| Objective:1.To investigate the effect of hypoxia inducible factor-1a(HIF-1a)on glycolysis of esophageal squamous cell carcinoma Eca109 and KYSE150 cells under hypoxia.2.It is preliminarily analyzed that Dihydroartemisinin(DHA)participates in the regulation of HIF-la and then affects the glycolysis of esophageal squamous cell carcinoma under hypoxia.Methods:1.The data of GSE17353 gene chip was downloaded from Gene Expression Omnibus database(GEO).The chip included 4 hypoxia samples and 4 normoxia samples.The differentially expressed genes(DEGs)between normoxia samples and hypoxia samples were analyzed.DEGs was introduced into DAVID for functional enrichment analysis,and the core genes were screened by Cytoscape software.2.The expression of HIF-la in esophageal carcinoma was analyzed by UALCAN database,and the correlation between HIF-1a and pathological features such as sample type,sex,race,tumor grade,tumor subtype,body weight and age were analyzed.The tissue samples of 30 patients with esophageal squamous cell carcinoma diagnosed clinically in the Department of Thoracic surgery of the affiliated Hospital of North Sichuan Medical College from 2018 to 2019 were collected.The expression of HIF-la in 30 pairs of esophageal squamous cell carcinoma and matched normal esophageal epithelial tissues was detected by real-time PCR,and the correlation between the expression of HIF-la and the clinicopathological features of the patients was analyzed.3.Using TCGA-ESCA dataset,a variety of bioinformatics tools(TIMER,cBioPortal,MEXPRESS)were used to explore the potential role of HIF-la in the expression and pathogenesis of ESCA.The network map of HIF-la and its 50 related genes was constructed by using GeneMANIA database,and the functional enrichment analysis was carried out by DAVID in order to explore more biological functions of HIF-la involved in regulation.4.Esophageal squamous cell carcinoma Eca109 and KYSE150 cells were cultured under normoxia and hypoxia(1%02),respectively,and the hypoxia time was set at 0h,12h,24h,36h and 48h.Cell growth was observed by inverted phase contrast microscope.Glucose uptake was measured by glucose detection kit.Lactic acid production was detected by lactic acid detection kit,and the expression levels of HIF-1a and glycolysis related genes(PDK1,HK2)were detected by real-time PCR and western blot experiments.5.The expression of HIF-1a was knocked down by siRNA transfection and cultured under hypoxia(1%O2)for 48h.Glucose uptake was detected by glucose detection kit,lactic acid production was detected by lactic acid detection kit,the expression levels of HIF-la and glycolysis related genes(PDK1,HK2)in cells were detected by real-time PCR and westernblot experiments,and cell proliferation was detected by CCK8 experiments.6.Esophageal squamous cell carcinoma Eca109 and KYSE150 cells were treated with Dimethyl sulfoxide(DMSO)or DHA respectively.Under hypoxia,glucose uptake was detected by glucose detection kit,lactic acid production was detected by lactic acid detection kit,and the expression levels of HIF-la and glycolysis related genes(PDK1,HK2)were detected by western blot experiments.Results:1.Through the analysis of GSE17353 microarray data,a total of 233 differentially expressed genes were screened,including 57 up-regulated genes and 176 down-regulated genes.Functional enrichment analysis showed that the DEGs were mainly enriched in hypoxia-inducible factor-1 signal pathway,cancer pathway,glycolysis metabolism pathway,and mainly involved in cell hypoxia response,glycolysis metabolism,redox reaction and other biological processes.10 Hub genes were screened by String and Cytoscape software,of which HIF-1a was in the first place.2.The results of online analysis of UALCAN database showed that HIF-1a was highly expressed in patients with esophageal carcinoma,and its expression level was related to clinicopathological parameters such as age,sex,race,tumor stage and so on.The expression of HIF-la in 30 pairs of cancer tissues and paracancerous tissues of ESCC patients was detected by real-time PCR.It was found that the expression level of HIF-1a in ESCA tissues was significantly higher than that in normal esophageal epithelial tissues(P<0.05).3.Through a variety of bioinformatics tools,it was found that the expression of HIF-la was significantly correlated with the abundance of ESCA immune cells and the expression of immune markers;there was a relationship between the methylation of ESCA gene promoter and the expression of HIF-la;and there was a certain mutation of HIF-la in ESCA.Functional enrichment analysis of HIF-la and its related genes showed that these genes were mainly concentrated in hypoxia inducible factor-1 signal pathway,glycolysis signal pathway,cancer signal pathway and so on.4.Real-time PCR and western blot experiments showed that the expression of HIF-1a in esophageal squamous cell carcinoma Eca109 and KYSE150 cells increased significantly after hypoxia(P<0.05),indicating that the hypoxia model was established successfully.The results of glucose uptake and lactic acid production showed that the glucose uptake and lactic acid production of Eca109 and KYSE150 cells under hypoxia were higher than those under normoxia,and the expression levels of glycolysis related genes(HK2 and PDK1)were significantly increased(P<0.05).The results showed that the cells changed from tight arrangement to lose spindle after hypoxia,and the glucose uptake and lactic acid production of esophageal squamous cell carcinoma Eca109 and KYSE150 cells under hypoxia were significantly higher than those under normoxia(P<0.05).5.Under hypoxia,HIF-la knockout significantly decreased glucose uptake and lactic acid production in esophageal squamous cell carcinoma Eca109 and KYSE150 cells,and significantly decreased glycolysis-related genes HK2,PDK1 mRNA and protein expression(P<0.05).CCK8 experiments showed that HIF-la knockout significantly decreased the proliferation ability of esophageal squamous cell carcinoma Eca109 and KYSE150 cells(P<0.05).6.Under hypoxia,DHA significantly decreased glucose uptake and lactic acid production in esophageal squamous cell carcinoma Eca109 and KYSE150 cells,and significantly inhibited the expression of HIF-la,HK2 and PDK1.Conclusions:1.HIF-la plays an important role in the regulation of hypoxia and is highly expressed in esophageal carcinoma.It is related to the clinicopathological parameters such as age,sex,race and tumor stage,and participates in the regulation of esophageal carcinoma gene mutation,methylation,immune infiltration and other biological functions.2.HIF-la promotes glycolysis of esophageal squamous cell carcinoma cells by up-regulating the expression of HK2 and PDK1 genes under hypoxia.3.Under hypoxia,DHA will significantly inhibit the glycolysis metabolism of esophageal squamous cell carcinoma cells.The possible mechanism is that it inhibits glycolysis metabolism by inhibiting HIF-1a and down-regulating the expression levels of HK2 and PDK1.4.HIF-la and its downstream glycolysis key genes HK2 and PDK1 may be the targets of DHA,but further research and experimental verification are needed. |
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