| Objective:(1)To investigate the molecular mechanism of lactate regulating the proliferation and metastasis of colon cancer cells by mediating p53 lactylation,and to provide a new idea for anti-tumor therapy targeting lactate metabolism.(2)To investigate the influence of lactylation site K291 on the nuclear translocation and stability of p53 protein,and to provide a new idea for the functional study of protein lactylation site.Methods:(1)Human colon cancer tissues and corresponding paracancer tissues were collected,and the pathological morphology was confirmed by hematoxylin-eosin staining(HE).Lactate levels in human colon cancer tissues and paracancer tissues were detected by lactate detection kit.Protein lactylation expression in colon cancer tissues was detected by Western blot.The expression of protein lactylation and p53 in colon cancer tissues were detected by immunohistochemical staining.Localization of lactylation and p53 in colon cancer tissues was observed by immunofluorescence staining.The presence of p53 lactylation in colon cancer tissue was detected by immunoprecipitation.(2)According to different treatment methods,the colon cancer cells were divided into the following groups: low glucose condition group(5 m M glucose culture system),high glucose condition group(25 m M glucose culture system),lactic acid group(20m M),sodium lactate group(20 m M),Oxamate group(20 m M)and 2-DG group(20m M).Lactate levels was detected by lactate detection kit,proliferation ability of colon cancer cells was detected by CCK-8,migration and invasion ability of colon cancer cells were detected by scratch test and Transwell test。Protein lactylation expression in colon cancer cells was detected by Western blot.The presence of p53 lactylation in cells was detected by immunocoprecipitate,and the expression of p300 protein was detected by Western blot.In addition,p300 inhibitor(C646)was added into the 25 m M glucose culture system of colon cancer cells,and the change of the p53 lactylation in the cells was detected by immunoprecipitation.(3)According to different treatment methods,the colon cancer cells were divided into three groups: low sugar condition group(5 m M glucose culture system),high sugar condition group(25 m M glucose culture system)and Oxamate group(20 m M).The subcellular localization of lactylation and p53 in colon cancer cells was observed by immunofluorescence staining.In addition,cytoplasmic protein and nuclear protein were extracted by nuclear plasma separation kit,and the expression of p53 was detected by Western blot.Based on these groups,colon cancer cells were treated with actinomycin(CHX).The cells were collected at 0,1,2 and 4 h time points to extract protein,and the expression of p53 was detected by Western blot.In addition,based on the same groups,colon cancer cells were treated with MG-132,the cells were collected at 0,1,2 and 4 h time points to extract protein,and the expression of p53 was detected by Western blot.(4)The p53 protein was enriched and purified in the high glucose and lactate groups of colon cancer cells by immunoprecipitation,and then the lactylation modification sites of p53 protein were analyzed qualitatively and semi-quantitatively by LC-MS/MS and database search.Subsequently,wild-type p53 plasmids(wild group)and mutant plasmids with Flag tags(mutant group)were constructed and overexpressed in cells.The Flag-p53 lactylation in wild group and mutant group was detected by immunoprecipitation.Transwell assay was used to detect cell migration and invasion in wild and mutant groups.The subcellular localization of lactylation and Flag-p53 in wild and mutant cells was observed by immunofluorescence staining.Wild group and mutant group were treated with actinomycin(CHX).Cell proteins were extracted at 0and 4 h time points,and the expression of Flag-p53 was detected by Western blot.In addition,wild group and mutant group cells were treated with MG-132.Cell proteins were extracted at 0 and 8 h time points,and the expression of Flag-p53 was detected by Western blot.Results:(1)Compared with the corresponding paracancular tissues,human colon cancer tumor tissues showed cancerous cell morphology,and lactate levels were significantly increased.The protein lactylation level of colon cancer tumor cells was significantly increased,and the p53 level was also increased.The co-localization of lactylation and p53 appeared in colon cancer tumor tissue.In addition,p53 lactylaiton is present in colon cancer tumor tissues.(2)Compared with the low glucose group,the protein the lactate level,cell proliferation ability,migration ability and invasion ability of colon cancer cells in the high glucose group were significantly increased.Compared with the high glucose group,the cells of the Oxamate group and the 2-DG group showed decreased proliferation,migration and invasion ability.Protein lactylation level and p53 lactylation level in high glucose group,lactic acid group and sodium lactate group were significantly increased.The level of p300 protein in colon cancer cells increased slightly in the high glucose condition group.The level of protein lactylation,the level of p53 lactylation and the level of p300 protein in the colon cancer cells of Oxamate group and 2-DG group were significantly decreased.In addition,the level of p53 lactylation was also significantly decreased in the C646 group.(3)The p53 protein in the cells of the low glucose group was mainly located in the nucleus,the p53 protein in the cells of the high glucose group was mainly located in the cytoplasm,and the p53 protein in the cells of the Oxamate group was mainly located in the nucleus,but there was no significant difference in the subcellular localization of the lactylation protein in the three groups.Compared with the low glucose group and Oxamate group,the stability of p53 protein in the cells of the high glucose group was significantly reduced,and the increased degradation of p53 protein in the cells of the high glucose group occurred through the proteasome pathway.(4)LC-MS/MS analysis showed that there were multiple lactylation modification sites in p53 protein,and these sites were all on lysine residues,among which the lactylation modification at K291 and K370 site was the most abundant.The wild-type p53 plasmid and mutant p53 plasmid with Flag tag could be stably overexpressed in cells.Compared with the wild group,the Flag-p53 lactylation in the mutant group was significantly decreased.Compared with the wild group,the number of migrating cells in the mutant group was significantly reduced.In the wild group,Flag-p53 protein was mainly located in the cytoplasm,while in the mutant group,Flag-p53 protein was increased in the nucleus.The stability of Flag-p53 protein in wild group and mutant group did not change significantly at 0～4 h.In addition,there was no significant change in the degradation of Flag-p53 protein by proteasome pathway between the wild group and the mutant group at 0～8 h.Conclusion: Lactate promotes the proliferation and metastasis of colon cancer cells by mediating p53 lactylation.p53 lactylation at K291 site causes p53 to accumulate in the cytoplasm and decrease in the nucleus. |
