LncRNA 016908 Functional And Mechanistic Studies Of Regulating Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells Affecting Osteoporosis Pathogenesis

Objectives:Osteoporosis(OP)is a systemic bone disease characterized by a reduced bone mass and degradation of the bone tissue microarchitecture,followed by an increased bone fragility and a drastic increase in the risk of fracture,which is a leading cause of deformity and death in the elderly.With more than 200 million OP patients worldwide,a new OP fracture occurs every 3 seconds,on average.The number of OP patients will increase exponentially with the aging of the population,which will cause a heavy burden to the country and society.Despite the high incidence and great harm of OP,it is difficult to reveal its pathogenesis,and OP has become an urgent global health issue.Therefore,continuous and in-depth research on the molecular mechanisms of OP pathogenesis is of great strategic importance to improve the quality of life of its patients and to enhance their healthy life expectancy.The osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)plays a major role in OP development,while lncRNAs play an important part in the osteogenic differentiation of bone marrow mesenchymal stem cells.As exosomes host a variety of lncRNAs,they may be involved in the regulation of the osteogenic differentiation of bone marrow mesenchymal stem cells.This project aims to investigate whether plasma exosomes of osteoporosis patients carry lncRNAs that regulate the osteogenic differentiation of BMSCs,and to clarify the function and mechanism of the target gene lncRNA 016908 in regulating the osteogenic differentiation of BMSCs,to lay the theoretical foundation and provide new ideas for the diagnosis and treatment of osteoporosis.Methods:Part ?:Plasma exosomes were extracted from osteoporotic patients and patients with a normal bone mass.Transmission electron microscopy was used to observe the morphological characteristics of exosomes.Western blot was used to detect the expression of exosome marker proteins CD63,CD81,and TSG101.The exosome particle size and distribution were analyzed using Nanoparticle Tracking Analysis(NTA).After successful exosome identification,RNA was extracted,and differentially expressed lncRNAs were screened using high-throughput sequencing;the clinical sample size was expanded and qRT-PCR was used to verify the expression of lncRNAS again,and the target lncRNAS with a significantly high expression were screened.Part ?:The biological functions of the target gene lncRNA 016908 were verified in BMSCs using gene function acquisition and deletion experiments and alizarin red staining.BMSCs were incubated with exosomes expressing lncRNA 016908 to verify the effect of exosomes carrying lncRNA 016908 on the biological functions of BMSCs.A vector overexpressing lncRNA 016908 was constructed and a lncRNA 016908 intervention siRNA sequence was synthesized and transfected into BMSCs to induce their osteogenic differentiation.QRT-PCR and Western blot were performed to detect the expression of genes related to osteogenic differentiation;alizarin red staining was performed to detect the calcium deposition of BMSCs after osteogenic differentiation.The downstream transcriptome and the downstream proteome were analyzed in BMSCs overexpressing lncRNA 016908,and the bone metabolism-related signaling pathways that may be regulated by lncRNA 016908 were explored.Part ?:Target lncRNA 016908 was localized in bone marrow mesenchymal stem cells using nucleoplasmic isolation and FISH experiments.RNA pull-down experiments were used to pull proteins that might bind to the target gene lncRNA 016908,and after mass spectrometry identification,the target proteins related to bone metabolism and aging were selected.Then,RIP experiments were performed on reverse pull-down proteins that bind to the target RNA and qRT-PCR was performed to verify the enrichment of the target gene lncRNA 016908.Eukaryotic translation initiation factor SA-2(EIFSA2),the target gene pulled using RNA pull-down,was verified to have a biological function in regulating osteogenic differentiation of bone marrow mesenchymal stem cells using gene function acquisition,deletion experiments,and alizarin red staining.An EIF5A2 overexpression vector was constructed,the intervention sequence of EIF5A2 was synthesized,and BMSCs were transfected to induce osteogenic differentiation.QRT-PCR and Western blot were performed to detect the expression of osteogenic differentiation-related genes;alizarin red staining assay was performed to detect the calcium deposition in BMSCs after osteogenic differentiation.The rescue assay was performed to verify whether lncRNA 016908 regulates the osteogenic differentiation of BMSCs by targeting EIF5A2.The overexpression of lncRNA 016908 was accompanied by the silencing of EIF5A2.The silencing of lncRNA 016908 was accompanied by an overexpression of EIF5A2.QRT-PCR and Western blot were used to detect osteogenic differentiation-related gene expression;alizarin red staining assay was used to detect the calcium deposition after the osteogenic differentiation of BMSCs.Results:Part ?:A large number of plasma-derived exosomes were successfully obtained,and their size,morphology,particle size,and surface markers were consistent with the exosome characteristics obtained using NTA,transmission electron microscopy methods,and western blot.After high-throughput sequencing,a large number of differentially expressed lncRNAs were screened,among which lncRNA 016908 was significantly highly expressed and verified to be stably highly expressed in the plasma exosomes of osteoporotic patients,using qRT-PCR.Part ?:Exosomes have the function of regulating the osteogenic differentiation of BMSCs.LncRNA 016908 can inhibit the osteogenic differentiation of BMSCs:with the overexpression of lncRNA 016908,there was a decreased expression of osteogenic differentiation-related genes OPN,OCN,and RUNX2,a reduced calcium deposition,and a weakened osteogenic differentiation of BMSCs.After intervention in the expression of lncRNA 016908,we observed an increased expression of osteogenic differentiation-related genes OPN,OCN,and RUNX2,an increased calcium deposition,and an enhanced osteogenic differentiation of BMSCs.Part ?:LncRNA 016908 is mainly localized in the nucleus of bone marrow mesenchymal stem cells;in this project,protein EIF5A2,which binds to the target gene lncRNA 016908,was pulled using the RNA pull-down assay;and then it was further reverse verified that a large amount of lncRNA 016908 was enriched in EIF5A2 using a RIP assay and qRT-PCR.EIF5A2 has the function of inhibiting bone marrow mesenchymal osteogenic differentiation of BMSCs;thus,the overexpression of EIF5A2 inhibited BMSCs.After the overexpression to EIF5A2,there was a decreased expression of the osteogenic differentiation-related genes OPN,OCN,and RUNX2,a reduced calcium deposition,and a reduced osteogenic differentiation of BMSCs;after intervention in the EIF5A2 expression,there was an increased expression of the osteogenic differentiation-related genes OPN,OCN,and RUNX2,an increased calcium deposition,and an enhanced osteogenic differentiation of BMSCs.The silencing of EIF5A2 could rescue the diminished osteogenic differentiation ability of BMSCs caused by the overexpression of lncRNA 016908,thus enhancing their osteogenic differentiation ability.The overexpression of EIF5A2 could reverse the enhanced osteogenic differentiation ability of BMSCs caused by silencing lncRNA 016908,leading to a reduced osteogenic differentiation ability.Conclusions:1.Exosomes carrying lncRNA 016908 regulate the osteogenic differentiation of bone marrow mesenchymal stem cells.2.LncRNA 016908 inhibits the osteogenic differentiation of bone marrow mesenchymal stem cells.3.EIF5A2 inhibits the osteogenic differentiation of bone marrow mesenchymal stem cells.4.LncRNA 016908 can inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells by targeting EIF5A2,which in turn,affects osteoporosis pathogenesis.