| Objective: Hepatocellular carcinoma(HCC)is one of the most malignant tumors worldwide.Diagnosing HCC is not difficult;however,HCC treatment does not yield the expected effects.Hence,studying the key molecular mechanism of HCC development is a high priority for discovering an effective treatment.HCC development is accompanied by cell energy metabolism that changes from oxidative phosphorylation to aerobic glycolysis,and which is termed the Warburg effect.Therefore,the rapid growth and cell activity of HCC are closely related to its glycolytic state.The characteristics of rapid growth and proliferation imply that HCC cells require much energy and sufficient material for synthesizing biological macromolecules.Currently,the question of how HCC cells obtain sufficient energy to maintain rapid proliferation under low nutritional status has not been answered.In conditions of hypoxia and low nutrition in particular,autophagy is a protective mechanism for HCC cells.Recent studies have shown that autophagy can promote HCC cell survival and maintain proliferation by influencing lipid metabolism in hypoxic environments.autophagy can promote glucose uptake and utilization,and that inhibiting autophagy caused an obvious decrease in glucose uptake.How does autophagy regulate glucose metabolism?MCTs play an important role in lactic acid transport and clear H+ in cancer cells.whether the Wnt pathway is involved in HCC metabolism is unclear.In the present study,we explored the mechanism of autophagy in influencing glycolysis,invasion,and metastasis in HCC cells.Autophagy enhances glucose uptake and lactic acid production by upregulating MCT1 expression and the activation of Wnt/?-catenin signaling.Therefore,we investigated the mechanism of autophagy-regulated glycolysis in HCC cells.Our findings provide an experimental and theoretical basis for the further study of autophagy in HCC development and treatment.Methods:(1)1.Eighty-five pairs of HCC tissues and the adjacent tissues were acquired from the Liaoning Cancer Hospital and Institute.The tissues were frozen in liquid nitrogen and then immediately stored at-80?.The hospital ethics committee approved the protocol,and all patients provided gave consent for the utilization of their tissue samples in this study.(2)Accordingly,we cultured Hep G2 and SMMC-7721 cells with EBSS,which contains neither nutrients nor serum,for 6h to induce autophagy.After the 6h starvation,there was significant autophagy in the control cells compared with the cells treated with the autophagy inhibitor 3-MA.Western blotting showed that both LC3-II expression and the LC3-?/LC3-? ratio were increased after starvation.Immunofluorescence showed significantly increased LC3 protein accumulation after starvation.Transmission electron microscopy revealed more characteristic autophagosomes in the starved cells.(3)We detected the impact of autophagy on HCC cell invasion and migration via Transwell assays.Cells that had been starved for 6h had significantly increased invasive and migration ability compared with the control and the cells treated with 3-MA.MMP2 and MMP9 were also increased in the autophagy-induced cells.MMP2 and MMP9 are the members of matrix metalloproteinases.Their function is involved in ECM degradation,which is the one of the key prcocess in tumor metastasis.The secretion of MMP2 and other specific factors about invasion were found to depend on autophagy.(4)We examined whether autophagy can promote HCC cell glycolysis.The glucose and lactate assays showed that autophagy significantly increased HCC cell glucose consumption and lactate production rates,while inhibition of autophagy by 3-MA significantly decreased them.Next,we probed the expression of several key enzymes of glycolysis,and found that autophagy promoted glycolysis by upregulating MCT1 in the HCC cells.Real-time PCR used to detect the expression of glycolysis key enzymes in the HCC cells revealed significantly increased MCT1 in the starved cells;cells that had been treated with 3-MA had decreased enzyme expression,and western blotting yielded similar results.To further investigate the role of MCT1 in autophagy-induced HCC glycolysis,we detected the effect of glycolysis when MCT1 knockout in autophagy induction.The konckdown effect of three MCT1 si RNAs was performed by wetern blot.The results showed that si RNA3 could knock down the MCT1 expression effectively.Then we found that MCT1 knockdown decreased consumption and lactate production rates when induction of autophagy by EBSS,which revealed MCT1 could participate in autophagy-induced glycolysis in HCC cells.(5)Wnt/?-catenin signaling plays an oncogenic role in many cancers;recent research has reported that Wnt/?-catenin is also involved in autophagy in human non–small cell lung cancer cells.We therefore detected whether autophagy can upregulate MCT1 via Wnt/?-catenin signaling in HCC cells.Western blotting showed increased ?-catenin expression following 6h starvation with EBSS as compared with the control,while ?-catenin expression was decreased in the 3-MA–treated cells.Furthermore,?-catenin activity is determined by its phosphorylation status and cellular localization.We therefore examined ?-catenin phosphorylation at Ser33 in HCC cells cultured with EBSS or 3-MA for 6h.The starved cells had decreased ?-catenin phosphorylation,while the autophagy-inhibited cells had increased ?-catenin phosphorylation.Immunofluorescence examination of the cellular localization of ?-catenin showed that autophagy increased ?-catenin nuclear accumulation after 6h starvation?(6)To further determine the role ?-catenin plays in autophagy-promoted glycolysis in HCC cells,we transfected HCC cells with ?-catenin si RNAs for 48 h to decrease ?-catenin expression.The ?-catenin levels were measured using real-time PCR and western blotting to evaluate transfection efficiency.We then examined the effect of ?-catenin on autophagy-induced glycolysis in the HCC cells.Western blotting showed that the ?-catenin knockdown decreased MCT1 expression significantly,and decreased glucose consumption and lactate production rates.These findings indicate that autophagy upregulates MCT1 and induces HCC cell glycolysis by activating Wnt/?-catenin signaling.Moreover,more researches indicated ?-catenin was involved in the regulation of target gene expression by promoting gene transcription.To further investigate the mechanism,the Top-Luc Flash reporter and p RL-TK(negative control)were performed a special luciferase experiment.Our results indicated that induction of autophagy by EBSS increased luciferase activity significantly in both Hep G2 and SMMC-7721 cells.(7)We examined MCT1 expression in 85 HCC tissues via western blotting and real-time PCR,which showed that both MCT1 protein and m RNA were highly expressed in HCC tissues compared with the adjacent tissues.Immunohistochemical analysis indicated that MCT1 was mainly expressed in the cell membrane in the HCC tissues,and that its expression was increased in the HCC tissues.LC3 is a marker protein in autophagy;some studies have shown that it is highly expressed in HCC tissues;Accordingly,we used the Pearson correlation to analyze the relationship between the expression of MCT1 m RNA and LC3 B m RNA in the 85 HCC tissues.Moreover,we divided the 85 tissues into 2 groups by lymph node metastases.And we we analysed the relative MCT1 expression in metastatic cases(n=11)versus non-metastatic cases(n=74).Additionally,we then analysed the correlation between autophagy marker LC3 B and metastasis.Conclusion: Overall,autophagy activation promotes metastasis and glycolysis in HCC cells.Autophagy induces MCT1 expression by activating Wnt/?-catenin signaling.This study makes the connection between autophagy and glucose metabolism in HCC cells and may provide a potential therapeutic target for HCC. |
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