Itchy E3 Ubiquitin Protein Ligase Research Articles

Knockdown of hsa_circ_0059955 Induces Apoptosis and Cell Cycle Arrest in Nucleus Pulposus Cells via Inhibiting Itchy E3 Ubiquitin Protein Ligase.

BackgroundCircular RNAs (circRNAs) play an important role in the progression of intervertebral disc (IVD) degeneration (IVDD). Using bioinformatics analysis, we have found that the expression of circRNA hsa_circ_0059955 was significantly downregulated in IVDD tissues. However, the relevant mechanism of hsa_circ_0059955 in the progression of IVDD remains unclear.MethodsCCK-8 and flow cytometry assays were used to evaluate cell proliferation and apoptosis. In addition, Western blot assay was used to detect the expressions of ITCH, p73, CDK2 in nucleus pulposus (NP) cells. Moreover, a puncture-induced IVDD rat model was established to explore the role of hsa_circ_0059955 in IVDD.ResultsThe level of hsa_circ_0059955 was significantly decreased in IVDD tissues from IVDD patients. Itchy E3 ubiquitin protein ligase (ITCH) is the host gene of hsa_circ_0059955, and downregulation of hsa_circ_0059955 significantly decreased the expression of ITCH in NP cells. In addition, downregulation of hsa_circ_0059955 markedly inhibited proliferation and induced apoptosis and cell cycle arrest in NP cells. Moreover, in vivo study illustrated that overexpression of hsa_circ_0059955 ameliorated IVDD in rats.ConclusionDownregulation of hsa_circ_0059955 could induce apoptosis and cell cycle arrest in NP cells in vitro, while overexpression of hsa_circ_0059955 attenuated the IVDD in a puncture-induced rat model in vivo. Therefore, hsa_circ_0059955 might serve as a therapeutic target for the treatment of IVDD.

Extracellular vesicle-carried microRNA-27b derived from mesenchymal stem cells accelerates cutaneous wound healing via E3 ubiquitin ligase ITCH.

Mesenchymal stem cells (MSCs) have been highlighted as promising candidate cells in relation to cutaneous wound healing. The current study aimed to investigate whether MSC‐derived extracellular vesicles (EVs) could transfer microRNA‐27b (miR‐27b) to influence cutaneous wound healing. The miR‐27b expression was examined in the established cutaneous wound mouse model, and its correlation with the wound healing rate was evaluated by Pearson's correlation analysis. The identified human umbilical cord MSC‐derived EVs were co‐cultured with human immortal keratinocyte line HaCaT and human skin fibroblasts (HSFs). The mice with cutaneous wound received injections of MSC‐derived EVs. The effects of EVs or miR‐27b loaded on wound healing and cellular functions were analysed via gain‐ and loss‐of‐function approaches in the co‐culture system. Dual‐luciferase reporter gene assay was employed to verify the relationship between miR‐27b and Itchy E3 ubiquitin protein ligase (ITCH). Rescue experiments were conducted to investigate the underlying mechanisms associated with the ITCH/JUNB/inositol‐requiring enzyme 1α (IRE1α) axis. miR‐27b was down‐regulated in the mouse model, with its expression found to be positively correlated with the wound healing rate. Abundant miR‐27b was detected in the MSC‐derived EVs, while EV‐transferred miR‐27b improved cutaneous wound healing in mice and improved proliferation and migration of HaCaT cells and HSFs in vitro. As a target of miR‐27b, ITCH was found to repress cell proliferation and migration. ITCH enhanced the JUNB ubiquitination and degradation, ultimately inhibiting JUNB and IRE1α expressions and restraining wound healing. Collectively, MSC‐derived EVs transferring miR‐27b can promote cutaneous wound healing via ITCH/JUNB/IRE1α signalling, providing insight with clinical implications.

Circular RNA ITCH suppresses proliferation, invasion, and glycolysis of ovarian cancer cells by up-regulating CDH1 via sponging miR-106a

BackgroundAccumulating data suggested that circular RNAs (circRNAs) played important roles in the development of human cancer. However, the potential mechanism of circRNAs in ovarian cancer remains unclear.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the levels of circRNA itchy E3 ubiquitin protein ligase (circ-ITCH), microRNA-106a (miR-106a) and E-cadherin (CDH1). Cell Counting Kit-8 (CCK-8) and Transwell assay were carried out to measure cell proliferation and invasion. Glucose consumption, lactate production, and ATP level were assessed by the glucose, lactate, and ATP assay kits, respectively. Cell apoptosis was detected by Flow cytometry. The binding sites were predicted by StarBase v.2.0 or microT-CDS and verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assays. CDH1 protein level was determined by western blot. The functional role of circ-ITCH was measured by xenograft tumor model in vivo.ResultsCirc-ITCH was down-regulated in ovarian cancer and positively correlated with 5-year overall survival of patients with ovarian cancer. RNase R digestion assay confirmed that circ-ITCH was more stable than its linear mRNA form. Moreover, circ-ITCH was mainly distributed in the cytoplasm of ovarian cancer cells.Functionally, circ-ITCH overexpression hindered proliferation, invasion, glycolysis and promoted apoptosis of ovarian cancer cells. Besides, circ-ITCH overexpression inhibited ovarian cancer cell progression by targeting miR-106a. Additionally, CDH1 was a target of miR-106a, and the protein level of CDH1 was negatively regulated by miR-106a. Similarly, CDH1 knockdown recovered the inhibition effects of miR-106a inhibitor or circ-ITCH overexpression on the progression of ovarian cancer cells. Importantly, circ-ITCH up-regulated the protein level of CDH1 by sponging miR-106a in ovarian cancer cells. Circ-ITCH overexpression suppressed the growth of ovarian cancer cells in vivo.ConclusionCirc-ITCH suppressed proliferation, invasion, glycolysis, and promoted apoptosis of ovarian cancer cells by modulating the miR-106a/CDH1 axis.

MIR-520f Regulated Itch Expression and Promoted Cell Proliferation in Human Melanoma Cells.

Accumulating evidence suggests that abnormal expression and dysfunction of microRNA is involved in development of cancers. However, the function of miR-520f especially in human melanoma remains elusive. In the current study, the underlying function of miR-520f in human melanoma was investigated. Our study demonstrated that the miR-520f level in human melanoma cell lines and clinical tissues was increased. Overexpression of miR-520f promoted cell proliferation by using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation, anchorage-independent growth assay, and 5-bromo-2-deoxyuridine assays. Furthermore, we revealed that miR-520f could interact with circular RNA Itchy E3 ubiquitin protein ligase (ITCH) 3′-untranslated region and suppress ITCH expression in human melanoma cells. The inhibitory effect of miR-520f-in could be partially restored by knockdown of ITCH in human melanoma cells. In summary, this study provides novel insights into miR-520f act as a crucial role in the regulation of human melanoma cell growth via regulating ITCH, which might be a potential biomarker and therapeutic target of human melanoma.

Ring Finger Protein 11 acts on ligand-activated EGFR via the direct interaction with the UIM region of ANKRD13 protein family.

RING finger protein 11 (RNF11) is an evolutionary conserved Really Interesting New Gene E3 ligase that is overexpressed in several human tumours. Although several reports have highlighted its involvement in crucial cellular processes, the mechanistic details underlying its function are still poorly understood. Utilizing stable isotope labelling by amino acids in culture (SILAC)-based proteomics analysis, we identified 51 proteins that co-immunoprecipitate with wild-type RNF11 and/or with its catalytically inactive mutant. We focused our attention on the interaction of RNF11 with Ankyrin repeat domain-containing protein 13 (ANKRD13)s family. Members of the ANKRD13 family contain ubiquitin-interacting motifs (UIM) that recognize the Lys-63-linked ubiquitin (Ub) chains appended to Epidermal growth factor receptor (EGFR) soon after ligand binding. We show that ANKRD13A, ANKRD13B and ANKRD13D form a complex with RNF11 invivo and that the UIMs are required for complex formation. However, at odds with the conventional UIM binding mode, Ub modification of RNF11 is not required for the interaction with ANKRD13 proteins. We also show that the interaction between ANKRD13A and RNF11 is modulated by the EGF stimulus and that a complex formed by ANKRD13A, RNF11 and activated EGFR is transiently assembled in the early phases of receptor endocytosis. Moreover, loss of function of the E3 ligases Itchy E3 ubiquitin-protein ligase (ITCH) or RNF11, respectively, abrogates or increases the ubiquitination of endogenous ANKRD13A, affecting its ability to bind activated EGFR. We propose a model whereby the ANKRD13 proteins act as molecular scaffolds that promote the transient formation of a complex between the activated EGFR and the E3 ligases ITCH and RNF11. By regulating the ubiquitination status of ANKRD13A and consequently its endocytic adaptor function, RNF11 promotes sorting of the activated EGFR for lysosomal degradation.

Potential of circular RNA itchy E3 ubiquitin protein ligase as a biomarker and treatment target for multiple myeloma.

BackgroundThis study aimed to investigate the association of circular RNA itchy E3 ubiquitin protein ligase (circ-ITCH) expression with disease risk, clinical characteristics, progression-free survival (PFS) and overall survival (OS) of multiple myeloma (MM), and to explore the influence of circ-ITCH overexpression on MM cell activities in vitro.MethodsBone marrow samples from 92 MM patients and 30 healthy controls were collected, and circ-ITCH expression was detected by quantitative polymerase chain reaction. PFS and OS in MM patients were calculated. Circ-ITCH in human MM cell lines and normal bone marrow mononuclear cells (BMMCs) were detected. Circ-ITCH overexpression and control overexpression plasmids were transfected to U226 cell line, and cell proliferation as well as apoptosis were assessed.ResultsCirc-ITCH expression was under-expressed in MM patients compared to healthy controls. And receiver operating characteristic curve displayed that circ-ITCH could distinguish MM patients from healthy controls [area under curve: 0.809 (95% CI: 0.722–0.895)]. Additionally, circ-ITCH high expression was associated with decreased International Staging System (ISS) stage in MM patients. Kaplan-Meier curves and Cox’s regression analysis displayed that circ-ITCH expression was positively correlated with PFS and OS. In vitro, circ-ITCH expression was lower in MM cell lines (including RPMI8226, U226 and NCI-H929) compared to normal BMMCs. In U226 cells, cell proliferation was decreased but apoptosis was elevated by circ-ITCH overexpression.ConclusionsCirc-ITCH might serve as a potential biomarker and treatment target for MM.

RETRACTED ARTICLE: Circular RNA ITCH suppressed prostate cancer progression by increasing HOXB13 expression via spongy miR-17-5p

BackgroundCircular RNA Itchy E3 ubiquitin protein ligase (Circ-ITCH) is significantly down-regulated in various kinds of tumors, however, the mechanisms of action and functions of circITCH gene in prostate cancer (PC) are still under investigation. The mail goal of this research was to study the functional role of Circ-ITCH gene in prostate cancer and to illuminate the function role of circ-ITCH gene in prostate cancer by targeting miR-17-5p/HOXB13.MethodsRT-qPCR was applied to measure the expression level of circ-ITCH and miR-17-5p in PC cell lines and tissues. CCK-8, colony formation, Brdu incorporation labeling and flow cytometry assays were applied to detect the effects of circ-ITCH and miR-17-5p on proliferation and cell apoptosis. Target gene prediction and screening, luciferase reporter gene assays were utilized to assess downstream target genes of miR-17-5p and Circ-ITCH. The protein and expression of HOXB13 gene were measured by Western blotting and RT-qPCR.ResultsCircITCH was significantly reduced in PC cell lines and tissues. Low circITCH expression level was highly related with preoperative PSA, tumor stage and Gleason score. Overexpression of circITCH can inhibit the malignant phenotype of prostate cancer. There was a high negative relationship between the expression level of microRNA-17-5p and circITCH in PC tissues, however, there existed a positive relationship between the expression of HOXB13 and circITCH. CircITCH acted as a sponge of miR-17-5p to increase HOXB13 gene expression. In addition, miR-17-5p overexpression or HOXB13 silencing can reduce the carcinogenic effects of circICCH in prostate cancer.ConclusionCircITCH promoted prostate cancer progression by regulating the HOXB13/miR-17-5p axis, and circITCH have a potential usage as therapeutic target for PC tumors.

Gα13-mediated LATS1 down-regulation contributes to epithelial-mesenchymal transition in ovarian cancer.

Gα13, a heterotrimeric G-protein of the Gα12/13 subfamily, is associated with aggressive phenotypes in various human cancers. However, the mechanisms by which Gα13 promotes cancer progression have not been fully elucidated. Here, we demonstrate that the activation of Gα13 induces epithelial-mesenchymal transition in ovarian cancer (OvCa) cells through down-regulation of large tumor suppressor kinase (LATS) 1, a critical component of the Hippo signaling pathway. A synthetic biology approach using a mutant GPCR and chimeric G-protein revealed that Gα13-regulated phosphorylation of LATS1 at serine 909 within its activation loop induced recruitment of the itchy E3 ubiquitin protein ligase to trigger LATS1 degradation. Our findings uncover novel mechanisms through which Gα13 activation induces dysregulation of the Hippo signaling pathway, which leads to aggressive cancer phenotypes, and thereby identify a potential target for preventing the metastatic spread of OvCa.-Yagi, H., Onoyama, I., Asanoma, K., Hori, E., Yasunaga, M., Kodama, K., Kijima, M., Ohgami, T., Kaneki, E., Okugawa, K., Yahata, H., Kato, K. Gα13-mediated LATS1 down-regulation contributes to epithelial-mesenchymal transition in ovarian cancer.

Downregulated circular RNA itchy E3 ubiquitin protein ligase correlates with advanced pathologic T stage, high lymph node metastasis risk and poor survivals in prostate cancer patients.

This study aimed to explore the correlation of circular RNA itchy E3 ubiquitin protein ligase (circ-ITCH) with pathological features as well as its predictive value on prognosis in prostate cancer patients. Three hundred and twenty-four patients with prostate cancer underwent radical prostatectomy were consecutively included in this retrospective study, and patients' specimens of tumor as well as paired adjacent tissues were collected for detection of circ-ITCH expression. Patients' baseline characteristics and survival data were obtained from follow-up records, and correlation of circ-ITCH with patients' pathological features and survival was determined. Circ-ITCH expression was decreased in tumor tissue compared with paired adjacent tissues (P< 0.001), and it presented with good value in distinguishing tumor tissues from paired adjacent tissues with area under curve (AUC) of 0.812 (95%CI: 0.780-0.845). Circ-ITCH low expression was associated with advanced pathologic T stage (P= 0.002) and high risk of lymph node metastasis (P= 0.047) in prostate cancer patients. As for prognosis, patients with circ-ITCH high expression had longer disease-free survival (DFS) (P< 0.001) and overall survival (OS) (P< 0.001). Additionally, circ-ITCH (high vs. low) independently predicted better survival, while Gleason score (> 7 vs. ⩽ 7) and surgical margin status (positive vs. negative) independently predicted worse survival in prostate patients underwent radical prostatectomy. Circ-ITCH is downregulated in prostate cancer tissues, and its low expression correlates with advanced pathologic T stage, high lymph mode metastasis risk and poor survival in prostate cancer patients underwent radical prostatectomy.

Upregulation of Circular RNA Itchy E3 Ubiquitin Protein Ligase Inhibits Cell Proliferation and Promotes Cell Apoptosis Through Targeting MiR-197 in Prostate Cancer.

Objective:This study aimed to investigate the effect of circular RNA itchy E3 ubiquitin protein ligase on cell proliferation and apoptosis and to explore its target micro-RNAs in prostate cancer cells.Methods:Circular RNA itchy E3 ubiquitin protein ligase expression in human prostate cancer cells and normal prostate epithelial cells was determined by real time-quantitative polymerase chain reaction assay. Circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids (circular RNA itchy E3 ubiquitin protein ligase(+) group and control overexpression plasmids group were transfected with PC-3 cells. Rescue experiment was performed by transfection of circular RNA itchy E3 ubiquitin protein ligase overexpression and micro-197 overexpression plasmids (circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids/micro RNA (+) group) into PC-3 cells. Cell Counting Kit-8 and annexin V/propidium iodide assays were conducted to evaluate cell proliferation and apoptosis, respectively. Western blot was performed to determine the expressions of apoptotic-related markers.Results:Circular RNA itchy E3 ubiquitin protein ligase expression was decreased in DU 145, 22RV1, VCaP, and PC-3 cells compared to RWPE cells. In PC-3 cells, cell proliferation rate was reduced in circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids group compared to control overexpression plasmids group at 48 hours and 72 hours. Cell apoptosis rate was elevated in circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids group compared to control overexpression plasmids group at 48 hours, and Western blot showed the similar results. Micro RNA-197 but not micro RNA-31 or micro RNA-432 was the target micro-RNA of circular RNA itchy E3 ubiquitin protein ligase. In rescue experiments, cell proliferation rate was elevated, but apoptosis rate was reduced in circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids/micro RNA (+) group compared to circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids group, indicating that circular RNA itchy E3 ubiquitin protein ligase upregulation inhibited cell proliferation but promoted apoptosis through downregulating micro RNA-197.Conclusion:Circular RNA itchy E3 ubiquitin protein ligase upregulation suppresses cell proliferation but promotes apoptosis through targeting micro RNA-197 in prostate cancer. Our study may provide a new insight for the treatment of prostate cancer.

Circ-ITCH correlates with small tumor size, decreased FIGO stage and prolonged overall survival, and it inhibits cells proliferation while promotes cells apoptosis in epithelial ovarian cancer.

The aim of this study was to evaluate the correlation of circular RNA itchy E3 ubiquitin protein ligase (Circ-ITCH) expression with clinicopathological features and survival in patients with epithelial ovarian cancer (EOC), and to explore its effect on cells proliferation as well as apoptosis in EOC cells. Seventy-seven EOC patients underwent surgery were retrospectively reviewed. Tumor tissues and paired adjacent tissues samples were collected, and Circ-ITCH expression was evaluated by quantitative polymerase chain reaction (qPCR). Blank mimic, Circ-ITCH mimic, blank inhibitor and Circ-ITCH inhibitor plasmids were transfected into SKOV3 cells and OVCAR-3 cells, and Counting Kit-8 (CCK-8) was performed to assess cells proliferation and Annexin V (AV)/propidium iodide (PI) was conducted to detect cells apoptosis. Median value of Circ-ITCH relative expression was 0.697 (0.367-1.106) in tumor tissues, which was lower compared with paired adjacent tissues (1.690 (0.867-2.813)) (P< 0.001), and it negatively correlated with tumor size (P= 0.005) as well as International Federation of Gynecology and Obstetrics (FIGO) stage (P< 0.001) in EOC patients. Multivariate Cox's analysis revealed that high Circ-ITCH expression was an independent predictive factor for favorable OS in EOC patients. Moreover, further in vitro experiments disclosed that Circ-ITCH expression was decreased in several EOC cell lines compared with normal ovarian epithelial cell line, and it inhibited cells proliferation while promoted cells apoptosis in SKOV3 and OVCAR-3 cells. Circ-ITCH correlates with small tumor size, decreased FIGO stage and prolonged OS, and it inhibits cells proliferation while promotes cells apoptosis in EOC.

Circular RNA ITCH suppresses proliferation and promotes apoptosis in human epithelial ovarian cancer cells by sponging miR-10a-α.

To explore the effect of Circular RNA Itchy E3 ubiquitin protein ligase (Circ-ITCH) on regulating epithelial ovarian cancer (EOC) cells proliferation and apoptosis, as well as its potential target microRNAs (miR) in vitro. Circ-ITCH mimic, blank mimic, Circ-ITCH inhibitor and blank inhibitor plasmids were transfected into SKOV3 cells. Rescue experiments were carried out by transfecting blank mimic, miR-10a mimic, Circ-ITCH mimic and miR-10a mimic/Circ-ITCH mimic plasmids into SKOV3 cells. Quantitative polymerase chain reaction (qPCR) was performed to detect RNA expression. Cells proliferation was determined by Cell Counting Kit-8 (CCK-8) assay, and cells apoptosis rate was detected using Annexin V (AV) / propidium iodide (PI) assay. Circ-ITCH expression was decreased in Human EOC cell lines SKOV3, A-2780, OVCAR-3 and HO-8910 compared with human normal ovarian epithelial cell line IOSE80, and the most significant reduction of Circ-ITCH expression was presented in SKOV3 cells. Cells proliferation was suppressed by Circ-ITCH mimic transfection and promoted by Circ-ITCH inhibitor transfection in SKOV3 cells. Cells apoptosis was enhanced by Circ-ITCH mimic transfection and inhibited by Circ-ITCH inhibitor transfection in SKOV3 cells. In addition, Circ-ITCH adversely regulated miR-10a expression in SKOV3 cells but not miR-4251 or miR-6505. Rescue experiments were subsequently performed, which exhibited that Circ-ITCH adversely regulated miR-10a expression, whereas miR-10a did not affect Circ-ITCH expression. And most importantly, miR-10a mimic attenuated the effect of Circ-ITCH on reducing proliferation and promoting apoptosis in SKOV3 cells. Circ-ITCH suppresses cells proliferation and promotes cells apoptosis via sponging miR-10a in EOC cells.

Circular RNA circ-ITCH inhibits bladder cancer progression by sponging miR-17/miR-224 and regulating p21, PTEN expression

BackgroundCirc-ITCH is a circRNA generated from several exons of itchy E3 ubiquitin protein ligase (ITCH) and tumor suppressor served as a sponge for certain miRNAs targeting their parental transcripts of ITCH. However, the role of circ-ITCH in bladder cancer (BCa) was not reported. In the present study, we investigated the role of circ-ITCH in BCa.MethodsQuantitative real-time PCR was used to detect the expression of circ-ITCH and survival analysis was adopted to explore the association between circ-ITCH expression and the prognosis of BCa. BCa cells were stably transfected with lentivirus approach and cell proliferation, migration, invasion, cell cycle and cell apoptosis, as well as tumorigenesis in nude mice were performed to assess the effect of circ-ITCH in BCa. Biotin-coupled probe pull down assay, Biotin-coupled miRNA capture, Fluorescence in situ hybridization and Luciferase reporter assay were conducted to confirm the relationship between the circ-ITCH and the microRNA.ResultsIn the present study, we found that circ-ITCH, is down-regulated in BCa tissues and cell lines. BCa patients with low circ-ITCH expression had shortened survival. Enforced- expression of circ-ITCH inhibited cells proliferation, migration, invasion and metastasis both in vitro and in vivo. Mechanistically, we demonstrated that circ-ITCH up-regulates the expression of miR-17 and miR-224 target gene p21 and PTEN through ‘sponging’ miR-17 and miR-224, which suppressed the aggressive biological behaviors of BCa.Conclusionscirc-ITCH acts as a tumor suppressor by a novel circ-ITCH/miR-17, miR-224/p21, PTEN axis, which may provide a potential biomarker and therapeutic target for the management of BCa.

Phosphorylation of the E3 ubiquitin protein ligase ITCH diminishes binding to its cognate E2 ubiquitin ligase

Heightened and extended inflammation underlies the pathogenesis of many disorders, including inflammatory bowel disease, sepsis, and inflammatory arthritis. Ubiquitin networks help dictate the strength and duration of inflammatory signaling. In innate immunity, the itchy E3 ubiquitin protein ligase (ITCH)-A20 ubiquitin-editing complex inhibits receptor-interacting Ser/Thr kinase (RIPK) activation by removing Lys-63-linked polyubiquitinated chains from key proteins in the nuclear factor kappa B (NF-κB) signaling pathway. The complex then attaches polyubiquitinated chains to these proteins to target them for lysosomal or proteasomal destruction. ITCH is phosphorylated and thereby inhibited by inhibitor of nuclear factor kappa B kinase subunit beta (IKKβ) to fine-tune the inflammatory response to the strength of the offending signal. However, the biochemical mechanism by which E3 ubiquitination is impaired by IKK-driven phosphorylation remains unclear. Here, we report that this phosphorylation impedes ITCH binding to its cognate E2 ubiquitin ligase, UbcH7. Using CRISPR-Cas9 genetic knockout to mimic the ITCH-UbcH7-inhibited state, we further show that genetic UbcH7 deficiency phenocopies ITCH phosphorylation in regulating RIPK2 ubiquitination. We conclude that phosphorylation can disrupt the binding of an E3 ubiquitin ligase to an E2-conjugating enzyme, leading to prolonged inflammatory signaling. To our knowledge, this is the first report of E3 ubiquitin ligase phosphorylation inhibiting E3 ligase activity by impairing E2-E3 complex formation.

Cinnamaldehyde induces cell apoptosis mediated by a novel circular RNA hsa_circ_0043256 in non-small cell lung cancer

Cinnamaldehyde (CA), the primary chemical component of the Chinese traditional herb Cinnamomum cassia, is an effective cytotoxic agent against various human cancers. Our previous study indicated that CA could trigger apoptosis in three kinds of non-small cell lung cancer (NSCLC) cells. However, CA mechanism of action in NSCLC has not been unveiled completely. Herein, we showed that a novel circular RNA hsa_circ_0043256 was upregulated in NSCLC cells in response to CA treatment, as detected by microarray and real-time PCR. Hsa_circ_0043256 could inhibit cell proliferation and induce apoptosis, while hsa_circ_0043256 knock-down could promote cell proliferation and restrain apoptosis induced by CA. Bioinformatics analysis predicted that hsa_circ_0043256 could work as a miR-1252 sponge, which could in turn directly target a vital negative regulator of Canonical Wnt signaling, Itchy E3 ubiquitin protein ligase (ITCH), as validated by dual-luciferase assay. Western blot results further confirmed that hsa_circ_0043256 could upregulate ITCH expression, whereas miR-1252 could partially abolish this effect. Interestingly, hsa_circ_0043256 knock-down could weaken Wnt/β-catenin pathway inhibition induced by CA. Finally, we discovered that CA induced apoptosis and meanwhile upregulated hsa_circ_0043256 expression in vivo. Immunohistochemical analysis revealed that ITCH expression was positively association with hsa_circ_0043256 levels. Above all, we characterized a new mechanism mediated by hsa_circ_0043256/miR-1252/ITCH axis in CA function against NSCLC, providing a novel insight into lung cancer therapy.

The anergy induction of M3 muscarinic acetylcholine receptor-reactive CD4+ T cells suppresses experimental sialadenitis-like Sjögren's syndrome.

Autoreactive CD4+ T cells are involved in the pathogenesis of Sjögren's syndrome (SS). The aim of the present study was to clarify the dominant T cell epitopes of M3 muscarinic acetylcholine receptor (M3R) and to establish a new antigen-specific therapy for SS using an experimental mouse model. Production of cytokines from M3R-reactive CD4+ T cells, after culture with various M3R peptides, was analyzed by enzyme-linked immunosorbent assay. Adoptive cell transfer was performed using splenocytes from M3R(-/-) mice that were immunized with M3R peptides or phosphate buffered saline plus H37Ra as a control. Rag1(-/-) mice were inoculated with the splenocytes and examined for the development of sialadenitis. Altered peptide ligands (APLs) of the T cell epitopes, with substitutions in amino acid residues at T cell receptor contact sites, were synthesized, and the ability of the APLs to suppress sialadenitis was evaluated. The mechanisms underlying such effects were assessed. CD4+ M3R-reactive T cells produced interleukin-17 (IL-17) and interferon-γ (IFNγ) in response to the N-terminal 1 (N1) and 1st extracellular loop peptides of M3R, and Rag1(-/-) mice that received N1- and/or 1st peptide-immunized splenocytes developed sialadenitis. Among the designed APLs, N1-APL7 (N→S at amino acid 15) significantly suppressed IFNγ production in vitro, and also suppressed sialadenitis in vivo. Levels of early growth response 2 in CD4+ T cells from the cervical lymph nodes of N1-APL7-treated mice were significantly higher than those of control mice, and cell proliferation was reversed by administration of exogenous IL-2. Levels of the anergy-related molecules itchy homolog E3 ubiquitin-protein ligase, Casitas B-lineage lymphoma b, gene related to anergy in lymphocytes, and Deltex-1 were significantly higher in CD4+ T cells cultured with N1-APL7. The major T cell epitopes were from the N1 and 1st peptide regions. Moreover, N1-APL7, selected as the antagonistic APL in vitro, also suppressed sialadenitis through the induction of anergy. This is a potentially useful strategy for regulating pathogenic T cell infiltration in SS.

Investigation of the roles of interleukin 6 and transforming growth factor β signaling in Th17/Treg imbalance of Hashimoto's thyroiditis

Objective To investigate the role of interleukin(IL)-6/transforming growth factor β(TGF-β)signaling in T helper cell 17(Th17)/regulatory T cells(Treg)imbalance of Hashimoto's thyroiditis(HT). Methods Thirty-eight patients with HT and thirty-two healthy controls were studied. Plasma concentration of IL-6 and TGF-β was measured by enzyme-linked immunosorbent assay. Expression levels of retinoid related orphan receptor γt(RORγt), forkhead box P3(FOXP3), Smad3, Smad4, transforming growth factor β inducible early response gene 1(TIEG1), itchy E3 ubiquitin protein ligase(Itch), suppressor of cytokine signaling 1(SOCS1), and SOCS3 mRNA were determined by realtime PCR. The proportions of CD4+ IL-17A+ T cells(Th17), CD4+ CD25+ FOXP3+ regulatory T cells(Treg)and mean fluorescence intensity for transforming growth factor β receptor type Ⅱ(TGF-βRⅡ)and phosphated signal transducer and activator of transcription 3(pSTAT3)protein in CD4+ T cells were analyzed by flow cytometry. Quantitative methylation specific PCR was used to evaluate methylation status of CpG islands in SOCS1 exon2, and three potential bind sites for signal transducer and activator of transcription 3(STAT3)in 5'-untraslated region(5'-UTR)of SOCS3 in CD4+ T cells. In-vitro culture of CD4+ T cells isolated from healthy donors was used to identify the effects of IL-6/TGF-β signaling on differentiation of Th17/Treg and methylation status of SOCS1 and SOCS3 genes after being stimulated for 72 hours. Results (1)In patients with HT, the proportion of Th17 cells and expression levels of RORγt mRNA were significantly elevated(P 0.05). (4)Compared with blank controls, Treg increased remarkably while Th17 and the ration of Th17/Treg decreased significantly after TGF-β stimulation for 72 h(P 0.05). Conclusions Aberrant IL-6/TGF-β signaling may be one of the important factors that contribute to imbalance of Th17/Treg in HT. (Chin J Endocrinol Metab, 2015, 31: 320-326) Key words: Interleukin 6; STAT3; Hashimoto's thyroiditis; Th17; Regulatory T cells

Endogenous spartin (SPG20) is recruited to endosomes and lipid droplets and interacts with the ubiquitin E3 ligases AIP4 and AIP5

The synopsis of this paper contained an error in nomenclature. In line 12 (right-hand column), ‘WWP2’ is incorrectly shown as a synonym for the ubiquitin E3 ligase AIP4; the synonym should have been presented as ‘ITCH’ (itchy E3 ubiquitin protein ligase homologue).