Expression Of Siglec-15 In Osteosarcoma And Its Effect On The Occurrence And Development Of Osteosarcoma

Purpose:Our study aimed to investigate the expression of Sialic acid-bound immunoglobulin lectin 15(Siglec-15)in osteosarcoma cells and the effect of Siglec-15 on proliferation,apoptosis and pyroptosis of osteosarcoma cells and its possible mechanism,and the relationship among Siglec-15,autophagy,invasion and migration in osteosarcoma cells,and the possible mechanism of Siglec-15 affecting the invasion and migration of osteosarcoma cells.Methods:The pathological tissues of 52 patients with pathologically confirmed osteosarcoma in the Affiliated Hospital of Qingdao University were obtained to make pathological sections,and the expression of Siglec-15 was detected.The correlation between the expression of Siglec-15 and the total survival(OS)time and clinicopathological parameters of 52 patients with osteosarcoma was analyzed.The expression of Siglec-15 in human osteosarcoma cell lines was detected by q RT-PCR and Western blotting.In vitro,Cell Counting Kit-8(CCK-8)assay,Colony formation experiment,EdU proliferation kit,TUNEL apoptosis kit and Western blotting were used to analyze the proliferation,apoptosis and pyroptosis of osteosarcoma cells.In vivo,the tumor size was compared between the siNC group and the siSiglec-15 group.Immunohistochemical staining(IHC),EdU proliferation kit,TUNEL apoptosis kit and Western blotting analyze the effects of Siglec-15 knockdown on the proliferation,apoptosis and pyroptosis of osteosarcoma cells.In addition,the underlying signal pathways of Siglec-15 involved in osteosarcoma cell proliferation,apoptosis and pyroptosis were detected by bioinformatics analysis,and the correlation was verified.IHC was used to analyze the expression of Siglec-15 and Beclin-1 in primary focus and pulmonary metastatic of osteosarcoma.χ~2test was used to analyze the correlation between Siglec-15,Beclin-1 and clinicopathological parameters of osteosarcoma patients.Short hairpin RNA(shRNA)constructed lentivirus and siRNA was transfected into KHOS and U2OS osteosarcoma cells.Western blotting was used to detect the changes of EMT markers and autophagy-related markers after down-regulation of Siglec-15 and Beclin-1 and overexpression of Beclin-1.Transwell assay,Phalloidin staining and Wound healing assay were used to detect the changes of cell invasion and migration ability and cytoskeleton rearrangement in shSiglec-15 group and siBeclin-1 group.IHC and transmission electron microscopy(TEM)were used to analyze the effect of down-regulation of Siglec-15 on autophagy of osteosarcoma cells.The effects of overexpression of Beclin-1 on autophagy,invasion and migration of osteosarcoma cells were analyzed by Transwell assay,Phalloidin staining and immunofluorescence(IF)assay.KHOS cells of siNC group and shSiglec-15 group were injected into tail vein of nude mice,and the differences of lung metastases were compared.Hematoxylin-eosin staining(HE)was used to compare the difference of pulmonary metastatic nodules between the two groups.IHC was used to compare the expression of EMT and autophagy-related proteins between the two groups.SPSS 26 and Graph Pad Prism 8 software were used for statistical analysis.The correlation between Siglec-15 and OS was analyzed by Kaplan-Meier,and the relationship between Siglec-15 and clinicopathological parameters was analyzed byχ~2test.ANOVA and Bonferroni multivariate comparative analysis were used.t-test was used to compare the two groups.P<0.05 was considered that the difference was statistically significant.Results:The expression of Siglec-15 was positive in 16 of 52 osteosarcoma tissue sections.The expression of Siglec-15 was negatively correlated with the OS of osteosarcoma patients,and had statistical difference with the Enneking stage.The results of Western blotting showed that in Siglec-15 knockdown group,the expression of Cyclin D1,Bcl-2 and p-STAT3 decreased,while the expression of cleaved caspase-3 and GSDME-N increased.Down-regulation of Siglec-15 significantly inhibited the proliferation of osteosarcoma cells,and promoted the apoptosis of osteosarcoma cells.In addition,on the basis of down-regulation of Siglec-15,down-regulated STAT3,cleaved caspase-3 and GSDME-N expression was further increased,while the expression of Bcl-2and Cyclin D1 was further decreased.Moreover,over-expression of Bcl-2 significantly reversed the expression of cleaved caspase-3 and GSDME-N after down-regulation of Siglec-15.In vivo,down-regulation of Siglec-15 expression could inhibit the growth of tumor in mice.In siSiglec-15 group,the expression of Cyclin D1,Bcl-2 and p-STAT3decreased,while the expression of cleaved caspase-3 and GSDME-N increased.Siglec-15and Beclin-1 are relatively highly expressed both in lung metastases and the patients who presented with pulmonary metastasis of osteosarcoma,and there was a correlation between Siglec-15 and Beclin-1 in pulmonary metastasis.Down-regulation of Siglec-15resulted in decreased expression of N-cadherin,Vimentin,MMP-9,increased expression of E-cadherin and LC3II,decreased expression of Beclin-1 and ATG14,and increased expression of p62.Besides,the number and ability of invasion and migration of KHOS and U2OS cells was decreased,and the cytoskeleton rearrangement was inhibited;autophagosomes increased and LC3 expression increased.Down-regulation of Beclin-1resulted in decreased the expression of N-cadherin,Vimentin and MMP-9,increased expression of E-cadherin,decreased the number and ability of invasion and migration of KHOS and U2OS cells,and reduced cytoskeleton rearrangement.After overexpression of Beclin-1,the expression of Siglec-15 had no significant change,while the expression of N-cadherin,Vimentin and MMP-9 increased,while the expression of E-cadherin decreased.Co-immunoprecipitation(CO-IP)of Siglec-15 in Beclin-1 and KHOS cells was achieved.Overexpression of Beclin-1 could rescue the inhibitory effect of Siglec-15knockdown on autophagy,invasion and migration of KHOS osteosarcoma cells.Down-regulation of Siglec-15 could inhibit lung metastasis of osteosarcoma in nude mice.Conclusion:Siglec-15 expressed in osteosarcoma.Down-regulation of Siglec-15expression can significantly inhibit the proliferation of osteosarcoma cells and promote the apoptosis and pyroptosis of osteosarcoma cells.Down-regulation of STAT3 can further inhibit the proliferation of osteosarcoma cells after down-regulation of Siglec-15and promote apoptosis and pyroptosis of osteosarcoma cells.Overexpression of Bcl-2 can reverse apoptosis and pyroptosis of osteosarcoma cells after Siglec-15 knockdown.Down-regulation of Siglec-15 inhibited osteosarcoma cell proliferation and promotes apoptosis and pyroptosis by targeting the Siglec-15/STAT3/Bcl-2 pathway.Siglec-15 is related to lung metastasis of osteosarcoma and can promote lung metastasis of osteosarcoma.Siglec-15-induced autophagy of osteosarcoma cells and promote invasion and migration of osteosarcoma via EMT and targeting the Siglec-15/Beclin-1/ATG14pathway,and provide new ideas for the treatment of osteosarcoma.