| Objective:Osteosarcoma is the most common malignant bone tumor,which occurs in adolescents,with high malignancy,early metastasis and poor prognosis,threatening human health.The current treatment plan for osteosarcoma mainly consists of three parts:preoperative chemotherapy-surgical resection-postoperative chemotherapy.Surgical treatment(amputation or limb-preserving surgery)is still the treatment modality for osteosarcoma,although the 5-year survival rate has increased significantly.However,the presence of mu Lti-drμg resistant gene expression,enhanced cellular DNA damage repair,tumor microenvironment-related drug resistance,and non-coding RNA dysfunction can contribute to a decrease in sensitivity to chemotherapeutic drugs in osteosarcoma,which adversely affects the treatment of osteosarcoma,and pulmonary metastasis is an important cause of death in osteosarcoma.Osteosarcoma stem cells are a small number of osteosarcoma cells with the ability of self-renewal and multiple differentiation,which play an important role in the development and progression of osteosarcoma,and the presence of osteosarcoma stem cells is one of the important factors affecting tumorigenesis-development-recurrence-drug resistance.Therefore,it is of great theoretical significance and clinical value to study the biological properties and related mechanisms of osteosarcoma stem cells in depth.Stannocalcin 2(STC2)is widely expressed in breast,colorectal,gastric,esophageal,prostate,kidney,liver,bone,ovary,lung and other tumor cells and tumor tissues,with autocrine or paracrine functions and the ability to interact with metal ions.STC2 expression is regulated at both transcriptional and post-transcriptional levels and is significantly stimulated especially under various stress conditions,such as endoplasmic reticulum stress,nutrient deprivation and hypoxic state.In pan-cancer analysis,STC2 overexpression was negatively correlated with overall survival,disease-free survival,and disease-specific survival.STC2 was closely associated with the tumor immune microenvironment,including immune cell infiltration,immune checkpoint genes,mismatch repair genes,tumor mutational load,and microsatellite instability.STC2was significantly and negatively correlated with sensitivity or resistance to multiple drugs.STC2 as a In this experiment,we investigated the relationship between STC2and osteosarcoma through clinical specimens to determine the expression differences and clinicopathological significance of STC2,followed by in vitro investigation of its phenotype on osteosarcoma stem cell proliferation and chemotherapy sensitivity from the perspective of molecular cell biology,and explored its potential mechanism.Finally,it was further validated by in vivo animal experiments.Material and methods:In this study,the expression of STC2 in human tissues was firstly detected by immunohistochemistry and western blot,and the correlation between STC2 and clinicopathological parameters of osteosarcoma patients was analyzed.Flow cytometric sorting was applied to isolate osteosarcoma stem cells from the osteosarcoma cell line U2OS,and the expression of STC2 and stemness indexes SOX2,OCT4 and NANOG in stem cells was also examined.Lentiviral transfection of OSCs and OSSCs was used to construct cell models expressing and knocking down STC2.The changes of STC2 on cell sphere-forming ability,proliferation ability,apoptosis,colony formation and migration invasion ability were detected by cell sphere-forming assay,CCK8 cell proliferation assay,apoptosis,clone-forming ability and transwell assay,respectively.The cisplatin IC50 values of OSCs and OSSCs were detected using CCK8,and the inhibition rate of each group of cells by itself 1/2 cisplatin IC50 concentration was examined(CCK8 method),and the flow apoptosis assay was also done.Next,in situ immunofluorescence was applied to observe the expression and localization of METTL3 and STC2 by two-photon confocal microscopy.RIP technique was applied to experimentally verify the direct binding effect of METTL3 and STC2.Me RIP was applied to detect the methylation level of m~6A in OSCs and OSSCs before and after overexpression of METTL3.The decay changes of STC2 were detected by RNA stability assay.The expression changes of STC2,SOX2,OCT4,NANOG,ABCG2,PI3K,p-PI3K,AKT and p-AKT proteins were detected by Western blot method.By constructing a tumor-forming animal model in nude mice,after further testing overexpression and knockdown of STC2,tumor growth curves were plotted according to tumor formation in vivo,and the difference in weight ratio of tumor formation was compared by weighing.The microscopic changes of tumor formation were observed by HE staining,and the expression of STC2 and KI67 were detected by immunohistochemical staining.The animal model of tumorigenic chemotherapy in nude mice was constructed to detect the difference of tumorigenesis in different groups,and Tunel assay was performed to detect the apoptosis.Results:In this study,STC2 showed high expression in osteosarcoma tissues and OSSCs,and the expression level of STC2 was closely related to the Enneking stage and the degree of tumor cell differentiation.OSSCs were successfully isolated from the osteosarcoma cell line U2OS,and osteosarcoma stem cells highly expressed SOX2,OCT4,and NANOG(all of the above p<0.05).Sphere-forming assay showed that overexpression of STC2 promoted the sphere-forming ability of OSSCs and OSCs,and knockdown inhibited this ability.CCK8 proliferation assay showed that overexpression of STC2 promoted the proliferation ability of OSSCs and OSCs,and knockdown inhibited this ability.Apoptosis assays showed that overexpression of STC2 inhibited the apoptotic ability of OSSCs and OSCs,and knockdown promoted this ability.Colony-forming assays showed that overexpression of STC2 promoted the colony-forming ability of OSSCs and OSCs,and knockdown inhibited this ability,while transwell assays showed that overexpression of STC2 promoted the migration ability of OSSCs and OSCs,and knockdown inhibited this ability.Immunofluorescence assays showed that METTL3 was localized in the nucleus and cell membrane,and STC2 was localized in the cytoplasm.IC50 assays showed that the IC50 value of cisplatin was greater in OSSCs than in OSCs.CCK8 and apoptosis assays showed that the knockdown group was more sensitive to cisplatin than the overexpression group at the same concentration of cisplatin.The results of Me RIP assay showed that the m~6A methylation level of STC2 was enhanced after overexpression of METTL3.SOX2,OCT4,NANOG,ABCG,p-PI3K,p-AKT expression was increased after overexpression of STC2 and decreased after knockdown.(p<0.05 for all of the above).The tumor-forming experiments in nude mice showed that the tumor formation rate was slowed down and the size and weight of the tumor became smaller after the stable knockdown of STC2.The expression of KI67 was reduced in the knockdown STC2 group in paraffin sections of tumor-forming tissues.The results of tumorigenic chemotherapy model in nude mice showed that the tumor was the smallest and the slowest growing in the stable knockdown STC2+cisplatin chemotherapy group,and the highest apoptosis rate was observed in the stable knockdown STC2+cisplatin chemotherapy group in the Tunel assay(p<0.05 for all of the above).Conclusion:1.The expression of STC2 is higher in osteosarcoma tissues than in normal tissues adjacent to the tumor.2.The expression of STC2 is closely related to the clinical stage and tumor malignancy of patients,i.e.the higher the expression of STC2,the more malignant the tumor is,and the clinical stage tends to be late and prone to metastasis.3.Osteosarcoma stem cells were successfully isolated from osteosarcoma cell line U2OS,and the protein expression levels of STC2 and stemness indexes SOX2,OCT4and NANOG were increased compared with parental cells.4.Silencing STC2 can:(i)inhibit self-renewal,proliferation,migration of OSSCs;(ii)promote apoptosis of OSSCs;and(iii)improve the sensitivity to chemotherapy with cisplatin.5.METTL3 promotes self-renewal,proliferation,migration and invasion of OSSCs by increasing the stability of STC2 m RNA,inhibits apoptosis of OSSCs,and promotes reduced sensitivity of OSSCs to cisplatin.6.In vivo animal experiments demonstrated that knockdown of STC2 inhibited the proliferation of osteosarcoma cells and increased their chemosensitivity to cisplatin by a mechanism related to the fact that low expression of STC2 reduced the self-renewal replication of osteosarcoma stem cells. |
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