| Background and objective: Osteosarcoma?OS?is a highly malignant tumor,and 10%-20% patients have developed metastases at the time of diagnosis.OS metastasis is a complicated pathological process,which has involved multiple signal transduction pathways;therefore,the high metastasis and high invasion related mechanisms of OS have long been the concerned topics.In recent years,people attempt to illustrate the mechanism of malignant tumor metastasis through exploring the roles of epithelial mesenchymal transition?EMT?and the related proteins.Typically,EMT is referred to as the biological process in which the epithelial cells are transformed into cells with mesenchymal phenotype through a specific program.EMT can result in loss of cell-cell connection and the acquisition of a migratory phenotype,thus inducing basilar membrane invasion and finally leading to metastasis.OS is the most common bone malignant tumor of mesenchymal origin.It is suggested in previous studies that,EMT only takes place during the epithelium-derived malignant tumor cell metastasis process;however,in-depth experimental results verify that the mesenchymal-derived sarcoma cells or stem cells can also change the EMT-related protein expression and enhance metastasis.Research discovers that,the tumor tissues of metastatic OS patients are associated with relatively high N-cadherin and Snail protein expression,while relatively low E-cadherin protein expression [150].Meanwhile,in multiple OS cell lines,cells can regulate the EMT-related protein expression to enhance the cell invasion capacity [140][145],which suggests that EMT is not only the key step in epithelium-derived tumor metastasis.Besides,it is suggested that there may also be EMT or EMT-related proteins expression changes in the mesenchymal cell-derived OS,which may thereby participate in the formation mechanism of OS metastasis.Currently,it is indicated that the nature of EMT is highly possible to be a biological phenomenon in which tumor cells acquire the metastatic capacity through signal transduction [157].These findings reveal that,there may be deeper transcriptional and epigenetic variations during the process of EMT-related proteins expression changes;as a result,the roles of EMT-related proteins in OS cell metastasis should be further explored.Existing studies suggest that,some low-molecular-weight?LMW?multiple chemokines and related receptors,which can attract the white blood cells to migrate to the infection site,have participated in the invasion and metastasis of tumor cells,such as breast cancer,gastric cancer,colorectal cancer,pancreatic ductal carcinoma and liver cancer [158-159].There is a large amount of macrophages in the tumor microenvironment?TME?of OS,which can induce CXCL6 expression through secreting TNF-?,TNF-?,IL-l? and IL-1? [154].In the meantime,Li [120] et al.discovered in their research that CXCL6 was highly expressed in the serum of OS patients;besides,in vitro experiment proved that,the addition of recombinant human CXCL6 could remarkably promote the proliferation of OS cells.The CXCL6 receptors are CXCR1 and CXCR2,the binding capacity of CXCL6 with CXCR2 is superior to that with CXCR1,and it is the only potent inducer of CXCR2 internalization [155].Apart from participating in tumor angiogenesis,CXCR2 is closely related to the EMT process of multiple tumor cells [121-123].Among the multiple signaling pathways in which CXCR2 promoting EMT,the PI3K/Akt signaling pathway is the most extensively studied.On the other hand,?-catenin is the core of the Wnt/?-catenin signaling pathway,which is discovered in research to form the complex with E-cadherin.The ?-catenin-E-cadherin complex can regulate EMT,thus playing a vital role in processes such as tumor cell proliferation,invasion and migration.At present,the PI3K/Akt signaling pathway is suggested to suppress the GSK-3? activity to promote the nuclear transport of ?-catenin;in other words,the PI3K/Akt signaling pathway can promote the activation of the Wnt/?-catenin signaling pathway [147,148].Such findings reveal that,investigating the interaction and regulatory mechanisms of the PI3K/Akt signaling pathway with other signaling pathways during tumor cell metastasis process may provide new clues for illustrating the mechanism by which chemokine regulates OS cell metastasis.To sum up,there is a large amount of macrophages in the OS TME,which can promote tumor cells to secrete CXCL6;besides,CXCL6 is highly expressed in the serum of OS patients,which can specifically and efficiently bind with CXCR2 with EMT-inducing effect.Thus,it can be speculated that,chemokine CXCL6 and its receptor CXCR2 can regulate the EMT-related protein expression during OS genesis and development,which can improve the migration and invasion capacities of OS cells,thus promoting OS metastasis.On this basis,this project had selected the chemokine CXCL6 and its receptor CXCR2 as the objects of study to explore their roles in OS cell migration and invasion,as well as the effects of EMT-related proteins.Besides,the roles of the PI3K/Akt and ?-catenin signaling pathways in the CXCL6-mediated metastasis were also investigated,with an aim to provide related theoretical and practical foundation for the early diagnosis and treatment of OS recurrence and metastasis. This experiment had determined the correlations of expression of chemokine CXCL6 and its receptor CXCR2 with the OS metastasis capacity using 4 kinds of OS cell lines with various metastasis capacities.Besides,the CXCL6 neutralizing antibody was used to observe the effects on the metastasis capacities of cell lines with strong metastasis capacities?MG63 and 143B?,as well as on the EMT-related proteins,so as to determine the potential relationship of the chemokine CXCL6 and its receptor CXCR2 with the OS EMT-related proteins.Meanwhile,the recombinant human CXCL6 was employed to verify whether it could affect the migration and invasion of Sa OS-2 and U2 OS cell lines with relatively weak metastasis capacities,as well as the expression of EMT-related proteins.Moreover,the influence of chemokine CXCL6 and its receptor CXCR2 on the growth and lung metastasis of OS in nude mice was also further verified in experiment in vivo.Finally,the PI3K/Akt and ?-catenin signaling pathway blockers and CXCR2 silencing were utilized to explore the mechanisms by which chemokine CXCL6 and its receptor CXCR2 induce metastasis in OS patients.Methods: 1.The R language software was used for statistical analysis on the OS data in The Cancer Genome Atlas?TCGA?,and the Fisher.test function was used for Fisher exact test,so as to analyze the correlations of CXCL6 and CXCR2 m RNA expression with metastasis in OS patients.2.CXCL6 and CXCR2 expression in 4 kinds of OS cell lines?MG63,143 B,Sa OS-2 and U2OS?were detected through RT-PCR and Western blotting,and the relationships of the migration and invasion capacities of the above OS cell lines with CXCL6 expression were detected through Transwell assay.3.The cell survival was detected in the recombinant human CXCL6-treated OS cells?MG63,143 B,Sa OS-2 and U2OS?through CCK-8 assay.4.MG63 and 143 B cells were treated with the CXCL6 neutralizing antibody;cell migration and invasion were detected through Transwell assay;the protein expression of E-cadherin,N-cadherin and Snail was examined through Western blotting;and the protein expression and distribution of E-cadherin and N-cadherin were determined by immunofluorescence assay.5.CXCR2 in Sa OS-2 and U2 OS cells was silenced through si RNA technique,cells were then treated with recombinant human CXCL6,cell migration and invasion were detected by Transwell assay,and the protein expression of E-cadherin and N-cadherin in cells were determined by immunofluorescence assay.Meanwhile,the protein expression of MMP-9,Snail,phospho-Akt Ser473,Akt and nuclear ?-catenin in cells was detected by Western blotting.Besides,the PI3K/Akt and ?-catenin signaling pathways were blocked with LY294002 and XAV939,respectively;cells were treated with recombinant human CXCL6;and cell migration,invasion and the expression of EMT-related proteins were examined using the Transwell assay and Western blotting.6.To verify the role of CXCL6 in promoting OS metastasis,the U2 OS cells with low CXCL6 expression and metastasis capacity were selected and infected with lentivirus;U2OS cells with high CXCL6 expression were constructed,screened,and used to establish the subcutaneous tumor-bearing nude mice model and caudal vein lung metastasis model.Subsequently,the CXCR2 antagonist SB225002 was injected intraperitoneally into the nude mice,and the effects of chemokine CXCL6 and its receptor CXCR2 on regulating tumor growth and proliferation were examined using the tumor growth curve,tumor mass,immunohistochemical method and Western blotting.In addition,lung metastasis was observed through lung tissue HE staining,so as to examine the regulatory effects of chemokine CXCL6 and its receptor CXCR2 on tumor metastasis.Results: 1.A total of 266 OS cases with clinical information as well as CXCL6 and CXCR2 m RNA expression information were extracted from TCGA?TCGA data version 20160128 for SARC?,among which,175 cases had metastasis information.Results of Fisher exact test suggested that,the m RNA expression level of CXCL6 was positively correlated with that of its receptor CXCR2?P=0.0006?;meanwhile,CXCL6 m RNA expression was markedly correlated with metastasis in patients?P=0.009645?.2.High CXCL6 and CXCR2 expression at both gene and protein levels could be discovered in the 4 kinds of OS cell lines,and the higher CXCL6 expression was indicative of stronger cell migration and invasion capacities.3.CCK-8 detection results indicated that,the MG63,143 B,Sa OS-2 and U2 OS cells treated with recombinant human CXCL6?100 ng/ml?for 48 h had apparently enhanced proliferation capacity compared with the blank control group?P?0.05?.4.CXCL6 neutralizing antibody could reduce the migration and invasion capacities of OS MG163 and 143 B cells,markedly down-regulate the expression of EMT-related proteins?Snail and N-cadherin?in cells,while evidently up-regulate that of E-cadherin.5.After silencing CXCR2 gene,the effects of the recombinant human CXCL6 on promoting the migration and invasion of Sa OS-2 and U2 OS cells were remarkably reduced;at the same time,the effects of recombinant human CXCL6 on the relative expression quantities of EMT-related proteins?E-cadherin and N-cadherin?as well as the signaling pathway-related proteins?p-Akt/Akt and nuclear ?-catenin?were also markedly decreased.After adding the signaling pathway blockers LY294002 and XAV939,the expression of MMP-9,Snail and N-cadherin in Sa OS-2 and U2 OS cells was notably down-regulated,while that of E-cadherin was evidently increased.6.Animal experimental results indicated that,subcutaneous injection of U2 OS cells with high CXCL6 expression could promote tumor growth,as well as the expression of proliferation cell nuclear antigen?PCNA?,p-Akt/Akt and nuclear ?-catenin in the tumor body.However,the relative expression quantities of cell proliferation proteins PCNA,p-Akt/Akt and nuclear ?-catenin were reduced after injection of CXCR2 antagonist.In the lung metastasis model,CXCL6 could remarkably promote the lung metastasis of tumor cells,while the lung metastases were evidently reduced after injection of CXCR2 antagonist.High CXCL6 expression could increase the tumor formation rate and lung metastasis rate,in the meantime of increasing the formation of lung tissue metastases.After giving CXCR2 antagonist,the lung metastasis rate was not obviously changed,but pathological experimental results suggested that the formation of lung tissue metastases was markedly reduced.Conclusions: 1.It was speculated based on TCGA data analysis,as well as the migration and invasion assays of 4 OS cells with differential CXCL6 expression that,expression of the OS chemokine CXCL6 might be related to the metastasis capacity.2.Applying the CXCL6 neutralizing antibody or silencing the chemokine receptor CXCR2 could suppress the OS cell chemokine CXCL6-mediated metastasis capacity,and change the expression of EMT-related proteins.These findings suggested that,the binding of chemokine CXCL6 with its receptor CXCR2 could regulate the expression of EMT-related proteins,thus inducing metastasis of OS cells.3.The PI3K/Akt and ?-catenin signaling pathway blockers could block the chemokine CXCL6-induced expression of EMT-related proteins,revealing that chemokine CXCL6 and its receptor CXCR2 might induce changes in EMT-related proteins expression through regulating the PI3K/Akt and ?-catenin signaling pathways,thus participating in the mechanism of OS metastasis.4.Experiment in vivo also verified that,chemokine CXCL6 and its receptor CXCR2 could affect the subcutaneous growth and lung metastasis of OS in nude mice,which further supported that,chemokine CXCL6 and its receptor CXCR2 were involved in OS metastasis through regulating the PI3K/Akt and ?-catenin signaling pathways. |
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