| Objective: Endometrial carcinoma(EC)is the most common cancer of female reproductive organs in the United States and the second most common cancer worldwide,next to cervical cancer.Women who have problems like obesity,diabetes,high blood pressure,receiving estrogen/tamoxifen therapy or genetic Lynch syndrome are at higher risk of contracting EC.Abnormal uterine bleeding is the most frequent symptom of EC,but many other disorders give rise to the same symptom.In some cases,endometrial cancers may reach an advanced stage before signs and symptoms can be noticed.Although the tumor marker CA125 may assist in the detection of EC,its concentration is more likely to be raised in type II or advanced stage cancers than earlier-stage cancers and a normal value does not exclude more advanced tumors.The 5-year survival rate of the patients who have distant metastasis slumps to 17%.Therefore,there is an urgent need to discover potential early diagnostic and prognostic candidates for the clinicians to refer to when adopting appropriate treatments.The gene expression omnibus(GEO)database is a large and comprehensive public gene expression data resource that contains a variety of tumor gene expression profile datasets.The Cancer Genome Atlas(TCGA)is a landmark cancer genomics program,that sequenced and molecularly characterized over many primary cancer and matched normal samples spanning 33 cancer types.It now contains genomic,epigenomic,transcriptomic,and proteomic data.Based on the common differentially expressed genes(DEGs)screened by the integrated analysis of the endometrial cancer datasets from GEO and TCGA databases,this study aims to identify key gene module by bioinformatic methods,and to select key gene with prognostic value for validation at protein level.Its influence on the malignant biological behavior of endometrial cancer cells and its underlying molecular mechanisms were also investigated,offering new insights and theoretical basis for molecular diagnostics,precision treatment and prognostic prediction for endometrial cancer patients.Methods: 1.Limma R package was used to screen the differentially expressed genes between endometrial cancer and adjacent normal tissues in the GEO datasets(i.e.GSE63678,GSE17025)(differential expression criteria: P value <0.05 and fold change>2 times).Deseq2 R package was used to screen the differentially expressed genes between endometrial carcinoma and adjacent normal tissues in TCGA endometrial cancer cohort(identification criteria for differential expression are the same as above).ggplot2 R package was used to visualize the expression profile of differential expressed genes in those three datasets.Venn diagram was used to acquired the common differentially expressed genes from the aforementioned datasets.2.pheatmap R package was used to draw the cluster heat map of the expression profile of the common differentially expressed genes in each dataset.3.Cluster Profiler R package was used to analyze GO(gene ontology)gene function and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway enrichment analysis of the common DEGs.4.String database was used to establish a protein-protein interaction(PPI)network of common differentially expressed genes,and MCODE(Molecular Complex Detection)plug-in in Cytoscape was used to identify key subnetwork gene modules in the parent network.5.The top 20 genes with the highest degree in the sub-network gene module were selected to analyze the impact of their expression levels on the prognosis of patients with endometrial cancer using the Survminer R package.6.According to the analysis of survival outcome,the key gene UBE2C(Ubiquitin Conjugating Enzyme E2 C)was selected to verify its m RNA level in the TCGA endometrial cancer cohort.The relationship between its expression level and the clinicopathological parameters was also studied.7.Immunohistochemistry results showed the positive expression of UBE2 C in 72 cases of endometrial cancer,23 cases of endometrial atypical hyperplasia and 34 cases of normal endometrial tissues from our hospital.The relationship between expression level of UBE2 C and the clinical pathological parameters of patients as well as prognosis were both investigated.8.Two endometrial cancer cell lines,ISHIKAWA and HEC-1-B,were used for subsequent cell biology experiments.RNA interference technology was used to inhibit the expression of UBE2 C in the ISHIKAWA and HEC-1-B endometrial cancer cell lines.Through MTT,wound healing,Transwell,Annexin V-PI assay,flow cytometry etc.,the changes in biological behaviors such as the proliferation,migration,invasion,apoptosis and cell cycle of endometrial cancer cells in each group after interference with UBE2 C were detected.Western blot was used to detect the changes in the expression of PI3K/Akt signaling pathway node proteins PI3 K,p-PI3 K,Akt,and p-Akt after transfection,and the expression changes of the tumor suppressor gene TP53 protein was also detected.Results: 1.Venn diagram of the integrated analysis results of GEO and TCGA datasets showed that the detected differentially expressed genes summed up to 344.Among them,overexpressed genes totaled 170 and down-expressed genes totaled 174.2.The clustering heat map showed that the screened common differentially expressed genes could better divide tumor samples and normal samples into two categories.3.The up-regulated differentially expressed genes were mainly enriched in mitotic mitosis(GO: BP),spindle(GO: CC)and microtubule association(GO: MF)(all p less than 0.05).The downregulated common differentially expressed genes were mainly enriched in the positive regulation of developmental growth(GO: BP),extracellular matrix(GO: CC),and calcium channel activity(GO: MF)(all p less than 0.05).4.The Up-regulated common DEGs were mainly enriched in the cell cycle(hsa04110),oocyte meiosis(hsa04114),cell senescence(hsa04218),carbon metabolism(hsa01200),p53 signaling pathway(hsa04115)(p All are less than 0.05).The down-regulated common differentially expressed genes were mainly enriched in EGFR tyrosine kinase inhibitor resistance(hsa01521),JAK-STAT signaling pathway(hsa04630),calcium signaling pathway(hsa04020),melanoma(hsa05218),and Micro RNA in cancer(Hsa05206)(p are all less than 0.05).5.The PPI network of common DEGs was constructed,which had 284 nodes and 3697 edges.The MCODE plug-in identified the key sub-network module,which had 76 nodes and 2677 edges.6.The high expression of key genes CCNB2,CDC20,BUB1 B,UBE2C,AURKB,FOXM1,NCAPG,RRM2,TPX2,DLGAP5,CDCA8,CDC45,MKI67,BUB1 and KIF2 C in the sub-network module were significantly associated with poor prognosis of EC patients(p <0.05).7.Univariate Logistic regression showed that in the TCGA endometrial cancer cohort,the high expression level of UBE2 C was correlated with the FIGO advanced stage(stage IV vs.I,OR = 3.152),poor differentiation(poor differentiation vs.well-differentiation,OR =7.578),histopathological classification(serous adenocarcinoma vs.endometrioid adenocarcinoma,OR = 4.813),distant metastasis(with distant metastasis vs.no distant metastasis,OR = 4.043),with tumor state(with tumor vs.without tumor,OR = 2.548)(all p values <0.05).8.Immunohistochemistry results showed that UBE2 C was mainly expressed in the cytoplasm and cell membrane,with partly stained in the nucleus.The positive rate of UBE2 C expression in endometrial carcinoma(88.89%)was significantly higher than that of endometrial atypical hyperplasia(60.87%)and normal endometrial tissue(35.29%)(P<0.05).The high expression rate of UBE2 C in FIGO III?IV stage(88.89%)was significantly higher than that in FIGO I?II stage(53.70%)(p < 0.05).In addition,the positive rate of high expression in patients with lymph node metastasis(92.86%)was significantly higher than that of patients without lymph node metastasis(58.70%)(p <0.05).Kaplan-Meier method indicated endometrial carcinoma patients with overexpressed UBE2 C tended to live significantly shorter than those with downexpressed UBE2 C.UBE2C was an independent risk factor for the prognosis of EC patients(P=0.047)by Cox regression analysis.9.After knocking down UBE2 C,the results of cell biology experiments showed that after inhibiting the expression of UBE2 C,the proliferation,invasiveness,and migration of endometrial cancer cells were significantly reduced,while apoptosis increased.Flow cytometry results showed that UBE2 C promoted the transition of G2/M of endometrial cells.Western blot analysis showed that after inhibiting the expression of UBE2 C,the expression of the node protein p-PI3 K and p-Akt of the PI3K/Akt signaling pathway decreased,and the expression of the tumor suppressor gene p53 protein increased.Genomic analysis of the TCGA endometrial cancer cohort showed that TP53 mutation,copy number amplification,and hypomethylation all contributed to the high expression of UBE2 C.GO and KEGG analysis showed that UBE2 C and its coexpressed genes might act on DNA replication to affect the proliferation and apoptosis of endometrial cancer cells byConclusion: 1.Two GEO datasets(ie GSE63678,GSE17025)and TCGA UCEC cohort were used to screen 344 DEGs.KEGG analysis of these common DEGs were mainly enriched in cell cycle,oocyte meiosis,cell senescence,carbon metabolism and p53 signaling pathway.2.The abnormally high expression levels of 15 genes selected from the PPI(protein-protein interaction)network were significantly related to the poor prognosis of EC,namely CCNB2,CDC20,BUB1 B,UBE2C,AURKB,FOXM1,NCAPG,RRM2,TPX2,DLGAP5,CDCA8,CDC45,MKI67,BUB1,KIF2 C.3.UBE2 C was highly expressed in endometrial cancer,and its expression level was related to FIGO advanced stage and poor prognosis.Cox risk model showed that high expression of UBE2 C was an independent risk factor for poor prognosis.4.UBE2 C promoted the migration,invasion,proliferation and G2/M transition of endometrial cancer cells through PI3K/Akt signaling pathway,and inhibited cell apoptosis.Somatic mutation,increased copy number variations,and DNA hypomethylation all played contributive role in its overexpression. |
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