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*

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*

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*

OBJECTIVE: To investigate the therapeutic effects of massage on denervated skeletal muscle atrophy in rats and its mechanism. METHODS: Forty-eight male SD rats were randomly divided into model group (n=24) and massage group (n=24). Gastrocnemius muscle atrophy model was established by transecting the right tibial nerve of rat. On the second day after operation, the gastrocnemius muscle of the rats in the massage group was given manual intervention and the model group was not intervened. Six rats were sacrificed at the four time points of 0 d, 7 d, 14 d and 21 d. The gastrocnemius of the rats were obtained and measured the wet mass ratio after weighing. Cross-sectional area and diameter of the muscle fiber were measured after HE staining. The relative expressions of miR-23a, Akt, MuRF1 and MAFbx mRNA were tested with qPCR. RESULTS: Compared with 0 d, the wet weight ratio, cross-sectional area and diameter of gastrocnemius muscle showed a progressive decline in the model group and massage group. The wet weight ratio, cross-sectional area and diameter of gastrocnemius muscle in the massage group were higher than those in the model group on 7 d, 14 d and 21 d (P＜0.05, P＜0.01). Compared with 0 d, the expressions of MuRF1, MAFbx and Akt mRNA were increased first and then were decreased in the model group and massage group. The expression of MuRF1 mRNA in massage group was lower than that in model group on 7 d and 21 d (P＜0.05, P＜0.01). The expression of MAFbx mRNA in massage group was lower than that in model group on 7 d, 14 d and 21 d (P＜0.01, P＜0.05, P＜0.01). The expression of Akt mRNA in massage group was higher than that in model group on 7 d, 14 d and 21 d (P＜0.05, P＜0.01). Compared with 0 d, the expression of miR-23a mRNA was increased in the model group and massage group on 21 d, and the expression of miR-23a mRNA in massage group was higher than that in model group (P＜ 0.05). CONCLUSION Massage can delay the atrophy of denervated skeletal muscle. The mechanism may be related to up-regulation of the expression of miR-23a and Akt mRNA, down-regulation of the expressions of MuRF1 and MAFbx mRNA, inhibition of protein degradation rate, and reduction of skeletal muscle protein degradation.
Animals ; Male ; Massage ; MicroRNAs ; metabolism ; Muscle Fibers, Skeletal ; Muscle Proteins ; metabolism ; Muscle, Skeletal ; physiopathology ; Muscular Atrophy ; therapy ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Sprague-Dawley ; SKP Cullin F-Box Protein Ligases ; metabolism ; Tripartite Motif Proteins ; metabolism ; 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

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

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

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

Retinoic acid-inducible gene I (RIG-I) is an important pattern recognition receptor that detects viral RNA and triggers the production of type-I interferons through the downstream adaptor MAVS (also called IPS-1, CARDIF, or VISA). A series of structural studies have elaborated some of the mechanisms of dsRNA recognition and activation of RIG-I. Recent studies have proposed that K63-linked ubiquitination of, or unanchored K63-linked polyubiquitin binding to RIG-I positively regulates MAVS-mediated antiviral signaling. Conversely phosphorylation of RIG-I appears to play an inhibitory role in controlling RIG-I antiviral signal transduction. Here we performed a combined structural and biochemical study to further define the regulatory features of RIG-I signaling. ATP and dsRNA binding triggered dimerization of RIG-I with conformational rearrangements of the tandem CARD domains. Full length RIG-I appeared to form a complex with dsRNA in a 2:2 molar ratio. Compared with the previously reported crystal structures of RIG-I in inactive state, our electron microscopic structure of full length RIG-I in complex with blunt-ended dsRNA, for the first time, revealed an exposed active conformation of the CARD domains. Moreover, we found that purified recombinant RIG-I proteins could bind to the CARD domain of MAVS independently of dsRNA, while S8E and T170E phosphorylation-mimicking mutants of RIG-I were defective in binding E3 ligase TRIM25, unanchored K63-linked polyubiquitin, and MAVS regardless of dsRNA. These findings suggested that phosphorylation of RIG inhibited downstream signaling by impairing RIG-I binding with polyubiquitin and its interaction with MAVS.
Adaptor Proteins, Signal Transducing ; metabolism ; Adenosine Triphosphate ; metabolism ; DEAD Box Protein 58 ; DEAD-box RNA Helicases ; chemistry ; genetics ; metabolism ; Dimerization ; Humans ; Mutagenesis, Site-Directed ; Phosphorylation ; Polyubiquitin ; metabolism ; Protein Binding ; Protein Structure, Tertiary ; RNA, Double-Stranded ; metabolism ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics ; Signal Transduction ; Transcription Factors ; metabolism ; Tripartite Motif Proteins ; Ubiquitin-Protein Ligases ; metabolism ; Ubiquitination

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