Objective To study the stemness characteristics of uterine corpus endometrial carcinoma(UCEC)and its potential regulatory mechanism.Methods Transcriptome sequencing data of UCEC was obtained from The Cancer Genome Atlas.Gene expression profile was normalized by edgeR package in R3.5.1.A one-class logistic regression machine learning algorithm was employed to calculated the mRNA stemness index(mRNAsi)of each UCEC sample.Then,the prognostic significance of mRNAsi and candidate genes was evaluated by survminer and survival packages.The high-frequency sub-pathways mining approach(HiFreSP)was used to identify the prognosis-related sub-pathways enriched with differentially expressed genes(DEGs).Subsequently,a gene co-expression network was constructed using WGCNA package,and the key gene modules were analyzed.The clusterProfiler package was adopted to the function annotation of the modules highly correlated with mRNAsi.Finally,the Human Protein Atlas(HPA)was retrieved for immunohistochemical validation.Results The mRNAsi of UCEC samples was significantly higher than that of normal tissues(
Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Endometrial Neoplasms/genetics* ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Mad2 Proteins ; Multigene Family ; Neoplastic Stem Cells ; Prognosis ; Securin

OBJECTIVE: To screen the key genes related to the prognosis of lung adenocarcinoma through big data analysis and explore their clinical value and potential mechanism. METHODS: We analyzed GSE18842, GSE27262, and GSE33532 gene expression profile data obtained from the Gene Expression Omnibus (GEO). Bioinformatics methods were used to screen the differentially expressed genes in lung adenocarcinoma tissues and KEGG and GO enrichment analysis was performed, followed by PPI interaction network analysis, module analysis, differential expression analysis, and prognosis analysis. The expressions of MAD2L1 and TTK by immunohistochemistry were verified in 35 non-small cell lung cancer specimens and paired adjacent tissues. RESULTS: We identified a total of 256 genes that showed significant differential expressions in lung adenocarcinoma, including 66 up-regulated and 190 down-regulated genes. Thirty-two up-regulated core genes were screened by functional analysis, and among them 29 were shown to significantly correlate with a poor prognosis of patients with lung adenocarcinoma. All the 29 genes were highly expressed in lung adenocarcinoma tissues compared with normal lung tissues and were mainly enriched in cell cycle pathways. Seven of these key genes were closely related to the spindle assembly checkpoint (SAC) complex and responsible for regulating cell behavior in G2/M phase. We selected SAC-related proteins TTK and MAD2L1 to test their expressions in clinical tumor samples, and detected their overexpression in lung adenocarcinoma tissues as compared with the adjacent tissues. CONCLUSIONS Seven SAC complex-related genes, including TTK and MAD2L1, are overexpressed in lung adenocarcinoma tissues with close correlation with the prognosis of the patients.
Adenocarcinoma of Lung/genetics* ; Big Data ; Cell Cycle Proteins/genetics* ; Computational Biology ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms/genetics* ; M Phase Cell Cycle Checkpoints ; Mad2 Proteins/genetics* ; Protein-Serine-Threonine Kinases/genetics* ; Protein-Tyrosine Kinases/genetics*

OBJECTIVETo study the effect of alternative splicing form -MAD2beta of mitotic arrest deficient protein 2 (MAD2) on the formation of multidrug resistance in human gastric adenocarcinoma cell SGC7901.

METHODSRNA was extracted from a multidrug resistance cell line SGC7901/ADR. The full-length MAD2beta cDNA was obtained by RT-PCR and cloned into the pUCm-T vector, and then recombined into the eukaryotic expression vector pcDNA3.1 in forward direction. Subsequently, pcDNA3.1/MAD2beta vectors were then transfected into SGC7901 cells by lipofectamine. Sensitivity to drug was detected by MTT assay. Cell cycle alteration and intracellular fluorescence intensity were determined by FACS.

RESULTSA fragment of 0.53 Kb was obtained and confirmed by DNA sequencing which was a new alternative splicing form of MAD2 named as MAD2beta. pcDNA3.1/MAD2beta transfected SGC7901 cells (SGC7901/MAD2beta) were more resistant to ADR, VCR and MMC than the control cells (SGC7901/pcDNA3.1), and also ADR fluorescence intensity of SGC7901/MAD2beta cells was lower (P < 0.05) than that of SGC7901/pcDNA3.1 cells.

CONCLUSIONMAD2beta could increase the multidrug resistance of SGC7901 cell line.

Adenocarcinoma ; metabolism ; pathology ; Alternative Splicing ; Antibiotics, Antineoplastic ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Calcium-Binding Proteins ; biosynthesis ; genetics ; Cell Cycle Proteins ; Cell Line, Tumor ; DNA-Binding Proteins ; biosynthesis ; genetics ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; genetics ; Humans ; Mad2 Proteins ; Mitomycin ; pharmacology ; Repressor Proteins ; Smad2 Protein ; Stomach Neoplasms ; metabolism ; pathology ; Trans-Activators ; biosynthesis ; genetics ; Transfection ; Vincristine ; pharmacology

In addition to DNA repair pathways, cells utilize translesion DNA synthesis (TLS) to bypass DNA lesions during replication. During TLS, Y-family DNA polymerase (Polη, Polκ, Polı and Rev1) inserts specific nucleotide opposite preferred DNA lesions, and then Polζ consisting of two subunits, Rev3 and Rev7, carries out primer extension. Here, we report the complex structures of Rev3-Rev7-Rev1(CTD) and Rev3-Rev7-Rev1(CTD)-Polκ(RIR). These two structures demonstrate that Rev1(CTD) contains separate binding sites for Polκ and Rev7. Our BIAcore experiments provide additional support for the notion that the interaction between Rev3 and Rev7 increases the affinity of Rev7 and Rev1. We also verified through FRET experiment that Rev1, Rev3, Rev7 and Polκ form a stable quaternary complex in vivo, thereby suggesting an efficient switching mechanism where the "inserter" polymerase can be immediately replaced by an "extender" polymerase within the same quaternary complex.
Binding Sites ; Crystallography, X-Ray ; DNA Repair ; DNA-Binding Proteins ; chemistry ; genetics ; metabolism ; DNA-Directed DNA Polymerase ; chemistry ; genetics ; metabolism ; Fluorescence Resonance Energy Transfer ; Humans ; Mad2 Proteins ; Nuclear Proteins ; chemistry ; genetics ; metabolism ; Nucleotidyltransferases ; chemistry ; genetics ; metabolism ; Protein Binding ; Protein Structure, Quaternary ; Protein Structure, Tertiary ; Proteins ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics