MiR-431 promotes cardiomyocyte proliferation by targeting FBXO32 expression.

BACKGROUND: The induction of cardiomyocyte (CM) proliferation is a promising approach for cardiac regeneration following myocardial injury. MicroRNAs (miRNAs) have been reported to regulate CM proliferation. In particular, miR-431 expression decreases during cardiac development, according to Gene Expression Omnibus (GEO) microarray data. However, whether miR-431 regulates CM proliferation has not been thoroughly investigated. METHODS: We used integrated bioinformatics analysis of GEO datasets to identify the most significantly differentially expressed miRNAs. Real-time quantitative PCR and fluorescence in situ hybridization were performed to determine the miRNA expression patterns in hearts. Gain- and loss-of-function assays were conducted to detect the role of miRNA in CM proliferation. Additionally, we detected whether miR-431 affected CM proliferation in a myocardial infarction model. The TargetScan, miRDB and miRWalk online databases were used to predict the potential target genes of miRNAs. Luciferase reporter assays were used to study miRNA interactions with the targeting mRNA. RESULTS: First, we found a significant reduction in miR-431 levels during cardiac development. Then, by overexpression and inhibition of miR-431, we demonstrated that miR-431 promotes CM proliferation in vitro and in vivo, as determined by immunofluorescence assays of 5-ethynyl-2'-deoxyuridine (EdU), pH3, Aurora B and CM count, whereas miR-431 inhibition suppresses CM proliferation. Then, we found that miR-431 improved cardiac function post-myocardial infarction. In addition, we identified FBXO32 as a direct target gene of miR-431, with FBXO32 mRNA and protein expression being suppressed by miR-431. FBXO32 inhibited CM proliferation. Overexpression of FBXO32 blocks the enhanced effect of miR-431 on CM proliferation, suggesting that FBXO32 is a functional target of miR-431 during CM proliferation. CONCLUSION: In summary, miR-431 promotes CM proliferation by targeting FBXO32, providing a potential molecular target for preventing myocardial injury.

DNMT1-mediated regulating on FBXO32 promotes the progression of glioma cells through the regulation of SKP1 activity.

Glioma, a prevalent and serious form of brain cancer, is associated with dysregulation of DNA methylation, where DNA methyltransferase-1 (DNMT1) plays a significant role in glioma progression. However, the involvement of F-box protein 32 (FBXO32) in glioma and its regulation by DNMT1-mediated methylation remain poorly understood. In this study, we investigated FBXO32 expression in glioma cells with high DNMT1 expression using the online dataset and correlated it with patient survival. Then impact of elevated FBXO32 expression on cell proliferation, migration, and invasion was evaluated, along with the examination of EMT-related proteins. Furthermore, a xenograft model established by injecting glioma cells stably transfected with FBXO32 was used to evaluate tumor growth, volume, and weight. The ChIP assay was employed to study the interaction between DNMT1 and the FBXO32 promoter, revealing that DNMT1 negatively correlated with FBXO32 expression in glioma cells and promoted FBXO32 promoter methylation. Moreover, we investigated the interaction between FBXO32 and SKP1 using Co-IP and GST pulldown assays, discovering that FBXO32 acts as an E3 ubiquitin ligase and promotes SKP1 ubiquitination, leading to its degradation. Interestingly, our findings demonstrated that high FBXO32 expression was associated with improved overall survival in glioma patients. Knockdown of DNMT1 in glioma cells increased FBXO32 expression and suppressed malignant phenotypes, suggesting that FBXO32 functions as a tumor suppressor in glioma. In conclusion, this study reveals a novel regulatory mechanism involving DNMT1-mediated FBXO32 expression in glioma cells, where FBXO32 acts as an E3 ubiquitin ligase to degrade SKP1 via ubiquitination. This FBXO32-mediated regulation of SKP1 activity contributes to the progression of glioma cells. These findings provide important insights into the molecular mechanisms underlying glioma progression and may hold promise for the development of targeted therapies for glioma patients.

11-beta-hydroxysteroid dehydrogenase type 1 (HSD11B1) gene expression in muscle is linked to reduced skeletal muscle index in sarcopenic patients.

BACKGROUND: Glucocorticoids play a significant role in metabolic processes and pathways that impact muscle size, mass, and function. The expression of 11-beta-hydroxysteroid dehydrogenase type 1 (HSD11B1) has been previously described as a major regulator of skeletal muscle function in glucocorticoid-induced muscle atrophy and aging humans. Our study aimed to investigate glucocorticoid metabolism, including the expression of HSD11B1 in skeletal muscle, in patients with sarcopenia. METHODS: Muscle biopsies were taken from the vastus lateralis muscle of thirty-three patients over 60 years of age with hip fractures. Sarcopenia status was assessed according to the criteria of the European Working Group on Sarcopenia in Older People 2. Skeletal muscle mass was measured by bioelectrical impedance analysis. Cortisol and cortisone concentrations were measured in serum. Gene expression analysis of HSD11B1, NR3C1, FBXO32, and TRIM63 in muscle biopsies was performed. Serial cross sections of skeletal muscle were labeled with myosin heavy chain slow (fiber type-1) and fast (fiber type-2) antibodies. RESULTS: The study included 33 patients (21 women) with a mean age of 82.5 ± 6.3 years, 17 patients revealed sarcopenic (n = 16 non-sarcopenic). Serum cortisone concentrations were negatively correlated with muscle mass (ß = - 0.425; p = 0.034) and type-2 fiber diameter (ß = - 0.591; p = 0.003). Gene expression of HSD11B1 (ß = - 0.673; p = 0.008) showed a negative correlation with muscle mass in the sarcopenic group. A significant correlation was found for the non-sarcopenic group for NR3C1 (ß = 0.548; p = 0.028) and muscle mass. CONCLUSION: These findings suggest a pathogenetic role of HSD11B1 in sarcopenic muscle.

The FBXO32/ATR/ATM axis acts as a molecular switch to control the sensitivity of osteosarcoma cells to irradiation through its regulation of EXO1 expression.

Osteosarcoma (OS) is the most common primary bone cancer in children and adolescents. In clinical treatments, the insensitivity of OS to conventional radiotherapy regimens significantly contributes to poor patient prognosis and survival. EXO1 is responsible for DNA repair pathways and telomere maintenance. Meanwhile, ATM and ATR are considered switches because they can regulate the expression of EXO1. However, their expression and interaction in OS cells under irradiation (IR) remain unclear. This study aims to investigate the roles of FBXO32, ATM, ATR and EXO1 in OS radiotherapy insensitivity and poor patient prognosis and explore potential pathogenic mechanisms. Bioinformatics is employed to analyse differential gene expression and correlations with prognosis in OS. Cell counting kit 8 assay, clone formation assay, and flow cytometry are used to evaluate cell survival and apopotosis under IR. Co-IP assay is used to detect protein‒protein interactions. Bioinformatics analysis reveals that EXO1 is closely related to survival, apoptosis and poor prognosis in OS. Silencing of EXO1 suppresses cell proliferation and increases the sensitivity of OS cells. Molecular biological experiments show that ATM and ATR act as switches to regulate EXO1 expression under IR. Higher expression of EXO1, which is closely correlated with IR insensitivity and poorer prognosis, might be used as a prognostic indicator for OS. Phosphorylated ATM enhances the expression of EXO1, and phosphorylated ATR induces the degradation of EXO1. More importantly, FBXO32 degrades ATR via ubiquitination in a time-dependent manner. Our data may provide a reference for future research in the mechanisms, clinical diagnosis, and treatment of OS.

Promotive Effect of FBXO32 on the Odontoblastic Differentiation of Human Dental Pulp Stem Cells.

Odontoblastic differentiation of human dental pulp stem cells (hDPSCs) is crucial for the intricate formation and repair processes in dental pulp. Until now, the literature is not able to demonstrate the role of ubiquitination in the odontoblastic differentiation of hDPSCs. This study investigated the role of F-box-only protein 32 (FBXO32), an E3 ligase, in the odontoblastic differentiation of hDPSCs. The mRNA expression profile was obtained from ribonucleic acid sequencing (RNA-Seq) data and analyzed. Immunofluorescence and immunohistochemical staining identify the FBXO32 expression in human dental pulp and hDPSCs. Small-hairpin RNA lentivirus was used for FBXO32 knockdown and overexpression. Odontoblastic differentiation of hDPSCs was determined via alkaline phosphatase activity, Alizarin Red S staining, and mRNA and protein expression levels were detected using real-time quantitative polymerase chain reaction and Western blotting. Furthermore, subcutaneous transplantation in nude mice was performed to evaluate the role of FBXO32 in mineralization in vivo using histological analysis. FBXO32 expression was upregulated in the odontoblast differentiated hDPSCs as evidenced by RNA-Seq data analysis. FBXO32 was detected in hDPSCs and the odontoblast layer of the dental pulp. Increased FBXO32 expression in hDPSCs during odontoblastic differentiation was confirmed. Through lentivirus infection method, FBXO32 downregulation in hDPSCs attenuated odontoblastic differentiation in vitro and in vivo, whereas FBXO32 upregulation promoted the hDPSCs odontoblastic differentiation, without affecting proliferation and migration. This study demonstrated, for the first time, the promotive role of FBXO32 in regulating the odontoblastic differentiation of hDPSCs, thereby providing novel insights into the regulatory mechanisms during odontoblastic differentiation in hDPSCs.

Exosome-Mediated miR-29 Transfer Reduces Muscle Atrophy and Kidney Fibrosis in Mice.

Our previous study showed that miR-29 attenuates muscle wasting in chronic kidney disease. Other studies found that miR-29 has anti-fibrosis activity. We hypothesized that intramuscular injection of exosome-encapsulated miR-29 would counteract unilateral ureteral obstruction (UUO)-induced muscle wasting and renal fibrosis. We used an engineered exosome vector, which contains an exosomal membrane protein gene Lamp2b that was fused with the targeting peptide RVG (rabies viral glycoprotein peptide). RVG directs exosomes to organs that express the acetylcholine receptor, such as kidney. The intervention of Exo/miR29 increased muscle cross-sectional area and decreased UUO-induced upregulation of TRIM63/MuRF1 and FBXO32/atrogin-1. Interestingly, renal fibrosis was partially depressed in the UUO mice with intramuscular injection of Exo/miR29. This was confirmed by decreased TGF-ß, alpha-smooth muscle actin, fibronectin, and collagen 1A1 in the kidney of UUO mice. When we used fluorescently labeled Exo/miR29 to trace the Exo/miR route in vivo and found that fluorescence was visible in un-injected muscle and in kidneys. We found that miR-29 directly inhibits YY1 and TGF-ß3, which provided a possible mechanism for inhibition of muscle atrophy and renal fibrosis by Exo/miR29. We conclude that Exo/miR29 ameliorates skeletal muscle atrophy and attenuates kidney fibrosis by downregulating YY1 and TGF-ß pathway proteins.

Identification of potential target genes associated with the reversion of androgen-dependent skeletal muscle atrophy.

Muscle wasting or atrophy is extensively associated with human systemic diseases including diabetes, cancer, and kidney failure. Accumulating evidence from transcriptional profiles has noted that a common set of genes, termed atrogenes, is modulated in atrophying muscles. However, the transcriptional changes that trigger the reversion or attenuation of muscle atrophy have not been characterized at the molecular level until now. Here, we applied cDNA microarrays to investigate the transcriptional response of androgen-sensitive Levator ani muscle (LA) during atrophy reversion. Most of the differentially expressed genes behaved as atrogenes and responded to castration-induced atrophy. However, seven genes (APLN, DUSP5, IGF1, PIK3IP1, KLHL38, PI15, and MKL1) did not respond to castration but instead responded exclusively to testosterone replacement. Considering that almost all proteins encoded by these genes are associated with the reversion of atrophy and may function as regulators of cell proliferation/growth, our results provide new perspectives on the existence of anti-atrogenes.

Fine-mapping scan of bipolar disorder susceptibility loci in Latino pedigrees.

We previously identified bipolar disorder (BD) susceptibility loci on 8q24, 14q32, and 2q12-14 in a genome-wide nonparametric linkage screen in a Latino cohort. We now perform a fine mapping analysis using a dense map of additional SNPs to identify BD susceptibility genes within these regions. One thousand nine hundred and thirty-eight individuals with Latino ancestry (880 individuals with BD Type I or Schizoaffective, Bipolar Type) from 416 Latino pedigrees from the United States, Mexico, Costa Rica, and Guatemala were genotyped with 3,074 SNPs to provide dense coverage of the 8q24 (11.5 cM), 14q32 (7.5 cM), and 2q12-14 (6.5 cM) chromosomal loci. Single-marker association tests in the presence of linkage were performed using the LAMP software. The top linkage peak (rs7834818; LOD = 5.08, p = 3.30E - 5) and associated single marker (rs2280915, p = 2.70E - 12) were located within FBXO32 on 8q24. On chromosome 2, the top linkage peak (rs6750326; LOD = 5.06, p = 3.50E - 5) and associated single marker (rs11887088, p = 2.90E - 6) were located in intragenic regions near ACTR3 and DPP10. None of the additional markers in the region around chromosome 14q32 met significance levels for linkage or association. We identified six SNPs on 2q12-q14 and one SNP in FBXO32 on 8q24 that were significantly associated with BD in this Latino cohort.

MicroRNA-23a and MicroRNA-27a Mimic Exercise by Ameliorating CKD-Induced Muscle Atrophy.

Muscle atrophy is a frequent complication of CKD, and exercise can attenuate the process. This study investigated the role of microRNA-23a (miR-23a) and miR-27a in the regulation of muscle mass in mice with CKD. These miRs are located in a gene cluster that is regulated by the transcription factor NFAT. CKD mice expressed less miR-23a in muscle than controls, and resistance exercise (muscle overload) increased the levels of miR-23a and miR-27a in CKD mice. Injection of an adeno-associated virus encoding the miR-23a/27a/24-2 precursor RNA into the tibialis anterior muscles of normal and CKD mice led to increases in mature miR-23a and miR-27a but not miR-24-2 in the muscles of both cohorts. Overexpression of miR-23a/miR-27a in CKD mice attenuated muscle loss, improved grip strength, increased the phosphorylation of Akt and FoxO1, and decreased the activation of phosphatase and tensin homolog (PTEN) and FoxO1 and the expression of TRIM63/MuRF1 and FBXO32/atrogin-1 proteins. Provision of miR-23a/miR-27a also reduced myostatin expression and downstream SMAD-2/3 signaling, decreased activation of caspase-3 and -7, and increased the expression of markers of muscle regeneration. Lastly, in silico miR target analysis and luciferase reporter assays in primary satellite cells identified PTEN and caspase-7 as targets of miR-23a and FoxO1 as a target of miR-27a in muscle. These findings provide new insights about the roles of the miR-23a/27a-24-2 cluster in CKD-induced muscle atrophy in mice and suggest a mechanism by which exercise helps to maintain muscle mass.

Upregulation of Atrogin-1/FBXO32 is not necessary for cartilage destruction in mouse models of osteoarthritis.

OBJECTIVE: In a preliminary study, we found that recently identified catabolic regulators of osteoarthritis (OA), including hypoxia-inducible factor (HIF)-2α and members of the zinc-ZIP8-MTF1 axis, upregulate the E3 ubiquitin ligase, Atrogin-1 (encoded by Fbxo32), in chondrocytes. As the ubiquitination/proteasomal degradation pathways are tightly regulated to modulate the expression of catabolic factors in chondrocytes, we examined the in vivo functions of Atrogin-1 in mouse models of OA. METHODS: The mRNA and protein levels of Atrogin-1 and other regulators of OA were determined in primary cultured mouse chondrocytes, OA human cartilage, and OA cartilage from wild-type (WT) and Fbxo32-knockout (KO) mice subjected to destabilization of the medial meniscus or intra-articular (IA) injection of adenoviruses expressing HIF-2α (Ad-Epas1), ZIP8 (Ad-Zip8), or Atrogin-1 (Ad-Fbxo32). The effect of Atrogin-1 overexpression on the cartilage of WT mice was examined by IA injection of Ad-Fbxo32. RESULTS: Atrogin-1 mRNA levels in chondrocytes were markedly increased by treatment with interleukin-1ß, HIF-2α, and members of the zinc-ZIP8-MTF1 axis. Atrogin-1 protein levels were also increased in OA cartilage from humans and various mouse OA models. However, the forced overexpression of Atrogin-1 in chondrocytes did not modulate the expression of cartilage matrix molecules or matrix-degrading enzymes. Moreover, overexpression of Atrogin-1 in the mouse joint tissues failed to cause OA pathogenesis, and Fbxo32 knockout failed to affect post-traumatic OA cartilage destruction in mice. CONCLUSIONS: Although Atrogin-1 is upregulated in OA cartilage, overexpression of Atrogin-1 in the joint tissues or knockout of Fbxo32 does not affect OA cartilage destruction in mice.

FBXO32 Targets c-Myc for Proteasomal Degradation and Inhibits c-Myc Activity.

FBXO32 (MAFbx/Atrogin-1) is an E3 ubiquitin ligase that is markedly up-regulated in muscle atrophy. Although some data indicate that FBXO32 may play an important role in tumorigenesis, the molecular mechanism of FBXO32 in tumorigenesis has been poorly understood. Here, we present evidence that FBXO32 targets the oncogenic protein c-Myc for ubiquitination and degradation through the proteasome pathway. Phosphorylation of c-Myc at Thr-58 and Ser-62 is dispensable for FBXO32 to induce c-Myc degradation. Mutation of the lysine 326 in c-Myc reduces c-Myc ubiquitination and prevents the c-Myc degradation induced by FBXO32. Furthermore, overexpression of FBXO32 suppresses c-Myc activity and inhibits cell growth, but knockdown of FBXO32 enhances c-Myc activity and promotes cell growth. Finally, we show that FBXO32 is a direct downstream target of c-Myc, highlighting a negative feedback regulation loop between c-Myc and FBXO32. Thus, FBXO32 may function by targeting c-Myc. This work explains the function of FBXO32 and highlights its mechanisms in tumorigenesis.

n-3 Fatty acids preserve muscle mass and insulin sensitivity in a rat model of energy restriction.

In obese subjects, the loss of fat mass during energy restriction is often accompanied by a loss of muscle mass. The hypothesis that n-3 PUFA, which modulate protein homoeostasis via effects on insulin sensitivity, could contribute to maintain muscle mass during energy restriction was tested in rats fed a high-fat diet (4 weeks) rich in 18 : 1 n-9 (oleic acid, OLE-R), 18 : 3 n-3 (α-linolenic acid, ALA-R) or n-3 long-chain (LC-R) fatty acid and then energy restricted (8 weeks). A control group (OLE-ad libitum (AL)) was maintained with AL diet throughout the study. Rats were killed 10 min after an i.v. insulin injection. All energy-restricted rats lost weight and fat mass, but only the OLE-R group showed a significant muscle loss. The Gastrocnemius muscle was enriched with ALA in the ALA-R group and with LC-PUFA in the ALA-R and LC-R groups. The proteolytic ubiquitin-proteasome system was differentially affected by energy restriction, with MAFbx and muscle ring finger-1 mRNA levels being decreased in the LC-R group (-30 and -20 %, respectively). RAC-α serine/threonine-protein kinase and insulin receptor substrate 1 phosphorylation levels increased in the LC-R group (+70 %), together with insulin receptor mRNA (+50 %). The ALA-R group showed the same overall activation pattern as the LC-R group, although to a lesser extent. In conclusion, dietary n-3 PUFA prevent the loss of muscle mass associated with energy restriction, probably by an improvement in the insulin-signalling pathway activation, in relation to enrichment of plasma membranes in n-3 LC-PUFA.

DNA Hypomethylation in Intragenic and Intergenic Enhancer Chromatin of Muscle-Specific Genes Usually Correlates with their Expression.

Tissue-specific enhancers are critical for gene regulation. In this study, we help elucidate the contribution of muscle-associated differential DNA methylation to the enhancer activity of highly muscle-specific genes. By bioinformatic analysis of 44 muscle-associated genes, we show that preferential gene expression in skeletal muscle (SkM) correlates with SkM-specific intragenic and intergenic enhancer chromatin and overlapping foci of DNA hypomethylation. Some genes, e.g., CASQ1 and FBXO32, displayed broad regions of both SkM- and heart-specific enhancer chromatin but exhibited focal SkM-specific DNA hypomethylation. Half of the genes had SkM-specific super-enhancers. In contrast to simple enhancer/gene-expression correlations, a super-enhancer was associated with the myogenic MYOD1 gene in both SkM and myoblasts even though SkM has < 1 percent as much MYOD1 expression. Local chromatin differences in this super-enhancer probably contribute to the SkM/myoblast differential expression. Transfection assays confirmed the tissue-specificity of the 0.3-kb core enhancer within MYOD1's super-enhancer and demonstrated its repression by methylation of its three CG dinucleotides. Our study suggests that DNA hypomethylation increases enhancer tissue-specificity and that SkM super-enhancers sometimes are poised for physiologically important, rapid up-regulation.

FBXO32, a new TGF-ß/Smad signaling pathway target gene, is epigenetically inactivated in gastric cardia adenocarcinoma.

FBXO32 has recently been identified as a TGF-ß/Smad signaling pathway target gene, involved in regulating cell survival and may be transcriptionally silenced by epigenetic mechanisms in some kind of carcinomas. The present study was to investigate the role and promoter methylation status of FBXO32 in gastric cardia adenocarcinoma (GCA), and determine the prognostic significance of FBXO32 in GCA. Bisulfite Conversion-Specific and Methylation-Specific PCR, real-time RT-PCR and immunohistochemical staining methods were used to detect the methylation status and expression of FBXO32 in GCA samples. The frequency of FBXO32 methylation in GCA tumor tissues (44.6%) was significantly higher than that in corresponding normal tissues (3.6%) and was associated with TNM stage, pathological differentiation, distant metastasis or recurrence and upper gastrointestinal cancers (UGIC) family history. Decreased mRNA and protein expression of FBXO32 was observed in GCA tumor tissues and was associated with FBXO32 promoter methylation status. A positive correlation between FBXO32 and p-Smad2/3, Smad4 protein expression was also found in clinical specimens. GCA patients in stage III and IV, with positive UGIC family history, and hypermethylation and down-expression of FBXO32 were most likely to develop metastatic disease and also showed the worse survival. In all, aberrant hypermethylation of FBXO32 may be one of the mechanisms that lead to loss or down expression of the gene in GCA, FBXO32 may be a functional tumor suppressor and reactivation of FBXO32 gene may has therapeutic potential and may be used as a prognostic marker for GCA patients.

Aberrant methylation and decreased expression of the TGF-ß/Smad target gene FBXO32 in esophageal squamous cell carcinoma.

BACKGROUND: F-box protein 32 (FBXO32) (also known as atrogin-1), a member of the F-box protein family, has recently been identified as a transforming growth factor beta (TGF-ß)/Smad target gene involved in regulating cell survival, and it may be transcriptionally silenced by epigenetic mechanisms in some kinds of carcinomas, yet its role in esophageal squamous cell carcinoma (ESCC) has not been defined. METHODS: The role of FBXO32 in ESCC and the correlation of FBXO32 methylation with a series of pathologic parameters were studied in a large cohort of patients with ESCC. RESULTS: Decreased messenger RNA (mRNA) expression and protein expression of FBXO32 were observed in esophageal cancer cell lines, and the silencing of FBXO32 could be reversed by treatment with 5-aza-2'-deoxycytidine or trichostatin A in the TE13 cell line. In addition, aberrant methylation of FBXO32 and histone deacetylation was capable of suppressing FBXO32 mRNA and protein expression in TE13 cells. Decreased mRNA and protein expression of FBXO32 was observed in ESCC tumor tissues and was associated with FBXO32 promoter methylation status. A positive correlation between FBXO32 and phosphorylated SMAD family members 2 and 3 expression and Smad4 protein expression also was observed in clinical specimens. FBXO32 methylation status and protein expression were independently associated with survival in patients with ESCC. CONCLUSIONS: FBXO32 may be a functional tumor suppressor. Its inactivation through promoter methylation could play an important role in ESCC carcinogenesis, and reactivation of the FBXO32 gene may have therapeutic potential and might be used as a prognostic marker for patients with ESCC.

DNMT1-mediated epigenetic suppression of FBXO32 expression promoting cyclin dependent kinase 9 (CDK9) survival and esophageal cancer cell growth.

Esophageal cancer (EC) is a common and serious form of cancer, and while DNA methyltransferase-1 (DNMT1) promotes DNA methylation and carcinogenesis, the role of F-box protein 32 (FBXO32) in EC and its regulation by DNMT1-mediated methylation is still unclear. FBXO32 expression was examined in EC cells with high DNMT1 expression using GSE163735 dataset. RT-qPCR assessed FBXO32 expression in normal and EC cells, and impact of higher FBXO32 expression on cell proliferation, migration, and invasion was evaluated, along with EMT-related proteins. The xenograft model established by injecting EC cells transfected with FBXO32 was used to evaluate tumor growth, apoptosis, and tumor cells proliferation and metastasis. Chromatin immunoprecipitation (ChIP) assay was employed to study the interaction between DNMT1 and FBXO32. HitPredict, co-immunoprecipitation (Co-IP), and Glutathione-S-transferase (GST) pulldown assay analyzed the interaction between FBXO32 and cyclin dependent kinase 9 (CDK9). Finally, the ubiquitination assay identified CDK9 ubiquitination, and its half-life was measured using cycloheximide and confirmed through western blotting. DNMT1 negatively correlated with FBXO32 expression in esophageal cells. High FBXO32 expression was associated with better overall survival in patients. Knockdown of DNMT1 in EC cells increased FBXO32 expression and suppressed malignant phenotypes. FBXO32 repressed EC tumor growth and metastasis in mice. Enrichment of DNMT1 in FBXO32 promoter region led to increased DNA methylation and reduced transcription. Mechanistically, FBXO32 degraded CDK9 through promoting its ubiquitination.

Impact of fasting on the rhythmic expression of myogenic and metabolic factors in skeletal muscle of adult mice.

Circadian rhythms and metabolism are tightly integrated, and rhythmic expression of metabolic factors is common in homeostatic processes. We measured the temporal changes in the expression of myogenic regulatory factors and expression and activity level of molecules involved in protein metabolism in skeletal muscles and livers in mice and examined the impact of fasting. Tissues were collected over 24 h (at zeitgeber times ZT1, ZT5, ZT9, ZT13, ZT17, ZT21, and ZT1 the following day) from adult male C57Bl/6J mice that had been either freely fed or fasted for 24 h. In skeletal muscle, there was a robust rise in the mRNA expression of the myogenic regulatory factors MyoD and myogenin during dark hours which was strongly suppressed by fasting. Circadian pattern was observed for mRNA of MuRF1, Akt1, and ribosomal protein S6 in muscles in fed and fasted mice and for Fbxo32 in fed mice. Activity (phosphorylation) levels of Akt(Ser473) displayed temporal regulation in fasted (but not fed) mice and were high at ZT9. Fasting caused significant reductions in phosphorylation for both Akt and S6 in muscles, indicative of inactivation. Hepatic phosphorylated Akt(Ser473) and S6(Ser235/236) proteins did not exhibit daily rhythms. Fasting significantly reduced hepatic Akt(473) phosphorylation compared with fed levels, although (unlike in muscle) it did not affect S6(Ser235/236) phosphorylation. This in vivo circadian study addresses for the first time the signaling activities of key molecules related to protein turnover and their possible cross-regulation of expression of genes related to protein degradation.

The atypical ubiquitin ligase RNF31 stabilizes c-Myc via epigenetic inactivation of FBXO32 and promotes cancer development.

RNF31, an atypical E3 ubiquitin ligase of the RING-between-RING protein family, is one of the important components of the linear ubiquitin chain complex LUBAC. It plays a carcinogenic role in a variety of cancers by promoting cell proliferation, invasion and inhibiting apoptosis. However, the specific molecular mechanism by which RNF31 exerts its cancer-promoting effects is still unclear. By analyzing the expression profile of RNF31-depleted cancer cells, we found that loss of RNF31 significantly resulted in the inactivation of the c-Myc pathway. We further showed that RNF31 played an important role in the maintenance of c-Myc protein levels in cancer cells by extending the half-life of c-Myc protein and reducing its ubiquitination. c-Myc protein levels are tightly regulated by the ubiquitin proteasome, in which the E3 ligase FBXO32 is required to mediate its ubiquitin-dependent degradation. We found that RNF31 inhibited the transcription of FBXO32 through EZH2-mediated trimethylation of histone H3K27 in the FBXO32 promoter region, leading to the stabilization and activation of c-Myc protein. Under this circumstance, the expression of FBXO32 was significantly increased in RNF31-deficient cells, promoting the degradation of c-Myc protein, inhibiting cell proliferation and invasion, increasing cell apoptosis, and ultimately blocking the progression of tumors. Consistent with these results, the reduced malignancy phenotype caused by RNF31 deficiency could be partially reversed by overexpression of c-Myc or further knockdown of FBXO32. Together, our results reveal a key association between RNF31 and epigenetic inactivation of FBXO32 in cancer cells, and suggest that RNF31 may be a promising target for cancer therapy.

A novel likely pathogenic variant in the FBXO32 gene associated with dilated cardiomyopathy according to whole­exome sequencing.

BACKGROUND: Familial dilated cardiomyopathy (DCM) is a genetic heart disorder characterized by progressive heart failure and sudden cardiac death. Over 250 genes have been reported in association with DCM; nonetheless, the genetic cause of most DCM patients has been unknown. The goal of the present study was to determine the genetic etiology of familial DCM in an Iranian family. METHODS: Whole-exome sequencing was performed to identify the underlying variants in an Iranian consanguineous family with DCM. The presence of the candidate variant was confirmed and screened in available relatives by PCR and Sanger sequencing. The pathogenic effect of the candidate variant was assessed by bioinformatics analysis, homology modeling, and docking. RESULTS: One novel likely pathogenic deletion, c.884_886del: p.Lys295del, in F-box only protein 32 (muscle-specific ubiquitin-E3 ligase protein; FBXO32) was identified. Based on bioinformatics and modeling analysis, c.884_886del was the most probable cause of DCM in the studied family. CONCLUSIONS: Our findings indicate that variants in FBXO32 play a role in recessive DCM. Variants in FBXO32 may disturb the degradation of target proteins in the ubiquitin-proteasome system and lead to severe DCM. We suggest considering this gene variants in patients with recessively inherited DCM.

FBXO32 targets PHPT1 for ubiquitination to regulate the growth of EGFR mutant lung cancer.

BACKGROUND: Phosphohistidine phosphatase 1 (PHPT1) is an oncogene that has been reported to participate in multiple tumorigenic processes. As yet, however, the role of PHPT1 in lung cancer development remains uncharacterized. METHODS: RNA sequencing assay and 18 pairs of tumor and normal tissues from patients were analyzed to reveal the upregulation of PHPT1 in lung cancer, followed by confirming the biological function in vitro and in vivo. Next, Gene Set Enrichment Analysis, lung cancer samples, apoptosis assay, mass spectrometry experiments and western blotting were used to investigate the molecular mechanism underlying PHPT1 driven progression in epidermal growth factor receptor (EGFR)-mutant lung cancer. Finally, we performed cellular and animal experiments to explore the tumor suppressive function of F-box protein 32 (FBXO32). RESULTS: We found that PHPT1 is overexpressed in lung cancer patients and correlates with a poor overall survival. In addition, we found that the expression of PHPT1 is elevated in EGFR-mutant lung cancer cells and primary patient samples. Inhibition of PHPT1 expression in EGFR mutant lung cancer cells significantly decreased their proliferation and clonogenicity, and suppressed their in vitro tumor growth. Mechanistic studies revealed that activation of the ERK/MAPK pathway is driven by PHPT1. PHPT1 is required for maintaining drug resistance to erlotinib in EGFR mutant lung cancer cells. We found that FBXO32 acts as an E3 ubiquitin ligase for PHPT1, and that knockdown of FBXO32 leads to PHPT1 accumulation, activation of the ERK/MAPK pathway and promotion of the proliferation, clonogenicity and growth of lung cancer cells. CONCLUSIONS: Our findings indicate that PHPT1 may serve as a biomarker and therapeutic target for acquired erlotinib resistance in lung cancer patients carrying EGFR mutations.