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*

This study aims to explore the effect and mechanism of Danggui Buxue Decoction(DBD)-containing serum in alleviating the H9c2 cell injury caused by the exposure to intermittent low oxygen. H9c2 cells were assigned into five groups: control(CON) group, intermittent low oxygen(IH) group, intermittent low oxygen plus DBD-containing serum(IH+DBD) group, intermittent low oxygen plus the autophagy enhancer rapamycin(IH+RAPA) group, and intermittent low oxygen plus DBD-containing serum and the autophagy inhibitor 3-methyladenine(IH+DBD+3-MA) group. Monodansylcadaverine(MDC) staining was employed to detect the changes of autophagosomes. Cell counting kit-8(CCK-8) assay was employed to determine the activity of myocardial cells, and lactate dehydrogenase(LDH) and creatine kinase(CK) kits were used to measure the LDH and CK levels in the cell culture, which would reflect the degree of cell damage. TdT-mediated dUTP nick-end labeling(TUNEL) staining was used to detect the apoptosis of myocardial cells, and JC-1 fluorescence probe to detect the changes in mitochondrial membrane potential. Western blot was employed to determine the expression levels of the autophagy-related proteins microtubule-associated proteins light chain 3Ⅱ(LC3Ⅱ), microtubule-associated proteins light chain 3Ⅰ(LC3Ⅰ), P62, Parkin and apoptosis related proteins pro caspase-3, caspase-3, B-cell lymphoma-2(Bcl-2), Bcl-2-associated X(Bax). The results showed that compared with the CON group, the IH group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated expression of P62. In addition, the IH group showed decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, and decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. Compared with the IH group, the IH+DBD and IH+RAPA groups showed increased fluorescence intensity of MDC staining, increased LC3Ⅱ/LC3Ⅰ ratio, up-regulated Parkin expression, and down-regulated P62 expression. In addition, the two groups showed increased cell survival rate, reduced content of LDH and CK in the culture medium, decreased number of TUNEL positive cells, and increased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. The IH+DBD+3-MA and IH groups showed no significant differences in the above indicators. Compared with the IH+DBD group, the IH+DBD+3-MA group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated P62 expression. In addition, the group had decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios, and declined mitochon-drial membrane potential. To sum up, DBD could promote the mitophagy, inhibit the apoptosis, and alleviated the injury of H9c2 cells exposed to low oxygen.
Oxygen ; bcl-2-Associated X Protein/metabolism* ; Caspase 3/genetics* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Apoptosis ; Autophagy ; Ubiquitin-Protein Ligases ; Microtubule-Associated Proteins

This study investigated the mechanism of Danggui Shaoyao Powder(DSP) against mitophagy in rat model of Alzheimer's disease(AD) induced by streptozotocin(STZ) based on PTEN induced putative kinase 1(PINK1)-Parkin signaling pathway. The AD rat model was established by injecting STZ into the lateral ventricle, and the rats were divided into normal group, model group, DSP low-dose group(12 g·kg~(-1)·d~(-1)), DSP medium-dose group(24 g·kg~(-1)·d~(-1)), and DSP high-dose group(36 g·kg~(-1)·d~(-1)). Morris water maze test was used to detect the learning and memory function of the rats, and transmission electron microscopy and immunofluorescence were employed to detect mitophagy. The protein expression levels of PINK1, Parkin, LC3BⅠ/LC3BⅡ, and p62 were assayed by Western blot. Compared with the normal group, the model group showed a significant decrease in the learning and memory function(P<0.01), reduced protein expression of PINK1 and Parkin(P<0.05), increased protein expression of LC3BⅠ/LC3BⅡ and p62(P<0.05), and decreased occurrence of mitophagy(P<0.01). Compared with the model group, the DSP medium-and high-dose groups notably improved the learning and memory ability of AD rats, which mainly manifested as shortened escape latency, leng-thened time in target quadrants and elevated number of crossing the platform(P<0.05 or P<0.01), remarkably activated mitophagy(P<0.05), up-regulated the protein expression of PINK1 and Parkin, and down-regulated the protein expression of LC3BⅠ/LC3BⅡ and p62(P<0.05 or P<0.01). These results demonstrated that DSP might promote mitophagy mediated by PINK1-Parkin pathway to remove damaged mitochondria and improve mitochondrial function, thereby exerting a neuroprotective effect.
Rats ; Animals ; Mitophagy ; Alzheimer Disease/genetics* ; Powders ; Protein Kinases/metabolism* ; Ubiquitin-Protein Ligases/metabolism*

OBJECTIVE: To analyze the gene mutation profile in children with acute lymphocyte leukemia (ALL) and to explore its prognostic significance. METHODS: Clinical data of 249 primary pediatric ALL patients diagnosed and treated in the Department of Hematological Oncology of Wuhan Children's Hospital from January 2018 to December 2021 were analyzed retrospectively. Next-generation sequencing (NGS) was used to obtain gene mutation data and analyze the correlation between it and the prognosis of children with ALL. RESULTS: 227 (91.2%) were B-ALL, 22 (8.8%) were T-ALL among the 249 cases, and 178 (71.5%) were found to have gene mutations, of which 85 (34.1%) had ≥3 gene mutations. NRAS(23.7%), KRAS (22.9%),FLT3(11.2%), PTPN11(8.8%), CREBBP (7.2%), NOTCH1(6.4%) were the most frequently mutated genes, the mutations of KRAS, FLT3, PTPN11, CREBBP were mainly found in B-ALL, the mutations of NOTCH1 and FBXW7 were mainly found in T-ALL. The gene mutation incidence of T-ALL was significantly higher than that of B-ALL (χ²= 5.573，P＜0.05) and were more likely to have co-mutations (P＜0.05). The predicted 4-year EFS rate (47.9% vs 88.5%, P＜0.001) and OS rate (53.8% vs 94.1%, P＜0.001) in children with tp53 mutations were significantly lower than those of patients without tp53 mutations. Patients with NOTCH1 mutations had higher initial white blood cell count （128.64×10⁹/L vs 8.23×10⁹/L，P＜0.001）, and children with NOTCH1 mutations had a lower 4-year EFS rate than those of without mutations (71.5% vs 87.2%, P＝0.037). CONCLUSION Genetic mutations are prevalent in childhood ALL and mutations in tp53 and NOTCH1 are strong predictors of adverse outcomes in childhood ALL, with NGS contributing to the discovery of genetic mutations and timely adjustment of treatment regimens.
Child ; Humans ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics* ; Cell Cycle Proteins/genetics* ; Proto-Oncogene Proteins p21(ras)/genetics* ; Retrospective Studies ; Ubiquitin-Protein Ligases/genetics* ; Prognosis ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics* ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ; Mutation ; Lymphocytes

OBJECTIVE: To explore the role of a new blood-based, multiomics and multidimensional method for evaluating the efficacy of patients with lymphoma. METHODS: 10 ml peripheral blood was extracted from each patient, and the genomic copy number aberrations (CNA) and fragment size (FS) were evaluated by low-depth whole genome sequencing of cfDNA, and the level of a group of plasma tumor marker (PTM) were detected at the same time. The cancer efficacy score (CES) was obtained by standardized transformation of the value of above three numerical indexes, and the changes of CES before and after treatment were compared to evaluate the patient's response to the treatment regimen. RESULTS: A total of 35 patients' baseline data were collected, of which 23 cases (65.7%) had elevated CES values. 18 patients underwent the first time test. The results showed that the CES value of 9 patients with positive baseline CES decreased significantly at the first test, and the efficacy evaluation was PR, which was highly consistent with the imaging evaluation results of the same period. At the same time, the CNA variation spectrum of all patients were evaluated and it was found that 23 patients had partial amplification or deletion of chromosome fragments. The most common amplification site was 8q24.21, which contains important oncogenes such as MYC. The most common deletion sites were 1p36.32, 4q21.23, 6q21, 6q27, 14q32.33, and tumor suppressor-related genes such as PRDM1, ATG5, AIM1, FOXO3 and HACE1 were expressed in the above regions, so these deletions may be related to the occurrence and development of lymphoma. CONCLUSION With the advantages of more convenience, sensitivity and non-invasive, this multiomics and multidimensional efficacy detection method can evaluate the tumor load of patients with lymphoma at the molecular level, and make more accurate efficacy evaluation, which is expected to serve the clinic better.
Humans ; Multiomics ; Lymphoma/genetics* ; Cell-Free Nucleic Acids ; Genomics/methods* ; DNA Copy Number Variations ; Ubiquitin-Protein Ligases

Humans ; Mitochondria/genetics* ; Mutation/genetics* ; Parkinson Disease/genetics* ; Protein Kinases ; Ubiquitin-Protein Ligases

OBJECTIVES: This study aimed to explore the specific mechanism, mediated by the reactive oxygen species (ROS) and PINK1/Parkin pathway, of the mitochondrial autophagy of human periodontal ligament cells (hPDLCs) under starvation conditions. METHODS: hPDLCs were isolated and cultured from normal periodontal tissues. Earle's balanced salt solution (EBSS) was used to simulated a starvation environment and thus stimulate hPDLCs mitochondrial autophagy. N-Acetyl-L-cysteine (NAC) was used to inhibit ROS production to explore the role of ROS in hPDLC mitochondrial autophagy. Cyclosporin A was used to inhibit the PINK1/Parkin pathway to study the role of ROS and the PINK1/Parkin pathway in hPDLCs activation under starvation. The mitochondrial membrane potential was detected by flow cytometry with a JC-1 mitochondrial membrane potential detection kit. The morphological structure of mitochondria and the formation of mitochondrial autophagosome were observed by transmission electron microscopy. Mito tracker red cmxros and lyso tracker green staining were used to observe the localization of mitochondria and lysosomes. The formation intensity of ROS was detected with a DCFH-DA ROS fluorescent probe. The expression levels of mitochondrial autophagy genes (Tomm20 and Timm23) and the PINK1/Parkin pathway were detected by real-time quantitative polymerase chain reaction (RT-qPCR). The expression levels of mitochondrial autophagy proteins (Tomm20 and Timm23) and PINK1/Parkin protein were detected by Western blot. RESULTS: EBSS starvation for 30 min induced the strongest activation of hPDLCs mitochondrial autophagy, increased the expression of ROS, downregulated the expression of mitochondrial autophagy-related genes (Tomm20 and Timm23) (P<0.001), and upregulated the PINK1/Parkin pathway (P<0.001). After NACinhibited ROS production, mitochondrial autophagy was also inhibited. Meanwhile, the expression of Tomm20 and Timm23 was upregulated (P<0.001 and P<0.05), and the expression of the PINK1/parkin pathway (P<0.001 and P<0.05) was down regulated. When cyclosporin A inhibited the expression of the PINK1/Parkin pathway (P<0.05 and P<0.05), it reversed the mitochondrial autophagy of hPDLCs (P<0.001 and P<0.01) and also upregulated the expression of Tomm20 and Timm23 (P<0.001 and P<0.01). CONCLUSIONS ROS enhanced the mitochondrial autophagy of hPDLCs primarily through the PINK1/Parkin pathway under starvation conditions.
Humans ; Mitophagy/genetics* ; Reactive Oxygen Species/metabolism* ; Periodontal Ligament/metabolism* ; Cyclosporine ; Protein Kinases/metabolism* ; Ubiquitin-Protein Ligases/metabolism*

OBJECTIVE: To analyze the clinical and genetic characteristics of a boy featuring unexplained developmental delay, malnutrition and distinct facial appearance. METHODS: Physical examination was carried out for the child. Peripheral blood samples were collected from the child and his parents for the extraction of genomic DNA and trio-whole exome sequencing. Candidate variants were verified by Sanger sequencing. RESULTS: The patient had facial dysmorphism including nasal alae aplasia, scalp defect and teeth deformities, in addition with recurrent diarrhea due to pancreatic exocrine insufficiency. DNA sequencing revealed that he has harbored compound heterozygous variants of the UBR1 gene, namely c.3167C>G (p.S1056X) and c.1911+14C>G, which were inherited from his father and mother, respectively. Database search has suggested the c.3167C>G to be a novel nonsense variant and c.1911+14C>G a known splicing variant. Based on the guidelines of the American College of Medical Genetics and Genomics, the two variants were predicted to be pathogenic and likely pathogenic, respectively. CONCLUSION The child was diagnosed with Johanson-Blizzard syndrome due to the compound heterozygous variants of the UBR1 gene. Above finding has enriched the mutational spectrum of the UBR1 gene and provided a basis for genetic counseling for this family.
Child ; Humans ; Male ; Ectodermal Dysplasia/genetics* ; Pancreatic Diseases/genetics* ; Ubiquitin-Protein Ligases/genetics*

OBJECTIVE: To explore the genetic basis for a patient with intellectual disability. METHODS: Whole exome sequencing and Sanger sequencing were carried out for the patient. The result was verified in her family. RESULTS: DNA sequencing revealed that the patient has carried a heterozygous nonsense c.40C>T (p.Arg14X) variant of the TRIP12 gene, which was de novo in origin. The variant was unrecorded in the Human Gene Mutation Database. Based on the American College of Medical Genetics and Genomics standards and guidelines, the variant was predicted to be pathogenic (PVS1+ PS2+ PP3). CONCLUSION The patient was diagnosed with autosomal dominant intellectual disability due to heterozygous c.40C>T variant of the TRIP12 gene.
Carrier Proteins/genetics* ; Codon, Nonsense ; Female ; Heterozygote ; Humans ; Intellectual Disability/genetics* ; Ubiquitin-Protein Ligases/genetics* ; Whole Exome Sequencing

OBJECTIVE: To screen proteins interacting with ring finger protein 216(RNF216) through yeast two hybrid experiment, and further clarify the role of RNF216 in the pathogenesis of gonadotropin-releasing hormone deficiency. METHODS: A recombinant expression vector pGBKT7-RNF216 was constructed and transformed into yeast Y2HGold, which was hybridized with a human cDNA library in order to screen proteins interacting with RNF216. The interaction was verified in yeast Y2HGold. RESULTS: A recombinant expression vector pGBKT7-RNF216 was successfully constructed and expressed in yeast Y2HGold. Filamin B (FLNB) was identified by yeast two hybrid experiment, and their interaction was verified in yeast Y2HGold. CONCLUSION An interaction between FLNB and RNF216 was identified through yeast two hybrid experiment. RNF216 may affect the proliferation and migration of GnRH neurons by regulating FLNB or FLNB/FLNA heterodimers.
Gene Library ; Gonadotropin-Releasing Hormone/genetics* ; Humans ; Proteins ; Two-Hybrid System Techniques ; Ubiquitin-Protein Ligases/genetics*