The Research Of Expression And Mechanism Of PKMYT1 In Esophageal Squamous Cell Carcinoma

Background and objectivesMalignant tumor is an important risk factor threatening human life and health,and esophageal cancer is a major component of malignant tumor.At present,esophageal cancer is the ninth most common malignant tumor in the world,and it is also the sixth leading cause of cancer-related death.According to the pathological classification,esophageal cancer can be divided into esophageal squamous cell carcinoma and esophageal adenocarcinoma.China is one of the regions with the highest incidence of esophageal cancer in the world.In China,most of the patients with esophageal cancer are esophageal squamous cell carcinoma.Although multidisciplinary diagnosis and treatment is making continuous progress,including the improvement of new surgical methods,molecular targeted therapy,chemotherapy and radiotherapy,the five-year survival rate of esophageal cancer is still poor.Therefore,it is necessary and urgent to explore the carcinogenesis mechanism of esophageal cancer,which may provide new targets and breakthroughs for the treatment of esophageal cancer.Membrane-associated tyrosine/threonine 1 protein kinases(PKMYT1),a member of the Wee protein kinase family,play a negative regulatory role in the cell cycle by preventing cells from entering the mitotic phase in the G2/M phase.At present,the high expression of PKMYT1 has been found in many kinds of malignant tumors,but its mechanism in esophageal cancer is not clear.The purpose of this study is to explore the effect of PKMYT1 expression on the prognosis of patients with esophageal squamous cell carcinoma,its group role in the progression of esophageal cancer and the related mechanisms involved,so as to provide new ideas for the treatment of esophageal cancer.Methods1.By analyzing the data of esophageal cancer in the Gene Expression Profiling Interactive Analysis(GEPIA),the difference of PKMYT1 mRNA expression in cancer and paracancerous tissues was compared.At the same time,the correlation between PKMYT1 and related gene expression was analyzed by using its gene map information.2.The database of patients was established by collecting clinical fresh specimens and pathological sections,and the mRNA and protein expression of PKMYT1 in patients with esophageal cancer was analyzed by fluorescence quantitative PCR and immunohistochemistry.Through the collection of clinical data and follow-up,to explore the relationship between esophageal squamous cell carcinoma and the prognosis of patients with esophageal squamous cell carcinoma.3.Fluorescence quantitative PCR was used to detect the expression of PKMYT1 in esophageal squamous cell carcinoma cell lines and normal esophageal cells,and the esophageal squamous cell carcinoma cell lines with higher expression were selected for further study.4.RNAi interference technique was used to knock down the expression of PKMYT1 in esophageal cancer cell lines,and the interference effect was detected by fluorescence quantitative PCR and Western blot.5.After the decreased expression of PKMYT1 in esophageal cancer cell line,the changes of proliferation ability of esophageal cancer cells were studied by CCK8 and cell cloning assay.The changes of anti-apoptosis ability of esophageal cancer cells were detected by flow cytometry and Western blot was used to detect the changes of apoptosis-related proteins.6.The changes of motility and invasion of esophageal cancer cells were verified by scratch test and cell migration and invasion test.The expression of related proteins was detected by Western blot and immunofluorescence assay.Results1.In GEPIA database,the expression of PKMYT1 in esophageal carcinoma was significantly higher than that in adjacent tissues.At the same time,the expression of PKMYT1 was strongly correlated with the expression of AKT,TWIST and Ki67.2.The expression of PKMYT1 in esophageal squamous cell carcinoma was significantly higher than that in adjacent tissues(p<0.01).At the same time,patients with high expression of PKMYT1 had shorter disease-free survival and survival time than those with low expression.Based on the analysis of the clinical information of patients with esophageal squamous cell carcinoma,it was found that there was a significant relationship between PKMYT1 and lymph node metastasis,tumor differentiation grade and pathological stage of esophageal cancer.At the same time,univariate and multivariate analysis by Cox regression analysis showed that the expression of PKMYT1 was an independent risk factor for the prognosis of patients with esophageal squamous cell carcinoma.3.Through the analysis of the expression of PKMYT1 in esophageal squamous cell carcinoma cell lines and esophageal immortalized epithelial cells,it was found that the expression of PKMYT1 in esophageal squamous cell carcinoma lines was higher than that in normal esophageal cells,and the expression level was the highest in KYSE70 and KYSE450 cells.These two cell lines were selected for further experiment.4.After transfection of KYSE70 and KYSE450 cells by siRNA,it was found that the expression of PKMYT1 in the treated cells decreased.5.In esophageal cancer cell lines,the decreased expression of PKMYT1 inhibits cell proliferation,reduces the proportion of G2/M phase cells in esophageal cancer cells,and promotes cell apoptosis.6.The decreased expression of PKMYT1 inhibited the proliferation and invasion of esophageal cancer cells.The results of Western blotting and immunofluorescence assay showed that the expression of neuro-cadherin(NCAD),twisted protein(TWIST)and vimentin(VIM)decreased,while the expression of epithelial cadherin(ECAD)increased,indicating that the Epithelial-mesenchymal transition(EMT)of esophageal cancer cells was reversed.At the same time,we also detected the expression of AKT/mTOR signal pathway by Western blot,and found that the expression of AKT/mTOR pathway was also inhibited.ConclusionThere is a differential expression of PKMYT1 in esophageal squamous cell carcinoma and adjacent tissues,and the high expression of PKMYT1 is closely related to the poor prognosis of patients with esophageal squamous cell carcinoma.Reducing the expression level of PKMYT1 at the cellular level can inhibit the expression of AKT/mTOR signaling pathway,thus inhibit the proliferation of esophageal cancer cells and promote their apoptosis;at the same time,inhibit the migration and invasion of esophageal cancer cells,reverse the process of EMT,and weaken the function of esophageal cancer cells.PKMYT1 may be a new potential target in the treatment of esophageal squamous cell carcinoma.