| Background and ObjectiveIncreasing evidence has demonstrated that long non-codingRNAs(lncRNAs)play essential roles in tumor initiation and progression,and thus may be biomarkers for diagnosis,prognosis and treatment of tumor patients.The purpose of this study was to expolore differential expression of lncRNAs,to seek for novel prognosisrelated molecular biomarks in esophageal cancer(ESCA).Meanwhile,the function and molecular mechanisms of SLC2A1-AS1 in esophageal squamous cell carcinoma(ESCC)was investigated.MethodsTCGA database was used to investigate the differential expressions of lncRNA,miRNA and mRNA and their interactive regulatory network,and to seek for prognosis-related lncRNA.q RT-PCR was utilized to detect the expressions of SLC2A1-AS1,miR-378a-3p and SLC2A1 in ESCC tissues and cells.Luciferase experiment was performed to verify the interaction of transcription factor GLI3 and the promoter of SLC2A1-AS1.GLI3 siRNA or pc DNA3.1-GLI3 was transfected to ESCC cells,and q RT-PCR was employed to examine the expressions of GLI3 and SLC2A1-AS1.CCK-8,clonoy formation,Ed U staining,Flow cytometry and Transwell chamber were performed to detect cell proliferation,apoptosis,migration and invasion.Western blot and immunohistochemistry were utilized to detect the protein expressions.q RT-PCR and FISH assay for the localization of SLC2A1-AS1 in ESCC cells.Dual-fluorescence reporter assay system and Ago-RIP experiment were used to investigate the binding of SLC2A1-AS1 or SLC2A1 with miR-378a-3p.EC9706 xenografted models were established,and pc DNA3.1-SLC2A1-AS1 and chemically modified SLC2A1-AS1 siRNA was used to treat the mice.The measurement of tumor volume was carried out to evaluate the therapeutic efficacy twice every week.Statistical assay: The data was investigated using Graph Pad Prism 8.0 software.The comparison of two groups was carried out using t test and the comparison of samples more than three was performed using one way ANOVA.A P value less than 0.05 was considered as statistical significance.Results1.A total of 145 lncRNAs,112 miRNAs and 2000 mRNAs were differentially expressed in ESCA.2.There were 301 nodes in ceRNA network,which consisted of 40 lncRNAs,28 miRNAs and 233 mRNAs,which formed 677 relations.3.LncRNAs(WDFY3-AS2,CASC8,UGDH-AS1,RAP2C-AS1,AC007128.1,AC016205.1,AC092803.2 and AC079949.2)was tightly associated with overall survival rate of ESCA patients.4.The expression of SLC2A1-AS1 in ESCC tissues was significantly higher than that in normal tissues(P<0.0001).SLC2A1-AS1 at high level was closely associated with TNM staging,lymph node metastasis and poor prognosis of ESCC patients(P<0.05).GLI3 bound to the region of SLC2A1-AS1 promoter,and GLI3 upregulation promoted the transcription of SLC2A1-AS1,whereas GLI3 downregulation suppressed the transcription of SLC2A1-AS1.SLC2A1-AS1 downregulation suppressed GLI3 expression,whereas SLC2A1-AS1 overexpression promoted GLI3 expression in ESCC cells.5.SLC2A1-AS1 downregulation suppressed cell proliferation and invasion and induced apoptosis,coupled with increased E-cadherin level,reduced N-cadherin and Vimentin levels,decreased glucose consumption and lactate production as well as reduced levels of SLC2A1,HK2,PFKM,PKM and LDHA,whereas SLC2A1-AS1 overexpression triggered converse efficacy.6.The results of q RT-PCR and FISH demonstrated that SLC2A1-AS1 was mainly localized in cytoplasm of ESCC cells.Dual-fluorescence reporter assay system and Ago-RIP revealed that SLC2A1-AS1 sponged miR-378a-3p in ESCC cells.SLC2A1-AS1 downregulation increased miR-378a-3p level,whereas SLC2A1-AS1 upregulation suppressed miR-378a-3p level in ESCC cells.7.MiR-378a-3p displayed low level in ESCC tissues and cells,whereas SLC2A1 exhibited high level in ESCC tissues and cells,and their expressions were both associated with TNM staging and lymph node metastasis.SLC2A1 was a direct target of miR-378a-3p in ESCC cells.MiR-378a-3p mimic significantly reduced SLC2A1 level,whereas miR-378a-3p inhibitor markedly promoted SLC2A1 level.8.Overexpressions of SLC2A1-AS1 and SLC2A1 reversed the decrease of proliferation and invasion and the increase of cell apoptosis as well as the downregulation of glycolysis related proteins HK2,PFKM,PKM and LDHA expressions mediated by miR-378a-3p mimic,however,SLC2A1-AS1 and SLC2A1 downregulations evoked the contrary effects.9.In vivo animal experiment demonstrated that SLC2A1-AS1 siRNA suppressed tumor growth,downregulated SLC2A1-AS1 expression and increased miR-378a-3p level,coupled with the downregulations of SLC2A1,HK2,PFKM,PKM and LDHA proteins,whereas pc DNA3.1-SLC2A1-AS1 displayed the converse results.Conclusions1.Many differential expression lncRNAs were found in ESCA,and many lncRNAs was tightly associated with the prognosis of ESCA patients.2.GLI3 overexpression may trigger the overexpression of SLC2A1-AS1 in ESCC cells,and GLI3 and SLC2A1-AS1 may form feedback regulatory loop in ESCC cells.3.SLC2A1-AS1 is highly expressed in ESCC,its downregulation suppresses the proliferation and invasion,and induces apoptosis by suppressing glycolysis related protein expressions,conversely,its upregulation promotes the proliferation and invasion,and inhibiting apoptosis by inducing glycolysis related protein expressions.4.SLC2A1-AS1 upregulates SLC2A1 and glycolysis related proteins by suppressing miR-378a-3p level in ESCC cells,which will promote glycolysis and accelerate ESCC progression. |
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