OBJECTIVEOsteosarcoma is the most common type of malignant bone tumor in children and adolescents. The role of E3 ligases in tumorigenesis is currently a focus in tumor research. In the present study, we investigated the role of the E3 ligase tripartite motif 21 (TRIM21) in osteosarcoma cell proliferation.

METHODS3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays were used to assess osteosarcoma cell viability. U2-OS cells stably carrying a recombinant lentivirus expressing tetracycline-regulated TRIM21 were screened. Co-immunoprecipitation was coupled with LCMS/MS analysis to identify novel interacting partners of TRIM21. Co-immunoprecipitation and bimolecular fluorescence complementation (BIFC) were performed to validate the interactions between TRIM21 and its novel partner YWHAZ. A TRIM21-ΔRING construct was generated to test the effects of TRIM21 ligase activity on YWHAZ.

RESULTSTRIM21 positively regulated osteosarcoma cell proliferation. Overexpression of TRIM21 enhanced osteosarcoma cell tolerance toward various stresses. YWHAZ protein was identified as a novel interacting partner of TRIM21 and its expression levels were negatively regulated by TRIM21. The RING domain of TRIM21 was required for TRIM21 negative regulation of YWHAZ expression. However, overexpression of YWHAZ did not affect positive regulation of osteosarcoma cell proliferation by TRIM21.

CONCLUSIONOur results further clarify the molecular mechanisms underlying the pathogenesis of osteosarcoma.

14-3-3 Proteins ; genetics ; metabolism ; Cell Proliferation ; genetics ; Humans ; Osteosarcoma ; genetics ; Ribonucleoproteins ; genetics ; metabolism ; Tumor Cells, Cultured

OBJECTIVE: To investigate the changes of GATA-1 protein expression during erythroid differentiation of K562 cells under hypoxia and how GATA-1 can regulate erythroid differentiation by up-regulating the expression of miR-451a and inhibiting the expression of 14-3-3ζ. METHODS: K562 cells were divided into 2 groups: the normoxia group and the hypoxia group, after the induction of hemin for 96 h, the positive cells rate of the benzidine staining, the mRNA expression of γ-globin and the expression of CD235a were detected, and the success of the model was verified. The changes of GATA-1 and miR-451a expression in the above-mentioned 2 groups, the changes of miR-451a expression after over-expressed GATA-1 were detected by Western blot and qRT-PCR. The cells in normoxic group and hypoxia group were divided into negative control group (NC group) and miR-451a over-expression group respectively, and the degree of erythroid differentiation in the four groups was judged according to the corresponding erythroid differentiation indexes, and the expression of 14-3-3ζ was detected by Western blot after over-expressed miR-451a. RESULTS: The positive cell rate of benzidine staining, mRNA expression of γ-globin and the expression of CD235a after 96 h induction by K562 cells under hypoxia were significantly higher than 0 h, suggesting that the erythroid differentiation model of K562 cells under hypoxia was replicated successfully. The expression levels of GATA-1 protein and miR-451a in the hypoxic group were significantly higher than that in the normoxic group (P<0.05). The expression level of miR-451a in hypoxia group was significantly higher than that in NC group after overexpressed GATA-1 (P<0.05). After over-expressed of miR-451a under hypoxia, the positive cell rate of benzidine staining, the mRNA expression level of γ-globin and the expression of CD235a were significantly higher than those in NC group (P<0.05). The expression level of 14-3-3ζ protein in miR-451a over-expressed group was lower than that in NC group under hypoxia (P<0.05). CONCLUSION Hypoxia can significantly increase the expression of GATA-1 protein, and the increase of GATA-1 expression can up-regulate the expression of miR-451a, thereby inhibiting the expression of 14-3-3ζ protein, which hinders the cell proliferation in erythroid differentiation model of K562 cells and plays an important role in promoting erythroid differentiation.
14-3-3 Proteins ; Cell Differentiation ; Erythroid Cells/metabolism* ; GATA1 Transcription Factor/metabolism* ; Humans ; Hypoxia ; K562 Cells ; MicroRNAs/genetics*

PRAS40, a proline-rich Akt substrate of 40 kD, is 14-3-3 binding protein. To study the interaction between PRAS40 and 14-3-3 isoforms, We constructed the expression vector pEG-PRAS40 (DNA-binding plasmid) and pJG-PRAS40 (transcriptional activity plasmid) in yeast using gateway cloning technology, then the plasmid of pEG-PRAS40/pJG-PRAS40 was co-transformed into yeast EGY48 strain with each pJG-14-3-3 /pEG-14-3-3 isoform plasmid. The co-transformation were tested by nutrition limitation growth analysis, beta-galactosidase color assay was used to study the interaction degree between PRAS40 and 14-3-3 isoforms. We confirmed successfully the construction of pJG-PRAS40 and pEG-PRAS40 with enzyme digestion. four 14-3-3 isoforms were found interacting with PRAS40 using yeast two-hybrid assay, the interaction degree of Epsilon was stronger than beta and zeta, tau was the weakest. Our result will be used to further study the biological function of PRAS40 and 14-3-3 as new drug target.
14-3-3 Proteins ; genetics ; metabolism ; Adaptor Proteins, Signal Transducing ; Humans ; Phosphoproteins ; genetics ; metabolism ; Protein Binding ; Protein Isoforms ; genetics ; metabolism ; Two-Hybrid System Techniques

14-3-3 Proteins ; genetics ; metabolism ; Animals ; Brain ; pathology ; Cricetinae ; Internal Ribosome Entry Sites ; Open Reading Frames ; Prion Diseases ; genetics ; metabolism

14-3-3 proteins are important proteins in plants, as they regulate plant growth and development and the response to biotic or abiotic stresses. In this study, a 14-3-3 gene(GenBank accession: OM683281) was screened from the cDNA library of the medicinal species Salvia miltiorrhiza by yeast two-hybrid and cloned. The open reading frame(ORF) was 780 bp, encoding 259 amino a cids. Bioinformatics analysis predicted that the protein was a non-transmembrane protein with the molecular formula of C_(1287)H_(2046)N_(346)O_(422)S_9, relative molecular weight of 29.4 kDa, and no signal peptide. Homologous sequence alignment and phylogenetic tree analysis proved that the protein belonged to 14-3-3 family and had close genetic relationship with the 14-3-3 proteins from Arabidopsis thaliana, Oryza sativa, and Nicotiana tabacum. The 14-3-3 gene was ligated to the prokaryotic expression vector pGEX-4 T-1 and then transformed into Escherichia coli BL21 for the expression of recombinant protein. Real-time fluorescent quantitative PCR showed that the expression of this gene was different among roots, stems, leaves, and flowers of S. miltiorrhiza. To be specific, the highest expression was found in leaves, followed by stems, and the lowest expression was detected in flowers. S. miltiorrhiza plants were treated with 15% PEG(simulation of drought), and hormones salicylic acid, methyl jasmonate, and ethephon, respectively, and the expression of 14-3-3 gene peaked at the early stage of induction. Therefore, the gene can quickly respond to abiotic stresses such as drought and plant hormone treatments such as salicylic acid, jasmonic acid, and ethylene. This study lays the foundation for revealing the molecular mechanism of 14-3-3 protein regulating tanshinone biosynthesis and responding to biotic and abiotic stresses.
14-3-3 Proteins/metabolism* ; Amino Acid Sequence ; Cloning, Molecular ; Ethylenes/metabolism* ; Gene Expression Regulation, Plant ; Hormones/metabolism* ; Phylogeny ; Plant Growth Regulators/pharmacology* ; Plant Proteins/metabolism* ; Recombinant Proteins/genetics* ; Salicylic Acid/metabolism* ; Salvia miltiorrhiza/metabolism*

OBJECTIVETo investigate the protective immunity against Echinococcus granulosus in mice immunized with rEg14-3-3.

METHODSICR mice were subcutaneously immunized three times with rEg14-3-3, followed by the challenge with Echinococcus granulosus protoscoleces intraperitoneally and then sacrificed after six months of post-challenge to detect the proliferation of splenocytes by MTT assay, and to measure the secretion of IL-2, IL-4, IL-10, and IFN-γ by ELISA. The rate of reduced hydatid cyst and the levels of IgE, IgG and IgG subclasses in sera were examined.

RESULTSMice vaccinated with rEg14-3-3 and challenged with protoscoleces revealed significant protective immunity of 84.47%. ELISA analysis indicated that the immunized mice generated specific high levels of IgG and the prevailing isotypes of IgG were IgG1 and IgG2a. Splenocytes from mice immunized with rEg14-3-3 showed a significant proliferation response. The secretion of IFN-γ and IL-2 increased significantly in the vaccinated mice whereas there was no significant difference in IL-4 and IL-10 levels between vaccinated and control mice.

CONCLUSIONThe results indicate that the rEg14-3-3 vaccine could induce a high level of protective immunity as a promising vaccine candidate to prevent cystic echinococcosis.

14-3-3 Proteins ; genetics ; metabolism ; Animals ; Blotting, Western ; Cell Proliferation ; Cytokines ; genetics ; metabolism ; Echinococcosis ; prevention & control ; Echinococcus granulosus ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Regulation ; physiology ; Mice ; Spleen ; cytology ; Vaccines ; immunology

14-3-3zeta is related to many cancer survival cellular processes. In a previous study, we showed that silencing 14-3-3zeta decreases the resistance of hepatocellular carcinoma (HCC) to chemotherapy. In this study, we investigated whether silencing 14-3-3zeta affects the radioresistance of cancer stem-like cells (CSCs) in HCC. Knockdown of 14-3-3zeta decreased cell viability and the number of spheres by reducing radioresistance in CSCs after gamma-irradiation (IR). Furthermore, the levels of pro-apoptotic proteins were upregulated in CSCs via silencing 14-3-3zeta after IR. These results suggest that 14-3-3zeta knockdown enhances radio-induced apoptosis by reducing radioresistance in liver CSCs.
14-3-3 Proteins/genetics/*metabolism ; Antigens, CD/genetics/*metabolism ; Apoptosis Regulatory Proteins/genetics/metabolism ; Carcinoma, Hepatocellular/genetics/metabolism ; Cell Line, Tumor ; *Gamma Rays ; Glycoproteins/genetics/*metabolism ; Humans ; Liver Neoplasms/genetics/metabolism ; Neoplastic Stem Cells/metabolism/*radiation effects ; Peptides/genetics/*metabolism ; *Radiation Tolerance

YWHAE gene is located on chromosome 17p13.3, and its product 14-3-3epsilon protein belongs to 14-3-3 protein family. As a molecular scaffold, YWHAE participates in biological processes such as cell adhesion, cell cycle regulation, signal transduction and malignant transformation, and is closely related to many diseases. Overexpression of YWHAE in breast cancer can increase the ability of proliferation, migration and invasion of breast cancer cells. In gastric cancer, YWHAE acts as a negative regulator of MYC and CDC25B, which reduces their expression and inhibits the proliferation, migration, and invasion of gastric cancer cells, and enhances YWHAE-mediated transactivation of NF-κB through CagA. In colorectal cancer, YWHAE lncRNA, as a sponge molecule of miR-323a-3p and miR-532-5p, can compete for endogenous RNA through direct interaction with miR-323a-3p and miR-532-5p, thus up-regulating K-RAS/ERK/1/2 and PI3K-AKT signaling pathways and promoting the cell cycle progression of the colorectal cancer. YWHAE not only mediates tumorigenesis as a competitive endogenous RNA, but also affects gene expression through chromosome variation. For example, the FAM22B-YWHAE fusion gene caused by t(10; 17) (q22; p13) may be associated with the development of endometrial stromal sarcoma. At the same time, the fusion transcript of YWHAE and NUTM2B/E may also lead to the occurrence of endometrial stromal sarcoma. To understand the relationship between YWHAE, NUTM2A, and NUTM2B gene rearrangement/fusion and malignant tumor, YWHAE-FAM22 fusion gene/translocation and tumor, YWHAE gene polymorphism and mental illness, as well as the relationship between 17p13.3 region change and disease occurrence. It provides new idea and basis for understanding the effect of YWHAE gene molecular mechanism and genetic variation on the disease progression, and for the targeted for the diseases.
14-3-3 Proteins/metabolism* ; Breast Neoplasms/genetics* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Cell Transformation, Neoplastic/genetics* ; Colorectal Neoplasms/genetics* ; Endometrial Neoplasms ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; MicroRNAs/genetics* ; Phosphatidylinositol 3-Kinases/metabolism* ; Sarcoma, Endometrial Stromal/pathology* ; Stomach Neoplasms/genetics* ; Transcription Factors/genetics* ; Translocation, Genetic

OBJECTIVETo screen genes differentially expressed in two human giant-cell lung cancer lines of same origin but with different metastasis potentials.

METHODSSuppression subtractive hybridization (SSH) was done twice on two giant-cell lung cancer lines, PLA-801C and PLA-801D (hereafter abbreviated as C and D), of same origin but with low (C) and high (D) metastatic potentials. In the first round, SSH C was used as tester and D as driver, while in the second round, the tester and driver were interchanged. The sequences acquired from both rounds of SSH were spotted on glass slides respectively and screened by hybridizing with two-color fluorescence probes. Clones that had different expression levels on chips were also confirmed by RNA dot blot or Northern blot.

RESULTSThere were 16 sequences with high expression in C as compared to those in D, and 79 sequences with high expression in D compared to those in C. After sequencing, most of them were found to be highly homologous to those encoding the following proteins: (1) cytokines and their receptors; (2) kinases and related proteins; (3) other proteins including enzymes, heat shock proteins, receptors, proteins of cell skeleton and mitochondria, products of oncogenes, etc; (4) some proteins deduced from gene sequences with yet unknown functions.

CONCLUSIONThe alterations in expression of some known genes, including HSP70, AXL receptor tyrosine kinase and 14-3-3zeta, might have impact on metastasis of giant-cell lung cancer. Whether some differentially expressed genes newly revealed are metastasis-related needs further study.

14-3-3 Proteins ; metabolism ; Carcinoma, Giant Cell ; genetics ; metabolism ; secondary ; Cell Line, Tumor ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; HSP70 Heat-Shock Proteins ; metabolism ; Humans ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Neoplasm Metastasis ; Nucleic Acid Hybridization ; Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins ; Receptor Protein-Tyrosine Kinases ; metabolism

To investigate the function of ALK3 gene, the gene regulation and the signaling pathway related to ventricular septum defect during heart development. The model mice with ALK3 gene knock-out via alpha-MHC-Cre/lox P system were bred. The mRNA expression level of control group was compared with that of experiment group and ALK3 downstream genes were screened using PCR-select cDNA subtraction microarray. The mRNA of control group was extracted from E11.5 normal mouse hearts, and that of experiment group, from E11.5 hearts of mice with alpha-MHC Cre(+/-) ALK3(F/+) genotype. It was found that the mice with ALK3 gene knock-out produced heart defects involving the interventricular septum. The platelet-activating factors acetylhydrolase and the transcription factor Pax-8 and so on, were down-regulated. However, the Protein Tyrosine Kinase (PTK) of Focal Adhesion Kinase (FAK) subfamily and beta subtype protein 14-3-3 were up-regulated in the alpha-MHC Cre(+/-) ALK3(F/-) mice. These data provide support that ALK3 gene played an important role during heart development. The platelet-activating factors acetylhydrolase and Pax-8 genes could be important ALK3 downstream genes in the BMP signaling pathway during interventricular septum development. PTK and beta subtype protein 14-3-3 might be regulatory factors in this pathway.
1-Alkyl-2-acetylglycerophosphocholine Esterase ; genetics ; metabolism ; 14-3-3 Proteins ; genetics ; metabolism ; Animals ; Bone Morphogenetic Protein Receptors, Type I ; genetics ; metabolism ; Genotype ; Heart Septal Defects, Ventricular ; genetics ; Mice ; Mice, Knockout ; Oligonucleotide Array Sequence Analysis ; PAX8 Transcription Factor ; Paired Box Transcription Factors ; genetics ; metabolism ; Protein-Tyrosine Kinases ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; genetics ; physiology