| Osteosarcoma is a malignant tumor characterized by malignant mesenchymal cells and is characterized by producing bone or bone-like tissue cells.It is the most common primary malignant bone tumor in adolescents and children,and the incidence rate is 4-5/100 million.The disease progresses rapidly,is highly malignant,and is prone to distant metastases.Although the 5-year survival rate of patients undergoing radical surgery and neoadjuvant chemotherapy is 50%-70%,the 5-yeax survival rate of osteosarcoma patients has not increased significantly in the past 30 years and due to its rapid onset,80%-90%of patients have systemic eicrolesion metastasis at the time of diagnosis'The 5-year survival rate of patients who have metastasis is only 20%-30%,mainly due to the occurrence of lung metastasis and drug resistance.Because the mechanism of tihe occurrence and metastasis of osteosarcoma is not yet clear,it is of great significance and research value to explore the mechanism of the occurrence and metastasis of osteosarcoma,to improve the treatment effect and improve the survival rate of patients.Bone marrow stroma antigen 2(STAG2)is a subunit of the cohesin,which is composed of chromosomal structural protein l(Smcl),chromosomal structural protein 3(Smc3),adhesive protein complex RAD21,STAGI,or STAG2.It is composed of four core subunits and participates in the post-mitotic chromosome separation and the formation of sister chromatids to ensure accurate chromosome segregation and DNA repair.At the same time,the adhesion protein complex can also regulate gene expression through transcriptional regulatory factors and DNA looping.It has been shown that down-regulation of STAG2 expression may lead to the down-regulation of the expression of other three core subunits,which results in a decrease or deletion of the function of the adhesion protein complex,which in turn leads to abnormal chromosome numbers;and in many tumors there is sister chromatid condensation and The chromatid,which is caused by the maintenance of the disorder,separates prematurely and forms an aneuploidy.Aneuploidy of cells can easily lead to increased expression of some proto-oncogenes and decreased or absent expression of anti-oncogenes.It is speculated that STAG2 may act as a "caretaker"in vivo and is a tumor suppressor gene,while STAG2 gene Inactivation of endogenous mutations can lead to chromosomal instability,resulting in aneuploidy.In recent years,many documents and databases have shown that STAG2 has high-frequency mutations in osteosarcoma,bladder cancer,and hematological diseases.Therefore,the current research focuses on revealing the important role of STAG2 in the loss of mutations in osteosarcoma cells.Tumor immunotherapy is an important method of tumor treatment after surgery,radiotherapy,and chemotherapy in recent years and has been widely used in clinics.Cytotoxic T lymphocyte(CTL)can have effective and specific direct inhibition.Kill tumor cells and avoid the side effects of other treatments.Programmed death receptor(PD-1)is an imnnunosuppressive receptor expressed by CTL.Its structure contains an immunoreceptor tyrosine-dependent inhibitory motif that functions as an antagonist of antigen receptors.The Programmed Death ligand(PD-L1)is one of the ligands of PD-1,also known as CD274(Cluster of differentiation 274)or B7-H1(B7 homolog 1),and is a member of the B7 immunoglobulin family.When PD-L1 interacts with PD-1,it can activate the intracellular CTL inhibitory signal,causing apoptosis of tumor antigen T cells and triggering immunosuppressive effects.For cancer patients,the negative imnunoregulatory effects of this signaling pathway can weaken or even kill CTL cells,allowing tumor cells to escape from offline immune surveillance and mediating tumor cell immune escape.PD-L1 is widely expressed on the surface of hematopoietic and nonhematopoietic tumor cell membranes,and is significantly up-regulated in many cancer tissues compared to normal tissues.Until now,the immunoregulatory mechanisms of PD-1 protein and PD·L1 protein exPression in cells have not been fully understood.To sunm up,the expression of PD-1/PD?L1 protein is mainly affected by protein signal pathways,including P13K/Akt signaling pathway,JAK/STAT signaling pathway MEK/Erk signaling pathway,NPM/ALK pathway,and MAPK pathway,etc.STAT3 is related to IRF-1.Shen et al.found that PD-1 and PD-L1 were up-regulated in lung metastasis of osteosarcoma,while the expression of PD-1 and PD-L1 in the cartilage tissue of normal mice was lower in the non-metastatic Saos-2 tumors.The extremely low amount indicates that the expression of PD-1/PD?L1 1s closely related to the metastasis of osteosarcoma.However,the impact of biological functions such as STAG2 and osteosarcoma metastasis and tumor immune escape is not clear.Therefore,we intend to study U2-OS of osteosarcoma cell line as the research object,to explore the influence of STAG2 deletion in osteosarcoma,and to elucidate the signaling pathways and specific mechanisms,so as to provide new molecular targets for the diagnosis and treatment of osteosarcoma.Treatment strategy.METHODS1.STAG2 mutates at high frequencies in osteosarcoma tissue and results in the loss of STAG2.According to the sequencing of each system tumor genome in the public database(cCOSMIC database),the analysis showed that the STAG2 mutation rate in the osteosarcoma was 8.84%5 and the mutation was mainly concentrated in exon 8,leading to the loss of STAG2 protein expression.Second,the CRISPR-Cas9 technique ambiguously knocked out the STAG2 high-frequency mutation exons,mimicking the STAG2 mutation in osteosarcoma in vivo.1.PCR genotyping results showed that exon 8 of STAG2 in the monoclonal genome was ambiguously knocked out.2.WESTERN-BLOT results showed that STAG2 expression was lost in osteosarcoma cells knocked out exon 8.Third,deletion of STAG2 inhibits the proliferation of osteosarcoma cells,causes G2/M phase arrest of osteosarcoma cells,and inhibits PI3k/AKT pathway and cell cycle-related protein expression.1.The results of the cell cycle showed that the proportion of G2/M phase in the KO group was higher than that in the WT group(P<0.001),the proportion in the G0/G1 phase was lower than that in the WT group(0.001),and the proportion in the S phase was lower than that in the WT group(P<0.05).).2.The results of CCK8 showed that the cell proliferation ability of Knockout(KO)group at 48h,72h5 96h,120h was lower than that of wild type(WT)group,P value was P<0.001,P=0.0067,P= 0.0174,P=0.0065.Western-Blot results showed that deletion of STAG2 inhibited the expression of cell cycle-associated protein and downstream key protein(mTOR)of PI3K/Akt signaling pathway.Fourth,the loss of STAG2 leads to osteogenic sarcoma cells susceptible to EMT transformation,increased invasiveness.Western-Blot results showed that the gray-scale value of the cell migration-associated protein N-Cadherin in osteosarcoma cells of KO group was significantly up-regulated while the expression of E-Cadherin was significantly decreased.Transwell results showed that the number of cells in the Transwell group and WT group in the KO group increased significantly(P<0.05).5.Deletion of STAG2 increases the resistance of osteosarcoma cells to cisplatin(DDP).1.CCK8 experiment showed that the cell viability of KO group was higher than that of WT group at 0.5ug/ml,1ug/ml,1.5ug/ml,and 2ug/ml DDP concentrations(P<0.001).Flow cytometry results of PI/AnexinV double staining showed that the apoptosis rate of KO group was lower than that of WT group at 1ug/ml,2ug/ml,3ug/ml cisplatin DDP concentration(P<0.001)..STAG2 deletion caused the endogenous and up-regulated expression of PD-L1 in osteosarcoma cells.1.The results of real-time fluorescence quantitative PCR and Western-Blot showed that the endogenous PD-L1 expression of osteosarcoma cells in STAG2 KO group was up-regulated compared with WT group.2.Immunofluorescence staining flow technique showed that the PD-l1 protein expression was up-regulated on osteosarcoma cell membrane in KO group.Conclusion:1.STAG2 has high-frequency mutations in osteosarcomas,leading to loss of expression;2.In vitro experiments showed that STAG2 deletion inhibited the proliferation of osteosarcoma cells and caused G2/M phase arrest of osteosarcoma cells,and inhibited the expression of PI3K/Akt pathway and cell cycle related proteins.3.Deletion of STAG2 leads to EMT transformation and enhanced invasion of osteosarcoma cells.4.The deletion of STAG2 increased the resistance of osteosarcoma cells to cisplatin(DDP);5.STAG2 deletion caused the endogenous and membrane PD-L1 expression of osteosarcoma cells to be upregulated,which was prone to immunosuppression and immune evasion. |
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