The Effect Of PKMYT1 On Radiosensitivity Of Lung Adenocarcinoma

Objective:Lung adenocarcinoma is one of the most common types of lung cancer,and its incidence is increasing significantly.Radiation therapy is an effective treatment method for lung adenocarcinoma.However,different patients with lung adenocarcinoma may show different degrees of radiation resistance,which affects the effect of radiotherapy and leads to treatment failure.Therefore,the development of radiosensitizers for lung adenocarcinoma is of great significance for the clinical treatment of lung adenocarcinoma.The purpose of this study was to screen out core genes related to the prognosis of lung adenocarcinoma by bioinformatics methods and then select a potential gene from these core genes,which may be a potential radiosensitivity target for lung adenocarcinoma and to performed basic experiments to verify it.Methods:1.Select the lung adenocarcinoma gene chips GSE32863,GSE33532 and GSE43458 from the GEO website.GEO2R was used to screen genes that were up-regulated in lung adenocarcinoma tissue compared to normal lung tissue adjacent to the cancer in each chip,and then Venn was used to screen genes that were up-regulated in three datasets.Perform Gene Ontology?GO?and Kyoto Encyclopedia of Gene and Genome?KEGG?analysis of these genes on the DAVID website.Use STRING to obtain the protein-protein interaction network?PPI?between genes and then use Cytoscape to check the potential correlations between these genes and use MCODE to analyze important modules in the protein interaction network to obtain core genes.Kaplan Meier-plotter was used to analyze the effect of core genes on the survival of patients with lung adenocarcinoma.GEPIA website was used to analyze the differential expression of core genes between lung adenocarcinoma tissue and normal lung tissue.2.Collect 30 pairs of lung adenocarcinoma tissues and adjacent normal lung tissues that underwent surgery in the First Affiliated Hospital of China Medical University.Western blot and Quantitative PCR?Q-PCR?experiments were used to detect the expression difference of PKMYT1 in lung adenocarcinoma and adjacent tissues.Kaplan-Meier was used to analyze the relationship between the expression of PKMYT1 and the survival prognosis and clinical characteristics of patients with lung adenocarcinoma.Lung adenocarcinoma cell lines A549,H1299,H1975,H441,and H1650 were cultured in vitro.Western blot was used to detect the expression of PKMYT1 in five cell lines.The H1299and A549 cell lines with the highest PKMYT1 expression were selected for subsequent experiments.Plasmid transfection reduced the expression of PKMYT1 in H1299 and A549 cells.Western blot and Q-PCR experiments were used to detect the expression of PKMYT1 in the two cells after transfection.The effects of reducing PKMYT1expression on the radiosensitivity of tumor cells were tested by clone formation experiments,and the mechanism of PKMYT1 affecting the radiosensitivity of tumor cells was investigated by examining the effect of reducing PKMYT1 expression on cell cycle at 0 and 4Gy doses.Results:1.This study used bioinformatics to screen 14 genes?PKMYT1,TTK,CHEK1,CDC20,PTTG1,MCM2,CDC25C,MCM4,CCNB1,CDC45,MAD2L1,CCNB2,BUB1 and CCNA2?from lung adenocarcinoma chips GSE33532,GSE43458 and GSE32863,which are closely related to the poor prognosis of lung adenocarcinoma?P<0.05?.These 14 genes may be viable prognostic biomarkers and potential therapeutic targets for lung adenocarcinoma.After understanding literature studies related to these 14genes,we found that PKMYT1 is a target that inhibits G₂ checkpoints and mitotic disasters,so PKMYT1 is likely to be a potential target for radiosensitivity.However,no research has explored the relationship between PKMYT1 and tumor radiation sensitivity.Therefore,we chose PKMYT1 as the research gene to carry out subsequent basic experiments to observe its effect on tumor radiosensitivity.2.Western blot and Q-PCR results of lung adenocarcinoma tissue samples showed that the expression of PKMYT1 in lung adenocarcinoma tissue was significantly higher than that in normal lung tissue adjacent to the cancer?P<0.05,P<0.05?.Combined with the previous bioinformatics analysis results,we can conclude that PKMYT1 is highly expressed in lung adenocarcinoma,which may be closely related to the occurrence and development of lung adenocarcinoma.The high expression of PKMYT1 is significantly associated with poor overall survival?OS?and progression-free survival?FPS?in male,female,M0 stage,smoking and surgical margins negative lung adenocarcinoma patients.Based on the results of Western blot,we selected H1299 and A549 with the highest PKMYT1 expression for transfection of the PKMYT1 silent plasmid.Western blot and Q-PCR results showed that the expression of PKMYT1 in the cells transfected with the silenced plasmid was significantly reduced?P<0.05,P<0.05?.The results of the colony formation experiments showed that tumor cells that reduced PKMYT1 expression were more sensitive to radiation?P<0.05?.The results of cell cycle experiments showed that after decreasing the expression of PKMYT1,the percentage of G₂-M phase cells in the A549 and H1299 cell cycle was reduced compared with the control group?P<0.05?,and the proportion of G₂-M phase cells after irradiation was also significantly reduced,eliminating G₂-M blocking caused by radiation?P<0.05?.Conclusion:This study used bioinformatics analysis to identify 14 important genes?PKMYT1,TTK,CHEK1,CDC20,PTTG1,MCM2,CDC25C,MCM4,CCNB1,CDC45,MAD2L1,CCNB2,BUB1 and CCNA2?associated with poor prognosis of lung adenocarcinoma.These 14 genes may be viable prognostic biomarkers and potential therapeutic targets for lung adenocarcinoma.This study demonstrated for the first time that reducing the expression of PKMYT1 can eliminate radiation-induced G₂-M block and increase the radiosensitivity of tumor cells.