Chemical induction of the interaction between AIMP2-DX2 and Siah1 to enhance ubiquitination.

AIMP2-DX2 (hereafter DX2) is an oncogenic variant of aminoacyl-tRNA synthetase-interacting multifunctional protein 2 (AIMP2) that mediates tumorigenic interactions with various factors involved in cancer. Reducing the levels of DX2 can effectively inhibit tumorigenesis. We previously reported that DX2 can be degraded through Siah1-mediated ubiquitination. In this study, we identified a compound, SDL01, which enhanced the interaction between DX2 and Siah1, thereby facilitating the ubiquitin-dependent degradation of DX2. SDL01 was found to bind to the pocket surrounding the N-terminal flexible region and GST domain of DX2, causing a conformational change that stabilized its interaction with Siah1. Our findings demonstrate that protein-protein interactions (PPIs) can be modulated through chemically induced conformational changes.

SIAH1 facilitates the migration and invasion of gastric cancer cells through promoting the ubiquitination and degradation of RECK.

Siah E3 ubiquitin protein ligase 1 (SIAH1) has been reported to participate in the development of several human cancers, including gastric cancer. However, the effect and mechanism of SIAH1 on the migration and invasion of gastric cancer cells need be further explored. Here, we first analyzed the clinical value of SIAH1 in gastric cancer, and found that SIAH1 was up-regulated in gastric cancer and associated with a poor prognosis. In addition, silencing of SIAH1 significantly inhibited the migration and invasion of gastric cancer cells through inhibiting the expression of matrix metalloproteinase-9 (MMP9), while overexpression of SIAH1 had the opposite effect. Molecularly, we provided the evidence that reversion-inducing cysteine-rich protein with Kazal motifs (RECK) was a potential substrate of SIAH1. We determined that SIAH1 could destabilize RECK through promoting its ubiquitination and degradation via proteasome pathway. We also found RECK was involved in SIAH1-regulated gastric cancer cell migration and invasion. In conclusion, SIAH1 is up-regulated in gastric cancer, which promotes the migration and invasion of gastric cancer cells through regulating RECK-MMP9 pathway.

Monomethyl fumarate attenuates lung Ischemia/Reperfusion injury by disrupting the GAPDH/Siah1 signaling cascade.

Monomethyl fumarate (MMF), a potent anti-inflammatory agent used to treat multiple sclerosis, has demonstrated efficacy in various inflammatory and ischemia/reperfusion (IR) models; however, its impact on IR-induced acute lung injury (ALI) has not been explored. We investigated, for the first time, whether MMF attenuates lung IR injury through inhibition of the GAPDH/Siah1 signaling pathway. Rats were subjected to IR injury using an isolated perfused lung model, and proximity ligation assays were employed to evaluate the presence and distribution of the GAPDH/Siah1 complex. In vitro studies involved pretreating human primary alveolar epithelial cells (HPAECs) with MMF and/or inducing GAPDH overexpression or silencing, followed by exposure to hypoxia-reoxygenation. The findings revealed significantly reduced lung damage indicators, including edema, proinflammatory cytokines, oxidative stress and apoptosis, in MMF-treated rats. Notably, MMF treatment inhibited GAPDH/Siah1 complex formation and nuclear translocation, indicating that disruption of the GAPDH/Siah1 cascade was the primary cause of these improvements. Our in vitro studies on pretreated HPAECs corroborate these in vivo findings, further strengthening this interpretation. Our study results suggest that the protective effects of MMF against lung IR injury may be attributed, at least in part, to its ability to disrupt the GAPDH/Siah1 signaling cascade, thereby attenuating inflammatory and apoptotic responses. Given these encouraging results, MMF has emerged as a promising therapeutic candidate for the management of lung IR injury.

Reactive Oxygen Species Induction by Hepatitis B Virus: Implications for Viral Replication in p53-Positive Human Hepatoma Cells.

Hepatitis B virus (HBV) infects approximately 300 million people worldwide, causing chronic infections. The HBV X protein (HBx) is crucial for viral replication and induces reactive oxygen species (ROS), leading to cellular damage. This study explores the relationship between HBx-induced ROS, p53 activation, and HBV replication. Using HepG2 and Hep3B cell lines that express the HBV receptor NTCP, we compared ROS generation and HBV replication relative to p53 status. Results indicated that HBV infection significantly increased ROS levels in p53-positive HepG2-NTCP cells compared to p53-deficient Hep3B-NTCP cells. Knockdown of p53 reduced ROS levels and enhanced HBV replication in HepG2-NTCP cells, whereas p53 overexpression increased ROS and inhibited HBV replication in Hep3B-NTCP cells. The ROS scavenger N-acetyl-L-cysteine (NAC) reversed these effects. The study also found that ROS-induced degradation of the HBx is mediated by the E3 ligase Siah-1, which is activated by p53. Mutations in p53 or inhibition of its transcriptional activity prevented ROS-mediated HBx degradation and HBV inhibition. These findings reveal a p53-dependent negative feedback loop where HBx-induced ROS increases p53 levels, leading to Siah-1-mediated HBx degradation and HBV replication inhibition. This study offers insights into the molecular mechanisms of HBV replication and identifies potential therapeutic targets involving ROS and p53 pathways.

SIAH1 modulates antiviral immune responses by targeting deubiquitinase USP19.

Tight control of the type I interferon (IFN) signaling pathway is critical for maintaining host innate immune responses, and the ubiquitination and deubiquitination of signaling molecules are essential for signal transduction. Deubiquitinase ubiquitin-specific protein 19 (USP19) is known to be involved in deubiquitinating Beclin1, TRAF3, and TRIF for downregulation of the type I IFN signaling. Here, we show that SIAH1, a cellular E3 ubiquitin ligase that is involved in multicellular pathway, is a potent positive regulator of virus-mediated type I IFN signaling that maintains homeostasis within the antiviral immune response by targeting USP19. In the early stages of virus infection, stabilized SIAH1 directly interacts with the USP19 and simultaneously mediates K27-linked ubiquitination of 489, 490, and 610 residues of USP19 for proteasomal degradation. Additionally, we found that USP19 specifically interacts with MAVS and deubiquitinates K63-linked ubiquitinated MAVS for negative regulation of type I IFN signaling. Ultimately, we identified that SIAH1-mediated degradation of USP19 reversed USP19-mediated deubiquitination of MAVS, Beclin1, TRAF3, and TRIF, resulting in the activation of antiviral immune responses. Taken together, these findings provide new insights into the molecular mechanism of USP19 and SIAH1, and suggest a critical role of SIAH1 in antiviral immune response and homeostasis.

Proteome-wide identification and functional analysis of ubiquitinated proteins in Hepa1-6 cells by knockdown of E3 ubiquitin ligase SIAH1.

Background: Hepatocellular carcinoma (HCC) is an aggressive malignancy that poses a serious threat to human life. The conventional therapies for HCC cannot substantially improve overall survival (OS), disease duration, and prognosis. Therefore, it is important to study the underlying mechanism of HCC and seek better methods for HCC prevention and treatment. Ubiquitination is a post-translational modification that modulates great cellular function by cooperating with E1, E2, and E3 ligases. Yet, the ubiquitination and lysine residues in HCC are still elusive. Seven in absentia homolog 1 (SIAH1), as an important E3 ubiquitin ligase, regulates ubiquitin-mediated proteolysis to function as a tumor suppressor in HCC. In the present study, we downregulated SIAH1 in the mouse HCC cell line Hepa1-6 and studied its function by using proteome-wide identification. Methods: SIAH1 was knocked down by SIAH1 short hairpin RNA (shRNA) in mouse HCC cell line Hepa1-6 cells, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was conducted to analyze the ubiquitinated proteins. Functional analysis was performed using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment. Results: The systematic profiling showed a total of 550 differently expressed proteins (DEPs), including 263 upregulated DEPs and 287 downregulated DEPs. Considering the amino acid sequences around the modified lysine residues, seven proteins were identified as conserved ubiquitination motifs in the peptides. The ubiquitinated proteins were mainly distributed in the cytoplasm, nucleus, and plasma membrane. Functional analysis suggested that the ubiquitinated proteins were mostly enriched in the nucleus, cytoplasm, and extracellular space; in addition, the ubiquitinated proteins were mostly attributed to the protein binding, and disease. The ubiquitinated proteins modulate HCC by mapping lysine modification sites. Conclusions: The use of high-throughput characterization to identify novel and specific targets associated with SIAH1 is of great significance in terms of functional weight. The results obtained in this paper from the analysis of proteomic data provided novel insights into ubiquitination regulation in HCC, which pave the way for further research and mechanism discovery of HCC.

The CK1ε/SIAH1 axis regulates AXIN1 stability in colorectal cancer cells.

Casein kinase 1ε (CK1ε) and axis inhibitor 1 (AXIN1) are crucial components of the ß-catenin destruction complex in canonical Wnt signaling. CK1ε has been shown to interact with AXIN1, but its physiological function and role in tumorigenesis remain unknown. In this study, we found that CK1δ/ε inhibitors significantly enhanced AXIN1 protein level in colorectal cancer (CRC) cells through targeting CK1ε. Mechanistically, CK1ε promoted AXIN1 degradation by the ubiquitin-proteasome pathway by promoting the interaction of E3 ubiquitin-protein ligase SIAH1 with AXIN1. Genetic or pharmacological inhibition of CK1ε and knockdown of SIAH1 downregulated the expression of Wnt/ß-catenin-dependent genes, suppressed the viability of CRC cells, and restrained tumorigenesis and progression of CRC in vitro and in vivo. In summary, our results demonstrate that CK1ε exerted its oncogenic role in CRC occurrence and progression by regulating the stability of AXIN1. These findings reveal a novel mechanism by which CK1ε regulates the Wnt/ß-catenin signaling pathway and highlight the therapeutic potential of targeting the CK1ε/SIAH1 axis in CRC.

Nrf2 Inhibits GAPDH/Siah1 Axis to Reduce Inflammatory Reactions and Proliferation of Microglia After Simulating Spinal Cord Injury.

OBJECTIVE: To explore the effect of nuclear factor erythroid 2-related factor 2 (Nrf 2) on microglial inflammatory response and proliferation after spinal cord injury (SCI) through the glyceraldehyde phosphate dehydrogenase (GAPDH) / Seven in absentia homolog 1 (Siah 1) signaling pathway. METHODS: Human microglia HMC3 was induced by lipopolysaccharide (LPS) to establish a SCI cell model. Microglia morphology after LPS stimulation was observed by transmission electron microscope (TEM), and cellular Nrf2, GAPDH/Siah1 pathway expression and cell viability were determined. Subsequently, the Nrf2 overexpression plasmid was transfected into microglia to observe changes in cell viability and GAPDH/Siah1 pathway expression. RESULTS: Microglia, mostly amoeba-like, were found to have enlarged cell bodies after LPS stimulation, with an increased number of cell branches, highly expressed Nrf2, GAPDH and Siah1, and decreased cell viability (P<0.05). Up-regulating Nrf2 inhibited the GAPDH/Siah1 axis, decreased inflammatory responses, and enhanced activity in post-SCI microglia (P<0.05). CONCLUSION: Up-regulating Nrf2 expression can reverse the inflammatory reaction of microglia after LPS stimulation and enhance their activity by inhibiting the GAPDH/Siah1 axis.

Hydrogen Peroxide Inhibits Hepatitis B Virus Replication by Downregulating HBx Levels via Siah-1-Mediated Proteasomal Degradation in Human Hepatoma Cells.

The hepatitis B virus (HBV) is constantly exposed to significant oxidative stress characterized by elevated levels of reactive oxygen species (ROS), such as H2O2, during infection in hepatocytes of patients. In this study, we demonstrated that H2O2 inhibits HBV replication in a p53-dependent fashion in human hepatoma cell lines expressing sodium taurocholate cotransporting polypeptide. Interestingly, H2O2 failed to inhibit the replication of an HBV X protein (HBx)-null HBV mutant, but this defect was successfully complemented by ectopic expression of HBx. Additionally, H2O2 upregulated p53 levels, leading to increased expression of seven in absentia homolog 1 (Siah-1) levels. Siah-1, an E3 ligase, induced the ubiquitination-dependent proteasomal degradation of HBx. The inhibitory effect of H2O2 was nearly abolished not only by treatment with a representative antioxidant, N-acetyl-L-cysteine but also by knockdown of either p53 or Siah-1 using specific short hairpin RNA, confirming the role of p53 and Siah-1 in the inhibition of HBV replication by H2O2. The present study provides insights into the mechanism that regulates HBV replication under conditions of oxidative stress in patients.

All-trans Retinoic Acid Inhibits Hepatitis B Virus Replication by Downregulating HBx Levels via Siah-1-Mediated Proteasomal Degradation.

All-trans retinoic acid (ATRA), the most biologically active metabolite of vitamin A, is known to abolish the potential of HBx to downregulate the levels of p14, p16, and p21 and to stimulate cell growth during hepatitis B virus (HBV) infection, contributing to its chemopreventive and therapeutic effects against HBV-associated hepatocellular carcinoma. Here, we demonstrated that ATRA antagonizes HBx to inhibit HBV replication. For this effect, ATRA individually or in combination with HBx upregulated p53 levels, resulting in upregulation of seven in absentia homolog 1 (Siah-1) levels. Siah-1, an E3 ligase, induces ubiquitination and proteasomal degradation of HBx in the presence of ATRA. The ability of ATRA to induce Siah-1-mediated HBx degradation and the subsequent inhibition of HBV replication was proven in an in vitro HBV replication model. The effects of ATRA became invalid when either p53 or Siah-1 was knocked down by a specific shRNA, providing direct evidence for the role of p53 and Siah-1 in the negative regulation of HBV replication by ATRA.

Structural Basis of the Interaction between Human Axin2 and SIAH1 in the Wnt/ß-Catenin Signaling Pathway.

The scaffolding protein Axin is an important regulator of the Wnt signaling pathway, and its dysfunction is closely related to carcinogenesis. Axin could affect the assembly and dissociation of the ß-catenin destruction complex. It can be regulated by phosphorylation, poly-ADP-ribosylation, and ubiquitination. The E3 ubiquitin ligase SIAH1 participates in the Wnt pathway by targeting various components for degradation. SIAH1 is also implicated in the regulation of Axin2 degradation, but the specific mechanism remains unclear. Here, we verified that the Axin2-GSK3 binding domain (GBD) was sufficient for SIAH1 binding by the GST pull-down assay. Our crystal structure of the Axin2/SIAH1 complex at 2.53 Å resolution reveals that one Axin2 molecule binds to one SIAH1 molecule via its GBD. These interactions critically depend on a highly conserved peptide 361EMTPVEPA368 within the Axin2-GBD, which forms a loop and binds to a deep groove formed by ß1, ß2, and ß3 of SIAH1 by the N-terminal hydrophilic amino acids Arg361 and Thr363 and the C-terminal VxP motif. The novel binding mode indicates a promising drug-binding site for regulating Wnt/ß-catenin signaling.

SIAH1 ubiquitination-modified HMGCR inhibits lung cancer progression and promotes drug sensitivity through cholesterol synthesis.

BACKGROUNDS: Lung cancer is one of the most frequently diagnosed cancers and the leading cause of cancer-related deaths worldwide. Deep understanding of chemoresistance will lead to remarkable progress in lung cancer treatment strategy. Cholesterol accumulation was associated with cisplatin resistance in lung cancer treatment. And we found the degree of cisplatin resistance was correlated with the expression of the cholesterol synthesis HMGCR. METHODS: We analyzed a group of 42 lung cancer patients who received cisplatin treatment after lung resection surgery. The expression of HMGCR and its correlation with cholesterol in lung cancer cell lines were determined by qRT-PCR and ELISA analyses. We focus on the function and mechanism of HMGCR in lung cancer and reveal that knockdown of HMGCR expression inhibits the proliferation, colony formation, and migration of lung cancer cell lines in vitro or in vivo and dramatically enhances the efficacy of cisplatin. RESULTS: Through mechanism studies, we illustrate that SIAH1, an E3 ubiquitin-protein ligase, ubiquitination modifies HMGCR and inhibits efflux protein activity via regulating cholesterol synthesis. In vivo experiments showed that SIAH1 overexpression or using HMGCR knockdown retard tumor growth and enhanced the efficacy of cisplatin. In summary, HMGCR affects cholesterol metabolism by regulating key enzymes in cholesterol synthesis, thereby reducing drug sensitivity. CONCLUSION: This study indicates that lung cancer patients with lower HMGCR levels may lead to a better prognosis and provide a potential treatment by SIAH1 overexpression for lung cancer patients with cisplatin resistance.

Intrahippocampal injection of IL-1ß upregulates Siah1-mediated degradation of synaptophysin by activation of the ERK signaling in male rat.

Interleukin-1ß (IL-1ß) has been described to exert important effect on synapses in the brain. Here, we explored if the synapses in the hippocampus would be adversely affected following intracerebral IL-1ß injection and, if so, to clarify the underlying molecular mechanisms. Adult male Sprague-Dawley rats were divided into control, IL-1ß, IL-1ß + PD98059, and IL-1ß + MG132 groups and then sacrificed for detection of synaptophysin (syn) protein level, synaptosome glutamate release, and synapse ultrastructure by western blotting, glutamate kit and electron microscopy, respectively. These rats were tested by Morris water maze for learning and memory ability. It was determined by western blotting whether IL-1ß exerted the effect of on syn and siah1 expression in primary neurons via extracellular regulated protein kinases (ERK) signaling pathway. Intrahippocampal injection of IL-1ß in male rats and sacrificed at 8d resulted in a significant decrease in syn protein, damage of synapse structure, and abnormal release of neurotransmitters glutamate. ERK inhibitor and proteosome inhibitor treatment reversed the above changes induced by IL-1ß both in vivo and in vitro. In primary cultured neurons incubated with IL-1ß, the expression level of synaptophysin was significantly downregulated coupled with abnormal glutamate release. Furthermore, use of PD98059 had confirmed that ERK signaling pathway was implicated in synaptic disorders caused by IL-1ß treatment. The present results suggest that exogenous IL-1ß can suppress syn protein level and glutamate release. A possible mechanism for this is that IL-1ß induces syn degradation that is regulated by the E3 ligase siah1 via the ERK signaling pathway.

LINC00963 promotes the malignancy and metastasis of lung adenocarcinoma by stabilizing Zeb1 and exosomes-induced M2 macrophage polarization.

BACKGROUND: Long intergenic non-coding RNA 00963 (LINC00963) is an oncogenic lncRNA in human cancers. However, little is known on how it impacts the pathogenesis of lung adenocarcinoma (LUAD). METHODS: Biological effects on proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) were examined by CCK-8, colony formation, EdU incorporation, transwell, and immunofluorescence assays, respectively. Macrophage polarization was evaluated by flow cytometry. Ubiquitination of Zeb1 was examined by co-immunoprecipitation. The location of LINC00963 in LUAD tissues and cell lines was tested by FISH assay. The LINC00963/HNRNPA2B1/Siah1 mRNA complex interaction was verified using RNA pull-down and immunoprecipitation assays. The exact roles of LINC00963 were further validated in metastasis and xenograft models. RESULTS: Higher LINC00963 expression in LUAD patients positively correlated with shorter overall survival, higher stages, and metastasis. LINC00963 mainly localized in the cytoplasm and aggravated malignant phenotypes of LUAD cells in vitro and metastasis in vivo. Mechanistically, LINC00963 directly interacted HNRNPA2B1 protein to trigger the degradation of Siah1 mRNA, which inhibited the ubiquitination and degradation of Zeb1. Moreover, exosomal LINC00963 derived from LUAD cells induced M2 macrophage polarization and promoted LUAD growth and metastasis. CONCLUSION: By stabilizing Zeb1 in cancer cells and delivering exosomes to induce M2 macrophage polarization, LINC00963 promoted the malignancy and metastasis of LUAD. Targeting LINC00963 may become a valuable therapeutic target for LUAD.

SIAH proteins regulate the degradation and intra-mitochondrial aggregation of PINK1: Implications for mitochondrial pathology in Parkinson's disease.

Parkinson's disease (PD) is characterized by degeneration of neurons, particularly dopaminergic neurons in the substantia nigra. PD brains show accumulation of α-synuclein in Lewy bodies and accumulation of dysfunctional mitochondria. However, the mechanisms leading to mitochondrial pathology in sporadic PD are poorly understood. PINK1 is a key for mitophagy activation and recycling of unfit mitochondria. The activation of mitophagy depends on the accumulation of uncleaved PINK1 at the outer mitochondrial membrane and activation of a cascade of protein ubiquitination at the surface of the organelle. We have now found that SIAH3, a member of the SIAH proteins but lacking ubiquitin-ligase activity, is increased in PD brains and cerebrospinal fluid and in neurons treated with α-synuclein preformed fibrils (α-SynPFF). We also observed that SIAH3 is aggregated together with PINK1 in the mitochondria of PD brains. SIAH3 directly interacts with PINK1, leading to their intra-mitochondrial aggregation in cells and neurons and triggering a cascade of toxicity with PINK1 inactivation along with mitochondrial depolarization and neuronal death. We also found that SIAH1 interacts with PINK1 and promotes ubiquitination and proteasomal degradation of PINK1. Similar to the dimerization of SIAH1/SIAH2, SIAH3 interacts with SIAH1, promoting its translocation to mitochondria and preventing its ubiquitin-ligase activity toward PINK1. Our results support the notion that the increase in SIAH3 and intra-mitochondrial aggregation of SIAH3-PINK1 may mediate α-synuclein pathology by promoting proteotoxicity and preventing the elimination of dysfunctional mitochondria. We consider it possible that PINK1 activity is decreased in sporadic PD, which impedes proper mitochondrial renewal in the disease.

SIAH1-mediated RPS3 ubiquitination contributes to chemosensitivity in epithelial ovarian cancer.

The E3 ligase SIAH1 is deregulated in human cancers and correlated with poor prognosis, but its contributions to chemoresistance in epithelial ovarian cancer (EOC) are not evident. Herein we found that SIAH1 was decreased in EOC tumour tissues and cell lines and negatively correlated with the RPS3 levels. SIAH1 overexpression suppressed tumour cell growth, colony formation, invasion, metastasis, and cisplatin resistance in vivo and in vitro. SIAH1 promoted RPS3 ubiquitination and degradation using the RING-finger domain, and these steps were required for RPS3 localization to the cytoplasm, which led to subsequent NF-κB inactivation and thereby conferred chemosensitivity. Moreover, ectopic expression of RPS3 or depletion of RPS3 ubiquitination mediated by SIAH1 via the K214R mutant significantly impaired cisplatin-induced tumour suppression in cells stably expressing SIAH1. Together, our findings reveal a tumour suppressor function of SIAH1 and provide evidence showing that the SIAH1-RPS3-NF-κB axis may act as an appealing strategy for tackling treatment resistance in EOC.

Protective effects of calcyclin-binding protein against pulmonary vascular remodeling in flow-associated pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension associated with congenital heart disease (CHD-PAH) is recognized as a cancer-like disease with a proliferative and pro-migratory phenotype in pulmonary artery smooth muscle cells (PASMCs). Calcyclin-binding protein/Siah-1-interacting protein (CacyBP/SIP) has been implicated in the progression of various cancers; however, it has not been previously studied in the context of CHD-PAH. Here, we aimed to examine the function of CacyBP/SIP in CHD-PAH and explore its potential as a novel regulatory target for the disease. METHODS: The expression of CacyBP/SIP in PASMCs was evaluated both in the pulmonary arterioles of patients with CHD-PAH and in high-flow-induced PAH rats. The effects of CacyBP/SIP on pulmonary vascular remodeling and PASMC phenotypic switch, proliferation, and migration were investigated. LY294002 (MedChemExpress, NJ, USA) was used to block the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway to explore changes in PASMC dysfunction induced by low CacyBP/SIP levels. Hemodynamics and pulmonary arterial remodeling were further explored in rats after short-interfering RNA-mediated decrease of CacyBP/SIP expression. RESULTS: CacyBP/SIP expression was markedly reduced both in the remodeled pulmonary arterioles of patients with CHD-PAH and in high-flow-induced PAH rats. Low CacyBP/SIP expression promoted hPASMC phenotypic switch, proliferation, and migration via PI3K/AKT pathway activation. Our results indicated that CacyBP/SIP protected against pulmonary vascular remodeling through amelioration of hPASMC dysfunction in CHD-PAH. Moreover, after inhibition of CacyBP/SIP expression in vivo, we observed increased right ventricular hypertrophy index, poor hemodynamics, and severe vascular remodeling. CONCLUSIONS: CacyBP/SIP regulates hPASMC dysfunction, and its increased expression may ameliorate progression of CHD-PAH.

Siah1 promotes the proliferation of NSCLC cells through ubiquitinating and stabilizing Notch1.

Seven in absentia homolog 1 (Siah1) has been shown plays important roles in the pathogenesis and development of multiple cancers. However, the functions and mechanisms of Siah1 in non-small cell lung cancer (NSCLC) remain unclear. In our study, we found that knock down of Siah1 could inhibit the proliferation of NSCLC cells, while over-expression of Siah1 had the opposite effects. Molecularly, the bioinformatics analysis determined that notch receptor 1 (Notch1) might be the potential target of Siah1. Subsequently, we identified that Siah1 acted as an E3 ligase to promote the ubiquitination and stabilization of Notch1 through the proteasome pathway. Furthermore, the results showed that the Siah1 expression was directly correlated with CTR9 in human NSCLC tissues. Finally, Siah1 could promote Akt phosphorylation through regulating Notch1, thus promoting the proliferation of NSCLC cells. In conclusion, our study demonstrated that Siah1 acts as an oncogene, can ubiquitinate and stabilize Notch1 by proteasome pathway, which promotes Akt phosphorylation and ultimately leads to NSCLC cell proliferation.

Circ-CCNB1 Modulates Trophoblast Proliferation and Invasion in Spontaneous Abortion by Regulating miR-223/SIAH1 axis.

CONTEXT: Spontaneous abortion (SA) is a common disorder in early pregnancy. Circular RNAs (circRNAs) have been reported to exert important regulatory effects on trophoblast function and embryo development. OBJECTIVE: The aim of this study was to explore whether and how circRNAs regulate trophoblast function in SA during early pregnancy. METHODS: Cell proliferation, 5-bromo-2-deoxyuridine (BrdU) staining, Transwell, immunofluorescence, Western blot, RNA pull-down, and dual luciferase reporter assays were performed to investigate the effect of circRNA cyclin B1 (circ-CCNB1) on trophoblast function in HTR-8/SVneo and JEG-3 cells. RESULTS: An in vitro study demonstrated that upregulation of circ-CCNB1 significantly inhibited trophoblast proliferation and invasion compared with the controls using HTR-8/SVneo and JEG-3 cells, respectively. Moreover, miR-223 was downregulated in the villous tissues of patients with SA and was further predicted and shown to negatively interact with circ-CCNB1, which is involved in trophoblast proliferation and invasion. Using bioinformatics tools and subsequent RNA pull-down and dual luciferase assays, we found that miR-223 directly targets seven in absentia homolog-1 (SIAH1) and that upregulation of miR-223 decreased circ-CCNB1-induced SIAH1 expression levels in HTR-8/SVneo cells. Interestingly, upregulation of circ-CCNB1 suppressed trophoblast proliferation and invasion through inhibition of CCNB1 nuclear translocation induced by SIAH1. Downregulation of SIAH1 enhanced circ-CCNB1-suppressed CCNB1 nuclear protein expression in trophoblast cells. CONCLUSION: Circ-CCNB1 served as a modulator of trophoblast proliferation and invasion by sponging miR-223, thus forming a regulatory network of circ-CCNB1/miR-223/SIAH1 in modulating CCNB1 nuclear translocation, which enabled us to elucidate the molecular mechanisms involved in normal embryo implantation or in SA.

FRK inhibits glioblastoma progression via phosphorylating YAP and inducing its ubiquitylation and degradation by Siah1.

BACKGROUND: We previously report that yes-associated protein (YAP), the core downstream effector of Hippo pathway, promotes the malignant progression of glioblastoma (GBM). However, although classical regulatory mechanisms of YAP are well explored, how YAP is modulated by the Hippo-independent manner remains poorly understood. Meanwhile, the nonreceptor tyrosine kinase Fyn-related kinase (FRK), which exhibits low expression and possesses tumor suppressor effects in GBM, is reported to be involved in regulation of protein phosphorylation. Here, we examined whether FRK could impede tumor progression by modulating YAP activities. METHODS: Human GBM cells and intracranial GBM model were used to assess the effects of FRK and YAP on the malignant biological behaviors of GBM. Immunoblotting and immunohistochemistry were used to detect the expression of core proteins in GBM tissues. Co-immunoprecipitation, proximity ligation assay, luciferase assay and ubiquitination assay were utilized to determine the protein-protein interactions and related molecular mechanisms. RESULTS: The expression levels of FRK and YAP were inversely correlated with each other in glioma tissues. In addition, FRK promoted the ubiquitination and degradation of YAP, leading to tumor suppression in vitro and in vivo. Mechanistically, FRK interacted with and phosphorylated YAP on Tyr391/407/444, which recruited the classical E3 ubiquitin ligase Siah1 to catalyze ubiquitination and eventually degradation of YAP. Siah1 is required for YAP destabilization initiated by FRK. CONCLUSIONS: We identify a novel mechanism by which FRK orchestrates tumor-suppression effect through phosphorylating YAP and inducing its ubiquitination by Siah1. FRK-Siah1-YAP signaling axis may serve as a potential therapeutic target for GBM treatment.