RING Finger Protein Research Articles

Rnf43 is “lord of the ring” finger proteins in remyelination

Oligodendrocyte precursor cell differentiation into myelinating oligodendrocytes is critical for remyelination in the central nervous system after injury. In this issue of Neuron, Niu etal. (2021) detail a novel role for ring finger protein Rnf43, which is expressed in response to injury and is essential to promote remyelination invivo.

HPIP protooncogene differentially regulates metabolic adaptation and cell fate in breast cancer cells under glucose stress via AMPK and RNF2 dependent pathways

While cancer cells rewire metabolic pathways to sustain growth and survival under metabolic stress in solid tumors, the molecular mechanisms underlying these processes remain largely unknown. In this study, cancer cells switched from survival to death during the early to late phases of metabolic stress by employing a novel signaling switch from AMP activated protein kinase (AMPK)-Forkhead box O3 (FOXO3a)-hematopoietic PBX1-interacting protein (HPIP) to the ring finger protein 2 (RNF2)-HPIP-ubiquitin (Ub) pathway. Acute metabolic stress induced proto-oncogene HPIP expression in an AMPK-FOXO3a-dependent manner in breast cancer (BC) cells. HPIP depletion reduced cell survival and tumor formation in mouse xenografts, which was accompanied by diminished intracellular ATP levels and increased apoptosis in BC cells in response to metabolic (glucose) stress. Glutamine flux (13C-labeled) analysis further suggested that HPIP rewired glutamine metabolism by controlling the expression of the solute carrier family 1 member 5 (SLC1A5) and glutaminase (GLS) genes by acting as a coactivator of MYC to ensure cell survival upon glucose deprivation. However, in response to chronic glucose stress, HPIP was ubiquitinated by the E3-Ub ligase, RNF2, and was concomitantly degraded by the proteasome-mediated pathway, ensuring apoptosis. In support of these data, clinical analyses further indicated that elevated levels of HPIP correlated with AMPK activation in BC. Taken together, these data suggest that HPIP is a signal coordinator during metabolic stress and thus serves as a potential therapeutic target in BC.

A PRC1-RNF2 knockout punch for cancer.

Shedding light on epigenetic mechanisms controlling anti-tumor immune responses, a new study shows that the tumor-intrinsic ring finger protein 2 (RNF2), the catalytic subunit of Polycomb repressor complex 1 (PRC1), acts as a negative regulator of a collaborative NK and CD4+ T cell anti-tumor immune response against breast cancer.

MCM3AP-AS1 regulates proliferation, apoptosis, migration, and invasion of breast cancer cells via binding with ZFP36.

BackgroundPrevious studies suggest that long noncoding RNA (lncRNA) maintenance complex component 3 associated protein (MCM3AP) antisense RNA 1 (MCM3AP-AS1) has a wide range of functions in several cancers. However, its expression and functions in breast cancer are unclear.MethodsReverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of MCM3AP-AS1. MTT, colony formation and anchorage-independent growth assay were used to examine the effect of MCM3AP-AS1 on growth of breast cancer cells. TUNEL and flow cytometry assay were applied to investigate the role of MCM3AP-AS1 on cell apoptosis. Wound heading and transwell matric penetration assay were used to detect the role of MCM3AP-AS1 on cell motility. RNA pull-down and RIP assay were used to examine the interaction of MCM3AP-AS1 and ZFP36.ResultsWe found that lncRNA MCM3AP-AS1 was significantly upregulated in breast cancer, which correlated with patients’ clinicopathological characteristics. Downregulation of MCM3AP-AS1 substantially inhibited the proliferation, apoptosis, migration, and invasion of breast cancer cells. Further analysis clarified that MCM3AP-AS1 binds with the RNA-binding protein ZFP36 ring finger protein (ZFP36) to regulate the levels of cyclin D1 (CCND1), c-myc (MYC), and matrix metalloproteinase 1 (MMP1).ConclusionsOur findings show lncRNA MCM3AP-AS1 might be a prognostic factor and a promising therapeutic target for breast cancer therapy.

Protease-activated receptor 2 stabilizes Bcl-xL and regulates EGFR-targeted therapy response in colorectal cancer.

The Bcl-2 homolog Bcl-xL is emerging as a key factor in tumorigenesis due to its prominent pro-survival and cell death-independent functions. However, the regulation of Bcl-xL by microenvironment and its implication in cancer therapy of colorectal carcinoma (CRC) are unclear. Here, we demonstrated that Bcl-xL expression was positively associated with protease-activated receptor 2 (PAR2) in CRC. Activation of PAR2 stabilized Bcl-xL protein in a proteasome-dependent manner, whereas E3 ligase RING finger protein 152 (RNF152) accelerated the ubiquitination and degradation of Bcl-xL. RNF152 silencing by specific siRNAs rescued the expression of Bcl-xL in PAR2-deficient cells. Moreover, RNF152 physically interacted with Bcl-xL, which was disturbed by PAR2 activation. Further studies with serial mutation of Bcl-xL revealed that phosphorylation of Bcl-xL at S145 reduced its binding affinity for RNF152 and stabilized Bcl-xL. Importantly, inhibition of PAR2 signaling by its gene silencing or specific chemical inhibitors increased apoptosis induced by different EGFR-targeted therapies. In patient-derived xenograft model, inhibition of PAR2 increased the response of CRC to different EGFR-targeted therapies. These results indicate that PAR2 stabilizes Bcl-xL by altering RNF152 signaling and that PAR2 inhibition sensitizes CRC to EGFR-targeted therapies in vivo.

Ring finger protein 213 assembles into a sensor for ISGylated proteins with antimicrobial activity

ISG15 is an interferon-stimulated, ubiquitin-like protein that can conjugate to substrate proteins (ISGylation) to counteract microbial infection, but the underlying mechanisms remain elusive. Here, we use a virus-like particle trapping technology to identify ISG15-binding proteins and discover Ring Finger Protein 213 (RNF213) as an ISG15 interactor and cellular sensor of ISGylated proteins. RNF213 is a poorly characterized, interferon-induced megaprotein that is frequently mutated in Moyamoya disease, a rare cerebrovascular disorder. We report that interferon induces ISGylation and oligomerization of RNF213 on lipid droplets, where it acts as a sensor for ISGylated proteins. We show that RNF213 has broad antimicrobial activity in vitro and in vivo, counteracting infection with Listeria monocytogenes, herpes simplex virus 1, human respiratory syncytial virus and coxsackievirus B3, and we observe a striking co-localization of RNF213 with intracellular bacteria. Together, our findings provide molecular insights into the ISGylation pathway and reveal RNF213 as a key antimicrobial effector.

Tumor necrosis factor alpha regulates myogenesis to inhibit differentiation and promote proliferation in satellite cells

TNF-α and NF-κB signaling is involved in the wasting of skeletal muscle in various conditions, in addition to cancer cachexia. TNF-α and NF-κB signaling promotes the expression level of muscle RING finger protein 1, a ubiquitin ligase, causing muscle degradation. Several studies have indicated that of TNF-α and NF-κB signaling suppresses muscle differentiation by reducing the levels of MyoD protein. On the other hand, TNF-α and NF-κB is required for myoblast proliferation. Thus, the role of TNF-α and NF-κB signaling in the process of myogenesis and regeneration of skeletal muscle is not completely elucidated. Here, we reported that TNF-α reduced the width of single fibers of skeletal muscle in an organ culture model. TNF-α and p65 repressed the transactivation of MyoD and suppressed myoblast differentiation. In addition, TNF-α increased the number of satellite cells, and NF-κB signaling was promoted at the proliferation stage during skeletal muscle regeneration in vivo. TNF-α and NF-κB signaling regulate myogenesis to inhibit differentiation and promote proliferation in satellite cells.

LISTERIN E3 Ubiquitin Ligase and Ribosome-Associated Quality Control (RQC) Mechanism.

According to cellular demands, ribosomes synthesize and maintain the desired pool of proteins inside the cell. However, sometimes due to defects in ribosomal machinery and faulty mRNAs, these nascent polypeptides are constantly under threat to become non-functional. In such conditions, cells acquire the help of ribosome-associated quality control mechanisms (RQC) to eliminate such aberrant nascent proteins. The primary regulator of RQC is RING domain containing LISTERIN E3 ubiquitin ligase, which is associated with ribosomes and alleviates non-stop proteins-associated stress in cells. Mouse RING finger protein E3 ubiquitin ligase LISTERIN is crucial for embryonic development, and a loss in its function causes neurodegeneration. LISTERIN is overexpressed in the mouse brain and spinal cord regions, and its perturbed functions generate neurological and motor deficits, but the mechanism of the same is unclear. Overall, LISTERIN is crucial for brain health and brain development. The present article systematically describes the detailed nature, molecular functions, and cellular physiological characterization of LISTERIN E3 ubiquitin ligase. Improve comprehension of LISTERIN's neurological roles may uncover pathways linked with neurodegeneration, which in turn might elucidate a promising novel therapeutic intervention against human neurodegenerative diseases.

A Novel Role for RNF126 in the Promotion of G2 Arrest via Interaction With 14-3-3σ

Cell cycle checkpoints and DNA repair are important for cell survival after exogenous DNA damage. Both rapid blockage of G2 to M phase transition in the cell cycle and the maintenance of relatively slow G2 arrest are critical to protect cells from lethal ionizing radiation (IR). Checkpoint kinase 1 is pivotal in blocking the transition from G2 to M phases in response to IR. The 14-3-3σ protein is important for IR-induced G2 arrest maintenance in which p53-dependent 14-3-3σ transcription is involved. It has been demonstrated that Ring finger protein 126 (RNF126), an E3 ligase, is required to upregulate checkpoint kinase 1 expression. Thus, our goal was to study the role of RNF126 in the G2/M phase checkpoint. The transition from G2 to M phases and G2 accumulation in response to IR were determined by flow cytometry through staining with phospho-histone H3 (pS10) antibody and propidium iodide, respectively. The interaction of RNF126 and 14-3-3σ was determined by GST-pulldown and coimmunoprecipitation assays. The stability of RNF126 and 14-3-3σ was determined by cycloheximide-based stability assay and ubiquitination detection by coimmunoprecipitation. The sequestering of cyclin-dependent kinase 1 and cyclin B1 from the nucleus was determined by immunofluorescence staining. RNF126 knockdown had no impact on the IR-induced transient blockage of G2 to M but impaired IR-induced G2 arrest maintenance in cells with or without wild-type p53. Mechanistically, RNF126 binds 14-3-3σ and prevents both proteins from ubiquitination-mediated degradation. Last, RNF126 is required for enforcing the cytoplasmic sequestration of cyclin B1 and cyclin-dependent kinase 1 proteins in response to IR. RNF126 promotes G2 arrest via interaction with 14-3-3σ in response to IR. Our study revealed a novel role for RNF126 in promoting G2 arrest, providing a new target for cancer treatment.

Preliminary Observations on Skeletal Muscle Adaptation and Plasticity in Homer 2-/- Mice.

Homer represents a diversified family of scaffold and transduction proteins made up of several isoforms. Here, we present preliminary observations on skeletal muscle adaptation and plasticity in a transgenic model of Homer 2-/- mouse using a multifaceted approach entailing morphometry, quantitative RT-PCR (Reverse Transcription PCR), confocal immunofluorescence, and electrophysiology. Morphometry shows that Soleus muscle (SOL), at variance with Extensor digitorum longus muscle (EDL) and Flexor digitorum brevis muscle (FDB), displays sizable reduction of fibre cross-sectional area compared to the WT counterparts. In SOL of Homer 2-/- mice, quantitative RT-PCR indicated the upregulation of Atrogin-1 and Muscle ring finger protein 1 (MuRF1) genes, and confocal immunofluorescence showed the decrease of neuromuscular junction (NMJ) Homer content. Electrophysiological measurements of isolated FDB fibres from Homer 2-/- mice detected the exclusive presence of the adult ε-nAChR isoform excluding denervation. As for NMJ morphology, data were not conclusive, and further work is needed to ascertain whether the null Homer 2 phenotype induces any endplate remodelling. Within the context of adaptation and plasticity, the present data show that Homer 2 is a co-regulator of the normotrophic status in a muscle specific fashion.

Whole Exome Sequencing in a Series of Patients with a Clinical Diagnosis of Tuberous Sclerosis Not Confirmed by Targeted TSC1/TSC2 Sequencing.

Background: Approximately fifteen percent of patients with tuberous sclerosis complex (TSC) phenotype do not have any genetic disease-causing mutations which could be responsible for the development of TSC. The lack of a proper diagnosis significantly affects the quality of life for these patients and their families. Methods: The aim of our study was to use Whole Exome Sequencing (WES) in order to identify the genes responsible for the phenotype of nine patients with clinical signs of TSC, but without confirmed tuberous sclerosis complex 1/ tuberous sclerosis complex 2 (TSC1/TSC2) mutations using routine molecular genetic diagnostic tools. Results: We found previously overlooked heterozygous nonsense mutations in TSC1, and a heterozygous intronic variant in TSC2. In one patient, two heterozygous missense variants were found in polycystic kidney and hepatic disease 1 (PKHD1), confirming polycystic kidney disease type 4. A heterozygous missense mutation in solute carrier family 12 member 5 (SLC12A5) was found in one patient, which is linked to cause susceptibility to idiopathic generalized epilepsy type 14. Heterozygous nonsense variant ring finger protein 213 (RNF213) was identified in one patient, which is associated with susceptibility to Moyamoya disease type 2. In the remaining three patients WES could not reveal any variants clinically relevant to the described phenotypes. Conclusion: Patients without appropriate diagnosis due to the lack of sensitivity of the currently used routine diagnostic methods can significantly profit from the wider application of next generation sequencing technologies in order to identify genes and variants responsible for their symptoms.

Beyond proteins: Ubiquitylation of lipopolysaccharide to fight bacteria.

Mechanisms and functions of protein ubiquitylation and LPS ubiquitylation. LPS ubiquitylation serves as a scaffold to recruit E3 ligases for the ubiquitylation of the membrane of bacteria. Polyubiquitin coat on the bacterial cell surface is one type of "eat-me" signal recognized by the host cells. The abbreviations used in the figure are Pro, Protein; Ub, Ubiquitin; E1, Ubiquitin-activating enzymes; E2, Ubiquitin-conjugating enzymes; E3, Ubiquitin-ligating enzymes; LPS, lipopolysaccharide; RNF213, Ring Finger Protein 213; and LUBAC, linear ubiquitin chain assembly complex.

Insufficiency of non-canonical PRC1 synergizes with JAK2V617F in the development of myelofibrosis.

Insufficiency of polycomb repressive complex 2 (PRC2), which trimethylates histone H3 at lysine 27, is frequently found in primary myelofibrosis and promotes the development of JAK2V617F-induced myelofibrosis in mice by enhancing the production of dysplastic megakaryocytes. Polycomb group ring finger protein 1 (Pcgf1) is a component of PRC1.1, a non-canonical PRC1 that monoubiquitylates H2A at lysine 119 (H2AK119ub1). We herein investigated the impact of PRC1.1 insufficiency on myelofibrosis. The deletion of Pcgf1 in JAK2V617F mice strongly promoted the development of lethal myelofibrosis accompanied by a block in erythroid differentiation. Transcriptome and chromatin immunoprecipitation sequence analyses showed the de-repression of PRC1.1 target genes in Pcgf1-deficient JAK2V617F hematopoietic progenitors and revealed Hoxa cluster genes as direct targets. The deletion of Pcgf1 in JAK2V617F hematopoietic stem and progenitor cells (HSPCs), as well as the overexpression of Hoxa9, restored the attenuated proliferation of JAK2V617F progenitors. The overexpression of Hoxa9 also enhanced JAK2V617F-mediated myelofibrosis. The expression of PRC2 target genes identified in PRC2-insufficient JAK2V617F HSPCs was not largely altered in Pcgf1-deleted JAK2V617F HSPCs. The present results revealed a tumor suppressor function for PRC1.1 in myelofibrosis and suggest that PRC1.1 insufficiency has a different impact from that of PRC2 insufficiency on the pathogenesis of myelofibrosis.

Identification of the similarly expressed genes in patients with polycystic ovary syndrome and transsexuals.

Polycystic ovary syndrome (PCOS) is a common female infertility, which may be caused by excessive androgen, but its mechanism remains unknown. Transsexuals are women who take androgen drugs for a long time, and gradually have male signs. Their ovaries may have received high concentrations of androgen, which leads to the failure of ovarian reproductive function. Therefore, we searched the relevant data of PCOS and transsexuals in gene expression omnibus database, used limma package to identify the most similarly genes, and then analyzed the possible mechanism of PCOS through gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. Then, the protein-protein interaction network was constructed by searching the String database, and the top 5 hub genes were identified by the cytohubba plug-in of Cytoscape. Finally, ubiquitin conjugating enzyme E2 E1 (UBE2E1), ubiquitin C (UBC), transcription elongation factor B subunit 1 (TCEB1), ubiquitin conjugating enzyme E2 N (UBE2N), and ring finger protein 7 (RNF7) genes were identified as the most similarly expressed genes between PCOS and Transsexuals. They may cause the ubiquitination of androgen receptor and eventually lead to sinus follicular growth arrest. In conclusion, 5 Central genes were identified in PCOS and transsexuals. These genes can be used as targets for early diagnosis or treatment of PCOS.

LGR4: Not Just for Wnt Anymore?

Leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4) is best known for its role in regulating the ability of cells to respond to Wnt ligands. In this well-known role, LGR4 serves as a receptor for R-spondins and forms a complex with the ubiquitin E3 ligases ring finger protein 43 (RNF43) and zinc and ring finger 3 (ZNRF3). RNF43 and ZNRF3 ubiquitinate Frizzleds (FZD), which are a family of ten WNT receptors. This ubiquitination decreases FZD receptor levels on the cell surface, reducing Wnt ligands' ability to activate signaling. While there were some previous indications of Wnt-independent functions of LGR4, this WNT-centric view has remained predominant. In this issue of Cancer Research, Yue and colleagues report that LGR4 also functions to regulate signaling through the EGF receptor. This work was stimulated by observing that while high levels of LGR4 expression in breast tumors correlated with poor patient outcomes, LGR4 levels did not correlate with a well-established Wnt-associated gene signature in these same patients. In contrast, high levels of Lgr4 expression strongly correlated with EGFR signaling. Reducing Lgr4 expression also inhibited signaling through the EGFR, potentially via regulation of the Casitas B-lineage lymphoma ubiquitin E3 ligase. Consistent with this model, LGR4 could be coimmunoprecipitated with a complex that contained EGFR and was capable of inhibiting EGFR ubiquitination. The implications of this work and how it challenges our understanding of the contributions of Wnt signaling and EGFR signaling in cancer are discussed as our several interesting future directions.See related article by Yue et al., p. 4441.

Ubiquitination of ATF6 by disease-associated RNF186 promotes the innate receptor-induced unfolded protein response.

Properly balancing microbial responses by the innate immune system through pattern recognition receptors (PRRs) is critical for intestinal immune homeostasis. Ring finger protein 186 (RNF186) genetic variants are associated with inflammatory bowel disease (IBD). However, functions for the E3 ubiquitin ligase RNF186 are incompletely defined. We found that upon stimulation of the PRR nucleotide-binding oligomerization domain containing 2 (NOD2) in human macrophages, RNF186 localized to the ER, formed a complex with ER stress sensors, ubiquitinated the ER stress sensor activating transcription factor 6 (ATF6), and promoted the unfolded protein response (UPR). These events, in turn, led to downstream signaling, cytokine secretion, and antimicrobial pathway induction. Importantly, RNF186-mediated ubiquitination of K152 on ATF6 was required for these outcomes, highlighting a key role for ATF6 ubiquitination in PRR-initiated functions. Human macrophages transfected with the rare RNF186-A64T IBD risk variant and macrophages from common rs6426833 RNF186 IBD risk carriers demonstrated reduced NOD2-induced outcomes, which were restored by rescuing UPR signaling. Mice deficient in RNF186 or ATF6 demonstrated a reduced UPR in colonic tissues, increased weight loss, and less effective clearance of bacteria with dextran sodium sulfate-induced injury and upon oral challenge with Salmonella Typhimurium. Therefore, we identified that RNF186 was required for PRR-induced, UPR-associated signaling leading to key macrophage functions; defined that RNF186-mediated ubiquitination of ATF6 was essential for these functions; and elucidated how RNF186 IBD risk variants modulated these outcomes.

Role of RNF213 polymorphism in defining quasi-moyamoya disease and definitive moyamoya disease.

Quasi-moyamoya disease (QMMD) is moyamoya disease (MMD) associated with additional underlying diseases. Although the ring finger protein 213 (RNF213) c.14576G>A mutation is highly correlated with MMD in the Asian population, its relationship to QMMD is unclear. Therefore, in this study the authors sought to investigate the RNF213 c.14576G>A mutation in the genetic diagnosis and classification of QMMD. This case-control study was conducted among four core hospitals. A screening system for the RNF213 c.14576G>A mutation based on high-resolution melting curve analysis was designed. The prevalence of RNF213 c.14576G>A was investigated in 76 patients with MMD and 10 patients with QMMD. There were no significant differences in age, sex, family history, and mode of onset between the two groups. Underlying diseases presenting in patients with QMMD were hyperthyroidism (n = 6), neurofibromatosis type 1 (n = 2), Sjögren's syndrome (n = 1), and meningitis (n =1). The RNF213 c.14576G>A mutation was found in 64 patients (84.2%) with MMD and 8 patients (80%) with QMMD; no significant difference in mutation frequency was observed between cohorts. There are two forms of QMMD, one in which the vascular abnormality is associated with an underlying disease, and the other in which MMD is coincidentally complicated by an unrelated underlying disease. It has been suggested that the presence or absence of the RNF213 c.14576G>A mutation may be useful in distinguishing between these disease types.

The E3 Ubiquitin Ligase Ring Finger Protein 5 Ameliorates NASH Through Ubiquitin-Mediated Degradation of 3-Hydroxy-3-Methylglutaryl CoA Reductase Degradation Protein 1.

NAFLD is the most prevalent chronic liver disease worldwide, but no effective pharmacological therapeutics are available for clinical use. NASH is the more severe stage of NAFLD. During this progress, dysregulation of endoplasmic reticulum (ER)-related pathways and proteins is one of the predominant hallmarks. We aimed to reveal the role of ring finger protein 5 (RNF5), an ER-localized E3 ubiquitin-protein ligase, in NASH and to explore its underlying mechanism. We first inspected the expression level of RNF5 and found that it was markedly decreased in livers with NASH in multiple species including humans. We then introduced adenoviruses for Rnf5 overexpression or knockdown into primary mouse hepatocytes and found that palmitic acid/oleic acid (PAOA)-induced lipid accumulation and inflammation in hepatocytes were markedly attenuated by Rnf5 overexpression but exacerbated by Rnf5 gene silencing. Hepatocyte-specific Rnf5 knockout significantly exacerbated hepatic steatosis, inflammatory response, and fibrosis in mice challenged with diet-induced NASH. Mechanistically, we identified 3-hydroxy-3-methylglutaryl CoA reductase degradation protein 1 (HRD1) as a binding partner of RNF5 by systematic interactomics analysis. RNF5 directly bound to HRD1 and promoted its lysine 48 (K48)-linked and K33-linked ubiquitination and subsequent proteasomal degradation. Furthermore, Hrd1 overexpression significantly exacerbated PAOA-induced lipid accumulation and inflammation, and short hairpin RNA-mediated Hrd1 knockdown exerted the opposite effects. Notably, Hrd1 knockdown significantly diminished PAOA-induced lipid deposition, and up-regulation of related genes resulted from Rnf5 ablation in hepatocytes. These data indicate that RNF5 inhibits NASH progression by targeting HRD1 in the ubiquitin-mediated proteasomal pathway. Targeting the RNF5-HRD1 axis may provide insights into the pathogenesis of NASH and pave the way for developing strategies for NASH prevention and treatment.

Circ-RNF111 aggravates the malignancy of gastric cancer through miR-876-3p-dependent regulation of KLF12

BackgroundThe aberrant expression of circular RNAs (circRNAs) plays vital roles in the advancement of human cancers, including gastric cancer (GC). In this study, the functions of circRNA ring finger protein 111 (circ-RNF111) in GC were investigated.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) assay was performed for the levels of circ-RNF111, microRNA-876-3p (miR-876-3p) and krueppel-like factor 12 (KLF12) mRNA. RNase R assay was conducted for the feature of circ-RNF111. Cell Counting Kit-8 (CCK-8) assay, colony formation assay, wound-healing assay, and transwell assay were applied for cell viability, colony formation, migration, and invasion, respectively. Flow cytometry analysis was used to analyze cell apoptosis and cell cycle process. The glycolysis level was examined using specific commercial kits. Western blot assay was carried out to measure the protein levels of hexokinase 2 (HK-2) and KLF12. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were employed to verify the combination between miR-876-3p and circ-RNF111 or KLF12. Murine xenograft model was constructed for the role of circ-RNF111 in vivo. Immunohistochemistry (IHC) was used for KLF12 level.ResultsCirc-RNF111 was higher expressed in GC tissues and cells than normal tissues and cells. Silencing of circ-RNF111 restrained cell viability, colony formation, migration, invasion, cell cycle process and glycolysis and induced apoptosis in GC cells in vitro. Circ-RNF111 positively regulated KLF12 expression via absorbing miR-876-3p. MiR-876-3p downregulation reversed the impacts of circ-RNF111 silencing on GC cell malignant phenotypes. MiR-876-3p overexpression repressed GC cell growth, metastasis and glycolysis, inhibited apoptosis and arrested cell cycle, while KLF12 elevation weakened the effects. Besides, circ-RNF111 knockdown inhibited tumor growth in vivo.ConclusionCirc-RNF111 knockdown relieved the development of GC by regulating miR-876-3p/KLF12 axis.

Negative Regulation of RNF90 on RNA Virus-Triggered Antiviral Immune Responses Targeting MAVS.

The antiviral innate immunity is the first line of host defense against viral infection. Mitochondrial antiviral signaling protein (MAVS, also named Cardif/IPS-1/VISA) is a critical protein in RNA virus-induced antiviral signaling pathways. Our previous research suggested that E3 ubiquitin-protein ligases RING-finger protein (RNF90) negatively regulate cellular antiviral responses by targeting STING for degradation, though its role in RNA virus infection remains unknown. This study demonstrated that RNF90 negatively regulated RNA virus-triggered antiviral innate immune responses in RNF90-silenced PMA-THP1 cells, RNF90-deficient cells (including HaCaTs, MEFs, and BMDMs), and RNF90-deficient mice. However, RNF90 regulated RNA virus-triggered antiviral innate immune responses independent of STING. RNF90 promoted K48-linked ubiquitination of MAVS and its proteasome-dependent degradation, leading to the inhibition of innate immune responses. Altogether, our findings suggested a novel function and mechanism of RNF90 in antiviral innate immunity.