The Role And Mechanism Of ATF4 In Regulating PHGDH And SHMT2 In Endometrial Cancer Cells

Objective:Endometrial cancer is often associated with disturbances in energy metabolism.Therefore,it is of great significance to explore the mechanism of energy metabolism in endometrial cancer cells for the treatment of endometrial cancer.As one of the eight non-essential amino acids in the human body,serine can participate in various physiological activities in the human body,and can provide one carbon unit,glycine,etc.for cell proliferation.There are two main ways of serine uptake in the human body: Ⅰself-synthesizing route;Ⅱ uptake through the external environment.There are growing evidence that serine plays an important role in tumor progression.Phosphoglycerate dehydrogenase is the first and rate-limiting enzyme in serine synthesis.Serine hydroxymethyltransferase 2 is mainly distributed in mitochondria and can convert glycine to serine.Studies had found that restricting serine synthesis in colorectal cancer cells combined with diets lacking serine glycine can inhibit tumor growth,but the study did not specifically reveal when serine mechanism of action following synthesis inhibition in combination with a lack of serine-glycine in the diet.Therefore,this topic mainly interpreted the mechanism of ATF4 in endometrial cancer after serine-glycine-deficient medium,provided a new idea for serine research,and also provided a new method and strategy for the treatment of endometrial cancer.Methods:1.Using TCGA database to analyze the difference of PHGDH expression in human endometrial cancer tissue and paracancerous tissue,and then specifically analyze the difference of PHGDH expression in cancer tissue and paracancerous tissue of 13 pairs of patients.Then,by analyzing the overall progression survival and progression-free survival of endometrial cancer patients,the relationship between PHGDH and the prognosis of endometrial cancer patients was verified.2.Quantitative Real-time PCR and Western Blot experiments were used to detect the expression levels of key enzymes for serine synthesis in endometrial cancer cells.3.After silencing PHGDH by si RNA transfection,we tested its effect on AN3 CA.Subsequently,AN3 CA and Ishikawa cells were treated with the PHGDH inhibitor NCT-503,and HEC-1-A cells with low PHGDH expression were used as a control to detect their effects on PHGDH-dependent cancer cells,and were detected by Annexin V-FITC/PI double staining.The apoptosis of NCT-503 in these three cell lines was detected,and the expression of apoptosis related proteins was detected by Western Blot.4.After silencing PHGDH by si RNA transfection,the effect of PHGDH inhibition on AN3 CA was examined.Subsequently,AN3 CA and Ishikawa cells were treated with the PHGDH inhibitor NCT-503,and the effect of NCT-503 on PHGDH-dependent endometrial cancer cells was examined using HEC-1-A cells with low PHGDH expression as a control.5.To investigate the mechanism of PHGDH inhibiting cell proliferation.The effect of inhibiting PHGDH on ROS was detected by DCFH-DA method,and after pretreatment with ROS scavenger TOC,DCFH-DA method was used to detect the effect of inhibiting PHGDH on ROS in endometrial cancer cells.6.To investigate the effect of inhibiting serine synthesis combined with the lack of serine glycine in the diet on ROS.DCFH-DA method was used to detect the effect of inhibiting PHGDH combined with lack of serine glycine on ROS.After pretreatment with TOC,the DCFH-DA method was used to detect the effect of its effect on ROS;its effect on the activity of endometrial cancer cells was detected by MTT method;its effect on AN3 CA cell apoptosis was detected by Annexin V-FITC/PI and Western blot method.7.To investigate the effect of inhibiting the resynthesis of serine and the replenishment of serine in combination with the lack of serine glycine in the diet on the viability of endometrial cancer cells.The effect of adding NCT-503 and SHIN1 in combination with lack of serine glycine in the diet on endometrial cancer cells was detected by MTT method,and the effect of adding TOC on endometrial cancer cells was detected after pretreatment with TOC.8.To explore whether p53 can regulate PHGDH in endometrial cancer cells.First,the expression of p53 protein in five endometrial cancer cells was detected.Subsequently,after the HEC-1-A and HEC-1-B cells with high p53 expression were treated with the p53 inhibitor Pifithrin α for 24 h,the effect on PHGDH when p53 was inhibited was examined,and AN3 CA and Ishikawa with low p53 expression were selected.After the cells were treated with the p53 inducer Nutlin-3a for 24 h,the effect on PHGDH when p53 was induced was examined.9.To investigate whether ATF4 can regulate PHGDH in endometrial cancer cells.AN3 CA and Ishikawa cells were treated with ATF4 si RNA,and then the proteins and m RNA of PHGDH and SHMT2 after silencing ATF4 were detected by Western Blot and Quantitative Real-time PCR.For further verification,we then transfected two cell lines with ATF4 high-expressing plasmids,and the same Western Blot and Quantitative Real-time PCR methods were used to detect the protein and m RNA expression levels of PHGDH and SHMT2 when the expression of ATF4 increased.Variety,we detected changes in the m RNA and protein expression levels of ATF4,PHGDH and SHMT2 when serine-glycine was deleted.Conclusions:Inhibition of PHGDH can increase ROS,thereby inhibiting the proliferation of endometrial cancer cells and inducing the apoptosis of endometrial cancer cells.Inhibition of serine synthesis in a serine-glycine-deficient medium environment effectively inhibited the proliferation of endometrial cancer cells,whereas the ROS scavenger TOC reversed this phenomenon.Deletion of serine-glycine leaded to an increase in ATF4 protein,which in turn leads to an increase in the expression of PHGDH and SHMT2 proteins,thereby promoting the intracellular serine synthesis pathway to maintain tumor cell growth.