| BackgroundEsophageal cancer(EC)ranks 8th among the most common malignant tumors in the world.It is a kind of malignant tumor known for its high incidence and high fatality rate.The mortality rate of esophageal cancer ranks sixth in the world.About 300000 people die of esophageal cancer every year.In China,the incidence of esophageal cancer ranks eighth in the incidence of malignant tumors,and about 150000 people die from esophageal cancer every year.The main pathological classification of esophageal cancer in China is esophageal squamous cell carcinoma(ESCC).The occurrence of esophageal cancer is a multi-factor process,as well as the characteristics of malignant occurrence,poor prognosis and low survival rate of esophageal cancer,which makes the clinical diagnosis and treatment of esophageal cancer even more difficult.Fundamental research around the occurrence and development of esophageal squamous cell carcinoma,genomics and proteomics have been applied to conduct a comprehensive study of esophageal squamous cell carcinoma,such as screening of genetic genes related to the occurrence and development of esophageal squamous cell carcinoma,epigenetic regulation of lncRNA,miRNA and methylation,protein molecules related to the occurrence and development of esophageal squamous cell carcinoma and screening of biological targets for clinical treatment.However,the feasibility and effectiveness of these studies in diagnosis and treatment are still not satisfactory.Therefore,it is one of the focuses of efforts to find more effective and feasible early diagnosis targets and molecular therapy targets for esophageal squamous cell carcinoma.Cyclin-dependent kinase 16(CDK16),also known as PCTAIRE1,is a member of the cyclin-dependent kinase family.Early studies of CDK16 have found that its main physiological functions are involved in vesicular transport,synaptic growth and spermatogenesis.It is gradually found that the high expression of CDK16 in solid tumor cells such as lung cancer,ovarian cancer,liver cancer,etc.can cause abnormal regulation of the cell cycle of tumor cells,enhanced proliferation ability,weakened apoptosis ability,and tumor cell radiotherapy tolerance.Studies have found that CDK16 can interact with p53 and p27,thus promoting tumor progression.These evidences suggest that cdk16 may be related to the occurrence and development of tumor.It is a new perspective on which specific regulatory mechanism of CDK16 exists in tumors.In the preliminary screening of genes relat to esophageal squamous cell carcinoma,large-scale screening is conducted for members of the CDK family using the database,and immunohistochemical experimental data show that CDK16 may play a role in the occurrence and progression of esophageal squamous cell carcinoma.And through the relevant literature research,it is found that the study of CDK16 in esophageal cancer has not been reported.Therefore,in order to further understand the role of CDK16 in the occurrence and development of esophageal squamous cell carcinoma,firstly,we discussed the relationship between the expression of CDK16 in the pathological tissues of esophageal squamous cell carcinoma and its clinical characteristics;then,we discussed whether CDK16 is involved in the cell biological processes such as the growth,proliferation and migration of esophageal squamous cell carcinoma.Finally,the proteins that CDK16 may interact with are explored by bioinformatics analysis.This study explores the pathogenesis of esophageal squamous cell carcinoma from a new point of view,and provides relevant basis for clinical treatment or prognosis evaluation of esophageal squamous cell carcinoma.MethodPart I Expression of CDK16 in esophageal carcinoma and its relationship with clinicopathological features of esophageal carcinoma1.CBioPortal databaseis is used to analyze the expression of CDK16 mRNA in esophageal cancer and the gene mutation of CDK16 gene in esophageal cancer.2.Immunohistochemistry is used to detect the expression of CDK16 protein in esoph ageal squamous cell carcinoma and adj acent tissues.Chi-square test is used to ana lyze the relationship between CDK16 protein and histopathological features of es ophageal squamous cell carcinoma.Part ? Effects of CDK16 knock-down on the growth,proliferation and migration of esophageal squamous cell carcinoma.1.qPCR and Western Blot techniques are used to detect the expression of CDK16 mRNA and protein in EC-1,Eca-109,TE-1,KYSE-30,KYSE-150,KYSE-450,KYSE-510 esophageal squamous cell carcinoma cell lines and N1217 normal esophageal epithelial cells,and to screen the cell lines with high expression of CDK16 mRNA and protein compared with normal esophageal epithelial cells.2.CDK16 siRNA transient transfection is used to knock down CDK16,in EC-1 and KYSE-510 cells to study the effect of CDK16 gene knockdown on the growth,proliferation and migration of esophageal squamous cell carcinoma.We divided the cells into EC-1-NC control group,EC-1-siRNA interference group,KYSE-510-NC control group and KYSE-510-siRNA interference group.The changes of cell growth and proliferation in each group are detected by CCK-8 method and MTT method,and the changes of cell migration ability are detected by scratch test and Transwell test.Part III Predicting protein interacting with CDK16 using bioinformatics analysis1.Five databases,STRING,UniHi,IntAct,HitPredict and genemania,are selected to screen the proteins that may interact with CDK16.The VENN map is used to screen the CDK16 interacting proteins from two or more databases for the second time.2.The screening results are analyzed by GO enrichment analysis and KEGG enrichment analysis by DAVID tool,and the CDK16 target proteins are screened again(P<0.05).3.The selected proteins are analyzed by STRING database,and the interaction between proteins is established.The PPI network diagram is established in Cytoscape software.Finally,the significant key genes are screened out from the differential genes by MCC algorithm for further analysis.4.Finally,the database is used to analyze the difference in the expression of the most significant key genes between esophageal cancer and normal esophageal tissues.Results1.CBioPortal results showed that there are 8%(15/185)CDK16 gene changes in 185 cases of esophageal cancer,mainly up-regulated CDK16 mRNA.2.The results of immunohistochemistry show that the expression level of CDK16 protein in esophageal squamous cell carcinoma is significantly higher than that in adjacent tissues.In the analysis of the relationship between the expression level of CDK16 protein and pathological features,it is found that the high expression of CDK16 in esophageal squamous cell carcinoma is related to the degree of differentiation,lymph node metastasis and TNM stage.3.The expression level of CDK16 in EC-1,Eca-109,TE-1,KYSE-150,KYSE-450 and KYSE-510 esophageal squamous cell lines is significantly higher than that in N1217 normal esophageal epithelial cells.The expression level of CDK16 is relatively high in two esophageal squamous cell carcinoma cell lines KYSE-510?EC-1.4.Two cell lines KYSE-510 and EC-1 are selected to use CDK16 siRNA transient.After knocking down the expression level of CDK16,the ability of cell growth,proliferation and migration is inhibited.5.Ten proteins that may interact with CDK16,such as YWHAH,YWHAZ,YWHAG,YWHAB,YWHAE,YWHAQ,XPO1,PPP2R2A,KSR1 and HDAC4,are screened by bioinformatics analysis.6.Through bioinformatics analysis,it is found that the expression levels of YWHAH and XPO1 in esophageal squamous cell carcinoma are different from those in normal esophageal tissues.Conclusion1.CDK16 express at a high level in both esophageal squamous cell carcinoma and esophageal squamous cell carcinoma.2.CDK16 can promote the growth,proliferation and migration of esophageal squamous cell carcinoma.3.Bioinformatics analysis shows that CDK16 may interact with YWHAH and XPO1 proteins,which may have an impact on the progression of esophageal squamous cell carcinoma. |
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