| BackgroundOsteosarcoma(OS)is a primary malignant tumor that occurs in adolescents.The most common symptom is local pain at the epiphyseal end of the long shaft.The incidence rate of OS in adolescents is high,reaching 8-11/million one year.The onset of the disease presents a male gender advantage,with males being affected 1.4 times more frequently than females.At present,the main clinical treatment for OS is surgery combined with radiotherapy and chemotherapy,but the effect is not good,especially for patients with lung metastasis,the prognosis is extremely poor.The pathogenesis mainly attributed to OS is still unclear.MiRNA is a type of non coding small RNA molecule(18-25 nucleotides)that contains stem ring structure which is processed by Dicer.MiRNA are widely expressed in animals and plants.Due to its function of inhibiting target mRNA transcription,translation,or being able to cleave target mRNA and promote its degradation,miRNAs are considered to play an important role in regulating developmental processes.MiRNA is encoded by the genome of higher eukaryotes,and it guides the RISC to degrade mRNA or hinder its translation by pairing with the target gene mRNA base.MiRNAs are quite conserved in species evolution,and miRNAs found in plants,animals,and fungi are only expressed in specific tissues and developmental stages.The tissue specificity and timing of miRNAs determine the functional specificity of tissues and cells,indicating that miRNAs play multiple roles in regulating cell growth and development.In recent years,it has been found that changes in miRNA expression in vivo are involved in the physiological processes of disease occurrence and development,and abnormal miRNA expression is also related to the occurrence and development of tumors.Mesenchymal stem cells(MSC)are an undifferentiated adult stem cell population that can self renew and differentiate into multiple lineages.MSCs have been clearly proven to have good therapeutic effects in diseases such as osteogenesis imperfecta,fractures,traumatic brain injury,stroke,and myocardial infarction.In the past few decades,many basic and clinical studies have shown that MSC are a promising therapeutic regeneration strategy because they are easy to isolate,low immunogenicity,and create a favorable environment for tissue regeneration and cell repair.MSCs can be attributed to damaged tissues and undergo subsequent transdifferentiation to repair and replace damaged cells,thereby promoting tissue repair.At present,research suggests that MSCs mainly achieve therapeutic effects in the body through paracrine signal transduction;MSCs can release bioactive molecules,affecting the proliferation,migration,and survival of adjacent cells.Research reports that MSC culture medium can promote tissue repair and regeneration.Exosomes are key bioactive vesicles responsible for the paracrine function of MSC;Exosomes regulate many physiological processes by influencing the survival,proliferation,migration and gene expression of receptor cells,and by reprogramming the pathological process of targeting cell behavior.Exosomes can promote tissue repair by delivering various functional proteins,RNA,and soluble cytokines.Therefore,this study attempts to identify active miRNAs in the extracellular vesicles secreted by bone MSC and explore their mechanism of inhibiting the proliferation,migration,and invasion of OS.ObjectiveThis study explores the molecular mechanisms and signaling pathways about miRNAs original exosome secreted by MSC inhibit the proliferation,migration,and invasion of OS.Method1.Using the latest version of bioinformatics analysis software,including R language,Gene Ontology(cell component),KEGG,protein interaction network,EVmiRNA,etc.,screen the genes(miR-22,Twistl,CADM1)and signal pathways(EMT)related to the pathogenesis of OS.2.Three human OS tissue samples and adjacent healthy tissues were obtained,MSC,MG63,and SAOS cell lines were cultured.Exosome were extracted using high-speed centrifugation.The accuracy of bioinformatics analysis results was verified using PCR and western blotting and differential expression of miR-22,Twistl,and CADM1 between tissues and cells was detected.3.Co-culture the supernatant of MSC culture medium with MG63 to explore its effects on the proliferation,migration,and invasion of MG63.Exosome derived from MSC(MSC-exo)were extracted using ultra high speed centrifugation,and GW4869 was added to detect the effects of MSC-exo and MSC-exo+GW4869 on the proliferation,migration,and invasion of MG63.4.MG63 were transfected with miR-22 mimic,miR-22 inhibitor,Twist1 plasmid and RNA interferon by RNA transfection technology(lipo2000).Detect the vitality value of transfected cells(MTS experiment),proliferation ability(CCK-8,plate cloning,EdU),invasion ability(transwell),migration ability(transwell,scratch experiment).At the same time,western blotting was used to detect related proteins in the EMT(Vimentin,N-cadherin,E-cadherin)pathway.PKH-26 detects whether labeled MSC-exo can be uptake by MG63.PCR was used to detect mature miR-22 and precursor miR-22(pri-miR-22,pre-miR-22)in MG63.Detect the effect of MSC-exo transfected with miR-22 mimic and miR-22 inhibitor on the proliferation,migration,and invasion of MG63.In vivo experiments were conducted to verify the effect of MSC-exo on the proliferation of MG63 in mice.Results1.Bioinformatics analysis results showed that miR-22,Twistl,CADM1,and EMT signaling pathways were enriched in gene chip data of patients with OS.2.miR-22 and CADM1 exhibit differential low expression in OS patient tissues and MG63,SAOS cell lines,while they exhibit differential high expression in healthy tissues and MSC cell lines.Twistl exhibits differential high expression in OS patient tissues and MG63,SAOS cell lines,while it exhibits differential low expression in healthy tissues and MSC cell lines.The differential expression of miR-22,Twistl,and CADM1 in the MG63 cell line was significantly higher than that in the SAOS cell line,so the MG63 cell line was selected for subsequent experiments.3.The supernatant of bone MSC culture medium co-cultured with MG63 can inhibit MG63 cell proliferation,migration,and invasion.The extraction of MSC-exo by ultrafast centrifugation can inhibit the proliferation,migration,and invasion of MG63 cells.After adding GW4869 to the supernatant of MSC medium,the inhibitory effect of MSC-exo on MG63 cell proliferation,migration,and invasion was weakened.4.MSC-exo with high expression of miR-22 is more effective than MSC-exo in inhibiting the proliferation,migration,and invasion of MG63 cells,promoting the expression of E-cadherin and inhibiting the expression of Vimentin and N-cadherin.MSC-exo with low expression of miR-22 has a lower inhibitory effect on MG63 cell proliferation,migration,and invasion compared to MSC-exo,inhibiting the expression of E-cadherin and promoting the expression of Vimentin and N-cadherin.The Twistl salvage experiment showed that high expression of Twistl can reverse the effect in MG63 cell proliferation,migration,and invasion caused by high expression of miR-22,while low expression of Twistl can reverse the increase effect in MG63 cell proliferation,migration,and invasion caused by low expression of miR-22.The MSC-exo labeled with PKH-26 detection can be uptake by MG63 cells and increases the expression of mature miR-22 in MG63 cells,while there is no significant change in the expression levels of pri-miR-22 and pre miR-22.The Luciferase reporter gene experiment confirmed that miR-22 has binding sites with Twistl.The chromatin coprecipitation experiment confirmed that Twistl has binding sites with CADM1.In vivo experiments showed that the tumor volume of MG63 cells transfected with miR-22 mimic was significantly reduced in mice,while the tumor volume of MG63 cells infiltrating MSC-exo was even smaller in mice.Conclusion1.The miR-22,Twistl,CADM1,and EMT signaling pathways may play important roles in the occurrence and development of OS in patients.2.The differential expression of miR-22,Twistl,and CADM1 in tissues of OS patients and MG63 cell lines may be involved in the pathological and physiological processes of OS.3.MSC-exo inhibits the proliferation,migration,and invasion of MG63 cells.4.MSC-exo carries a large amount of mature miR-22 that are UP-taken by MG63 cells,and inhibits OS proliferation,migration,and invasion by regulating the EMT signaling pathway through the miR-22/Twistl/CADM1 axis. |
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