The Molecular Mechanism Of Cofilin1 Palmitoylation In Regulating The Migration And Invasion Of HeLa Cell

BackgroundCofilin1 is a regulator of actin movement.It promotes pseudopodia formation by regulating the reorganization of the cytoskeleton actin and then participates in the movement of tumor cells,thus playing an indispensable role in tumor cell metastasis and invasion.Studies have shown that palmitoylation of protein may affect the proliferation or migration of tumor cells by changing the function of the modified protein.In the early stage of this project,it was found that Cofilin1 is a potential palmitoylation modified protein by analyzing the protein palmitoylation of cervical cancer and its adjacent tissues.Looking at the literature,it is found that the molecular function and potential regulatory mechanism of Cofilin1palmitoylation in cervical cancer are unknown.Therefore,this project takes the palmitoylation modification of Cofilin1 as the entry point.First,determine the palmitoylation modification site of the protein,identify the palmitoylation modification enzyme and depalmitoylation modification enzyme that modify it,and then study the effect of Cofilin1.The role of palmitoylation in cervical cancer migration and invasion lays a foundation for exploring the molecular mechanism of Cofilin1 palmitoylation modification on cervical cancer migration and invasion.ObjectiveExplore the molecular mechanism of the palmitoylation modification of Cofilin1 to regulate the migration and invasion of cervical cancer He La cells.Methods1.Identification of Cofilin1 palmitoylation modification:ABE（Acyl-biotinyl exchange）,RAC（Resin-assisted Capture）,Western blot experiment to detect Cofilin1palmitoylation modification.2.Identification of Cofilin1 palmitoylation modification sites:construction of mutant plasmids,cell transfection,ABE,RAC,Western blot experiments to detect Cofilin1palmitoylation modification sites.3.Identification of Cofilin1 palmitoylation modification enzymes:cell transfection,ABE,RAC,Western blot,real-time PCR（Real-Time PCR）experiments to screen Cofilin1palmitoylation modification enzymes.4.Identification of Cofilin1 depalmitoylation modification enzymes:cell transfection,ABE,RAC,Western blot,Co-Immunoprecipitation,and cell immunofluorescence experiments to screen Cofilin1 depalmitoylation modification enzymes.5.The effect of palmitoylation of Cofilin1 on cell migration and invasion:cell scratching,Transwell migration and Transwell invasion experiments detect the effect of palmitoylation of Cofilin1 on the migration and invasion of He La cells.Results1.Through cell transfection,ABE,RAC,Western blot experiments,the results showed that the NH₂OH treatment group Cofilin1 palmitoylation level was significantly higher than the control group;through ABE,Western blot experiments,the results showed that with the addition of 2-BP With the prolongation of the treatment time,the palmitoylation level signal of Cofilin1 gradually weakened,and the inhibitory effect was the most obvious after 10 h of drug treatment.The results showed that Cofilin1 is a palmitoylation modified protein.2.Through protein purification and mass spectrometry analysis experiments,the results showed that the palmitoylation percentage of Cys³⁹ was 4.29%,the palmitoylation percentage of Cys⁸⁰ was 14.71%,and the percentage of palmitoylation modification of Cys¹³⁹ was 12.77%,the palmitoylation percentage of Cys¹⁴⁷ is 10.87%.Through cell transfection,ABE,RAC,Western blot experiments,the results showed that the palmitoylation level of Cys80 site mutation decreased significantly,and the palmitoylation level of the three-site mutant 3C/A（C39A/C139A/C147A）was slightly There is a decrease;the four-site mutant 4C/A（C39A/C80A/C139A/C147A）has the most significant decrease in the level of palmitoylation modification,and the difference is statistically significant compared with the control group（P<0.05）;the results show that Cys³⁹,Cys⁸⁰,Cys¹³⁹,Cys¹⁴⁷are all palmitoylation modification sites,and the palmitoylation modification level at Cys⁸⁰site is the highest.3.Through cell transfection,ABE,RAC,Western blot experiments,the results showed that ZDHHC4,ZDHHC5,ZDHHC6 can significantly increase the palmitoylation modification level of Cofilin1,and the difference is statistically significant compared with the control group（P<0.05）;Through real-time fluorescent quantitative PCR experiments,the results showed that the expression levels of ZDHHC4,ZDHHC5,and ZDHHC6 were higher than those of other controls.The results showed that ZDHHC4,ZDHHC5,and ZDHHC6 are the palmitoylation modification enzymes of Cofilin1.4.Through cell transfection,ABE,RAC,Western blot experiments,the results showed that PPT2 can significantly reduce the palmitoylation level of Cofilin1,compared with the control group,the difference is statistically significant（P<0.05）;through immunoprecipitation,Western blot experiments,the results showed that PPT2 can bind to Cofilin1 in He La cells;cell immunofluorescence experiments showed that PPT2 can co-localize with Cofilin1 in He La cells,and the results showed that PPT2 is a depalmitoylation modification enzyme of Cofilin1.5.Through cell scratch and Transwell migration experiments,the results showed that overexpression of mutant C80A and depalmitoylation modification enzyme PPT2,the healing rate and the number of penetration of He La cells were significantly increased.Compared with the control group,the difference was statistically significant.Significance（P<0.05）;through the Transwell invasion experiment,the results showed that overexpression of mutant C80A and depalmitoylation modification enzyme PPT2,the number of He La cells penetrating the membrane was significantly increased,compared with the control group,the difference was statistically significant（P<0.05）;The results show that the reduced level of palmitoylation of Cofilin1 can significantly promote the migration and invasion of He La cells.ConclusionCofilin1 is a palmitoylated protein,and its palmitoylation sites are Cys³⁹,Cys⁸⁰,Cys¹³⁹,Cys¹⁴⁷;ZDHHC4,ZDHHC5,ZDHHC6 are the palmitoylation modification enzymes of Cofilin1;PPT2 is the depalmitoylation modification enzyme of Cofilin1;Cofilin1Palmitoylation has the ability to regulate the migration and invasion of cervical cancer He La cells.