OBJECTIVE: To analyze the relationship between the promoter methylation status of Tripartite-motif protein 58 (TRIM58) and its mRNA expression level in acute myeloid leukemia (AML), and to explore the expression and regulation of TRIM58 in AML. METHODS: The bisulfite sequencing PCR (BSP) and quantitative real-time PCR (qPCR) technologies were used to detect the promoter methylation status and expression levels of TRIM58 mRNA in primary CD34⁺ and CD34^- AML cells and the AML cell lines KG1a and K562 were determined. RESULTS: The expression of TRIM58 mRNA in CD34⁺ cells was down-regulated in 10 AML patients, while that in CD34^- cells was down-regulated in 12 cases. Differences in the promoter methylation level of TRIM58 were statistically significant between AML group and normal control group (P<0.05). Additionally, the expression of TRIM58 mRNA was down-regulated in cell lines KG1a and K562, and up-regulated after decitabine treatment. CONCLUSION The down-regulation of mRNA expression of TRIM58 in AML cells may be related to its promoter methylation status.
DNA Methylation ; Decitabine ; Humans ; Leukemia, Myeloid, Acute/metabolism* ; RNA, Messenger/metabolism* ; Tripartite Motif Proteins/metabolism*

Muscular dystrophy (MD), a group of inherited disorders characterized by progressive skeletal muscle wasting and weakness, can be classified into several groups according to Mendelian inheritance patterns and clinical features. Many genes related to MD have been identified and cloned by genetic linkage analysis and positional cloning strategy. Our understanding of the molecular mechanisms giving rise to muscular dystrophy have made a progress by the functional analysis of proteins encoded by candidate genes for MD. This article reviews genes and their functional mechanisms in the pathogenesis of muscular dystrophy.
Calpain ; genetics ; Dystrophin ; genetics ; Humans ; Lamin Type A ; genetics ; Muscle Proteins ; genetics ; Muscular Dystrophies ; etiology ; genetics ; Myostatin ; Transforming Growth Factor beta ; genetics ; Tripartite Motif Proteins ; Ubiquitin-Protein Ligases ; genetics

BACKGROUND: The hepatitis B virus (HBV) vaccine has been efficiently used for decades. However, hepatocellular carcinoma caused by HBV is still prevalent globally. We previously reported that interferon (IFN)-induced tripartite motif-containing 25 (TRIM25) inhibited HBV replication by increasing the IFN expression, and this study aimed to further clarify the anti-HBV mechanism of TRIM25. METHODS: The TRIM25-mediated degradation of hepatitis B virus X (HBx) protein was determined by detecting the expression of HBx in TRIM25-overexpressed or knocked-out HepG2 or HepG2-NTCP cells via Western blotting. Co-immunoprecipitation was performed to confirm the interaction between TRIM25 and HBx, and colocalization of TRIM25 and HBx was identified via immunofluorescence; HBV e-antigen and HBV surface antigen were qualified by using an enzyme-linked immunosorbent assay (ELISA) kit from Kehua Biotech. TRIM25 mRNA, pregenomic RNA (pgRNA), and HBV DNA were detected by quantitative real-time polymerase chain reaction. The retinoic acid-inducible gene I (RIG-I) and pgRNA interaction was verified by RNA-binding protein immunoprecipitation assay. RESULTS: We found that TRIM25 promoted HBx degradation, and confirmed that TRIM25 could enhance the K90-site ubiquitination of HBx as well as promote HBx degradation by the proteasome pathway. Interestingly, apart from the Really Interesting New Gene (RING) domain, the SPRY domain of TRIM25 was also indispensable for HBx degradation. In addition, we found that the expression of TRIM25 increased the recognition of HBV pgRNA by interacting with RIG-I, which further increased the IFN production, and SPRY, but not the RING domain is critical in this process. CONCLUSIONS The study found that TRIM25 interacted with HBx and promoted HBx-K90-site ubiquitination, which led to HBx degradation. On the other hand, TRIM25 may function as an adaptor, which enhanced the recognition of pgRNA by RIG-I, thereby further promoting IFN production. Our study can contribute to a better understanding of host-virus interaction.
Humans ; Hepatitis B virus ; DEAD Box Protein 58/metabolism* ; RNA ; Liver Neoplasms ; Virus Replication ; Tripartite Motif Proteins/genetics* ; Transcription Factors ; Ubiquitin-Protein Ligases/genetics*

PURPOSE: Tripartite-motif-containing protein 56 (TRIM56) has been found to exhibit a broad antiviral activity, depending upon E3 ligase activity. Here, we attempted to evaluate the function of TRIM56 in multiple myeloma (MM) and its underlying molecular basis. MATERIALS AND METHODS: TRIM56 expression at the mRNA and protein level was measured by qRT PCR and western blot analysis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry analysis was performed to investigate the effect of TRIM56 on MM cell proliferation and apoptosis. The concentrations of interferon (IFN)-β, interleukin (IL)-6, and tumor necrosis factor-α in MM cell culture supernatants were detected with respective commercial ELISA kits. Western blot was employed to determine the effect of TRIM56 on toll-like receptor 3 (TLR3)/toll-IL-1 receptor (TIR) domain-containing adaptor inducing IFN-β (TRIF) signaling pathway. RESULTS: TRIM56 expression was prominently decreased in MM cells. Poly (dA:dT)-induced TRIM56 overexpression in U266 cells suppressed proliferation, induced apoptosis, and enhanced inflammatory cytokine production, while TRIM56 knockdown improved growth, diminished apoptosis, and inhibited inflammatory cytokine secretion in RPMI8226 cells. Moreover, TRIM56 knockdown blocked TLR3 signaling pathway. Furthermore, poly (I:C), a TLR3 agonist, markedly abolished TRIM56 depletion-induced increase of proliferation, decrease of apoptosis, and reduction of inflammatory factor in MM cells. CONCLUSION: TRIM56 may act as a tumor suppressor in MM through activation of TLR3/TRIF signaling pathway, contributing to a better understanding of the molecular mechanism of TRIM56 involvement in MM pathogenesis and providing a promising therapy strategy for patients with MM.
Adaptor Proteins, Vesicular Transport/metabolism ; Apoptosis/drug effects ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cytokines/secretion ; Disease Progression ; Down-Regulation/drug effects ; Gene Knockdown Techniques ; Humans ; Multiple Myeloma/metabolism ; Multiple Myeloma/pathology ; Poly I-C/pharmacology ; Signal Transduction/drug effects ; Toll-Like Receptor 3/metabolism ; Tripartite Motif Proteins/deficiency ; Tripartite Motif Proteins/metabolism ; Ubiquitin-Protein Ligases/deficiency ; Ubiquitin-Protein Ligases/metabolism

OBJECTIVES: To screen the DNA methylation loci associated with the age of Han males in northern China and to construct an age estimation model. METHODS: Twenty-one candidate methylation loci were screened. The DNA methylation levels of 476 blood samples from Chinese Han males were detected for 21 amplicons using EpiTYPER technology platform, and data on 153 DNA methylation loci were obtained. RESULTS: After correlation analysis, 8 age-related DNA methylation loci were finally screened. CpG1, CpG2, CpG4, CpG7, CpG8 were located on TRIM59, RASSF5, Clorf132, CSNK1D, ELOVL2,CpG5, CpG6 on PDE4C, and CpG3 on chr17:21452808. Based on the 8 loci, 352 samples were used for model construction. A multivariate linear regression age estimation model was constructed （R²=0.93）, with mean absolute deviation （MAD） of 2.69 years old. When 109 samples were used for model validation, the MAD was 3.80 years old. The test was repeated 3 times in 15 new samples, with MADs of 4.08, 4.68 and 3.93 years old, respectively. CONCLUSIONS The age estimation model on Han males in northern China constructed in this study can be used to estimate the age of victims and suspects and to narrow the scope of investigation, and therefore has practical application value.
Adaptor Proteins, Signal Transducing ; Apoptosis Regulatory Proteins ; Asian People ; Child, Preschool ; China ; CpG Islands ; DNA Methylation ; Humans ; Intracellular Signaling Peptides and Proteins ; Linear Models ; Male ; Membrane Proteins ; Metalloproteins ; Monomeric GTP-Binding Proteins ; Tripartite Motif Proteins

This study was aimed to investigate the changes of muscle protein synthesis and degradation under different movement conditions, so as to provide theoretical basis for muscle atrophy mechanism. Sprague Dawley (SD) rats were randomly divided into control, endurance training (treadmill training), hind limb overhanging and eccentric training (treadmill training, angle -16º) groups. The gastrocnemius muscles of rats were taken and weighed. The muscle was sectioned, and HE staining was employed to determine the cell's cross-sectional area. Protein expression of p-Akt was measured by immunohistochemistry; and the expressions of MuRF1 and FoxO1 were determined by Western blot. The results showed that, compared with control group, hind limb overhanging and eccentric training groups exhibited decreased muscle weight and cross-sectional area, but endurance training group did not show any changes. The expressions of p-Akt in endurance and eccentric training groups, not in hind limb overhanging group, were significantly higher than that in control group. Compared with that of control, MuRF1 protein remained unchanged in endurance training groups, but was increased in eccentric training and hind limb overhanging groups; FoxO1 protein was decreased in endurance training group, but was increased in eccentric training and hind limb overhanging groups. These results indicate that movement (endurance and eccentric training) can activate Akt expression, but does not increase muscle weight, whereas eccentric training and hind limb overhanging can increase the expressions of MuRF1 and FoxO1, and induce amyotrophy, suggesting MuRF1 and FoxO1 are major determinant factors in muscle atrophy.
Animals ; Forkhead Transcription Factors ; physiology ; Hindlimb Suspension ; Muscle Proteins ; physiology ; Muscle, Skeletal ; physiology ; Muscular Atrophy ; physiopathology ; Nerve Tissue Proteins ; physiology ; Physical Conditioning, Animal ; Proto-Oncogene Proteins c-akt ; physiology ; Rats ; Rats, Sprague-Dawley ; Tripartite Motif Proteins ; Ubiquitin-Protein Ligases ; physiology

OBJECTIVETo investigate the effects of AICAR on the activity of transcription factor FOXO1 and expression of ubiquitin ligase MuRF1 in rat cardiomyocytes, and explore the possible role of AMP-activated protein kinase (AMPK) in proteolysis pathways.

METHODSIn vitro cultured neonatal rat cardiac myocytes were treated with AICAR, and Western blotting was used to detect the phosphorylation of FOXO1 and expression of MuRF1 in the cells.

RESULTSAICAR activated AMPK in rat cardiac myocytes. Activated AMPK significantly inhibited the phosphorylation of FOXO1 and increased MuRF1 protein expression.

CONCLUSIONAMPK may regulate proteolysis by activating FOXO1 transcription factor and up-regulating MuRF1 expression.

AMP-Activated Protein Kinases ; metabolism ; Aminoimidazole Carboxamide ; analogs & derivatives ; pharmacology ; Animals ; Cells, Cultured ; Forkhead Transcription Factors ; metabolism ; Muscle Proteins ; metabolism ; Myocytes, Cardiac ; metabolism ; Nerve Tissue Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley ; Ribonucleotides ; pharmacology ; Tripartite Motif Proteins ; Ubiquitin-Protein Ligases ; metabolism

OBJECTIVETo investigate the expression of tripartite-motif protein 25 (TRIM25) and pyruvate kinase M2 (PKM2) protein in non-small cell lung cancer (NSCLC) and explore their role in the occurrence and progression of NSCLC.

METHODSThe expressions of TRIM25 and PKM2 protein were detected in 60 NSCLC specimens and 20 adjacent normal lung tissue (>5 cm from the lesions) with immunofluorescence histochemical method and in 10 fresh specimens of NSCLC with Western blotting. The results were analyzed in relation with the clinicopathological features of the patients.

RESULTSThe positivity rates of TRIM25 expression was 45% in the 60 lung carcinoma specimens, significantly higher than that in the 20 normal lung tissues (10%, P=0.005). TRIM25 protein was expressed in 28.6% of lung adenocarcinoma tissues and in 59.4% of squamous carcinoma tissues (P=0.017). TRIM25 protein expression was positively correlated with the TNM stages and lymph node metastasis of NSCLC (P<0.05). The expressions of PKM2 protein in 60 cases of lung carcinoma was 73.3%,while in 20 cases of normal lung tissues the expressions was 30%(P=0.001). The positivity rates of PKM2 expression differed significantly between lung adenocarcinoma and squamous carcinoma (57.1% vs 87.5%, P=0.008). An inverse correlation was noted between TRIM25 and PKM2 expressions (P=0.026).

CONCLUSIONTRIM25 and PKM2 protein may participate in the occurrence and progression of NSCLC, and their expressions are inversely correlated.

Adenocarcinoma ; enzymology ; Carcinoma, Non-Small-Cell Lung ; enzymology ; Carcinoma, Squamous Cell ; enzymology ; Carrier Proteins ; metabolism ; Humans ; Lung ; pathology ; Lung Neoplasms ; enzymology ; Lymphatic Metastasis ; Membrane Proteins ; metabolism ; Thyroid Hormones ; metabolism ; Transcription Factors ; metabolism ; Tripartite Motif Proteins ; Ubiquitin-Protein Ligases ; metabolism

OBJECTIVETo study muscle atrophy F-box (MAFbx) and muscle ring finger 1 (MuRF1) mRNA expression and its relationship with muscular contraction following free muscle transfer.

METHODSThe gracilis muscle was orthotopic transferred in adult rat to establish the animal model. The muscle at the unoperated side was used as control. The expression of MAFbx and MuRF1 mRNA, the muscle contraction and muscle function were measured by real-time PCR and multiple function physiological device. The relationship among the expression of MAFbx and MuRF1 mRNA, the muscle contraction and muscle function was analyzed.

RESULTSAfter muscle free transfer, muscle wet weight reservation, the maximum contraction and tetanus strength reduce first and increased later, but still lower than those at control side. The expression of MAFbx and MuRF1 mRNA reached peak level 3 - 4 weeks after muscle transfer which was 7.1 and 4.1 times as that at control side. It decreased later, but still higher than that at control side, showing a significant difference between them (P< 0. 05).

CONCLUSIONSPersistent over-expression of MAFbx and MuRF1 mRNA after muscle transfer has a close relationship with muscle atrophy and muscle dysfunction. MAFbx and MuRF1 can be used as markers for early muscle atrophy, and also as potential target for drug treatment of muscle atrophy.

Animals ; Female ; Muscle Contraction ; Muscle Proteins ; genetics ; Muscle, Skeletal ; pathology ; Muscular Atrophy ; genetics ; metabolism ; pathology ; RING Finger Domains ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; SKP Cullin F-Box Protein Ligases ; genetics ; Tripartite Motif Proteins ; Ubiquitin-Protein Ligases ; genetics

OBJECTIVE: To investigate the effect of the tripartite motif containing 31 (TRIM31) gene silencing on the proliferation and apoptosis of multiple myeloma cells and its possible mechanism. METHODS: The normal bone marrow plasma cells (nPCs) were selected as control, and the mRNA and protein expression levels of TRIM31 in human multiple myeloma cell lines (U266, RPMI-8226, NCI-H929 and KMS-11) were detected by RT-qPCR and Western blot. Recombinant lentivirol vector containing shRNA-TRIM31 and its negative control were used to infect U266 cells respectively, and the mRNA expression level of TRIM31 in infected cells was detected by RT-qPCR. Then cell proliferation, colony forming and apoptosis were analyzed by CCK-8, soft agar assay, and flow cytometry, respectively. The protein expression levels of TRIM31, cleaved-caspase-3, BCL-2, Bax, p-Akt (Ser473), Akt and PI3K (p110α) were evaluated by Western blot. In addition, the PI3K/Akt signaling pathway-specific inhibitor LY294002 and TRIM31-shRNA lentivirus were used to interfere with U266 cells, and the cell proliferation, apoptosis, and protein expression of p-Akt (Ser473) and Akt were detected by CCK-8, flow cytometry and Western blot, respectively. RESULTS: Compared with nPCs, the expression levels of TRIM31 mRNA and protein in U266, RPMI-8226, NCI-H929 and KMS-11 cells were significantly increased (P<0.001), especially in U266 cells. After lentivirus infection, the levels of TRIM31 mRNA and protein in U266 cells were significantly decreased (P<0.001). TRIM31 silencing significantly inhibited the proliferation of U266 cells (P<0.05), attenuated the ability of cell cloning, improved cell apoptosis, up-regulated the protein expressions of cleaved-caspase-3 and Bas as well as down-regulated expressions of BCL-2, p-Akt (Ser473) and PI3K (p110α). There was no significant effect on Akt protein. Intervention of LY294002 significantly enhanced the inhibition on cell proliferation and the promotion on apoptosis mediated by TRIM31 gene silencing in U266 cells. CONCLUSION TRIM31 gene silencing can inhibit U266 cell proliferation and promote its apoptosis, which may be closely related to inhibition of PI3K/Akt signaling pathway.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Gene Silencing ; Humans ; Multiple Myeloma ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Tripartite Motif Proteins/genetics* ; Ubiquitin-Protein Ligases/genetics*