Effects Of Smac On Esophageal Squamous Cell Carcinoma Via Warburg Effect

Esophageal cancer is a malignant lesion formed by abnormal proliferation of the digestive tract epithelium.China is one of the countries with the highest incidence of ESCC in the world.With the aging of the population,the total morbidity and mortality of ESCC keeps rising.Although some progress has been made in the basic and clinical research of ESCC in recent years,the genetic basis and molecular mechanism of its occurrence have not been revealed,so researches on its pathogenesis can be used as the molecular basis for future targeted drug therapy.As an important organelle involved in cell energy metabolism and apoptosis,mitochondria play an important role in the development of tumor cells.The Smac protein is a pro-apoptotic regulator released from the mitochondria to the cytosol.Under the stimulation of apoptosis,it is released from the mitochondria into the cytoplasm,and the inhibition of Caspase is released by binding to the Inhibitor of apoptosis protein to induce apoptosis.Studies have shown that changes in energy metabolism of tumor cells affect the function of mitochondria,which in turn changes the life processes of cell proliferation,apoptosis and migration.The Warburg effect is a unique form of energy metabolism in tumor cells,which provides sufficient material,energy,and microenvironment conditions for tumor cell metastasis.However,themetabolic mode of tumor cells is not static.It will change with the changes of genes,microenvironment,drugs and other factors.Therefore,this study aims to find out whether the Smac gene can change the energy of ESCC cells.Metabolic pathways in turn affect the growth and survival of tumor cells.ObjectiveThis study will examine the effects Smac protein on cell proliferation,apoptosis,migration,and mitochondrial metabolism by overexpressing and si RNA interfering with the Smac gene in esophageal squamous cell carcinoma cells.Combining cell and animal experiments,the effects of Smac on esophageal squamous cell carcinoma were revealed via energy metabolism.Methods1.Western blot was used to detect the expression of Smac protein in ESCC tissues and cells.Smac overexpressed and si RNA interfered ESCC cell lines were constructed mediated by lentivirus.2.CCK-8 was used to detect the proliferation and survival of Smac-overexpressed and-interfered ESCC cells under oxidative damage condition.3.Mitochondrial membrane potential and Annexin V-FITC/PI double staining were employed to detect the apoptosis of Smac-overexpressed and-interfered ESCC cells.4.Transwell chamber was used to detect cell migration when Smac was overexpressed and interfered.5.The lactic acid release,glucose uptake and expression of reactive oxygen species in the cells were detected by spectrophotometer and chemical fluorescence probe method respectively.6.The changes of the second mitochondrial apoptosis pathway,the protein related to glycolysis process,N-cadherin,E-cadherin,Vimentin,P-AMPK and HIF-1α protein were detected by Western blot.7.The ESCC xenograft model was established.After tumor-bearing,the volume of the tumor mass and the weight of the nude mice were measured and recorded every 3 days.After 3 weeks,the tumor mass was weighed and the apoptosis level of four groups of transplanted tumor cells was detected by TUNEL method.8.The expression levels of N-cadherin,E-cadherin and Vimentin protein and glycolytic process related proteins LDH and HK2 in transplanted tumor tissues were detected by immunofluorescence assay.9.Chemical fluorescence probe method was used to detect the changes of ROS levels in the four groups of transplanted tumor tissues.ResultsPart Ⅰ: Effect of Smac expression changes on phenotype of esophageal squamous cell carcinoma1.The expression of Smac protein was down-regulated in ESCC tissues and cells,Eca109 and EC1 cell lines with Smac overexpression and si RNA interference were successfully constructed.2.The Smac overexpressing and si RNA interfering Eca109 and EC1 cells under oxidative stress,Smac overexpressing cell line survival rate was significantly smaller than the control group,Smac si RNA interference cell line survival rate was higher than the control group.Both mitochondrial membrane potential and flow cytometry showed that Smac overexpression promoted apoptosis of Eca109 and EC1 cells under oxidative stimulation,while Smac si RNA interfered with apoptosis of Eca109 and EC1 cells.At the same time,Western blot analysis showed that Smac overexpression inhibited the expression of apoptosis inhibitory proteins Bcl-2 and X-IAP,and the expression of apoptosis-executing protein Pro-Caspase 3 was also significantly decreased.3.The results of Transwell assay showed that the number of migration of Eca109 and EC1 cells overexpressed by Smac was decreased compared with the control group,and the number of migration of Eca109 and EC1 cells by Smac was increased.Western blot analysis showed that Smac overexpression increased theexpression of E-cadherin and Vimentin proteins in Eca109 and EC1 cells.Smac overexpressed Eca109 and EC1 cells had less lactate release than control cells,and glucose uptake was lower than normal Eca109 and EC1 cells.The amount of lactic acid released from Eca109 and EC1 cells si RNA interfered by Smac was significantly higher,and the absorption of glucose was also higher than that of the control group.4.Smac overexpression reduced ROS levels in Eca109 and EC1 cells,while Smac si RNA interference increased ROS levels in Eca109 and EC1 cells.Western blot analysis showed that Smac overexpression down-regulated the expression of HIF-1α and P-AMPK in Eca109 and EC1 cells,while Smac si RNA interference promoted the expression of HIF-1α and P-AMPK in Eca109 and EC1 cells.Part Ⅱ: Smac promotes cell migration in Eca109 xenografts through the Warburg effect1.Smac overexpression and si RNA interference had no significant effect on the size and growth rate of Eca109 xenografts.2.The results of immunofluorescence showed that Smac si RNA interference increased the expression of E-cadherin and Vimentin,which promoted the migration process in Eca109 xenografts,and increased the expression of key enzymes in the glycolysis process.Smac si RNA interference promotes ROS production in Eca109 xenografts.Conclusion1.The expression level of Smac protein in ESCC tissues and cells was significantly lower than that in normal tissue cells.2.Smac overexpression enhances the sensitivity of ESCC cells to oxidative stress.The low expression of Smac protein in ESCC cells reduces the sensitivity of cells to oxidative stimulation on the one hand,and promotes cell migration through changes in cellular energy metabolism.3.Smac overexpression and si RNA interference have little effect on the size and growth rate of Eca109 xenografts,but may affect the migration ability of tumor cells through the Warburg effect.