| Objective:Chemotherapy is one of the main methods of tumor treatment.Most chemotherapy drugs cause tumor cell death by damaging tumor cell DNA or inhibiting mitosis[1].However,during the chemotherapy,some tumor cells decrease their chemosensitivity when they adapt to the DNA damages.[2].Chemoresistance is a troublesome problem in the process of tumor treatment.The main reason is that the molecular mechanism is complicated.Apoptosis is a cellular stress response characterized by DNA fragmentation,nuclear pyknosis,cell shrinkage and plasma membrane blebbing.Tumor cell apoptosis is an important mechanism for chemotherapeutic drugs to exert anti-tumor effects[3].Forkhead protein FOXK2 is a member of the Forkhead box(FOX)transcription factor family,and its protein consists of 660 amino acids.In structure,FOXK2 and other family members have a highly conserved“winged-helix”DNA binding domain[4].FOXK2 plays an important role in cell survival[5],aerobic glycolysis[6],mitochondrial metabolism[5],autophagy[7]and so on.Under the insulin stimulation,knockdown of FOXK2 might lead to the upregulation of apoptosis related genes[5].It was reported that the CDK1-cyclin B kinase complex phosphorylated the Ser368 and Ser423 sites of FOXK2,and inhibited cell apoptosis by regulating FOXK2 protein stability[8].It was also shown that FOXK2 promoted the apoptosis of clear-cell renal cell carcinoma by regulating the epidermal growth factor receptor(EGFR)[9].Recent research revealed that CHK2 phosphorylated FOXK1 and FOXK2 under the stimulation of DNA damage,led to a increase of autophagy and a decrease of apoptosis in tumor cells[10].Therefore,it could be summarized that both FOXK2 protein itself and its phosphorylation modification played an important role in tumor cells apoptosis.Next we want to explore whether other protein post-translational modifications of FOXK2 similarly affect the apoptosis of tumor cells.As one of the important post-translational modification mechanisms,the acetylation modification plays a key role in cell cycle,DNA damage repair,metabolism,apoptosis,autophagy and other cellular activities[11,12].Multiple studies have shown that FOXK2regulated cell functions by the post-translational modifications of phosphorylation and SUMOylation.However,the acetylation of FOXK2 has not been reported.In this study,we first discovered that FOXK2 was an acetylated protein,and its deacetylase was SIRT1.The Sirtuin family of deacetylases plays an important role in aging-related diseases.The deacetylase activity of SIRT1 is nicotinamide adenine dinucleotide(NAD)dependent.SIRT1 plays important post-translational modification[17]mediated regulation roles in DNA damage repair[13],stress response[14],cell cycle[15]and mitochondrial metabolism[16].However,the molecular mechanism of SIRT1 mediated FOXK2 deacetylation to affect the chemosensitivity of tumor cells remains largely unknown.This is also the key scientific problem to be solved in this study.We speculate that the acetylation status of FOXK2 may affect the chemosensitivity of tumor cells,which will provide new ideas and targets for solving the chemoresistance.Methods:1.Study on FOXK2 acetylation.1.1 To explore whether FOXK2 can be acetylated in cells by immunoprecipitation and Western blot.1.2 The four acetyltransferases were overexpressed in the cells,and the acetyltransferase of FOXK2 was determined by immunoprecipitation and Western blot;To detect whether the two proteins interact in vitro by immunoprecipitation.1.3 SIRT1 was overexpressed in the cells,to confirm the deacetylase of FOXK2 by immunoprecipitation and Western blot;To detect whether the two proteins interact in vitro by immunoprecipitation;To detect whether the two proteins co-localize in the cells by immunofluorescence and confocal laser scanning microscope.1.4 By using immunoprecipitation,Western blot,and mass spectrometry,the acetylation site of FOXK2 was determined;To construct wild-type and mutated-type plasmids of FOXK2 acetylation site by molecular cloning.To determine the effect of this site on the level of FOXK2 acetylation in 293T and H1299.2.To detect the molecular mechanism of SIRT1 mediated FOXK2 deacetylation under the stimulation of DNA damage.2.1 Under Cisplatin stimulation,the changes on the interaction between FOXK2 and SIRT1 were detected by immunoprecipitation and Western blot.2.2 Under Cisplatin stimulation,the changes on the acetylation of FOXK2 were detected by immunoprecipitation and Western blot.2.3 Under Cisplatin stimulation,the nuclear distribution changes of the acetylated wild-type and dead-type FOXK2 were detected by protein nuclear-plasma separation,Western blot and immunofluorescence.2.4 Under Cisplatin stimulation,to detect whether the acetylation level of FOXK2depends on SIRT1 deacetylase activity by immunoprecipitation and Western blot.3.To construct H1299?HCT116 and Hela cell lines with FOXK2 stable knockdown by using lentiviral infection,and to detect the knockdown efficiency by Western blot;To further construct cell lines with stable overexpressed FOXK2-WT and FOXK2-K223R on the basis of the knockdown cell lines.4.To detect the effect of FOXK2 acetylation status on the chemosensitivity of tumor cells.4.1 Under the action of chemotherapy drugs,the CCK-8 cytotoxicity assay was performed to detect the survival difference in the tumor cell lines of sh FOXK2-ov NC,sh FOXK2-ov WT,and sh FOXK2-ov K223R.4.2 Under the action of chemotherapy drugs,flow cytometric analysis was performed to detect the apoptosis difference in the tumor cell lines of sh FOXK2-ov NC,sh FOXK2-ov WT,and sh FOXK2-ov K223R.4.3 Under the action of chemotherapy drugs,Western blot was performed to detect the protein expression differences of Cleaved-PARP and Cleaved-caspase3 in the tumor cell lines of sh FOXK2-ov NC,sh FOXK2-ov WT,and sh FOXK2-ov K223R.4.4 By tumor xenograft assay,the difference in chemosensitivity of the tumor cell lines of sh FOXK2-ov WT and sh FOXK2-ov K223R was detected in vivo.Results:1.FOXK2 was an acetylated protein.Its acetylase was CBP and its deacetylase was SIRT1.FOXK2 interacted with CBP and SIRT1 in vitro;Confocal laser scanning microscope showed that FOXK2 and SIRT1 co-localized in the nuclear.2.The mass spectrometry analysis results showed that the acetylation site of FOXK2 was Lys223;The wild-type and dead-type vector plasmids of the FOXK2acetylation site were successfully constructed.The acetylation level of the dead-type FOXK2 protein was significantly reduced in cells..3.Under the stimulation of DNA damage,the interaction between FOXK2 and SIRT1 was weakened;The acetylation of FOXK2 was enhanced and the distribution in the nucleus was significantly reduced;The immunofluorescence results showed that the nuclear distribution of dead-type FOXK2 at acetylation site was not reduced;The acetylation level of FOXK2 depended on SIRT1 deacetylase activity.4.H1299?HCT116 and Hela cell lines with FOXK2 stable knockdown were successfully constructed,and the knockdown efficiency was high;The cell lines with stable overexpressed FOXK2-WT and FOXK2-K223R on the basis of the knockdown were successfully constructed.5.Under the action of chemotherapy drugs,FOXK2 promoted the apoptosis of H1299?HCT116 and Hela cells;The acetylation state of FOXK2 promoted the apoptosis of H1299?HCT116 and Hela cells,and the dead-type FOXK2(deacetylated state)at the acetylation site could rescue the apoptosis of these tumor cells.Conclusion:1.For the first time,we discovered that the forkhead protein FOXK2was an acetylated protein,and SIRT1 deacetylated FOXK2 through K223.2.Under the stimulation of DNA damage,the interaction of FOXK2 and SIRT1 was weakened,resulting in the increased acetylation of FOXK2 and the decreased nuclear distribution.3.The acetylation state of FOXK2 enhanced the chemosensitivity of tumor cells,while the deacetylation state of FOXK2 weakened the chemosensitivity of tumor cells.Our research is expected to find new targets for tumor chemoresistance,and to provide a theoretical basis for solving the chemoresistance problem. |
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