| Background:Liver cancer is a kind of tumor with high incidence and high fatality rate.Although a lot of research has been done in the treatment of liver cancer in the past 30 years,the survival rate has improved slowly.Metastasis of liver cancer,as the most malignant biological behavior in tumorigenesis and development,seriously limits the treatment of patients and affects the prognosis.Therefore,it is urgent to understand the mechanism of liver cancer metastasis,and to find an effective breakthrough point to inhibit the metastasis of liver cancer and improve the prognosis of patients.Previous studies have shown that more than 95% of liver cancer experiences hypoxia as a result of the rapid growth of tumors.Hypoxia is common in many human tumor types and is associated with poor prognosis and decreased survival rate.Therefore,hypoxia microenvironment and the activation of hypoxia-related genes may be important factors for tumor metastasis.Glucose reprogramming is an important feature of tumor hypoxia microenvironment.When oxygen does not reach the tumor tissue,the tumor will experience hypoxia stress.In order to adapt to this hypoxia stress,tumor tissues often rely on glycolysis to cope with the reduction of oxygen by changing metabolic patterns such as reducing oxidative phosphorylation.For example,in the rate-limiting step of glycolysis,pyruvate is converted to acetyl coenzyme A through pyruvate dehydrogenase,into tricarboxylic acid cycle or into lactic acid through lactate dehydrogenase(LDHA).The decrease in the rate of pyruvate conversion to acetyl coenzyme A and the simultaneous increase of lactic acid production are essential for tumor growth and survival.More and more evidence shows that many metabolic enzymes,such as GLUT1,HK2,LDHA,PKM2,PDK1,play an important role in tumorigenesis and development.Therefore,glucose reprogramming is likely to be an important link affecting tumor metastasis.As a key enzyme in glucose metabolism,PDK regulates the flow of acetyl coenzyme A through mitochondria and controls the exchange of glycolysis and TCA,which is closely related to many types of tumors.In addition,the expression of PDK is closely related to tumor stage,degree of invasion and metastasis,and overall survival rate.PDK1 is one of the four subtypes of PDK.The increased expression of PDK1 is related to tumor stage,metastasis and poor prognosis of patients.It is important to pay attention to PDK1 in hypoxic microenvironment in order to understand the effect of glucose metabolism on metastasis.EMT(epithelial to mesenchymal transformation)is a process of obtaining mesenchymal characteristics in epithelial cancer cells,which endows cancer cells with enhanced invasion and migration ability,and promotes tumor spread and metastasis.Some classical EMT-related transcription factors,such as Twist,Zeb and Snail,can regulate the expression of EMT markers through transcription,and then complete the process of epithelial to mesenchymal transformation.It is clear that EMT and PDK1 play an important role in the invasion and metastasis of tumor cells in hypoxic microenvironment.However,the relationship between PDK1,a key enzyme of glucose metabolism in hypoxic microenvironment,and the regulation of EMT process is still unknown.The study of PDK1,its mediated changes in glucose metabolism and EMT-related signaling pathways induced by hypoxia in tumor cells can help us to understand the role of glucose metabolism in tumor invasion and metastasis.It provides a new idea for the treatment strategy of tumor metastasis.In conclusion,we propose the hypothesis that hepatoma cells reprogram glucose metabolism through metabolic enzyme PDK1 under the induction of hypoxic microenvironment,thus promoting the EMT process,enhancing the invasion and migration of tumor cells,and then promoting the metastasis of liver cancer.Inhibition of PDK1,can reverse the change of glucose metabolism,prevent EMT signaling pathway,reduce the invasion and migration of hepatoma cells,and inhibit the metastasis of liver cancer.Purpose:In this study,we selected hepatoma cell lines with different metastatic ability,treated with hypoxia environment,from the point of view of the role of PDK1,a key enzyme of glucose metabolism,in the invasion and metastasis of tumor cells,the EMT was taken as thebreakthrough point,to analyze the effect of glucose metabolism on the biological process of tumor metastasis.Methods:(1)To observe the effects of hypoxic microenvironment on the viability,proliferation,invasion and migration of low metastatic hepatoma cell line HepG2.MTT was used to detect the effect of hypoxia on the viability of HepG2 cells,colony formation assay was used to detect the effect of hypoxia on the proliferation of HepG2,and transwell was used to detect the effect of hypoxia on the invasion and migration of HepG2 cells.(2)To search for potential hypoxia-related core genes based on bioinformatics and data mining.Difference analysis was used to identify hypoxia-related differentially expressed genes,GO and KEGG enrichment analysis were used to annotate hypoxia-related differentially expressed genes,protein interaction network was constructed and network analysis was used to determine hypoxia-related core genes.Data mining reconfirmed the expression of core genes and clinical relevance.(3)To observe the effect of hypoxia on the expression of metabolic enzyme PDK1 and PDK1-mediated glucose metabolism.The protein expression of PDK1 was detected by Western blot,the mRNA expression of PDK1 was detected by real-time quantitative PCR,and glucose uptake,lactic acid production and ATP production were detected by kit.(4)To observe the effects of PDK1 on glucose metabolism,invasion and metastasis of hepatocellular carcinoma(HCC)under hypoxia.Under hypoxia,the cells were treated with DCA(PDK1 inhibitor)or transfected with shPDK1 plasmid.Glucose uptake,lactic acid production and ATP production were detected by kit,and cell viability was detected by MTT.Colony formation assay was used to detect the cell proliferation,and transwell was used to detect the invasion and migration of cells.(5)To investigate the role of EMT in the metastasis regulated by PDK1 under hypoxia.The expression of EMT related molecular markers(Twist1,E-cadherin,N-cadherin,vimentin)protein was detected by western blot at the basic level of low metastatic hepatoma cell line HepG2 and high metastatic hepatocellular carcinoma cell line HCCLM3,and the mRNA expression level of EMT related molecular marker was detected by real-time quantitative PCR.The protein expression of EMT-related molecular markers was detected by western blot in hypoxia-induced HepG2,and the mRNA expression of EMT-related molecular markers was detected by real-time quantitative PCR.Under hypoxia,Western blot,and real-time quantitative PCR were used to detect the expression of EMT related molecular markers in HepG2 cells with DCA(PDK1 inhibitor)treatment or shPDK1 plasmid transfection.Results:(1)After hypoxia treatment of low metastatic hepatoma cell line HepG2,the cell viability,proliferation,invasion and migration enhanced.(2)The results of hypoxia-related bioinformatics and data mining showed that 113 genes were up-regulated and 164 genes were down-regulated in hypoxia compared with normal oxygen,and were significantly enriched in HIF signaling pathway and glucose metabolism pathway.The construction of protein interaction network and related functional analysis identified 12 core genes.The expression of PDK1,PGK1 and SLC2A1 was highly correlated with hypoxia,while the expression of PDK1 was related to the stage,metastasis and poor survival time of HCC.(3)After hypoxia induction,the levels of PDK1 protein and mRNA in HepG2 cells increased,and glucose uptake,lactic acid production and ATP production increased.(4)Under hypoxia,DCA or shPDK1 inhibited PDK1,decrease glucose uptake,lactic acid production and ATP production,and the cell viability,proliferation,invasion and migration of HepG2 cells were weakened.(5)The protein and mRNA expression of mesenchymal marker(Twist1,N-cadherin,vimentin)in high metastasis cells(HCCLM3)was higher than that in low metastasis cells(HepG2).After hypoxia induction,the protein and mRNA expression of mesenchymal markers increased,while the protein and mRNA expression of epithelial markers decreased in low metastasis cells(HepG2).After PDK1 was inhibited by DCA or shPDK1,the protein and mRNA expression of mesenchymal marker decreased,while the protein and mRNA expression of epithelial marker increased.Conclusions:In summary,hepatoma cells induced by hypoxia microenvironment reprogram tumor glucose metabolism through metabolic enzyme PDK1,thus promote the EMT process,enhance the invasion and migration of tumor cells,and then promote the metastasis of liver cancer.Inhibition of PDK1,can reverse the change of glucose metabolism,prevent EMTsignaling pathway,reduce the invasion and migration of hepatoma cells,and inhibit the metastasis of liver cancer. |
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