Molecular Mechanism Of CHK2 Lactylation Regulating DNA Damage Response

Objective:Lung cancer is a malignant tumor with high morbidity and mortality worldwide,among which non-small cell lung cancer(NSCLC)accounts for about85%,with poor prognosis.At present,although great progress has been made in lung cancer surgery,radiotherapy,chemotherapy and molecular targeted therapy,the therapeutic effect is still not ideal,and the emergence of acquired drug resistance seriously hinders the therapeutic effect of lung cancer.DNA damage can affect the cell cycle,and checkpoint proteins can block the cell cycle and provide the repair time for DNA repair.DNA damage exceeds the repair ability and starts the apoptosis pathway.CHK2 is a key regulator of DNA damage response,an important element in its signal transduction pathway,and an important supervisor of various cell survival and disease processes,participating in important life activities such as apoptosis,cell cycle arrest and DNA damage repair.Lactylation modification is an important and novel post-translational modification of proteins.However,it has not been reported whether non-histone undergo lactylation modification.This topic is mainly to study whether non-histone CHK2 lactylation modification is involved in the regulation of cell drug resistance as the research object,and to study the role of a new protein post-translational modification-lactylation modification change.Method: 1.Flow cytometry was used to investigate the influence of sodium lactate treatment on Cisplatin induced apoptosis.After treatment with NALA(20mmol/L)and Cisplatin(30μmol/L)for 24 hours,samples were collected and divided into negative control group,PI group,FITC group and FITV-PI double-staining group.After treatment with the apoptosis kit,the cells were fully reacted at room temperature without light for 15 min.1xbinding buffer was added to detect cell apoptosis within 1 hour.2.Immunofluorescence technology was used to study whether sodium lactate and lactate dehydrogenase inhibitor affected the cell cycle.Cell slippage was placed in 6-well plates,and drug treatment was added after cell adherence.Lactate and lactate dehydrogenase inhibitor were added for 12 h,Cisplatin30μmol/L was added for 12 h,the medium was sucked clean after washing with PBS,the drug was withdrawn,cells were collected for 2h,antibody was fixed and applied overnight,and the proportion of p-h3 positive cells was observed under microscope after treatment the next day.3.Clone colony assay was used to verify whether sodium lactate and lactate dehydrogenase inhibitors could kill cisplatin in lung cancer cells.Cisplatin(0,0.5,1,1.5μmol/L),sodium lactate(10mmol/L)and lactate dehydrogenase inhibitor(10mmol/L)were added in the experiment,respectively.The number of clone colony cells was observed after 7-9 days of treatment,and the number of each clone cell was > 50 tractable plates.After fixed staining,photos were taken and image-J count analysis was performed.4.Co-ip and Western blot were used to test whether lactylation modification existed.293 T cells were transfected with CHK2,sodium lactate was 20mmol/L and lactate dehydrogenase inhibitor was 10mmol/L.After 24 hours of treatment,samples were collected for immunoprecipitation co-IP test and WB test to determine whether there was lactylation modification in CHK2.At the same time,after the successful construction of stable Sh LDHA2/B1 and Sh LDHA2/ B3 strains,CHK2 transfection was carried out to construct stable cell lines with LDHA and LDHB knockdown using specific sh RNA to determine the effect of lactic acid on THE lactation of CHK2.After 48 hours,the samples were collected for co-IP immunoprecipitation test,and the samples were added for WB test5.Western blot assay was used to explore the CHK2-mediated signaling pathway regulated by sodium lactate and lactate dehydrogenase inhibitors.NALA 20mmol/L,OX 10mmol/L,DNA damage inducer Cisplatin30μmol/L were added to 6-well plate cells after adherence,and samples were collected for 24 hours for wb experiment to detect key molecules of signaling pathway CHK2 and p53.Meanwhile,phosphorylated P-CHK2,P-p53 and Cleave PARP protein expression levels were detected6.Co-ip and Western blot were used to screen and identify lactylation modification enzymes and de-lactylation modification enzymes of CHK2.HDAC inhibitor TSA(1:1000)and SIRT family inhibitor NAM(1:100)were added to the cells for 24 hours after the cell plate was pasted to the wall,and then the samples were collected for immunoprecipitation experiment co-IP experiment and wb experiment.According to the analysis of the results,the type of eraser in the Lactylation modification of CHK2 was determined.Then,SIRT family plasmid and CHK2 plasmid were transfected into 293 T cells.After 48 hours,the cells were collected for immunoprecipitation experiment,co-IP experiment and then WB experiment.At the same time,the acetyltransferase library plasmid and CHK2 plasmid were transfected into 293 T cells,and the cells were collected 48 hours later for immunoprecipitation experiment,co-IP experiment and wb experiment.293 T cells were added with CBPi(1:1000 concentration)for 24 hours after they were affixed to the wall for co-IP and Western blot assay.Sh CBP stable was constructed and transfected into CHK2 48 hours later,the cells were recovered for Co-IP and Western blot assay.7.Screening and identifying the Lactylation modification sites of CHK2.After plasmid construction and point mutation design and synthesis of primers,PCR amplification,transformation,enzyme linking,DNA glue recovery,transfection,immunoprecipitation and Western blot were used to search for Lactylation modification points using DNA target plasmid as template.8.Western blot was used to explore the downstream signaling pathway of CHK2 after the Lactylation modification site mutation.A stable lung cancer cell line carrying CHK2 wild-type and K492/494 R mutation was constructed.NALA(20mmol/L)and Cisplatin(30μmol/L)were added after cell lamination and fixation,and the protein was recovered 24 hours after addition for WB experiment.The downstream molecules of P53,P-53 and Cleave PARP were detected.9.Use immunofluorescence technology to explore the effect of Lactylation modification on regulating cell cycle.Use the constructed lung cancer cell line carrying CHK2 wild type and K492/494 R mutation to stabilize the lung cancer cell line.Lactate or lactate dehydrogenase inhibitor was added for 12 h,and DNA damage inducer Cisplatin30μmol/L was added.After 12 h,the medium was aspirated,washed with PBS,the drug was withdrawn,and the cells were collected after adding ordinary medium for 2h.10.CCK-8 experiment was used to explore the effect of lactic acid modification of CHK2 on chemotherapy.The Cisplatin concentration was 0,20,40,60,80μmol/L and the Cisplatin concentration was 0,20,40,60,80μmol/L and the Cisplatin concentration was 0,20,40,60,80μmol/L.After 2-3 days of dose-adding,CCK8 kit was used to process the 4-hour on-machine detection results,and the TGCA database could be collected to analyze the influence of CHK2 on lung cancer survival rate.Result:1.Flow cytometry showed that the apoptosis rate of cisplatin + sodium lactate group was lower than that of cisplatin alone,indicating that sodium lactate treatment inhibited cisplatin induced apoptosis.2.Immunostaining for anti-phosphorylated histone 3-H3SER10 antibody(P-H3)showed that sodium lactate treatment resulted in decreased G2/M test site function and increased positive cells,while lactate dehydrogenase inhibitor treatment resulted in enhanced G2/M test site function,resulting in cell cycle arrest and reduced cells entering mitotic phase.These results indicate that sodium lactate and lactate dehydrogenase inhibitors affect G2/M cell cycle arrest.3.Sodium lactate can significantly enhance the resistance of lung cancer cells to chemotherapy drug cisplatin,and lactate dehydrogenase can significantly improve the killing effect of chemotherapy drug cisplatin on lung cancer cells,indicating the effect of sodium lactate and lactate dehydrogenase inhibitors on cisplatin killing lung cancer cells.4.As a non-histone protein,CHK2 can undergo Lactylation modification.Meanwhile,specific sh RNA was used to construct LDHA and LDHB knockdown stable cell lines,and CHK2 was transferred into these stable cell lines to detect the lactate level of CHK2.It was found that the combination of LDHA and LDHB significantly down-regulated the lactate level of CHK2,again indicating that the Lactylation modification of CHK2 could occur.5.Sodium lactate inhibited cisplatin induced CHK2-mediated P-p53 level and cleave PARP protein level,indicating that sodium lactate inhibited CHK2 activity and inhibited apoptosis.Lactate dehydrogenase inhibitors promote Cisplatin induced CHK2-mediated P-p53 levels and cleave PARP protein levels,suggesting that lactate dehydrogenase inhibitors promote CHK2 activity and apoptosis.The results showed that sodium lactate and lactate dehydrogenase inhibitors regulate the CHK2-mediated signaling pathway.6.The deacetylase inhibitors NAM(SIRT family inhibitors)and TSA(HDAC inhibitors),as well as the screening of acetyl transferase libraries and deacetylase libraries,showed that CBP had the activity of lactoyltransferase,while SIRT1 and 2had the activity of lactoyltransferase,which promoted the Lactylation modification and delactate modification of CHK2,respectively.The addition of NAM,a SIRT family inhibitor,increased the Lactylation modification level of CHK2,suggesting that the SIRT family was involved in regulating the Lactylation modification level of CHK2.The addition of CBP inhibitor CBPi showed that the lactate modification enzyme level of CHK2 decreased.The sh CBP stable strain was constructed,and cells were collected 48 hours after transfection with CHK2 for Co-IP and Western blot experiments.The Lactylation modification enzyme level of CHK2 cells in sh CBP stable strain decreased,confirming again that CBP was the lactate modification enzyme of CHK2.These results indicate that eraser of CHK2 is SIRT1/2,and writer of CHK2 is CBP.7.Plasmid construction and point mutation design were used to screen thelactate modification sites of CHK2,and K492/494 R was identified as the main lactate modification site of CHK2.8.The addition of sodium lactate to wild-type CHK2 induced by chemotherapy drug Cisplatin can decrease the protein levels of P-p53 and Cleave PARP,indicating that sodium lactate can affect the CHK2 signaling pathway by affecting the Lactylation modification level,and the enhancement of lactate modification level can inhibit cell apoptosis by inhibiting the function of CHK2.In k492/494 R mutant cells,we found that sodium lactate could not regulate the lactate level of the CHK2 Lactylation modification site,and had almost no effect on p-p53 and Cleave PARp protein levels after chemotherapy drug induction,and did not cause the decrease of p-p53 and Cleave PARP protein levels.9.Cells carrying CHK2 K492/494 R mutation increased cisplatin-induced G2/M cell cycle arrest and reduced mitotic cells;Sodium lactate inhibited G2/M cell cycle arrest induced by CISplatin in CHK2 wild-type cells,but did not inhibit G2/M cell cycle arrest induced by cisplatin in CHK2 K492/494 R mutant cells.Lactate dehydrogenase inhibitors enhanced G2/M cell cycle arrest induced by CHK2wild-type cisplatin,but did not further enhance G2/M cell cycle arrest in cells carrying CHK2 K492/494 R mutation,suggesting that CHK2 lactate modification regulates G2/M checkpoint.10.Compared with wild-type cells,cells carrying CHK2 KR mutation were more sensitive to chemotherapy drug cisplatin.Sodium lactate could significantly improve the tolerance of wild-type lung cancer cells to chemotherapy drugs,but had no significant effect on cells carrying CHK2 KR mutation.Lactate dehydrogenase inhibitors improve the killing effect of cisplatin on CHK2 wild-type lung cancer cells,but have no obvious effect on CHK2 KR mutant cells.These results indicate that CHK2 lactylation modification has a certain effect on the chemotherapy effect of tumor cells.The TCGA database analysis found that high expression CHK2 can significantly improve the survival of patients.Conclusion : CHK2 Lactylation modification can inhibit CHK2 activation,inhibit apoptosis and cell cycle arrest,and promote the tolerance of lung cancer cells to chemotherapy drugs.