| Introduction:Osteosarcoma(OS),with high propensity for local invasion and metastasis,is the most common primary bone malignancy.Though great improvement is achieved by a combination of surgical therapy and chemotherapy,the total prognosis in patients with metastatic or recurrent OS remains unsatisfactory.Therefore,further exploration of detailed mechanism and confirmation of potential molecular target are of large importance for the diagnosis and treatment of OS.Tumor microenvironment(TME)is comprised of extracelluar matrix and multiple-types of cells,including fibroblasts,immunocyte,endothelial cells,pericyte and so on.TME functions as key regulator in cancer progression.The interaction between tumor cells and other cells in TME facilitated tumor growth and metastasis.As an important component of TME,cancer-associated fibroblasts(CAFs),in ways like paracrine,contribute to tumor progression through multiple tactics such as promotion of tumor cell proliferation,extracellular matrix remodeling,angiogenesis,mediation of tumor-associated inflammatory responses,induction of epithelial-mesenchymal transition,and induction of immunosuppression.Recently,accumulate evidence has shown that CAFs are essential in the development of cancer,with a mechanism related closely to CAF-secreted exosomes.Exosomes,mostly be secreted by eukaryotic cells,are extracellular vesicles that are involved in intercellular communication.Exosomes,with component of proteins,DNA,RNA,etc.,influence tumor progression via its component be taken by tumor cells.CAFs secreted exosomes have been revealed to facilitate proliferation,invasion and metastasis in a variety of tumors.Wingless-type MMTV integration site family,member 2b(Wnt2b)is a member of the Wnt family.Wnt2 b can activate classic Wnt/β-catenin signaling pathway.Wnt signaling has been proved as being vital for embryonic development,tissue integrity,and stem cell activity.A release control of Wnt signaling might lead to numerous diseases like cancer.The proteins of Wnt family members can be carried and be transported by exosomes into target cells,thereby regulate downstream target genes directly or indirectly.As firstly discovered as a genomic tissue protein,Special AT-rich sequence binding protein 1(STAB1)has been well-accepted as an oncogene among malignancies.SATB1 interacts with certain proteins including transcriptional factors through its dimeric structural domain,and is extensively implicated in the development of various cancerous diseases.However,the role of SATB1 playing in OS has not been fully unveiled.In the current study,we aimed to explore whether exosomal-Wnt2 b,secreted by CAFs via a paracrine manner,could be transported to OS cells,thereby led to an activation of Wnt signaling and an elevation of SATB1 in OS cells,and finally promoted OS progression.Methods:Part I:1)Fresh OS tissues and adjacent normal tissues were collected.Cancer associated fibroblasts(CAFs)and normal tissue fibroblasts(NFs)in OS tissue and in adjacent normal tissues were extracted,identified by morphology,cellular immunofluorescence,and western blot assays,respectively.2)An ultracentrifugation method was used to extracted exosomes from CAFs and NFs cells,independently.A transmission electron microscopy,a nanoparticle tracking analysis(NTA),and specific maker identification by western blot assay were applied to verify the extracted exosomes.An exosomes-uptaken assay was traced by an immunofluorescence assay in osteosarcoma cells.3)The CAFs-exosome(CAFs-exo)and NFs-exosome(NFs-exo)were co-cultured with OS cell lines U2 OS and MG63,by using co-culture of equal volume of PBS and U2 OS or MG63 as a control,respectively.The proliferation,migration and invasion ability changes in OS cells were measured by a CCK8 assay,a wound healing assay and a transwell assay.4)OS cells were subcutaneously injected into the axilla of nude mice to construct subcutaneous transplantation tumor models.The constructed mice models were randomly divided into three groups.CAFs-exo,NFs-exo,and equal volume of PBS solution(control group)were timely injected into the constructed mice models.The size and weight of formatted tumors in each group were recorded accordingly.The expression of Ki67 in the formatted tumors was analyzed by an immunohistochemistry assay.5)OS cells were intravenously injected into nude mice to construct the lung-metastasis model.CAFs-exo,NFs-exo and isochoric PBS were intravenously injected into the formatted mice models.The numbers of formatted metastatic nudes were analyzed to determine the effect of CAFs-exo on lung metastasis in vivo.Part II:1)The expression of SATB1 in a human osteoblast cell line h FOB1.19 and in OS cell lines U2 OS and MG63 were measured by a Real-time PCR assay and a western blot assay,respectively.2)Specific small interfering RNA targeted SATB1(si SATB1)and overexpression plasmids loading SATB1(oe SATB1)were constructed,then be transfected into U2 OS and MG63 cells,by using si NC as well as vector as a control.The expression changes of SATB1 in U2 OS and MG63 cells were detected to verify the transfection efficacy by a Real-time PCR assay and a western blot assay.CCK8 assay,wound healing assay and transwell assay were performed to check the proliferation,migration and invasion ability changes.3)CAFs-exo,NFs-exo and PBS were co-cultured with OS cells,and the expression of SATB1 in the co-cultured OS cells was measured by a western blot assay.4)si SATB1 and si NC were transfected into the co-cultured OS cells,and the expression of SATB1 were determined by a western blot assay.CCK8 assay,wound healing assay and transwell assay were performed to check the proliferation,migration and invasion ability changes.Part III:1)An online bioinformatics analysis of OS-related GEO-datasets as well as a correlation analysis in collected clinical samples were used to screen the potential upstream SATB1regulators2)The differential expression of Wnt2 b in CAFs、NFs and in CAFs-exo、NFs-exo was detected by a western blot assay3)The differential expression of Wnt signal proteins Wnt2 b,β-catenin and c-Myc in CAFs-exo/NFs-exo co-cultured OS cells were measured by a western blot assay.4)Specific short hairpin RNA targeted Wnt2 b as well as a negative control(shNC)were transfected into CAFs,and the transfection efficacy was verified by a Real-time PCR assay and a western blot assay.The CAFs-exo was collected,and the expression of Wnt2 b in the collected exosomes was checked by a western blot assay.5)The collected CAFs-exo were co-cultured with U2 OS and MG63,and the expression of Wnt2 b,β-catenin and c-Myc in CAFs-exo/NFs-exo co-cultured OS cells were measured by a western blot assay.6)ICG001,a specific inhibitor of Wnt signal pathway,was added into the co-cultured OS cells,and a negative control was set.The expression of c-Myc and SATB1 in diverse ICG001-intervene OS cells were measured by a western blot assay.7)The isolated exosomes from shNC and sh Wnt2 b CAFs were co-cultured with U2 OS and MG63.A CCK8 assay,a wound healing assay and a transwell assay were performed to verify the proliferation,migration and invasion ability changes of OS cells.8)Clinical OS tissue specimens were collected,and the expression of Wnt2 b and SATB1 in OS tissue specimens were analyzed by an immunohistochemistry assay.The expression of Wnt2 b as well as SATB1 and the clinicopathological features in patients with OS was analyzed.Meanwhile,a Kaplan-Meier survival curve analysis was applied to assess the relationship between the abnormal expression levels of Wnt2 b as well as SATB1 and postoperative survival risk in patients with osteosarcoma.Results:1.The morphology of CAFs and NFs that were extracted from fresh OS and adjacent normal tissues is conformed to the morphological characteristics of fibroblasts as indicated by Inverted microscopy.α-SMA and FAP were high-expressed in CAFs but were low-expressed in NFs,while Vimentin was expressed both in CAFs and in NFs,as illustrated by an immunofluorescence assay and a western blot assay,indicated that CAFs and NFs cells were successfully extracted.Exosomes of CAFs and NFs were collected by a high-speed centrifugation method.The morphology,size and specific markers of the extracted exosomes were verified by transmission electron microscopy,NTA and a western blot assay.The detection findings suggested that the extraction of exosomes from CAFs and NFs cells was successful.Exosomes of CAFs and NFs were taken by OS cells as verified by a exosomes-tracing assay.Co-culture of CAFs-exo and OS cells facilitated proliferation,migration and invasion as demonstrated by a CCK8 assay,a wound healing assay and a transwell assay independently.The findings of in vivo subcutaneous transplantation tumor models and lung metastasis models indicated that CAFs-exo promoted OS tumor growth and lung metastasis.2.The outcomes of Real-time PCR and western blot suggested that the expression of SATB1 in U2 OS and MG63 was significantly higher than that in h FOB1.19.Downregulation of SATB1 decreased proliferation,migration and invasion in OS cells,and upregulation of SATB1 promoted proliferation,migration and invasion in OS cells.As illustrated by a western blot assay,the expression of SATB1 in CAFs-exo co-cultured OS cells was higher than that in NFs-exo co-cultured OS cells as well as in the control OS cells.Transfection of si SATB1 in the CAFs-exo co-cultured OS cells suppressed the CAFs-exo-mediated SATB1 expression,decreased the proliferation,migration and invasion ability of OS cells.3.As demonstrated by the bioinformatics analysis of GEO datasets and our clinically detection,the expression of Wnt2 b,a Wnt signaling pathway ligand,was positively correlated with SATB1.The expression of Wnt2 b in CAFs and CAFs-exo was remarkably higher than that in NFs and NFs-exo as indicated by a western blot assay.As displayed by a further western blot assay,the expression of Wnt2 b in CAFs-exo co-cultured OS cells was higher than that in control OS cells.No difference was found between NFs-exo co-cultured OS cells and control OS cells.It was also found thatβ-catenin and c-Myc were also elevated in CAFs-exo co-cultured OS cells but not in NFs-exo co-cultured OS cells as well as in control OS cells.Transfection of sh Wnt2 b decreased the expression of Wnt2 b in CAFs and in CAFs-exo.The CAFs-exo in sh Wnt2b-CAFs was co-cultured with OS cells,and the expression of Wnt2 b,β-catenin and c-Myc were obviously downregulated.And the phenomenon strongly suggested that CAFs-exo activated Wnt signal via transduction of Wnt2 b.ICG001 suppressed the expression of SATB1 in CAFs-exo co-cultured OS cells as determined by a western blot assay.All the findings suggested that CAFs-exo containing Wnt2 b could be taken by OS cells thereby activated Wnt signal pathway and facilitated the upregulation of SATB1 in OS cells.Downregulation of Wnt2 b in CAFs-exo decreased the proliferation,migration and invasion ability of CAFs-exo co-cultured OS cells.It was uncovered by an IHC assay that in OS tissues,the expression level of Wnt2 b and SATB1 was correlated with tumor staging and distant metastasis.High-expression of Wnt2 b and SATB1 was a risk factor of long-term survival in patients with osteosarcoma as determined by a Kaplan-Meier survival analysis.Conclusion:1.CAFs-exo could promote the proliferation,migration and invasion in OS cells2.Co-culture of CAFs-exo and OS cells promoted the upregulation of SATB1 in OS cells.Downregulation of SATB1 partially suppressed CAFs-mediated proliferation,migration and invasion in OS cells.3.The CAFs-exo containing Wnt2 b was taken by OS cells,thereby leading to an activation of Wnt signal pathway,promoted the upregulation of SATB1,and finally promoted the progression of OS. |
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