| BackgroundAccording to the 2020 Global Cancer Statistics Report,ovarian cancer(OC)is the second leading cause of death in gynecological malignancies worldwide.Although people have done a lot of work to clarify the cause,the molecular mechanism is still unclear.In order to identify candidate genes in the occurrence and progression of ovarian cancer,bioinformatics methods were used to screen differentially expressed genes(DEGs).The relationship between the expression of human monopolar spindle 1(MPS1),also known as serine/threonine and tyrosine kinase(TTK),and the survival and the prognosis of patients with serous ovarian cancer is not clear.This project aims to study the relationship between the expression of TTK and the prognosis of patients with ovarian cancer and its biological function in ovarian cancer.MethodsThe R language packages were used to obtain the mRNA transcriptome data of 362 ovarian cancer patients from the Cancer Genome Atlas(TCGA)and 180 normal people from the Genotype-Tissue Expression(GTEx).DEGs were screened by the tidyverse software package,the ggplot2 software package was used to visualize DEGs,and the clusterProfiler software package was used to perform Gene Ontology(GO)function annotations and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis on the selected DEGs.Search Tool for the Retrieval of Interacting Genes(STRING)was used to construct a protein-protein interaction network(PPI).Cytoscape’s Molecular Complex Detection(MCODE)app was used to identify the hub genes of the PPI network.Through the background investigation of each hub gene,the target gene to be studied was finally determined.Gene Expression Profiling Interactive Analysis(GEPIA2)and Kaplan-Meier Plotter were used to map the gene expression profile of the target gene TTK in various tumor tissues and the survival curves of lung adenocarcinoma,esophageal carcinoma,thyroid cancer,breast cancer,cervical squamous cell carcinoma and endometrial carcinoma.GEPIA2 was used to analyze the expression of TTK in ovarian cancer and normal ovarian tissues.Kaplan-Meier Plotter was used to analyze the correlation between TTK and the prognosis of patients with high grade serous ovarian cancer(HGSOC).Taking HGSOC as the research object,we collected tumor tissue specimens and clinicopathological information of HGSOC patients who did not undergo pre-operative chemotherapy in the Department of Obstetrics and Gynecology,Qilu Hospital,Shandong University.Immunohistochemistry was used to detect the expression level of TTK protein in HGSOC,and the median of immunohistochemical staining results was used as the cut-off value,which was used as the threshold for judging the high and low expression levels of TTK.Fisher exact test,Spearman’s rank correlation coefficient,log-rank test,and Cox risk regression model were used to analyze the relationship between TTK expression level and clinical characteristics,as well as the relationship between TTK expression level and patient survival prognosis,so as to study whether TTK is a prognostic factor of serous ovarian carcinoma.The expression of TTK mRNA and protein in ovarian cancer cell lines was detected by RT-PCR and Western Blot.The cell line HEY was transfected with lentivirus to construct an over-expression cell line,small interfering RNA(siRNA)was transfected into SKOV3 cell line,and then the transfection efficiency was verified from the mRNA and protein levels by Cell Counting Kit-8(CCK-8)assay,Colony formation assay,ethynyl deoxyuridine(EdU)proliferation assay.All experiments were performed 3 independent replicates.The results are expressed as mean± standard deviation(SD).Student’s t-test was used for two independent samples by GraphPad Prism 9.0.0,one-way ANOVA or two-way ANOVA were used for multiple samples which obeyed normal distribution,and Brown-Forsythe and Welch ANOVA test were used for non-normal distribution.Results1.A total of 996 DEGs genes were identified,of which 587 genes were up-regulated and 409 genes were down-regulated.The cell location in GO enrichment analysis showed that DEGs were mainly located in cell-cell junctions,collagen-containing extracellular matrix,tight junction,cornified envelope,and Golgi cis cisterna;molecular functions mainly focused on the signaling receptor activators activity,receptor ligand activity,serine hydrolase activity,serine-type endopeptidase activity,serine-type peptidase activity,in the biological process,it regulates epidermis development,nuclear division,skin development,chromosome segregation,cornification and so on.Pathway analysis showed that these genes were mainly enriched in signaling pathways such as calcium signaling pathway,cell cycle,neuroactive ligand-receptor interaction,pancreatic secretion,PI3K-Akt signaling pathway,etc.The first group of hub genes with the highest PPI score was selected for analysis,a total of 18.Through domestic and foreign literature searches,background investigations were conducted respectively,and the hub gene TTK was finally determined to be studied.Based on the analysis of the database,the expression of TTK is different in most tumors,and the expression of TTK in lung adenocarcinoma,esophageal carcinoma,thyroid cancer,breast cancer,cervical squamous cell carcinoma and endometrial carcinoma and other tumors is related to the prognosis of patients.High expression has a poor prognosis,and low expression has a good prognosis.The expression of TTK in ovarian cancer tissues and normal ovarian tissues is also different.Given that TTK is significantly related to the prognosis of patients in other malignant tumors,and the development of its inhibitors and the effect of phase I clinical trials,it has shown that some anti-tumor cell microtubule formation drugs are sensitized and the development of tumor vaccines is also accelerated the urgent need for the research of TTK in ovarian cancer,that is,the expression of TTK in ovarian cancer,its biological function,and the specific prognosis of patients with ovarian cancer need to be further verified.2.The database shows that in HGSOC patients,there is no significant correlation between TTK expression and overall survival(OS)(p=0.39),hazard ratio(HR)is 1.07,95%confidence interval(CI)is 0.92-1.25.Immunohistochemical experiments showed that the staining range of TTK protein was cytoplasm and nucleus,but mainly concentrated in the cytoplasm,and the high expression ratio was 42%(13/31).The protein expression of TTK in HGSOC tissue was significantly higher than that in the contralateral normal ovarian tissue.Fisher’s exact test results showed that the two groups of high and low TTK expression were in the patient’s age,C A125 level,tumor maximum diameter,and the Federation International Gynecology and Obstetrics(FIGO)staging,ascites,residual lesions,pelvic lymph node status,and patient survival status were not statistically significant(p>0.05).Spearman correlation analysis showed that TTK expression levels were negatively correlated with patient age,tumor maximum diameter,residual lesions,and positively correlated with CA125 levels and FIGO staging,but they were not statistically significant(all p>0.05).The results of univariate Kaplan-Meier analysis showed that TTK expression(p=0.036)and FIGO staging(p=0.008)were significantly correlated with OS in HGSOC patients.The results of univariate Kaplan-Meier analysis showed that TTK expression(p=0.036)and FIGO staging(p=0.008)were significantly correlated with OS in HGSOC patients.Univariate Cox risk regression model analysis also showed that TTK expression(p=0.044)and FIGO staging(p=0.030)were significantly related to the patient’s OS.There was no statistically significant correlation with patient age,CA125 expression level,tumor size,ascites,residual lesions,lymph node metastasis,and patient’s OS(p>0.05).The results of multivariate COX risk regression analysis showed that FIGO staging has a significant impact on the OS of HGSOC patients(p=0.035).The risk of death in HGSOC patients at stage Ⅲ-Ⅳ is 8.904 times that of stage I-II.It is an independent influencing factor,and TTK expression is not an independent influencing factor of OS in HGSOC patients3.The expression of mRNA and protein of TTK in ovarian cancer cell lines was verified by RT-PCR and Western Blot experiments.Transfect cells with lentivirus,construct an over-expression cell line,and transiently transfect the cells to silence the gene TTK.In the Cell Counting Kit-8(CCK-8)assay,Colony formation assay,ethynyl deoxyuridine(EdU)proliferation assay,it can be seen that overexpression of TTK can significantly promote cell proliferation,while silencing TTK has a significant inhibitory effect on cell proliferation.Conclusions1.Among the differentially expressed genes in ovarian cancer tissues and normal ovarian tissues,a total of 18 genes in BIRC5、CDC20、TPX2、UBE2C、KIAA0101、TTK、MELK、TOP2A、DLGAP5、RRM2、AURKB、NEK2、HJURP、CEP55、PBK、MKI67、KIF18B、DEPDC1 are potential biomarkers of ovarian cancer.2.TTK gene is more highly expressed in ovarian cancer tissues than in normal tissues.3.High TTK expression is associated with poor prognosis of patients4.High expression of TTK promotes cell proliferation,and low expression inhibits cell proliferation.5.TTK may be involved in the carcinogenesis of ovarian cancer.It is very likely to become a candidate target for the diagnosis and treatment of ovarian cancer. |
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