| Background and Purpose:Osteosarcoma is one of the most common and aggressive primary malignant bone tumor,and it occurs most frequently in children and adolescents.Due to advancement of various treatment strategies such as surgery,chemotherapy,and radiotherapy,the five-year survival rate in non-metastatic osteosarcoma patients has reached 60%.However,the overall survival rate has not markedly increased over the last three decades due to the molecular mechanisms underlying the pathogenesis and progression of osteosarcoma are poorly understood.Therefore,it is of great clinical significance to explore the molecular mechanisms related to the progression and pathogenesis of osteosarcoma and to identify more effective therapeutic targets.In recent years,the function of lnc RNA in the tumor development has gradually attracted the attention of scholars and has been widely studied.Numerous studies have shown that lnc RNA are involved in the regulation of immortal proliferation,immune evasion,angiogenesis,apoptosis resistance,and metastasis of many tumors.KCNQ1OT1 is a lnc RNA which reported to be aberrantly expressed in some tumors and involved in promoting tumor proliferation,metastasis and drug resistance.Although studies have shown that KCNQ1OT1 was up-regulated in osteosarcoma and was associated with poor prognosis of patients,the specific role and regulatory mechanism of KCNQ1OT1 in the development of osteosarcoma is still unclear.Therefore,the main research purpose of this subject is to explore the expression of KCNQ1OT1 in osteosarcoma and its relationship with clinical features,and to further explore the function of KCNQ1OT1 on the growth of osteosarcoma in vitro and in vivo and its specific regulatory mechanisms.Experimental Design:(1)In order to explore the relationship between the expression of KCNQ1OT1 and the clinical characteristics of osteosarcoma patients,we performed fluorescence in situ hybridization and statistical analysis.The expression patterns of KCNQ1OT1 in osteosarcoma cell lines and osteoblast cell line were analyzed by realtime quantitative polymerase chain reaction.(2)Stable KCNQ1OT1 knockdown U-2OS and 143 B cell lines were established.The cell counting kit-8 assay,Ed U assay,colony formation assay,cell cycle assay and cell apoptosis assay were used to explored the biological function of KCNQ1OT1 in the progression of osteosarcoma.A subcutaneous xenograft tumor model in nude mice was used to detect the effect of KCNQ1OT1 knockdown on the growth of osteosarcoma in vivo.(3)A metabolic flux analyzer was used to measure extracellular acidification rate and the oxygen consumption rate in KCNQ1OT-knockdown and control osteosarcoma cells.Western blotting and rescue experiments were used to confirmed that KCNQ1OT1 promoted aerobic glycolysis in osteosarcoma cells by regulating the expression of ALDOA.(4)The specific mechanism of KCNQ1OT1 regulating ALDOA expression was explored through database analysis,dual-luciferase reporter assay,RNA-binding protein immunoprecipitation assay,tissue immunofluorescence and rescue experiments.Results:(1)KCNQ1OT1 was significantly upregulated in osteosarcoma tissues or cell lines and was associated with poor Ennecking staging in patients.(2)Knockdown of KCNQ1OT1 significantly inhibited the growth of osteosarcoma in vitro and in vivo.(3)Knockdown of KCNQ1OT1 inhibited the aerobic glycolytic and facilitated the oxidative phosphorylation by down-regulating the expression of ALDOA in osteosarcoma.(4)miR-34c-5p inhibited ALDOA expression by directly targeting its 3’UTR.KCNQ1OT1 blocked the inhibitory effect of miR-34c-5p on ALDOA expression by competitively binding to miR-34c-5p.Conclusions:In conclusion,our study identified that KCNQ1OT1 is an oncogene that was highly expressed in osteosarcoma.KCNQ1OT1 promoted the growth of osteosarcoma by promoting aerobic glycolysis in osteosarcoma.Mechanistically,KCNQ1OT1 competes with a key glycolytic coding m RNA,ALDOA,for miR-34c-5p and relieves the inhibitory effect of miR-34c-5p on ALDOA,thereby leading to increased ALDOA expression and aerobic glycolysis.Our study demonstrated that the KCNQ1OT1/miR-34c-5p/ALDOA axis plays an important role in the glucose metabolism reprogramming and development of osteosarcoma,and suggested that KCNQ1OT1-miR-34c-5p/ALDOA axis may provide a new therapeutic target for OS treatment... |
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