the mechanism,the MMP in PDLSCs after stretching was impaired,and the increase of mitochondrial fission further led to the activation of mitophagy,which was confirmed by the rescue experiments of blocking mitophagy.The enhancement of anaerobic oxidation caused by mitophagy is also necessary for the rise of the expression level of ALP and RUNX2 under mechanical force.Conclusions1.Mechanical force negatively regulated the expression of force-sensitive lncRNA SNHG8.The reduction of SNHG8 can enhance the osteogenic differentiation of PDLSCs under mechanical force and osteogenic induction.2.The nucleus-located lncRNA SNHG8 was regulated by epigenetic processes.The expression of SNHG8 was regulated by the enrichment of H3K4me3 at the promoter.Meanwhile,SNHG8 could combine with EZH2,the crucial subunit of PRC2,to realize the regulation of related physiological processes.ChIP-seq showed that mechanical stimulation had a significant impact on the metabolic pathway of PDLSCs.3.The anaerobic oxidation was enhanced,and aerobic oxidation was decreased in PDLSCs under mechanical force.The oxidative profile changes were caused by the influence of mitophagy guided by histone methylation.Mitophagy not only regulated the oxidation pattern in PDLSCs under stretch but also plays a crucial role in the expression of ALP and RUNX2.