Mediator is a highly conserved large protein complex (25 proteins, >1000 kDa) and preeminently responsible for eukaryotic transcription, which contains a dissociable 'Cdk8 module'. Although increasing evidence demonstrates that Cdk8 module plays both positive and negative roles in transcription regulation, the detailed structure, and subunit organization, molecular mechanism how it regulates transcription remain elusive. Here we used single-particle electron microscopy to characterize the structure and subunit organization of the Cdk8 module and illuminated the substantial mobility of the Med13 subunit results in the structural flexibility. The Cdk8 module interaction with core Mediator is concurrent with active transcription in vivo. An interaction with the Cdk8 module induces core Mediator into very extended conformation in vitro, which is presumed to be an active functional state of Mediator. Taken together, our results illuminated the detailed architecture of Cdk8 module, and suggested the Cdk8 module could positively regulate transcription by modulating Mediator conformation.
Cyclin-Dependent Kinase 8 ; chemistry ; Humans ; Mediator Complex ; chemistry ; Models, Molecular ; Protein Conformation ; Protein Subunits ; chemistry ; Saccharomyces cerevisiae Proteins ; chemistry

PURPOSE: The peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that regulates expression of mediators of lipid metabolism and the inflammatory response. Thyroid hormone receptor-associated proteins 220 (TRAP220) is an essential component of the TRAP/Mediator complex. The objective of this study was to clarify whether PPARgamma or TRAP220 are significant prognostic markers in resectable colorectal cancer (CRC). MATERIALS AND METHODS: A total of 399 patients who underwent curative resection for CRC were enrolled. We investigated the presence of PPARgamma and TARP220 in CRC tissues and adjacent normal tissues by immunohistochemistry. Correlation between the expression of these factors and clinicopathologic features and survival was investigated. RESULTS: Median age of the patients was 63 years (range, 22 to 87 years), and median follow-up duration 61.1 months (range, 2 to 114 months). PPARgamma and TRAP220 expression showed significant correlation with depth of invasion (p=0.013 and p=0.001, respectively). Expression of TRAP220 also showed association with lymph node metastasis and TNM stage (p=0.001). Compared with patients with TRAP220 negative tumors, patients with TRAP220 positive tumors had longer 5-year disease-free survival (DFS) tendency (p=0.051). Patients who were PPARgamma positive combined with TRAP220 positive had a better 5-year DFS (64.8% vs. 79.3%, p=0.013). In multivariate analysis expression of both PPARgamma and TRAP220 significantly affected DFS (hazard ratio, 0.620; 95% confidence interval, 0.379 to 0.997; p=0.048). CONCLUSION: TRAP220 may be a valuable marker for nodal metastasis and TNM stage. Tumor co-expression of PPARgamma and TRAP220 represents a biomarker for good prognosis in CRC patients.
Colorectal Neoplasms* ; Disease-Free Survival ; Follow-Up Studies ; Humans ; Immunohistochemistry ; Lipid Metabolism ; Lymph Nodes ; Mediator Complex Subunit 1* ; Multivariate Analysis ; Neoplasm Metastasis ; Peroxisomes* ; PPAR gamma* ; Prognosis ; Thyroid Gland

MED1 is a key transcription co-activator subunit of the Mediator complex that is essential for RNA polymerase II-dependent transcription. MED1 functions as a co-activator for PPARs and other nuclear receptors and transcription factors, and plays an important role in lipid metabolism. To examine how MED1 might affect plasma lipids, plasma triglyceride, cholesterol levels, and lipoprotein profiles, were measured in MED1(deltaLiv) mice fasted for 24, 48 and 72 hours. Histological changes in liver sections from MED1(deltaLiv) mice after 72 hours of fasting were also examined using H&E staining. There was no fat accumulation in livers of MED1(deltaLiv) mice compared to MED1(fl/fl) and PPARalpha -/- control mice after 72 hours of fasting. Compared with MEDl(fl/fl) mice, plasma triglycerides in MED1(deltaLiv) mice were significantly increased after 24, 48 and 72 hours of fasting, and plasma cholesterol was significantly increased after 48 and 72 hours of fasting. Lipoprotein profiles were similar in fed MED1(fl/fl) and MED1(deltaLiv) mice. However, very low density lipoprotein (VLDL) was significantly increased in MED1(deltaLiv) mice after 24 hours of fasting. We conclude that, hyperlipidemia in MED1(deltaLiv) mice in response to fasting is due to the accumulation of VLDL, which suggests that MED1 plays a pivotal role in the regulation of plasma triglyceride and cholesterol levels.
Animals ; Cholesterol ; blood ; Fasting ; Hyperlipidemias ; blood ; Lipoproteins, VLDL ; blood ; Liver ; chemistry ; Mediator Complex Subunit 1 ; genetics ; physiology ; Mice ; Mice, Knockout ; Triglycerides ; blood

OBJECTIVETo study the relationship of GYPC and TRIP3 gene expression and the prognosis of acute lymphoblastic leukemia (ALL) in children in order to explore the molecular biological mechanisms of recurrence and remission of ALL.

METHODSThirty-eight newly diagnosed ALL children were enrolled. Of the 38 patients, 31 achieved complete remission (CR) and 12 relapsed. Semi-quantitative RT-PCR was employed to measure blood GYPC and TRIP3 gene expression. Twenty blood samples from normal children were used as controls.

RESULTSBlood GYPC expression in newly diagnosed ALL children was significantly higher than that in the control group (p<0.01) and the CR group (p<0.01). The expression of GYPC gene in the CR group was similar to that in the control group. Other than the control group (p<0.01) and the CR group (p<0.01), the GYPC expression of the relapse group was significantly higher than that in the newly diagnosed group (p<0.01). The CR group showed lower GYPC gene expression than the nonjremission group before treatment (p<0.05). Blood expression of TRIP3 gene in the newly diagnosed and the relapse groups was significantly lower than that in the control group (p<0.05). The CR group had increased TRIP3 gene expression compared with the control group (p<0.01) as well as the newly diagnosed and the relapse groups (p<0.01). Of the 38 newly diagnosed ALL children, the patients with positive TRIP3 expression showed higher remission rate than those with negative TRIP3 (p<0.05). The TRIP3 gene expression before treatment in patients who achieved CR was higher than that in non-remission patients (p<0.05).

CONCLUSIONSA high GYPC gene expression is associated with an unfavorable outcome, in contrast, a high TRIP3 gene expression is associated with a favorable outcome in childhood ALL.

Adolescent ; Child ; Child, Preschool ; Female ; Glycophorin ; genetics ; Humans ; Infant ; Male ; Mediator Complex Subunit 1 ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; mortality ; Prognosis ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription Factors ; genetics

Objective To investigate the expression and prognostic significance of mediator complex subunit 8 (MED8) in gastric cancer and its impact on the cell cycle.Methods The expression of MED8 in gastric cancer and adjacent tissues and its correlation with patients' prognosis were analyzed using public databases.A validation cohort of 104 patients who underwent radical resection for gastric cancer in the First Affiliated Hospital of Bengbu Medical College from June 2012 to July 2017 was included.The receiver operating characteristic curve was established to evaluate the predictive value of MED8 for postoperative 5-year survival.Bioinformatics tools were used to predict the biological roles of MED8 in gastric cancer.The effect of the MED8 level on the G1/S phase transition of gastric cancer cells (MGC-803) was analyzed via lentivirus transduction and flow cytometry.Western blotting was carried out to assess the impact of MED8 expression on the protein levels of cyclin-dependent kinase 4(Cdk4) and G1/S-specific cyclin-D1(CyclinD1) in MGC-803 cells.Results The high expression of MED8 in the gastric cancer tissue was associated with poor prognosis (P<0.001) and had prognostic significance (area under curve=0.733,P<0.001).Gene enrichment analysis suggested that MED8 may participate in the cell cycle process.Flow cytometry results revealed that the upregulation of MED8 expression promoted the transition of MGC-803 cells from the G1 phase to the S phase (P<0.001),while the downregulation of MED8 had the opposite effect (P<0.001).Western blotting showed increases in the protein levels of Cdk4 and CyclinD1 in MGC-803 cells with upregulated MED8 expression (all P<0.001),and decreases in the cells with downregulated MED8 expression (all P<0.001).Conclusion MED8 is highly expressed in gastric cancer and may affect its progression and prognosis by regulating the G1/S phase transition of gastric cancer cells.
Humans ; Stomach Neoplasms ; Prognosis ; Cell Proliferation ; Cell Cycle ; Mediator Complex/metabolism* ; Cell Line, Tumor

A boy aged 4 years and 2 months was found to have delayed language and motor development, instability of gait, poor eye contact, stereotyped behavior, and seizure at the age of 3 years. Physical examination showed special facial features, including plagiocephaly, blepharoptosis, wide nasal bridge, down-turned mouth corners at both sides, and low-set ears. There were only two knuckles at the little finger of the left hand. The anteroposterior and lateral films of the spine showed scoliosis; echocardiography showed ventricular septal defect; the Gesell Developmental Scale showed delayed language development and moderate intellectual disability; there were no abnormalities in the karyotype; genome-wide SNP arrays found a duplication in 12q24.21 region with a size of 1.03 Mb in chromosome 12, while this was not seen in his parents. The boy was diagnosed with MED13L syndrome. Point mutation, deletion, and duplication in the MED13L gene can lead to MED13L syndrome. The patients with different genotypes may have different phenotypes. Genome-wide SNP arrays may help with the diagnosis of this disease.
Child, Preschool ; Chromosome Deletion ; DNA Copy Number Variations ; Humans ; Intellectual Disability ; genetics ; Male ; Mediator Complex ; genetics ; Phenotype ; Polymorphism, Single Nucleotide ; Syndrome

The Mediator Complex plays key roles in activating gene transcription in eukaryotes. Mediator of RNA polymerase II transcription subunit 12 homolog (MED12) is a subunit of the Mediator Complex and regulates the activity of the complex. MED12 is involved in a variety of cellular activities, and mutations in MED12 gene impair MED12 activities and are associated with several diseases, including Opitz-Kaveggia syndrome, Lujan syndrome, uterine leiomyomas and prostate cancer. This review will discuss the biological function of MED12 and the relationship between MED12 mutations and diseases.
Agenesis of Corpus Callosum ; genetics ; Anus, Imperforate ; genetics ; Constipation ; genetics ; Craniofacial Abnormalities ; genetics ; Female ; Genetic Predisposition to Disease ; Humans ; Leiomyoma ; genetics ; Male ; Marfan Syndrome ; genetics ; Mediator Complex ; genetics ; metabolism ; Mental Retardation, X-Linked ; genetics ; Muscle Hypotonia ; congenital ; genetics ; Mutation ; Prostatic Neoplasms ; genetics ; Transcription, Genetic ; Uterine Neoplasms ; genetics

OBJECTIVE: To construct the eukaryotic expression vector of MJD1 with normal copies of CAG trinucleotide repetition and MJD1 with CAG trinucleotide repetition expansion mutation respectively, and to determine whether the polyglutamine expansion in ataxin-3 could lead to the formation of intranuclear aggregation. METHODS: The coding sequence of wild-type MJD1 and mutant MJD1 was amplified by PCR from pAS2-1-MJD20Q and pAS2-1-MJD68Q respectively. After being digested with BamH I and Hind III, the PCR products were inserted into pcDNA3. 1-Myc-His(-) B. The recombinant plasmids pcDNA3.1-Myc-His(-) B-MJD20Q and pcDNA3.1-Myc-His(-) B-MJD68Q were identified by enzyme digestion analysis and DNA sequencing. The recombinant plasmid was transfected into SH-SYSY cells and the expression of MJD1 in the transfected cells was analyzed by Western blot. The immunofluorescence of the transfected cells was examined using a confocal microscope to observe the formation of intranuclear aggregation. RESULTS: Enzyme digestion analysis and DNA sequencing showed that the target gene was cloned into pcDNA3. 1-Myc-His(-) B. The expression of MJD1 in the transfected cells was confirmed by Western blot; The SH-SY5Y cells transfected with pcDNA3. 1-Myc-His(-) B-MJD68Q showed the formation of intranuclear aggregation, but the cells transfected with pcDNA3.1-Myc-His(-) B-MJD20Q did not show such phenomenon. CONCLUSION The eukaryotic expression vectors of MJD1 has been successfully constructed; The polyglutamine expansion in ataxin-3 could lead to the formation of intranuclear aggregation.
Ataxin-1 ; Ataxin-3 ; Ataxins ; Base Sequence ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; Humans ; Mediator Complex ; Molecular Sequence Data ; Nerve Tissue Proteins ; biosynthesis ; genetics ; Neuroblastoma ; metabolism ; pathology ; Nuclear Proteins ; biosynthesis ; genetics ; Plasmids ; genetics ; Receptors, Thyroid Hormone ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Repressor Proteins ; biosynthesis ; genetics ; Transfection ; Tumor Cells, Cultured

OBJECTIVECytotoxin-associated protein (CagA) of H. pylori has been confirmed to be closely associated with gastric inflammation and tumorigenesis, but the mechanism behind it is little understood. In this study, we try to determine roles of CagA(+) strain in activating PI3K/Akt1 signaling pathway, and affecting expression of p21(WAF1/CIP1) and p27(KIP1), and also in releasing IL-8 in host cells.

METHODSAkt1 phosphorylation and IL-8 levels of CagA(+) and CagA⁻ strain infected AGS cells were detected by ELISAs. Two quantitative RT-PCRs were established to measure p21(WAF1/CIP1) and p27(KIP1) mRNA levels in the CagA(+) and CagA⁻ strain infected cells. LY294002, an inhibitor of PI3K/Akt pathway, was used to define effect of the pathway in IL-8 release.

RESULTSCagA(+) strain could induce an obvious elevation of Akt1 phosphorylation in the infected AGS cells while CagA? strain failed to do so. The CagA(+) H. pylori strain infected AGS cells showed significant drops both in p21(WAF1/CIP1) and p27(KIP1) mRNA levels, whereas the CagA⁻ H. pylori strain caused a remarkable increase in p21(WAF1/CIP1) mRNA without affecting p27(KIP1) gene transcription in the AGS cells. Both the CagA(+) and CagA⁻ H. pylori strains enabled AGS cells to produce close elevated levels of IL-8, and the LY294002 block resulted in unexpected elevations of IL-8 levels.

CONCLUSIONSCagA can activate PI3K/Akt1 pathway that plays an inhibitory role in IL-8 release in H. pylori infected AGS cells. Activation of PI3K/Akt1 pathway and subsequent negative regulation of p21(WAF1/CIP1) and p27(KIP1) expression might be involved in CagA-associated carcinogenesis.

Antigens, Bacterial ; genetics ; physiology ; Bacterial Proteins ; genetics ; physiology ; Cell Line ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; Cyclin-Dependent Kinase Inhibitor p27 ; Gastric Mucosa ; cytology ; enzymology ; microbiology ; Helicobacter pylori ; metabolism ; pathogenicity ; physiology ; Humans ; Interleukin-8 ; secretion ; Intracellular Signaling Peptides and Proteins ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Transcription, Genetic ; Virulence

PURPOSE: Lactobacillus casei (L. casei) is known to exert anti-proliferation effects on many types of cancer cells. However, the effect of L. casei on liver cancer has not been reported. Accordingly, the aim of this study was to determine the anti-cancer effect of L. casei extract on Huh7 cells. MATERIALS AND METHODS: L. casei ATCC393 extract was prepared and purified. After the treatment of L. casei extract on Huh7 cells, cell viability, cell cycle arrest and cell death were analyzed by flow cytometry. The expression levels of tumor necrosis factor-alpha receptor 1 (TNFR1) and death receptor 3 (DR3) mRNA related with extrinsic apoptosis were assessed by reverse transcription polymerase chain reaction. Additionally, P21 and P27 cell cycle proteins as well as Caspase-3, -8, -9, phospho-Bad and Bcl-2 apoptosis proteins were analyzed by western blot analysis. To determine the effect of L. casei extract on cancer stem-like cells, we analyzed changes in side population fraction through flow cytometry. RESULTS: The cell viability of Huh7 cells treated with L. casei extract was decreased by 77%, potentially owing to increases in the rates of Huh7 cells arrested in the G2/M phase (3% increase) and that underwent apoptosis (6% increase). The expression levels of TNFR1 and DR3 mRNA, as well as P21 and P27 cell cycle proteins, were increased. Meanwhile, the expressions of caspase-8, -9, phospho-Bad and Bcl-2 proteins decreased. However, in the case of side population cells, no remarkable changes were observed. CONCLUSION: L. casei extract exerts a potent anti-tumor effect on the viability of liver cancer cells, although not on cancer stem-like cells.
Apoptosis/drug effects ; Carcinoma, Hepatocellular/*pathology ; Caspase 8/metabolism ; Caspase 9/metabolism ; Cell Cycle Checkpoints/drug effects ; Cell Extracts/*pharmacology ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cyclin-Dependent Kinase Inhibitor p21/metabolism ; Cyclin-Dependent Kinase Inhibitor p27/metabolism ; Cytostatic Agents/*pharmacology ; Flow Cytometry ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Lactobacillus casei/*chemistry ; Liver Neoplasms/*pathology ; Proto-Oncogene Proteins c-bcl-2/metabolism ; RNA, Messenger/metabolism ; Receptors, Tumor Necrosis Factor, Member 25/metabolism ; Receptors, Tumor Necrosis Factor, Type I/metabolism ; bcl-Associated Death Protein/metabolism