Effects Of Glutamine And 3-phosphoglycerate Dehydrogenase On The Proliferation Of Cervical Cancer Hela Cells And Its Related Mechanism

Objective: This study aims to explore the effect of glutamine(Gln)deficiency on the proliferation,migration and apoptosis of Hela cells of cervical cancer and its related mechanisms.Furthermore,the expression of PHGDH and the effect of further silencing PHGDH on the proliferation of cervical cancer Hela cells and the related mechanism were further studied under the condition of glutamine deficiency.Methods:1.CCK8 method was used to detect the growth of Hela cells cultured under different concentrations(0/0.1/0.5/1/2/4mmol/L)Gln for 48 h,and the growth of cells in each group was detected after continuous culture for 48 h.2.Cell scratch experiment was used to observe the migration ability of Hela cells under different concentrations(0/0.1/0.5/1/2/4mmol/L)Gln.3.PE Annexin V Apoptosis Detection Kit I was used to detect the Apoptosis of Hela cells cultured at different concentrations(0/0.1/0.5/1/2/4mmol/L)Gln for 48 h.4.Flow cytometry was used to detect the ROS level in Hela cells cultured at different concentrations(0/0.1/0.5/1/2/4mmol/L)Gln for 48 h.5.The expression of PHGDH in Hela cells of cervical cancer was inhibited by lentivirus transfection RNA interference technology,and the PHGDHsh RNA gene silencing cell model was constructed.Fluorescence microscopy and Werstern blot were used to detect the transfection efficiency and the expression of PHGDH in cells after transfection.6.CCK8 method was used to detect Hela,Helavec and Helash-PHGDH at different concentrations(0/0.1/0.5/1/2/4mmol/L)Gln for 48 h and 96 h,and the growth of cells in each group was detected.7.PE Annexin V Apoptosis Detection Kit I was used to detect the Apoptosis of Hela,Helavec and Helash-PHGDH cells.8.Werstern blot was used to detect the expression of PHGDH protein in Hela cells cultured at different concentrations(0/0.1/0.5/1/2/4mmol/L)Gln for 48 h.9.Flow cytometry was used to detect ROS in Hela,Helavec and Helash-PHGDH cells cultured at different concentrations(0mmol/L /4mmol/L)Gln for 48 h.10.The GSH/GSSGTM Assay(premega)kit was used to detect the intracellular GSH/GSSG ratio of Hela and helash-phgdh at different concentrations(0mmol/L/4mmol/L)Gln for 48 h.11.The Total NADP and NADPH Assay kit(Abcom company)was used to detect the changes in the ratio of NADP/NADPH in Hela cells after 48 h culture at different concentrations(0/0.1/0.5/1/2/4mmol/L)Gln,as well as the ratio of NADP/NADPH in Hela,Helavec and Helash-PHGDH cells.Results:1.In the absence of glutamine,Hela cell growth was inhibited(P<0.05),and the lower glutamine concentration,the greater the inhibition on Hela cell growth;Hela cell growth inhibition was further aggravated after continuous culture for 48 h.2.In the scratch experiment,the wound healing rate of Hela cells decreased with the decrease of glutamine concentration in the culture medium(P<0.05).3.When glutamine concentration <0.5mmol/L,the number of Hela cells apoptosis was significantly increased(P<0.05).4.When glutamine <1mmol/L,the ROS level in Hela cells was significantly increased(P<0.05).5.CCK8 assay showed that the growth inhibition rate of Helash-PHGDH group was significantly higher than that of Helavec group and Hela group at the same Gln concentration(P<0.05),and there was no significant difference between Helavec group and Hela group.When glutamine was deficient,the growth of Hela cells was inhibited(P<0.05).After PHGDH was silenced,the growth of Hela cells in cervical cancer was further inhibited.6.The number of apoptosis in Helash-PHGDH group was significantly higher than that in Hela group and Helavec group(P<0.05).7.Werstern blot results showed that the expression of PHGDH protein in Hela cells was significantly increased when glutamine was deficient(P<0.05).8.The intracellular ROS level of Helash-PHGDH group was significantly higher than that of Helavec group and Hela group(P<0.05)under the condition of 4/0mmol/LGln,and there was no significant difference between Helavec group and Hela group.Compared with the 4mmol/L Gln culture environment,the 0mmol/L Gln culture condition also resulted in increased intracellular ROS level in each group(P<0.05).9.The intracellular GSH/GSSG ratios of the Hela/4mmol/L Gln group,Hela/0mmol/L Gln group,Helash/PHGDH/4mmol/L Gln group and hela-shphgdh /0mmol/L Gln group were 46.87 1.25,13.59 1.83,10.99 0.75,and 3.50 2.57,respectively,and the differences were statistically significant(P<0.05).10.When Gln<2mmol/L,the intracellular NADP/NADPH ratio of Hela group began to increase,and the difference was statistically significant(P<0.05),but there was no significant difference in the intracellular NADP/NADPH ratio of Hela,Helavec and Helash-PHGDH.Conclusions:1.Glutamine deficiency inhibits the proliferation,migration and apoptosis of Hela cells of cervical cancer by inducing the increase of ROS level in Hela cells of cervical cancer.2.PHGDH silencing can inhibit the proliferation of Hela cells in cervical cancer and induce their apoptosis.3.Silencing PHGDH further inhibited Hela cell proliferation under the condition of glutamine deficiency.4.Glutamine deficiency induces increased expression of PHGDH protein in Hela cells of cervical cancer,increased intracellular ROS level,decreased GSH/GSSG ratio,and increased NADP/NADPH ratio.After PHGDH silencing,the ROS level in Hela cells was further increased and the GSH/GSSG ratio was further decreased,but there was no significant change in the NADP/NADPH ratio in Hela cells.