| Background: Osteosarcoma is the most common primary bone malignancy in children and adolescents with highly malignant,aggressive and early metastasis.Neoadjuvant chemotherapy combined with extensive resection of tumor and postoperative chemotherapy is the main method for the treatment of osteosarcoma.The 5-year overall survival rate of osteosarcoma patients is about 70%.In recent years,the treatment of osteosarcoma is in a bottleneck stage,and the 5-year survival rate of patients has not been further improved.Therefore,it is necessary to further explore the pathogenesis and development of osteosarcoma to provide novel targets for improving the prognosis and treatment of patients with osteosarcoma.Abnormal glucose metabolism is closely related to the occurrence and development of malignant tumor,and malignant tumor cells are more prone to hyper-glycolysis.The glucose metabolism demand of tumor cells is significantly higher than normal.Even under aerobic condition,tumor cells choose to undergo glycolysis,consuming a large amount of glucose and generating lactic acid,which is known as the "Warburg effect".Abnormal glucose metabolism is closely related to the occurrence and development of tumors.Hyper-glycolysis can not only provide sufficient energy for the progression of tumors,but also provide material materials for the growth of tumor cells.The production of a large amount of lactic acid acidifies the microenvironment to promote malignant tumor invasion and metastasis and allow malignant tumor cells to escape immune killing.Macrophages play a significant role in the occurrence and metastasis of malignant tumors and in the regulation of tumor immune microenvironment.M2 macrophages are involved in tumor progression and metastasis through the secretion of carcinogenic cytokines,growth factors and proteases,while lactic acid is closely related to the functional polarization of macrophages.Long non-codingRNA(lncRNA)plays an important role in the proliferation,metastasis,drug resistance,apoptosis,substance metabolism,tumor immunity and macrophage polarization of malignant tumors.LncRNA UCA1 has been found to be involved in the progression of various types of malignant tumors,but its role in osteosarcoma remains to be further explored.Purpose:This study was to investigate the expression of LncRNA UCA1 in osteosarcoma and its relationship with clinicopathologic features.Further study on the progression and glycolysis of osteosarcoma as well as macrophage polarization and the related mechanisms.We hope to provide new potential targets for the diagnosis and therapies of osteosarcoma and provide the corresponding theoretical basics for clinical targeting LncRNA UCA1 therapy.Methods: Firstly,the differentially expressed LncRNA UCA1 was obtained by GEO dataset analysis and enrichment analysis was performed.RT-q PCR was used to detect the expression of LncRNA UCA1 in osteosarcoma tissue samples,and its relationship with the prognosis and clinicopathological characteristics of patients was analyzed.Secondly,the localization of LncRNA UCA1 in osteosarcoma cells was explored by FISH assay,and the effects of silencing and overexpression of LncRNA UCA1 on proliferation,migration and invasion,apoptosis,glycolysis and macrophage polarization of osteosarcoma cells were investigated in vitro cell experiments.The methods included CCK8 assay,cell colony formation assay,transwell assay,scratch assay,apoptosis assay,glucose uptake and lactic acid production assay and co-culture with macrophages,etc.Third,bioinformatics predicted the potential binding miRNAs of LncRNA UCA1,and dual luciferase gene reporting assay and RT-q PCR verified the binding relationship.Rescue experiments were conducted to verify whether the effects of LncRNA UCA1 on osteosarcoma cell proliferation,migration and invasion,apoptosis,glycolysis and macrophage polarization were dependent on miRNA-96 and miRNA-143 and were verified by subcutaneous tumor bearing experiments in nude mice.Finally,the common potential target gene HK2 of miRNA-96 and miRNA-143 was identified by bioinformatics prediction and double luciferase gene reporting assay,and the expression of HK2 in osteosarcoma tissues and cell lines was investigated by western blot assay.In addition,western blotting assay and RT-q PCR were used to determine whether LncRNA UCA1 regulated HK2 expression through miRNA-96/miRNA-143.Results: Firstly,the differentially expressed LncRNAs were obtained by using GEO data set,and RT-q PCR detection confirmed that LncRNA UCA1 was significantly highly expressed in osteosarcoma tissues,and enrichment analysis found that it was related to metabolic pathways.Kaplan-Meier survival analysis found that patients with high LncRNA UCA1 expression had a worse prognosis.Analysis of LncRNA UCA1 and its clinical characteristics showed that high expression of LncRNA UCA1 was mainly related to tumor size,recurrence and lung metastasis of patients.Secondly,the expression of LncRNA UCA1 in osteosarcoma cells was significantly higher than that in osteoblasts.Cell lines 143 b and HOS with the highest and lowest relative expression levels were selected for functional experiments.FISH experiments showed that LncRNA UCA1 was distributed in both cytoplasm and nucleus of osteosarcoma,but mainly in cytoplasm.After overexpression of LncRNA UCA1,the proliferation,invasion and migration of osteosarcoma cells were significantly enhanced,cell apoptosis was significantly decreased,glucose uptake was stronger,lactic acid production was significantly increased,and the polarization of M2 macrophages was promoted.However,inhibition of LncRNA UCA1 expression significantly weakened the proliferation,invasion and migration ability of osteosarcoma cells,increased apoptosis rate,weakened glucose uptake ability,significantly reduced lactic acid production,and inhibited the polarization of M2 macrophages.Third,bioinformatics and dual luciferase gene reporting experiments demonstrated that miR-96 and miR-143 could be targeted to LncRNA UCA1.Meanwhile,RT-q PCR experiments demonstrated that inhibition of LncRNA UCA1 could increase the expression of miR-96 and miR-143,while overexpression of LncRNA UCA1 could decrease the expression of miR-96 and miR-143.Overexpression and silencing of miR-96 and miR-143 had no significant effect on LncRNA UCA1.miR-96 and miR-143 were under-expressed in osteosarcoma tissues and cell lines.Salvage experiments showed that the reduction of miR-96 and miR-143 could significantly reverse the inhibitory effects of LncRNA UCA1 on osteosarcoma cell proliferation,invasion and migration,glucose uptake,lactic acid production,M2 macrophage polarization and cell apoptosis promotion,which were verified in nude mice.Increased miR-96 and miR-143 significantly reversed the effects of overexpressed LncRNA UCA1 on proliferation,invasion and migration of osteosarcoma cells,cell apoptosis,glucose uptake capacity,lactic acid production,and M2 macrophage polarization.Fourth,bioinformatics predicted that HK2 was a common potential target gene of miR-96 and miR-143.Dual luciferase gene reporting experiments confirmed the targeting binding relationship between miR-96 and miR-143 and HK2,and HK2 was significantly elevated in osteosarcoma tissues and cell lines.The HK2 expression of si-UCA1+ miR-96 inhibitor group and si-UCA1+ miR-143 inhibitor group was significantly increased compared with that of siUCA1 group.The expression of HK2 in oe-UCA1+ miR-96 mimics group and oe-UCA1+ miR-143 mimics group was significantly lower than that in oe-UCA1 group.Conclusions: LncRNA UCA1 was highly expressed in both osteosarcoma tissues and cell lines,and was positively correlated with poor prognosis,tumor size,recurrence,and lung metastasis.LncRNA UCA1 regulates the expression of HK2 by binding miR-96 and miR-143 through the competitive endogenousRNA(ceRNA)mechanism,regulates glycolysis,promotes the proliferation,invasion and migration of osteosarcoma,inhibits cell apoptosis and promotes M2 macrophage polarization.LncRNA UCA1 may provide potential therapeutic target for targeting osteosarcoma glucose metabolism and macrophage polarization. |
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