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Hepatology Communications Aug 2023The role of thioredoxin-interacting protein (TXNIP) in lipopolysaccharide-induced liver injury in mice has been reported, but the underlying mechanisms are poorly...
BACKGROUND
The role of thioredoxin-interacting protein (TXNIP) in lipopolysaccharide-induced liver injury in mice has been reported, but the underlying mechanisms are poorly understood.
METHODS
We overexpressed deubiquitinase in cells overexpressing TXNIP and then detected the level of TXNIP to screen out the deubiquitinase regulating TXNIP; the interaction between TXNIP and deubiquitinase was verified by coimmunoprecipitation. After knockdown of a deubiquitinase and overexpression of TXNIP in Huh7 and HepG2 cells, lipopolysaccharide was used to establish a cellular inflammatory model to explore the role of deubiquitinase and TXNIP in hepatocyte inflammation.
RESULTS
In this study, we discovered that ubiquitin-specific protease 5 (USP5) interacts with TXNIP and stabilizes it through deubiquitylation in Huh-7 and HepG2 cells after treatment with lipopolysaccharide. In lipopolysaccharide-treated Huh-7 and HepG2 cells, USP5 knockdown increased cell viability, reduced apoptosis, and decreased the expression of inflammatory factors, including NLRP3, IL-1β, IL-18, ASC, and procaspase-1. Overexpression of TXNIP reversed the phenotype induced by knockdown USP5.
CONCLUSIONS
In summary, USP5 promotes lipopolysaccharide-induced apoptosis and inflammatory response by stabilizing the TXNIP protein.
Topics: Apoptosis; Deubiquitinating Enzymes; Lipopolysaccharides; NLR Family, Pyrin Domain-Containing 3 Protein; Signal Transduction; Humans; Hep G2 Cells; Endopeptidases; Carrier Proteins
PubMed: 37534934
DOI: 10.1097/HC9.0000000000000193 -
Molecular Medicine (Cambridge, Mass.) Mar 2023Diabetes-related limb ischemia is a challenge for lower extremity amputation. Aurora Kinase A (AURKA) is an essential serine/threonine kinase for mitosis, while its role...
BACKGROUND
Diabetes-related limb ischemia is a challenge for lower extremity amputation. Aurora Kinase A (AURKA) is an essential serine/threonine kinase for mitosis, while its role in limb ischemia remains unclear.
METHOD
Human microvascular endothelial cells (HMEC-1) were cultured in high glucose (HG, 25 mmol/L D-glucose) and no additional growth factors (ND) medium to mimic diabetes and low growth factors deprivation as in vitro model. Diabetic C57BL/6 mice were induced by streptozotocin (STZ) administration. After seven days, ischemia was surgically performed by left unilateral femoral artery ligation on diabetic mice. The vector of adenovirus was utilized to overexpress AURKA in vitro and in vivo.
RESULTS
In our study, HG and ND-mediated downregulation of AURKA impaired the cell cycle progression, proliferation, migration, and tube formation ability of HMEC-1, which were rescued by overexpressed AURKA. Increased expression of vascular endothelial growth factor A (VEGFA) induced by overexpressed AURKA were likely regulatory molecules that coordinate these events. Mice with AURKA overexpression exhibited improved angiogenesis in response to VEGF in Matrigel plug assay, with increased capillary density and hemoglobin content. In diabetic limb ischemia mice, AURKA overexpression rescued blood perfusion and motor deficits, accompanied by the recovery of gastrocnemius muscles observed by H&E staining and positive Desmin staining. Moreover, AURKA overexpression rescued diabetes-related impairment of angiogenesis, arteriogenesis, and functional recovery in the ischemic limb. Signal pathway results revealed that VEGFR2/PI3K/AKT pathway might be involved in AURKA triggered angiogenesis procedure. In addition, AURKA overexpression impeded oxidative stress and subsequent following lipid peroxidation both in vitro and in vivo, indicating another protective mechanism of AURKA function in diabetic limb ischemia. The changes in lipid peroxidation biomarkers (lipid ROS, GPX4, SLC7A11, ALOX5, and ASLC4) in in vitro and in vivo were suggestive of the possible involvement of ferroptosis and interaction between AUKRA and ferroptosis in diabetic limb ischemia, which need further investigation.
CONCLUSIONS
These results implicated a potent role of AURKA in diabetes-related impairment of ischemia-mediated angiogenesis and implied a potential therapeutic target for ischemic diseases of diabetes.
Topics: Humans; Mice; Animals; Vascular Endothelial Growth Factor A; Aurora Kinase A; Diabetes Mellitus, Experimental; Endothelial Cells; Neovascularization, Physiologic; Phosphatidylinositol 3-Kinases; Hindlimb; Mice, Inbred C57BL; Ischemia; Muscle, Skeletal
PubMed: 36977984
DOI: 10.1186/s10020-023-00635-4 -
Cell Reports Dec 2021RNA-DNA hybrids are often associated with genome instability and also function as a cellular regulator in many biological processes. In this study, we show that...
RNA-DNA hybrids are often associated with genome instability and also function as a cellular regulator in many biological processes. In this study, we show that accumulated RNA-DNA hybrids cause multiple defects in budding yeast meiosis, including decreased sporulation efficiency and spore viability. Further analysis shows that these RNA-DNA hybrid foci colocalize with RPA/Rad51 foci on chromosomes. The efficient formation of RNA-DNA hybrid foci depends on Rad52 and ssDNA ends of meiotic DNA double-strand breaks (DSBs), and their number is correlated with DSB frequency. Interestingly, RNA-DNA hybrid foci and recombination foci show similar dynamics. The excessive accumulation of RNA-DNA hybrids around DSBs competes with Rad51/Dmc1, impairs homolog bias, and decreases crossover and noncrossover recombination. Furthermore, precocious removal of RNA-DNA hybrids by RNase H1 overexpression also impairs meiotic recombination similarly. Taken together, our results demonstrate that RNA-DNA hybrids form at ssDNA ends of DSBs to actively regulate meiotic recombination.
Topics: Cell Cycle Proteins; DNA Breaks, Double-Stranded; DNA Breaks, Single-Stranded; DNA, Fungal; DNA-Binding Proteins; Homologous Recombination; Meiosis; Nucleic Acid Conformation; Nucleic Acid Heteroduplexes; RNA, Fungal; Rad51 Recombinase; Replication Protein A; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 34879269
DOI: 10.1016/j.celrep.2021.110097 -
Overexpression of wild type RRAS2, without oncogenic mutations, drives chronic lymphocytic leukemia.Molecular Cancer Feb 2022Chronic lymphocytic leukemia (CLL) is the most frequent, and still incurable, form of leukemia in the Western World. It is widely accepted that cancer results from an...
BACKGROUND
Chronic lymphocytic leukemia (CLL) is the most frequent, and still incurable, form of leukemia in the Western World. It is widely accepted that cancer results from an evolutionary process shaped by the acquisition of driver mutations which confer selective growth advantage to cells that harbor them. Clear examples are missense mutations in classic RAS genes (KRAS, HRAS and NRAS) that underlie the development of approximately 13% of human cancers. Although autonomous B cell antigen receptor (BCR) signaling is involved and mutations in many tumor suppressor genes and oncogenes have been identified, an oncogenic driver gene has not still been identified for CLL.
METHODS
Conditional knock-in mice were generated to overexpress wild type RRAS2 and prove its driver role. RT-qPCR analysis of a human CLL sample cohort was carried out to measure RRAS2 transcriptional expression. Sanger DNA sequencing was used to identify a SNP in the 3'UTR region of RRAS2 in human CLL samples. RNAseq of murine CLL was carried out to identify activated pathways, molecular mechanisms and to pinpoint somatic mutations accompanying RRAS2 overexpression. Flow cytometry was used for phenotypic characterization and shRNA techniques to knockdown RRAS2 expression in human CLL.
RESULTS
RRAS2 mRNA is found overexpressed in its wild type form in 82% of the human CLL samples analyzed (n = 178, mean and median = 5-fold) as well as in the explored metadata. A single nucleotide polymorphism (rs8570) in the 3'UTR of the RRAS2 mRNA has been identified in CLL patients, linking higher expression of RRAS2 with more aggressive disease. Deliberate overexpression of wild type RRAS2 in mice, but not an oncogenic Q72L mutation in the coding sequence, provokes the development of CLL. Overexpression of wild type RRAS2 in mice is accompanied by a strong convergent selection of somatic mutations in genes that have been identified in human CLL. R-RAS2 protein is physically bound to the BCR and mediates BCR signals in CLL.
CONCLUSIONS
The results indicate that overexpression of wild type RRAS2 is behind the development of CLL.
Topics: Animals; Genes, ras; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Membrane Proteins; Mice; Monomeric GTP-Binding Proteins; Mutation; Receptors, Antigen, B-Cell; Signal Transduction
PubMed: 35120522
DOI: 10.1186/s12943-022-01496-x -
Journal of the Formosan Medical... Aug 2023The purpose of this study was to clarify the effect of ZC3H13 on the growth of papillary thyroid carcinoma (PTC).
PURPOSE
The purpose of this study was to clarify the effect of ZC3H13 on the growth of papillary thyroid carcinoma (PTC).
METHODS
Firstly, we used qRT-PCR and Western blot to compare the difference in the expression of ZC3H13 between normal thyroid epithelial cells and PTC cell lines. Then, ZC3H13 overexpression/knockout thyroid cancer cells were constructed by lentivirus transfection, and the effects of overexpression of ZC3H13 on the proliferation, migration and invasion of PTC cells were detected by CCK8 and transwell experiments. Lastly, MeRIP-qPCR, RIP and o Actinomycin D were used to verify that ZC3H13 regulated the expression of downstream target gene IQGAP1 through m6A modification.
RESULTS
ZC3H13 expression was decreased in PTC cell lines BCPAP, KTC-1, k1, HTH83, and TPC-1. Proliferation, invasion, and migration of PTC cells were inhibited by overexpressed ZC3H13 but increased by knockdown of ZC3H13. IQGAP1 expression was suppressed by ZC3H13 overexpression but enhanced by ZC3H13 knockdown. In ZC3H13-overexpressed PTC cells, the m6A level of IQGAP1 mRNA was increased, and the IQGAP1 mRNA expression was decreased with the increasing time of Actinomycin D treatment. YTHDF2 enriched more IQGAP1 mRNA than IgG and knockdown of YTHDF2 reversed the effect of ZC3H13 overexpression on IQGAP1 mRNA stability. The xenograft tumor experiment in nude mice confirmed that the overexpression of ZC3H13 inhibited tumor growth, while overexpression of IQGAP1 could reverse the inhibitory effect of ZC3H13 overexpression on tumor growth.
CONCLUSION
ZC3H13 mediates IQGAP1 mRNA degradation by promoting m6A modification of IQGAP1 mRNA, this provides a prospective therapeutic target for PTC.
Topics: Mice; Animals; Humans; Thyroid Cancer, Papillary; MicroRNAs; Mice, Nude; Dactinomycin; Cell Line, Tumor; Cell Proliferation; Neoplasm Invasiveness; Cell Movement; Thyroid Neoplasms; RNA, Messenger; Gene Expression Regulation, Neoplastic; Nuclear Proteins; RNA-Binding Proteins
PubMed: 36739231
DOI: 10.1016/j.jfma.2022.12.019 -
Chemical & Pharmaceutical Bulletin 2020C1q/tumor necrosis factor (TNF)-related protein 12 (CTRP12) is a secretory protein that participates in the regulation of glucose and lipid metabolism in obesity and...
C1q/tumor necrosis factor (TNF)-related protein 12 (CTRP12) is a secretory protein that participates in the regulation of glucose and lipid metabolism in obesity and diabetes. Its role in cardiovascular disease, particularly sepsis-induced cardiac injury, is unclear. Here, we stimulated cardiomyocytes with lipopolysaccharide (LPS) to establish an in vitro cardiomyocyte injury model and CTRP12 was overexpressed with an adenovirus delivery system. Overexpression of CTRP12 reduced the transcription and release of pro-inflammatory cytokines from LPS-stimulated cardiomyocytes, including TNFα, interleukin-1 (IL-1), and IL-6. Reactive oxygen species (ROS) level increased and the oxidation/redox system was disturbed in LPS-stimulated cardiomyocytes, as evident from the decrease in superoxide dismutase activity and an increase in reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and malondialdehyde level. CTRP12 overexpression decreased the increasing level of ROS and ameliorated the unbalance in the oxidation/redox system in LPS-stimulated cardiomyocytes. The viability of cardiomyocytes decreased after LPS stimulation, and the cells underwent apoptosis. CTRP12-overexpressing cardiomyocytes showed a decrease in the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive cells, and the ratio of B cell lymphoma (Bcl)-1/Bax in these cells was recovered. In comparison with the control group, LPS-stimulated cardiomyocytes showed reduced expression of nuclear factor E2-related factor 2 (NRF2), while CTRP12-overexpressing cardiomyocytes showed elevated NRF2 expression. Small-interfering RNA-mediated silencing of NRF2 expression in cardiomyocytes resulted in the inhibition of the protective effects of CTRP12. Thus, CTRP12 ameliorated injury in LPS-stimulated cardiomyocytes in an NRF2-dependent manner.
Topics: Adenoviridae; Animals; Cell Line; Cells, Cultured; Down-Regulation; Gene Transfer Techniques; Inflammation; Intercellular Signaling Peptides and Proteins; Interleukin-1; Interleukin-6; Lipopolysaccharides; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha; Up-Regulation
PubMed: 32009080
DOI: 10.1248/cpb.c19-00646 -
Cancer Medicine Jun 2023Gastric cancer (GC) is one of most common cancers worldwide. Several studies have suggested that Rab31 functions as a membrane vesicle transport regulator; however, the...
BACKGROUND
Gastric cancer (GC) is one of most common cancers worldwide. Several studies have suggested that Rab31 functions as a membrane vesicle transport regulator; however, the mechanism by which RAB31 regulates exosome secretion and promotes metastasis remains to be clarified.
METHODS
We examined the expression of RAB31 protein and mRNA in GC tissue samples via immunohistochemistry and reverse transcription-polymerase chain reaction assays, respectively. We elucidated the function of RAB31 in GC cells by constructing a cell model and a pulmonary metastatic model of GC with overexpression of RAB31. Protein mass spectrometry was used to identify the exosomal protein.
RESULTS
RAB31 expression increased at both the protein and mRNA levels with the development of GC. Cells overexpressing RAB31 showed an enhanced ability to migrate in both the in vitro cell model and the pulmonary metastatic model of GC. Exosome nanoparticle tracking analysis and electron microscopy revealed that the both the number and size of the exosomes secreted by GC cells were reduced when RAB31 expression was depleted. Injection of exosomes derived from RAB31 overexpressing cells promoted pulmonary metastasis in vivo. Analysis of the exosomal proteins revealed that PSMA1 was overexpressed in GC tissue in accordance with RAB31 expression. PSMA1 overexpression was highly associated with poor prognosis of GC patients.
CONCLUSION
Our findings revealed a key role for RAB31 in GC metastasis through regulation of exosome secretion.
Topics: Humans; Exosomes; Stomach Neoplasms; RNA, Messenger; Cell Line, Tumor; Cell Proliferation; MicroRNAs; Gene Expression Regulation, Neoplastic; Cell Movement; rab GTP-Binding Proteins
PubMed: 37222416
DOI: 10.1002/cam4.6007 -
Journal of Experimental & Clinical... Feb 2024Par-3 Family Cell Polarity Regulator (PARD3) is a cellular protein essential for asymmetric cell division and polarized growth. This study aimed to study the role of...
BACKGROUND
Par-3 Family Cell Polarity Regulator (PARD3) is a cellular protein essential for asymmetric cell division and polarized growth. This study aimed to study the role of PARD3 in hepatic tumorigenesis.
METHODS
The essential role of PARD3 in mediating hepatic tumorigenesis was assessed in diet-induced spontaneous liver tumour and syngeneic tumour models. The mechanism of PARD3 was delineated by bulk and single-cell RNA sequencing. The clinical significance of PARD3 was identified by tissue array analysis.
RESULTS
PARD3 was overexpressed in tumour tissues and PARD3 overexpression was positively correlated with high tumour stage as well as the poor prognosis in patients. In models of spontaneous liver cancer induced by choline-deficient, amino acid-defined (CDAA) and methionine-choline-deficient (MCD) diets, upregulation of PARD3 was induced specifically at the tumorigenesis stage rather than other early stages of liver disease progression. Site-directed knockout of PARD3 using an adeno-associated virus 8 (AAV8)-delivered CRISPR/Cas9 single-guide RNA (sgRNA) plasmid blocked hepatic tumorigenesis, while PARD3 overexpression accelerated liver tumour progression. In particular, single-cell sequencing analysis suggested that PARD3 was enriched in primitive tumour cells and its overexpression enhanced tumour-initiating cell (TICs). Overexpression of PARD3 maintained the self-renewal ability of the CD133 TIC population within hepatocellular carcinoma (HCC) cells and promoted the in vitro and in vivo tumorigenicity of CD133 TICs. Transcriptome analysis revealed that Sonic Hedgehog (SHH) signalling was activated in PARD3-overexpressing CD133 TICs. Mechanistically, PARD3 interacted with aPKC to further activate SHH signalling and downstream stemness-related genes. Suppression of SHH signalling and aPKC expression attenuated the in vitro and in vivo tumorigenicity of PARD3-overexpressing CD133 TICs. Tissue array analysis revealed that PARD3 expression was positively associated with the phosphorylation of aPKC, SOX2 and Gli1 and that the combination of these markers could be used to stratify HCC patients into two clusters with different clinicopathological characteristics and overall survival prognoses. The natural compound berberine was selected as a potent suppressor of PARD3 expression and could be used as a preventive agent for liver cancer that completely blocks diet-induced hepatic tumorigenesis in a PARD3-dependent manner.
CONCLUSION
This study revealed PARD3 as a potential preventive target of liver tumorigenesis via TIC regulation.
Topics: Humans; Adaptor Proteins, Signal Transducing; Carcinogenesis; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Line, Tumor; Cell Transformation, Neoplastic; Choline; Hedgehog Proteins; Liver Neoplasms; Neoplastic Stem Cells; RNA, Guide, CRISPR-Cas Systems
PubMed: 38317186
DOI: 10.1186/s13046-024-02967-3 -
BioMed Research International 2019Myoferlin (MYOF), initially identified in muscle cells, is a member of the Ferlin family involved in membrane fusion, membrane repair, and membrane trafficking.... (Review)
Review
Myoferlin (MYOF), initially identified in muscle cells, is a member of the Ferlin family involved in membrane fusion, membrane repair, and membrane trafficking. Dysfunction of this protein is associated with muscular dysfunction. Recently, a growing body of studies have identified MYOF as an oncogenic protein. It is overexpressed in a variety of human cancers and promotes tumorigenesis, tumor cell motility, proliferation, migration, epithelial to mesenchymal transition, angiogenesis as well as metastasis. Clinically, MYOF overexpression is associated with poor outcome in various cancers. It can serve as a prognostic marker of human malignant disease. MYOF drives the progression of cancer in various processes, including surface receptor transportation, endocytosis, exocytosis, intercellular communication, fit mitochondrial structure maintenance and cell metabolism. Depletion of MYOF demonstrates significant antitumor effects both in vitro and in vivo, suggesting that targeting MYOF may produce promising clinical benefits in the treatment of malignant disease. In the present article, we reviewed the physiological function of MYOF as well as its role in cancer, thus providing a general understanding for further exploration of this protein.
Topics: Animals; Calcium-Binding Proteins; Humans; Membrane Proteins; Mice; Models, Biological; Muscle Proteins; Neoplasms
PubMed: 31828126
DOI: 10.1155/2019/7365913 -
Viruses Oct 2022Prions replicate by a self-templating mechanism. Infidelity in the process can lead to the emergence of new infectious structures, referred to as variants or strains....
Prions replicate by a self-templating mechanism. Infidelity in the process can lead to the emergence of new infectious structures, referred to as variants or strains. The question of whether prions are prone to mis-templating is not completely answered. Our previous experiments with 23 variants of the yeast [] prion do not support broad mutability. However, it became clear recently that the heat shock protein Hsp104 can restrict [] strain variation. This raises the possibility that many transmutable variants of the prion may have been mistaken as faithful-propagating simply because the mutant structure was too sturdy or too frail to take root in the wild-type cell. Here, I alter the strength of Hsp104 in yeast, overexpressing wild-type Hsp104 or expressing the hypo-active Hsp104 mutant, and check if the new environments enable the variants to mutate. Two variants hitherto thought of as faithful-propagating are discovered to generate different structures, which are stabilized with the hypo-active chaperone. In contrast, most transmutable variants discovered in cells overexpressing Hsp104 have been correctly identified as such previously in wild-type cells without the overexpression. The majority of transmutable variants only mis-template the structure of VH, VK, or VL, which are the most frequently observed variants and do not spontaneously mutate. There are four additional variants that never give rise to different structures in all cell conditions tested. Therefore, quite a few [] variants are faithful-propagating, and even the transmutable ones do not freely evolve but can only change to limited structural types.
Topics: Saccharomyces cerevisiae; Prions; Peptide Termination Factors; Saccharomyces cerevisiae Proteins; Heat-Shock Proteins
PubMed: 36366434
DOI: 10.3390/v14112337