The Study Of Mutant PIK3CA Affecting Colorectal Tumor Cell Proliferation Via PI3K-MEK/PDK1-GPT2 Pathway

Objective:The phosphatidylinositol-3-kinase catalytic subunit α(PIK3CA)gene is mutated in many human cancers.This mutation affects PI3 K function and promotes tumor cell proliferation.However,its underlying mechanism remains unclear.First,this subject detects the changes in the levels of cell proliferation and apoptosis after the PIK3 CA gene mutation in HCT116 cells,then studies the signal pathway relationship between the mutant protein and cell proliferation,then uses different inhibitors to investigate the effect on the cells.Changes in levels of proliferation and apoptosis.Finally,observe the anti-tumor effect of each inhibitor in vivo,and provide more methods for clinical treatment.Methods:(1)Use CRISPR/Cas9 to construct PIK3 CA mutant HCT116 cells(MUT cells).(2)The contents of PIK3 CA and GPT2 in the constructed PIK3 CA mutant cells were detected by Western Blot method and RT-PCR.(3)Detect the glutamine-dependent characteristics of PIK3 CA mutant HCT116 by CCK-8 or flow cytometry.(4)Apply PI3 K,MEK,PDK1,AKT inhibitors to MUT cells,and detect the changes of key proteins in PIK3CA-related signaling pathways in MUT cells by Western Blot;(5)Western Blot was used to detect changes in the expression of P-Rex1 in PIK3 CA mutant HCT116,and the effect of adding different inhibitors to its expression.(6)Apply different inhibitors to MUT cells,to detect the effects of different inhibitors on the proliferation and apoptosis of the mutant cells by CCK-8 or flow cytometry.(7)Observe the tumor formation,proliferation ability and survival period of HCT116 cells and MUT cells in nude mice.(8)Use PI3 K,MEK,PDK1,MEK+PDK1 inhibitors in nude mice to observe the anti-tumor effect of each group of MUT cells and the survival period of nude mice.Results:(1)It was detected that the m RNA and protein expression levels of PIK3 CA in the constructed PIK3 CA mutant cells increased,and the m RNA and protein expression levels of GPT2 also increased(P<0.05).(2)When cultured under normal conditions,MUT cells proliferated faster than HCT116 cells and had less apoptosis than normal cells(P<0.05);under low serum environment,HCT116 cells increased apoptosis less than MUT cells(P<0.05).(3)When glutamine is removed,the proliferation ability of MUT cells is reduced,and the apoptosis is significantly higher than that of HCT116 cells(P<0.05).(4)Use PI3 K,MEK,PDK1,and AKT inhibitors to act on MUT cells to detect the decreased expression levels of key proteins ATF4 and GPT2 in PIK3CA-related signaling pathways(P<0.05).(5)The expression of P-Rex1 in MUT cells increased(P<0.05),and PI3 K inhibitor was added to reduce its expression(P<0.05),and there was no significant effect on its expression when PDK1 and MEK inhibitors were added.(6)PI3K,MEK,PDK1,MEK+PDK1 inhibitors have a significant inhibitory effect on the proliferation of MUT cells,and apoptosis is significantly increased(P<0.05).(7)The tumor growth of HCT116 cells and MUT cells in nude mice was significantly faster than normal cells,and the survival period was shorter(P<0.05).(8)After applying different inhibitors to nude mice inoculated with MUT cells,the MEK inhibitor alone and the combined inhibitor group significantly inhibited tumor growth,with better tumor suppression effects(P<0.05),and the nude mice had a long survival period,but PDK1 There was no significant difference between the inhibitor group and the normal group.Conclusion:1.The PIK3 CA mutation enhances the proliferation ability of HCT116 cells,and its proliferation is more dependent on glutamine.2.The mutated PIK3 CA can regulate the expression of GPT2 through the signal transduction molecule MEK or PDK1,thereby promoting the proliferation of MUT cells.The mutated PIK3 CA mediates GPT2 through the MEK/PDK1 dual pathway.Among them,PI3K-MEK-GPT2 is a new unreported signal pathway discovered in this study,and MEK inhibitors have better tumor suppressive effects in in vivo experiments.3.The above signal pathways can be blocked by PDK1 inhibitors or MEK inhibitors,thereby inhibiting the proliferation of MUT cells in vivo and in vitro,and the combined application of MEK and PDK1 inhibitors can most effectively inhibit the proliferation of MUT cells.It is worth noting that in nude mice,MEK inhibitors can effectively inhibit the growth of MUT tumors,but PDK1 inhibitors have no such effect.This may indicate that in animals,PI3 K is compared with PI3K-PDK1-GPT2 pathway.-The MEK-GPT2 pathway plays a more important role in the growth of MUT tumors.