Molecular Regulation Of Osteosarcomaderived Small Extracellular Vesicles On The Differentiation And Function Of Osteoclast In Tumor Microenvironment

Osteosarcoma(OS)is the most common primary bone cancer characterized by highly aggressive,progressive bone destruction,and high incidence of distant metastasis.OS most often occur in children and adolescents,with about 5%of osteosarcomas occurring in the jaw.Previous studies on OS mainly focused on tumor cells,while ignoring the effect of non-tumor cells such as osteoclasts in the microenvironment on tumor progression.Recent studies have shown that the number of osteoclasts is significantly higher in osteolytic destruction of OS than normal bone tissue,and inhibition of osteoclastogenesis can significantly reduce the bone destruction caused by OS.However,mechanisms governing osteoclastogenesis in OS are still unclear.Therefore,it is of great significance to explore the mechanism of osteoclast formation in OS microenvironment for the development of new drugs targeting osteoclasts in the tumor microenvironment.Small extracellular vesicles(s EVs)could mediate cellular communications by delivering bioactive molecules to recipient cells,participating in cellular information exchange and regulating the status and function of recipient cells.Studies have shown that s EVs are important molecular mediators that mediate the reprogramming of mesenchymal cells in the microenvironment of malignant tumors and thus promote tumor progression.At present,there are few studies on the regulation of s EVs on osteoclasts,and the mechanism is not clear.The aim of this study was to investigate the effect of OS cell-derived s EVs on osteoclast formation and function,the mechanism underlying the regulation of osteoclast formation and function by mi RNA delivered by OS cells's EVs,and the inhibitory effect of sulforaphane(SFN)on the level of mi RNA in OS cell-derived s EVs and osteoclast formation.In this paper,we will discuss from the following three parts.Part 1.Study on the role of Osteosarcoma-derived s EVs in osteoclastogenesisObjective:To investigate the role of s EVs secreted by OS cells in osteoclastogenesis and bone destruction.Methods:SEVs were extracted from OS cells by differential ultracentrifugation and identified by transmission electron microscopy(TEM),dynamic light scattering(DLS)and Western Blot.RAW264.7 cells were stimulated by s EVs,and the uptake of s EVs by RAW264.7 cells was observed by confocal microscopy.The effects of s EVs on the proliferation and migration of RAW264.7 cells were detected by CCK-8 and Transwell assay.For the osteoclastogenesis assay,RAW264.7 cells were induced by nuclear factor kappa B receptor activating factor ligand(RANKL)in vitro.Osteoclastogenesis and bone resorption were detected by TRAP staining,TRITC-phalloidin staining,and resorption pit assay.QRT-PCR and Western Blot were used to detect the expression of cathepsin K(CTSK)and matrix metalloproteinase-9(MMP9).Results:The results of TEM,DLS and Western Blot showed that the extracted s EVs were consistent with the characteristics of small extracellular vesicles.Confocal microscopy results showed that s EVs were absorbed by RAW264.7 cells.CCK-8 and Transwell assay showed that s EVs promoted the proliferation and migration of RAW264.7 cells.TRAP staining,TRITC-phalloidin staining,and resorption pit assay revealed that osteoclast formation and function were enhanced with the treatment of s EVs.QRT-PCR and Western Blot results showed that the treatment of s EVs increased the expression of CTSK and MMP9.Conclusion:s EVs secreted by OS cells could promote the formation and function of osteoclast.Part 2.Osteosarcoma-derived s EVs promote osteoclast differentiation and bone destruction through the delivery of mi R-19a-3pObjective:To investigate the mechanism of s EVs derived from OS cells promoting osteoclastogenesis and bone destruction.Methods:Bioinformatics analysis was applied to predict the related genes and targeted mi RNAs involved in osteoclastogenesis in OS.The dual luciferase reporter gene assay was used to verify the target binding of mi R-19a-3p with PTEN.RAW264.7 cells were transfected with mi RNA Oligo(mi R-19a-3p mimics-NC,mimics,inhibitor-NC,and inhibitor),and then induced with RANKL.Osteoclast formation and function were detected by TRAP staining,TRITC-phalloidin staining,and resorption pit assay.The expressions of CTSK,MMP9 and PTEN were detected by q RT-PCR and Western Blot.The activation of Akt signaling pathway was detected by Western Blot.After transfecting OS cells with mi RNA oligo,s EVs(s EVs ^oligo)was extracted.RAW264.7 cells were treated with s EVs ^oligoand induced with RANKL,and then osteoclast formation,function and expression of related molecules were detected.Results were verified by bone marrow macrophages(BMMs).Mice were subcutaneously injected with K7M2 cells to establish OS model.The levels of mi R-19a-3p in s EVs were detected in the blood of mice.The bone parameters of femur were analyzed by micro-CT scanning,hematoxylin-eosin(HE)staining.TRAP staining detected the distribution of osteoclasts in femur.Results:Bioinformatics analysis showed that mi R-19a-3p and PTEN may be involved in the enhancement of osteoclast formation and function in OS.Dual luciferase reporter gene assay revealed the target binding of mi R-19a-3p with PTEN.RAW264.7 cells were transfected with mi RNA Oligo and induced with RANKL.Results showed that the osteoclast formation,function,the expression of PTEN and phosphorylated Akt(p-Akt)were increased in mi R-19a-3p mimics group,while the results were opposite in mi R-19a-3p inhibitor group.In addition,RAW264.7 cells were stimulated by s EVs ^oligoand induced with RANKL.The results showed that the number of osteoclasts was increased,the bone resorption function was enhanced,the expression of PTEN was decreased and the expression of p-Akt was increased in the s EVs ^mimicsgroup,while the s EVs ^inhibitor group showed the opposite results.The results were verified by BMMS in vitro.The level of mi R-19a-3p in s EVs derived from OS mice blood was significantly higher than normal mice.Micro-CT and HE staining results displayed the osteopenia in femur of OS mice.TRAP staining results showed that the number of osteoclasts in OS mice was reduced.Conclusion:Mi R-19a-3p delivery via OS cell-derived s EVs promotes osteoclast formation and bone destruction through PTEN degradation and further activating PI3K/AKT signaling pathway.Part 3.SEVs are partly involved in regulation of SFN on osteoclast formation in OSObjective:To investigate the effect of SFN on osteoclastogenesis in OS and related mechanism.Methods:OS cells were stimulated by SFN and s EVs were extracted.RAW264.7 cells were treated with SFN-s EVs and induced with RANKL,and then osteoclast formation,function and expression of related molecules were detected.The effect of SFN on RAW264.7 cells and BMMS proliferation were measured by CCK8.For osteoclastogenesis,RAW264.7 cells and BMMs were induced with RANKL.The number and size of osteoclast were detected by TRAP staining and TRITC-phalloidin staining.The autophagosomes were observed by TEM.The pre-osteoclast cells were exposed to various concentrations of SFN at different time points to explore the effective concentration and the stage at which SFN mainly affected osteoclastogenesis.After the autophagy pathway was activated with rapamycin(RAP),the effect of SFN on osteoclast formation was detected.QRT-PCR and Western blot were conducted to detect the expressions of CTSK,MMP9,autophagy-related molecules Atg5-Atg12,Beclin1 and light chain 3(LC3).The JNK signaling was detected by Western blot.RAW264.7 cells were pretreated with anisomycin,the JNK signaling activator,and SP600125,the JNK signaling inhibitor,and then induced with RANKL.The expressions of Atg5-Atg12,Beclin1 and LC3 were detected by Western Blot.The bone destruction model was established by subcutaneous injection of LPS in the middle of the calvaria of mice,and SFN was intraperitoneally injected.Micro-CT and HE staining were used to detect the bone tissue morphology and the bone parameters of mice calvaria.Trap staining was used to detect the distribution of osteoclasts in mice calvaria.The expression and distribution of CTSK and LC3 in bone were detected by immunohistochemistry(IHC)and immunofluorescence(IF)staining.Results:QRT-PCR showed the decrease of mi R-19a-3p in SFN-s EVs.RAW264.7 cells were stimulated by SFN-s EVs and induced with RANKL,and the results showed that the number of osteoclasts was decreased significantly.SFN inhibited osteoclast formation in a dose-dependent manner.Combination with the results of osteoclastogenesis and CCK8 assay,SFN at 2.5?M effectively inhibited the formation of osteoclasts with little effect on the proliferation of pre-osteoclasts.Besides,SFN significantly inhibited osteoclast formation at the early period.The activation of autophagy by RAP almost reversed the SFN-elicited anti-osteoclastogenesis.The results of q RT-PCR and Western blot showed that SFN inhibited the expressions of CTSK,MMP9,Atg5-Atg12,Beclin1 and LC3,while RAP treatment decreased this inhibition effect.Furthermore,Western blot analysis revealed that SFN inhibited phosphorylated JNK(p-JNK)expression in a dose-dependent manner.The JNK activator anisomycin significantly promoted autophagy,whereas the inhibitor SP600125markedly suppressed autophagic activation in pre-osteoclasts.Micro-CT and HE staining results showed that the calvaria destruction was significant after subcutaneous injection of LPS,and SFN could protect against LPS-induced calvarial erosion.Trap staining results showed that SFN attenuated the effect of LPS-induced osteoclast increase.The results of IHC and IF staining showed that the expressions of CTSK and LC3 were increased in mice calvaria in the LPS group,while they were decreased after intraperitoneal injection of SFN.Conclusion:SFN indirectly inhibits osteoclastogenesis by decreasing the level of s EVs'mi R-19a-3p.Besides,SFN directly inhibits osteoclastogenesis through the suppression of the autophagic pathway and the JNK signaling participates in this process.Above all,our study shows that:1.SEVs secreted by OS cells could promote the formation and function of osteoclasts.2.Mi R-19a-3p delivery via OS cell-derived s EVs promotes osteoclast formation and bone destruction through phosphatase and tensin homolog(PTEN)degradation and further activating phosphatidylinositol 3-kinase(PI3K)/proteinkinaseB(AKT)signalingpathway.Inaddition,in vivoexperimentsshowedthe increased level of s EVs'mi R-19a-3p in blood of OS mice,and OS mice exhibitedosteopenia with increased number of osteoclasts.Herein,we demonstrated that blood s EVs'mi R-19a-3p derived from OS could be considered as a crucial factor in osteoclastogenesisand bone destruction,and we clarified the associated mechanism in vitro study.3.S FNcould inhibit osteoclastogenesis in OS by decreasing the level of s EVs'mi R-19a-3p derivedfrom OS cells and directly suppressing autophagic pathway of pre-osteoclast.Therefore,webelieve that OS cells's EVs are involved in regulation of osteoclastogenesis,and mi R-19a-3p in s EVs may become a new target for the prevention and treatment of OS bonedestruction and tumor progression.