FBXO45 levels regulated ferroptosis renal tubular epithelial cells in a model of diabetic nephropathy by PLK1.

Objective: This research aims to investigate the role and underlying biological mechanism of FBXO45 in regulating ferroptosis of renal fibrocytes in a diabetic nephropathy (DN) model. Methods: C57BL/6 mice were fed with a high-fat diet and injected with streptozotocin to induce diabetes. Human renal glomerular endothelial cells stimulated with d-glucose. Results: Serum FBXO45 mRNA expression was found to be down-regulated in patients with DN. There was a negative correlation between the expression of serum FBXO45 mRNA and serum α-SMA, Collagen I, and E-cadherin mRNA in patients with DN. Additionally, the expression of serum FBXO45 mRNA showed a negative correlation with blood sugar levels. Based on a 3D model prediction, it was observed that FBXO45 interacts with polo-like kinase 1 (PLK1) at GLY-271, ILE-226, GLY-166, LEU-165, ARG-245, and ASN-220, while PLK1 interacts with FBXO45 at TYR-417, ARG-516, HIS-489, TYR-485, GLN-536, and ARG-557. This interaction was confirmed through immunoprecipitation assay, which showed the interlinking of FBXO45 protein with PLK1 protein. Conclusions: These findings indicate that FBXO45 plays a role in mitigating ferroptosis in DN through the regulation of the PLK1/GPX4/SOX2 pathway. This highlights the potential of targeting FBXO45 as a therapeutic approach to ameliorate ferroptosis in DN.

Fbxo45 facilitates the malignant progression of breast cancer by targeting Bim for ubiquitination and degradation.

BACKGROUND: Breast cancer is one of the common malignancies in women. Evidence has demonstrated that FBXO45 plays a pivotal role in oncogenesis and progression. However, the role of FBXO45 in breast tumorigenesis remains elusive. Exploration of the regulatory mechanisms of FBXO45 in breast cancer development is pivotal for potential therapeutic interventions in patients with breast cancer. METHODS: Hence, we used numerous approaches to explore the functions of FBXO45 and its underlaying mechanisms in breast cancer pathogenesis, including CCK-8 assay, EdU assay, colony formation analysis, apoptosis assay, RT-PCR, Western blotting, immunoprecipitation, ubiquitination assay, and cycloheximide chase assay. RESULTS: We found that downregulation of FBXO45 inhibited cell proliferation, while upregulation of FBXO45 elevated cell proliferation in breast cancer. Silencing of FBXO45 induced cell apoptosis, whereas overexpression of FBXO45 inhibited cell apoptosis in breast cancer. Moreover, FBXO45 interacted with BIM and regulated its ubiquitination and degradation. Furthermore, knockdown of FBXO45 inhibited cell proliferation via regulation of BIM pathway. Notably, overexpression of FBXO45 facilitated tumor growth in mice. Strikingly, FBXO45 expression was associated with poor survival of breast cancer patients. CONCLUSION: Our study could provide the rational for targeting FBXO45 to obtain benefit for breast cancer patients. Altogether, modulating FBXO45/Bim axis could be a promising strategy for breast cancer therapy.

LncRNA MEG8 ameliorates Parkinson's disease neuro-inflammation through miR-485-3p/FBXO45 axis.

OBJECTIVE: Studies suggest that LncRNA maternally expressed 8, small nucleolar RNA host gene (MEG8) contributes to inflammatory regulation, while the function and potential mechanisms of MEG8 in Parkinson's disease (PD) are unknown. This study aimed to assess the clinical value and biological function of MEG8 in PD. METHODS: One hundred and two PD patients, eighty-six AD patients, and eighty healthy controls were enrolled in this study. Lipopolysaccharide (LPS)-induced microglia BV2 constructs an in vitro cell model. RT-qPCR was conducted to quantify the levels of MEG8, miR-485-3p, and FBXO45 in serum and cells. ROC curve was employed to examine the diagnostic value of MEG8 in PD. Serum and cellular pro-inflammatory factor secretion were quantified by ELISA. Dual-luciferase reporter and RIP assay to validate the targeting relationship between miR-485-3p and FBXO45. RESULTS: MEG8 and FBXO45 were significantly decreased in the serum of PD patients and LPS-induced bv2, while miR-485-3p was increased (P < 0.05). ROC curve confirmed that serum MEG8 has high sensitivity and specificity to identify PD patients from healthy controls and AD patients, respectively. Elevated MEG8 alleviated LPS-induced inflammatory factor overproduction compared with LPS-induced BV2 (P < 0.05), but this alleviating effect was eliminated by miR-485-3p (P < 0.05). The LPS-induced inflammatory response was suppressed by the low expression of miR-485-3p but significantly reversed by silencing of FBXO45. MEG8 was a sponge for miR-485-3p and inhibited its levels and promoted FBXO45 expression (P < 0.05). CONCLUSION: Elevated MEG8 is a potential diagnostic biomarker for PD and may mitigate inflammatory damage in PD via the miR-485-3p/FBXO45 axis.

Identification of F-Box/SPRY Domain-Containing Protein 1 (FBXO45) as a Prognostic Biomarker for TMPRSS2-ERG-Positive Primary Prostate Cancers.

BACKGROUND: F-box/SPRY domain-containing protein 1 (FBXO45) plays a crucial role in the regulation of apoptosis via the ubiquitylation and degradation of specific targets. Recent studies indicate the prognostic potential of FBXO45 in several cancers. However, its specific role in prostate carcinoma remains unclear. METHODS: A systematic analysis of FBXO45 mRNA expression in PCA was performed using The Cancer Genome Atlas database and a publicly available Gene Expression Omnibus progression PCA cohort. Subsequently, FBXO45 protein expression was assessed via immunohistochemical analysis of a comprehensive tissue microarray cohort. The expression data were correlated with the clinicopathological parameters and biochemical-free survival. The immunohistochemical analyses were stratified according to the TMPRSS2-ERG rearrangement status. To assess the impact of FBXO45 knockdown on the tumour proliferation capacity of cells and metastatic potential, transfection with antisense-oligonucleotides was conducted within a cell culture model. RESULTS: FBXO45 mRNA expression was associated with adverse clinicopathological parameters in the TCGA cohort and was enhanced throughout progression to distant metastasis. FBXO45 was associated with shortened biochemical-free survival, which was pronounced for the TMPRSS2-ERG-positive tumours. In vitro, FBXO45 knockdown led to a significant reduction in migration capacity in the PC3, DU145 and LNCaP cell cultures. CONCLUSIONS: Comprehensive expression analysis and functional data suggest FBXO45 as a prognostic biomarker in PCA.

CACNA1C-AS2 inhibits cell proliferation and suppresses cell migration and invasion via targeting FBXO45 and PI3K/AKT/mTOR pathways in glioma.

Glioma is the most common brain cancer with a poor prognosis, and its underlying molecular mechanisms still needs to be further explored. In the current study, we discovered that an antisense lncRNA, CACNA1C-AS2, suppressed growth, migration and invasion of glioma cells, suggesting that CACNA1C-AS2 functions as a tumor suppressor. Furthermore, we found that CACNA1C-AS2 negatively regulated Fbxo45 protein expression in glioma cells. Impressively, extensive experimental results revealed that Fbxo45 accelerated growth, migration and invasion of glioma cells. Clinically, increased Fbxo45 expression was observed in 75 human glioma tissue samples. Moreover, in vivo experiments also demonstrated that Fbxo45 overexpression enhanced tumor growth in mice. Especially, we further identified that Fbxo45 activated mTORC1 rather than mTORC2 through PI3K/AKT signaling to promote cell growth and motility in glioma cells. Rescue experiments also exhibited that CACNA1C-AS2 inhibited cell growth and motility partly through down-regulating Fbxo45 expression in glioma. Our results provide the novel insights into the critical role of CACNA1C-AS2/Fbxo45/mTOR axis involved in regulating glioma tumorigenesis and progression, and further indicate that CACNA1C-AS2 and Fbxo45 may be the potential biomarkers and therapeutic targets for glioma.

Fbxo45-mediated NP-STEP46 degradation via K6-linked ubiquitination sustains ERK activity in lung cancer.

Lung cancer is one of the most threatening malignant tumors to human health. Epidermal growth factor receptor (EGFR)-targeted therapy is a common and essential means for the clinical treatment of lung cancer. However, drug resistance has always affected the therapeutic effect and survival rate in non-small cell lung cancer (NSCLC). Tumor heterogeneity is a significant reason, yielding various drug resistance mechanisms, such as EGFR-dependent or -independent extracellular signal-regulated kinase 1 and/or 2 (ERK1/2) activation in NSCLC. To examine whether this aberrant activation of ERK1/2 is related to the loss of function of its specific phosphatase, a series of in vitro and in vivo assays were performed. We found that F-box/SPRY domain-containing protein 1 (Fbxo45) induces ubiquitination of NP-STEP46 , an active form of striatal-enriched protein tyrosine phosphatase, with a K6-linked poly-ubiquitin chain. This ubiquitination led to proteasome degradation in the nucleus, which then sustains the aberrant level of phosphorylated-ERK (pERK) and promotes tumor growth of NSCLC. Fbxo45 silencing can significantly inhibit cell proliferation and tumor growth. Moreover, NSCLC cells with silenced Fbxo45 showed great sensitivity to the EGFR tyrosine kinase inhibitor (TKI) afatinib. Here, we first report this critical pERK maintenance mechanism, which might be independent of the upstream kinase activity in NSCLC. We propose that inhibiting Fbxo45 may combat the issue of drug resistance in NSCLC patients, especially combining with EGFR-TKI therapy.

E3 ubiquitin ligase RBX1 drives the metastasis of triple negative breast cancer through a FBXO45-TWIST1-dependent degradation mechanism.

Triple-negative breast cancer (TNBC) patients are at high risk of recurrence and metastasis in the early stages, although receiving standard treatment. However, the underlying mechanism of TNBC remains unclear. Here, the critical effect of E3 ubiquitin ligase RBX1 in the metastasis of TNBC was reported for the first time. We discovered that RBX1 expression was evidently raised in the tissues of TNBC. Our clinical research displayed that high RBX1 expression was markedly related to poor distant invasion and survival. Functional analysis exhibited that RBX1 facilitated metastasis of TNBC cells through increasing EMT. Furthermore, we demonstrated that RBX1 knockdown increased the levels of the Twist family bHLH transcription factor 1 (TWIST1), is a significant regulator in the EMT process in some cancers. It can be observed an evident positive correlation between the TWIST1 and RBX1 levels, further confirming that EMT induced by RBX1 in TNBC cells is determined by TWIST1. Mechanistically, RBX1 modulates the expression of TWIST1 via modulating FBXO45, directly binding to FBXO45, and facilitating its degradation and ubiquitination. Briefly, our findings confirm that RBX1 is probably a new biomarker of TNBC carcinogenesis, thus suggesting that targeting the RBX1/FBXO45/TWIST1 axis may be an underlying strategy for TNBC treatment.

Diagnostic Performance of miR-485-3p in Patients with Parkinson's Disease and its Relationship with Neuroinflammation.

Parkinson's disease (PD) is one of the most common progressive neurodegenerative diseases. Some microRNAs (miRNAs) play critical roles in the development of many neurological diseases. This study aims to evaluate the clinical significance and biological function of miR-485-3p in the development and progression of PD. The expression of miR-485-3p in serum of PD patients was analyzed by quantitative real-time PCR (qRT-PCR). LPS-treated microglia BV2 cells were used to mimic neuroinflammation in the pathogenesis of PD. The levels of inflammatory cytokines, including IL-1ß, IL-6 and TNF-α, were detected by enzyme-linked immunosorbent assay (ELISA). The diagnosis value of miR-485-3p was evaluated by plotting receiver operating characteristic (ROC) curves. A luciferase reporter assay was performed to demonstrate the interaction between miR-485-3p and FBXO45. The results showed that miR-485-3p was significantly up-regulated in serum of PD patients compared with that in both Alzheimer's disease (AD) and healthy cases, and had diagnostic accuracy for PD screening. The activated microglia BV2 cells induced by LPS also had elevated miR-485-3p, and the knockdown of miR-485-3p inhibited the release of pro-inflammatory cytokines. FBXO protein 45 (FBXO45) served as a potential target of miR-485-3p, which was speculated to mediate the function of miR-485-3p. Our results suggest that the up-regulated expression of miR-485-3p in PD may be a novel diagnostic biomarker for PD. Reducing the expression level of miR-485-3p can inhibit the inflammatory responses of BV2 cells, which indicated that miR-485-3p, as a regulator of neuroinflammation, may have the potential as a therapeutic target in PD.

Elevated FBXO45 promotes liver tumorigenesis through enhancing IGF2BP1 ubiquitination and subsequent PLK1 upregulation.

Dysregulation of tumor-relevant proteins may contribute to human hepatocellular carcinoma (HCC) tumorigenesis. FBXO45 is an E3 ubiquitin ligase that is frequently elevated expression in human HCC. However, it remains unknown whether FBXO45 is associated with hepatocarcinogenesis and how to treat HCC patients with high FBXO45 expression. Here, IHC and qPCR analysis revealed that FBXO45 protein and mRNA were highly expressed in 54.3% (57 of 105) and 52.2% (132 of 253) of the HCC tissue samples, respectively. Highly expressed FBXO45 promoted liver tumorigenesis in transgenic mice. Mechanistically, FBXO45 promoted IGF2BP1 ubiquitination at the Lys190 and Lys450 sites and subsequent activation, leading to the upregulation of PLK1 expression and the induction of cell proliferation and liver tumorigenesis in vitro and in vivo. PLK1 inhibition or IGF2BP1 knockdown significantly blocked FBXO45-driven liver tumorigenesis in FBXO45 transgenic mice, primary cells, and HCCs. Furthermore, IHC analysis on HCC tissue samples revealed a positive association between the hyperexpression of FBXO45 and PLK1/IGF2BP1, and both had positive relationship with poor survival in HCC patients. Thus, FBXO45 plays an important role in promoting liver tumorigenesis through IGF2BP1 ubiquitination and activation, and subsequent PLK1 upregulation, suggesting a new strategy for treating HCC by targeting FBXO45/IGF2BP1/PLK1 axis.

IL-24 inhibits the malignancy of human glioblastoma cells via destabilization of Zeb1.

Glioblastoma (GBM) is the most common and fatal type of primary malignant tumours in the central nervous system. Cytokines such as interleukins (ILs) play an important role in GBM progression. Our present study found that IL-24 is down-regulated in GBM cells. Recombinant IL-24 (rIL-24) can suppress the in vitro migration and invasion of GBM cells while increase its chemo-sensitivity to temozolomide (TMZ) treatment. rIL-24 negatively regulates the expression of Zeb1, one well known transcription factors of epithelial to mesenchymal transition (EMT) of cancer cells. Over expression of Zeb1 can attenuate IL-24-suppressed malignancy of GBM cells. Mechanistically, IL-24 decreases the protein stability of Zeb1 while has no effect on its mRNA stability. It is due to that IL-24 can increase the expression of FBXO45, which can destabilize Zeb1 in cancer cells. Collectively, we reveal that IL-24 can suppress the malignancy of GBM cells via decreasing the expression of Zeb1. It suggests that targeted activation of IL-24 signals might be a potential therapy approach for GBM treatment.

Bioinformatics analysis identifying FBXO45 gene as a potential oncogene in esophageal cancer.

BACKGROUND: F-box protein 45 (FBXO45) is a member of the F-box protein family, and is reportedly involved in the progression of many diseases. However, its role in esophageal cancer (ESCA) remains unclear. METHODS: The expression, clinical characteristics, gene function, pathway, and correlation between the infiltration of different immune cells were analyzed using public data. The pan-cancer expression of FBXO45 was assessed using the TIMER2 database. The expression of FBXO45 in different tumor stages and histology subtypes were evaluated using the UALCAN database. The protein-protein interaction (PPI) network was constructed using the STRING database. Immune cell infiltration data were downloaded from the ImmuCellAI database. RESULTS: The top 300 genes most positively correlated with FBXO45 were screened into the enrichment analysis. The functional enrichment results showed that FBXO45 was mainly associated with proteasomal protein catabolic process and the regulation of DNA metabolic processing in the biological process (BP) category; spindle, chromosomal region, and focal adhesion in the cellular component category; and ATPase activity and ubiquitin-protein transferase activity terms in the molecular function category. FBXO45 was overexpressed in ESCA and other cancer types. FBXO45 expression was positively associated with the infiltration levels of immunosuppressive cells, such as CD8+ (cluster of differentiation 8+) T cells and NK (natural killer cell) cells, in ESCA. MYCBP2 and SKP1 were most associated with FBXO45. CONCLUSIONS: Our results suggested that FBXO45 is a potential oncogene in ESCA. Elevated FBXO45 expression indicates a relatively immunosuppressive microenvironment.

Association Study Reveals One Susceptibility Locus with Vitiligo in the Chinese Han Population.

Objective: The purpose of this study was to investigate associations between the 14 reported loci (from a meta-analysis of genome-wide association studies [GWAS] in the Caucasian population) and vitiligo in the Chinese Han population. Materials and Methods: In this study 14 single nucleotide polymorphisms (SNPs) at 14 different genetic loci were evaluated for their association with viteligo in a Chinese Han cohort, including 1472 cases and 1472 controls of by using the Sequenom MassArray iPLEX1 system. A Bonferroni adjustment was used for multiple comparisons and pBonferroni <0.0056 was considered statistically significant. Results: The T allele of the locus within the FBXO45-NRROS gene (3q29) was significantly associated with vitiligo (odds ratio = 1.22, 95% confidence interval: 1.10-1.36, p = 0.0001). Association at the genotype level was strong (p = 0.0007). The other SNPs were not associated with vitiligo (pBonferroni >0.0056). Conclusion: A SNP at the rs6583331 locus 3q29 is associated with the susceptibility of vitiligo in the Chinese Han population, which suggests that there is a common genetic factor predisposing to the development of vitiligo in the Chinese and Caucasian populations.

A genome-wide association study of coping behaviors suggests FBXO45 is associated with emotional expression.

Individuals use coping behaviors to deal with unpleasant daily events. Such behaviors can moderate or mediate the pathway between psychosocial stress and health-related outcomes. However, few studies have examined the associations between coping behaviors and genetic variants. We conducted a genome-wide association study (GWAS) on coping behaviors in 14088 participants aged 35 to 69 years as part of the Japan Multi-Institutional Collaborative Cohort Study. Five coping behaviors (emotional expression, emotional support seeking, positive reappraisal, problem solving and disengagement) were measured and analyzed. A GWAS analysis was performed using a mixed linear model adjusted for study area, age and sex. Variants with suggestive significance in the discovery phase (N = 6403) were further examined in the replication phase (N = 7685). We then combined variant-level association evidence into gene-level evidence using a gene-based analysis. The results showed a significant genetic contribution to emotional expression and disengagement, with an estimation that the 19.5% and 6.6% variance in the liability-scale was explained by common variants. In the discovery phase, 12 variants met suggestive significance (P < 1 × 10-6 ) for association with the coping behaviors and perceived stress. However, none of these associations were confirmed in the replication stage. In gene-based analysis, FBXO45, a gene with regulatory roles in synapse maturation, was significantly associated with emotional expression after multiple corrections (P < 3.1 × 10-6 ). In conclusion, our results showed the existence of up to 20% genetic contribution to coping behaviors. Moreover, our gene-based analysis using GWAS data suggests that genetic variations in FBXO45 are associated with emotional expression.

PAM forms an atypical SCF ubiquitin ligase complex that ubiquitinates and degrades NMNAT2.

PHR (PAM/Highwire/RPM-1) proteins are conserved RING E3 ubiquitin ligases that function in developmental processes, such as axon termination and synapse formation, as well as axon degeneration. At present, our understanding of how PHR proteins form ubiquitin ligase complexes remains incomplete. Although genetic studies indicate NMNAT2 is an important mediator of PHR protein function in axon degeneration, it remains unknown how PHR proteins inhibit NMNAT2. Here, we decipher the biochemical basis for how the human PHR protein PAM, also called MYCBP2, forms a noncanonical Skp/Cullin/F-box (SCF) complex that contains the F-box protein FBXO45 and SKP1 but lacks CUL1. We show FBXO45 does not simply function in substrate recognition but is important for assembly of the PAM/FBXO45/SKP1 complex. Interestingly, we demonstrate a novel role for SKP1 as an auxiliary component of the target recognition module that enhances binding of FBXO45 to NMNAT2. Finally, we provide biochemical evidence that PAM polyubiquitinates NMNAT2 and regulates NMNAT2 protein stability and degradation by the proteasome.

Novel rare variants in F-box protein 45 (FBXO45) in schizophrenia.

The ubiquitin ligase F-box protein 45 (FBXO45) is critical for synaptogenesis, neuronal migration, and synaptic transmission. FBXO45 is included in the 3q29 microdeletion region that confers a significant risk for schizophrenia, as shown by rare structural variant studies. Thus, FBXO45 is considered a prominent candidate for mediating schizophrenia pathogenesis. Here, we investigated rare, deleterious single nucleotide variants (SNVs) as well as small insertions and deletions (INDELs) in FBXO45 that may contribute to schizophrenia susceptibility. Using Sanger sequencing, we performed mutation screening in FBXO45 exon regions in 337 schizophrenia patients. Novel missense or nonsense variants were followed up with a genetic association study in an independent sample set of 601 schizophrenia patients and 916 controls, a case report for assessing the clinical consequence of the mutations, a pedigree study for measuring mutation inheritance in the proband's family, bioinformatics analyses for evaluating mutation effect on protein structure and function, and mRNA expression analysis for examining mutation transcriptional influence on FBXO45 expression. One heterozygous, novel, and rare missense mutation (R108C) was identified in a single schizophrenia patient and in his healthy mother. At age 20, this patient was diagnosed with paranoid schizophrenia and carried some clinical features of 3q29 deletion phenotypes, including premorbid IQ decline. With follow-up genotyping, this mutation was not found in either the schizophrenia group (0/601) or the healthy control group (0/916). Bioinformatics analyses predicted that R108C probably pathologically impacted the structure and function of the FBXO45 protein. The relative expression of FBXO45 in SCZ case with R108C mutation was relatively low when compared to 50 schizophrenia patients and 52 healthy controls. The R108C mutation in FBXO45 is a rare variant with a modest effect on schizophrenia risk that may disrupt the structure and function of the FBXO45 protein. Our findings also suggest that FBXO45 may be a new attractive candidate gene for schizophrenia.

Chromosomal microarray analysis of consecutive individuals with autism spectrum disorders or learning disability presenting for genetic services.

Chromosomal microarray analysis is now commonly used in clinical practice to identify copy number variants (CNVs) in the human genome. We report our experience with the use of the 105 K and 180K oligonucleotide microarrays in 215 consecutive patients referred with either autism or autism spectrum disorders (ASD) or developmental delay/learning disability for genetic services at the University of Kansas Medical Center during the past 4 years (2009-2012). Of the 215 patients [140 males and 75 females (male/female ratio=1.87); 65 with ASD and 150 with learning disability], abnormal microarray results were seen in 45 individuals (21%) with a total of 49 CNVs. Of these findings, 32 represented a known diagnostic CNV contributing to the clinical presentation and 17 represented non-diagnostic CNVs (variants of unknown significance). Thirteen patients with ASD had a total of 14 CNVs, 6 CNVs recognized as diagnostic and 8 as non-diagnostic. The most common chromosome involved in the ASD group was chromosome 15. For those with a learning disability, 32 patients had a total of 35 CNVs. Twenty-six of the 35 CNVs were classified as a known diagnostic CNV, usually a deletion (n=20). Nine CNVs were classified as an unknown non-diagnostic CNV, usually a duplication (n=8). For the learning disability subgroup, chromosomes 2 and 22 were most involved. Thirteen out of 65 patients (20%) with ASD had a CNV compared with 32 out of 150 patients (21%) with a learning disability. The frequency of chromosomal microarray abnormalities compared by subject group or gender was not statistically different. A higher percentage of individuals with a learning disability had clinical findings of seizures, dysmorphic features and microcephaly, but not statistically significant. While both groups contained more males than females, a significantly higher percentage of males were present in the ASD group.