The Mechanism Of Cystathionine Β-synthase Promote Growth Of Osteosarcoma Cells By NF-κB Signaling Pathway

Osteosarcoma（OS）,the most common primary bone tumor in children and adolescents,has a high tendency of local invasion and metastasis,which is related to genes,age,sex,height,hormone and other factors.Although the combination of surgery and chemotherapy has greatly improved the prognosis of patients with osteosarcoma,the prognosis of metastatic or recurrent osteosarcoma remains unsatisfactory.Therefore,it is urgent to explore the pathogenesis of osteosarcoma,find effective therapeutic targets,and carry out targeted and precise treatment.In mammals,hydrogen sulfide（H₂S）is mainly produced by cystathionine-β-synthase（CBS）,cystathionineγ-lyase（CSE）and 3-mercatopyruvate sulfurtransferase（3-MST）.H₂S is a’double-edged sword’in the tumor development,which is related to the concentration of H₂S.Therefore,we can explore the effect of H₂S on tumor growth by changing the concentration of H₂S via endogenously regulating the expression of H₂S-producing enzymes.Although the role of H₂S in various tumors has been extensively studied,the role and mechanism of H₂S in human osteosarcoma are still unclear.This paper explores the effect of overexpression/knockdown of the CBS gene on the growth of human osteosarcoma cells and its mechanism.To determine whether H₂S plays a role in human osteosarcoma,the content of H₂S in normal human osteoblast h FOB1.19 and five human osteosarcoma cells（143B,SJSA-1,HOS,MG63,U2OS）was detected by ELISA,which showed that the content of H₂S in human osteosarcoma cells,especially 143B and HOS cells,was higher than that in h FOB1.19.The expression of three H₂S-producing enzymes was detected by q PCR and Western blot from the m RNA and protein levels,respectively.The results showed that CBS’s m RNA and protein levels in five human osteosarcoma cells were higher than those in h FOB1.19.Therefore,143B and HOS cells with the highest expression of CBS were selected as the research objects for subsequent research.To explore the role of CBS in human osteosarcoma cells,human osteosarcoma cell lines with stable overexpression/knockdown of CBS were constructed.The constructed lentiviral vector was used to infect 143B and HOS cells,and the uninfected cells were killed by puromycin.The fluorescence effect of the cells was observed by fluorescence microscope and the expression of CBS was detected by q PCR and Western blot to verify whether the cells were successfully infected.The successfully constructed cells and normal uninfected cells were divided into two groups:the Control group,the Mock group and the CBS group;the Control group,the sh-Scb group,the sh-CBS-1 group,and the sh-CBS-2 group.Compared with the Control group,there was no significant difference in CBS content between the Mock and sh-Scb groups.The CBS content in the CBS group increased,and the CBS content in the sh-CBS-1 group and sh-CBS-2groups decreased.The effect and mechanism of CBS on the growth of human osteosarcoma cells were detected by a series of cell function experiments and molecular experiments in vitro.MTT assay and CCK8 assay showed that overexpression of CBS could promote the survival of human osteosarcoma cells,and knockdown of CBS could inhibit cell survival.Through the Tunel experiment and detection of the expression level of related apoptotic proteins,it was found that CBS overexpression could inhibit cell apoptosis,and knockdown of CBS could promote apoptosis.Western blot was used to detect the expression of pyroptosis protein.The results showed that knockdown of CBS could induce pyroptosis in human osteosarcoma cells,while overexpression of CBS had the opposite effect.Ed U assay,plate cloning assay and soft agar colony formation assay showed that overexpression of CBS could promote the proliferation and colony formation of human osteosarcoma cells.The knockdown of CBS had the opposite effect.Scratch test and Transwell test showed that overexpression of CBS could promote the migration ability of human osteosarcoma cells,and knockdown of CBS could inhibit the migration ability of human osteosarcoma cells.Through Invasion assay and detection of the expression level of EMT-related proteins,it was found that overexpression of CBS could promote the invasion ability of human osteosarcoma cells,and knockdown of CBS exerted an inhibitory effect.The mechanism of action was discussed.It was speculated that mi R-3918 targeting CBS may promote the growth of human osteosarcoma cells through the ROS-mediated NF-κB signaling pathway.A nude mouse osteosarcoma model was established in vivo,and the growth of tumors in each group was monitored in real-time by small living animal imaging technology.After 21 days of feeding,the nude mice were dissected,and the tumor tissues were taken,photographed and weighed.Ki67,CD31,Cleaved caspase 3,Cleaved GSDMD,Vimentin,and p-p65 were detected by immunohistochemical staining.The results showed that overexpression of CBS could promote tumor proliferation,angiogenesis,and invasion,inhibit tumor death,and accelerate tumor growth.In summary,both in vitro and in vivo experiments have shown that mi R-3918 targeting CBS can promote the survival,proliferation,cloning,colony formation,migration and invasion of human osteosarcoma cells through the NF-κB signaling pathway,and inhibit cell death,thereby regulating the growth of human osteosarcoma.