Circular RNA Itchy E3 Ubiquitin Research Articles

Circular RNA CircITCH (has-circ-0001141) suppresses hepatocellular carcinoma (HCC) progression by sponging miR-184

ABSTRACT Aberrant expression of circular RNA (circRNA) is involved in the occurrence of various diseases and tumor development, in which plays a vital role, including hepatocellular carcinoma (HCC). Nevertheless, the regulation mechanism and biological function of circITCH in hepatocellular carcinoma (HCC) remain unclear. The expression level of circular RNA itchy E3 ubiquitin protein ligase (circ-ITCH) was identified and validated by real-time polymerase-chain reaction (RT-qPCR) in HCC cell lines. The stability of circITCH was confirmed by Ribonuclease R (RNase R) assay. Subsequently, through silencing and overexpression of circITCH to investigate the functional roles of circITCH in HCC proliferation, invasion, and apoptosis. We also carried out bioinformatics analysis, luciferase reporter assays to define the relationship between microRNA (miR)-184 and circITCH. Moreover, xenograft mouse models and immunohistochemistry were employed to assess the function of circITCH in HCC. CircITCH (hsa_circ_0001141) was a stable circRNA and downregulated in HCC cells. Overexpression of circITCH inhibited cell proliferation, migration, invasion, and promoted apoptosis in vitro and in vivo, whereas knockdown of circITCH had the opposite effects. Mechanistically, miR-184 could be sponged by circITCH, and its overexpression could mitigate the suppressive effects of circITCH overexpression on HCC progression. Through biological website to predict the target genes of miR-184 may be combined. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to investigate mRNAs with significant functional enrichment and pathways, also which its relationship with HCC-related pathway and immune cells. Our findings reveal that circITCH served as a repressor to restrain HCC malignancy via miR-184. Therefore, circITCH may serve as a potential prognostic marker and therapeutic target for HCC. Abbreviations: HCC: hepatocellular carcinoma; CircRNA: Circular RNA; miRNA: MicroRNA; Circ-ITCH: circular RNA itchy E3 ubiquitin protein ligase; RT-qPCR: real-time polymerase-chain reaction; RNase R: Ribonuclease R; CeRNA: competing endogenous RNAs; SiRNA: small interfering RNA

Circ-ITCH overexpression promoted cell proliferation and migration in Hirschsprung disease through miR-146b-5p/RET axis.

Hirschsprung disease (HSCR) is a congenital intestinal disease caused by the abnormal proliferation and migration of enteric nerve cells (ENCC). Research suggested critical roles for circular RNA (circRNA) itchy E3 ubiquitin protein ligase (ITCH) in gastrointestinal malignancies progression. However, the function of circ-ITCH in HSCR remains poorly defined. The related genes expression in 30 HSCR patients and 30 controls without HSCR were detected using qRT-PCR. Cell proliferation was assessed by CCK-8 assay and EdU assay. Cell migration was detected with wound-healing assay and transwell assay. The interactions among circ-ITCH, miR-146b-5p, and RET were confirmed by Dual luciferase reporter assay. Circ-ITCH and RET expressions were downregulated in HSCR patients and cells, while the miR-146b-5p expression was upregulated. Circ-ITCH overexpression facilitated cell proliferation, migration, and activated MAPK pathway, which were reversed by circRNA-ITCH knockdown. Circ-ITCH negatively regulated miR-146b-5p expression. MiR-146b-5p overexpression abolished the promoting effects of circ-ITCH overexpression on cell proliferation and migration. MiR-146b-5p inhibited RET expression. RET overexpression eliminated the inhibitory effects of miR-146b-5p overexpression on cell proliferation and migration. Circ-ITCH overexpression facilitated cell proliferation and migration in HSCR by regulating miR-146b-5p/RET/MAPK axis. The expressions of Circ-ITCH and RET were markedly reduced in HSCR, while miR-146b-5p expression was increased in HSCR. Circ-ITCH overexpression enhanced the proliferative and migratory abilities of SH-SY5Y and 293T cells. Circ-ITCH negatively regulated miR-146b-5p expression.

Circular RNA ITCH attenuates the progression of nasopharyngeal carcinoma by inducing PTEN upregulation via miR-214.

Circular RNA itchy E3 ubiquitin protein ligase (circ-ITCH) has previously been reported to play a key role in carcinogenesis. Nevertheless, the role of circ-ITCH in nasopharyngeal carcinoma (NPC) remains to be explored. Gene expression analysis was performed using a quantitative real-time polymerase chain reaction, western blotting and immunohistochemistry. The role of circ-ITCH in NPC was explored using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, colony formation, transwell invasion, scratch healing and xenograft tumor assays. Furthermore, luciferase reporter assay was carried out to assess the interactions among circ-ITCH, microRNA-214 (miR-214) and phosphatase and tensin homolog (PTEN). The levels of circ-ITCH and PTEN were decreased, whereas the level of miR-214 was increased in NPC tissues collected from 28 subjects compared to normal nasopharynx tissues collected from 15 subjects. Moreover, a negative correlation between circ-ITCH and miR-214 expression and a positive correlation between circ-ITCH and PTEN expression were observed in NPC tissues. Downregulation of circ-ITCH expression was also observed in NPC cell lines. In addition, upregulation of circ-ITCH markedly inhibited NPC cell proliferation, migration and invasion. Furthermore, circ-ITCH was confirmed to exert its function by sponging miR-214. PTEN was found to be a direct target gene of miR-214 and its expression was negatively correlated with miR-214 expression in NPC tissues. Moreover, our results showed that the circ-ITCH/miR-214 axis regulated NPC proliferation, migration and invasion through regulating the expression of PTEN. Upregulation of circ-ITCH or PTEN blocked miR-214-mediated promotion of NPC tumorigenesis in vitro. Additionally, upregulation of circ-ITCH also suppressed NPC tumorigenesis in vivo. The present study demonstrated that circ-ITCH suppressed NPC tumorigenesis by upregulating PTEN expression through interacting with miR-214, thus proposing a novel mechanism for NPC inhibition.

CircITCH: A Circular RNA With Eminent Roles in the Carcinogenesis.

Circular RNAs (circRNAs) are a group of long non-coding RNAs with enclosed structure generated by back-splicing events. Numerous members of these transcripts have been shown to affect carcinogenesis. Circular RNA itchy E3 ubiquitin protein ligase (circITCH) is a circRNA created from back splicing events in ITCH gene, a protein coding gene on 20q11.22 region. ITCH has a role as a catalyzer for ubiquitination through both proteolytic and non-proteolytic routes. CircITCH is involved in the pathetiology of cancers through regulation of the linear isoform as well as serving as sponge for several microRNAs, namely miR-17, miR-224, miR-214, miR-93-5p, miR-22, miR-7, miR-106a, miR-10a, miR-145, miR-421, miR-224-5p, miR-197 and miR-199a-5p. CircITCH is also involved in the modulation of Wnt/β-catenin and PTEN/PI3K/AKT pathways. Except from a single study in osteosarcoma, circITCH has been found to exert tumor suppressor role in diverse cancers. In the present manuscript, we provided a comprehensive review of investigations that reported function of circITCH in the carcinogenesis.

Circular RNA ITCH suppresses metastasis of gastric cancer via regulating miR-199a-5p/Klotho axis

ABSTRACT Circular RNAs (circRNAs) are considered as a new regulatory factor in growth, metastasis and therapeutic resistance of human cancers. But the clinical significance and underlying mechanism of circular RNA ITCH (circ-ITCH) in gastric cancer (GC) remain unknown. In the present study, we found that circ-ITCH was down-regulated in GC cell lines, GC tissues and their serum-derived exosomes. The level of circ-ITCH was related to invasion depth. Functional assays showed that circ-ITCH overexpression inhibited the proliferation, migration, invasion and epithelial mesenchymal transition (EMT) of GC cells, whereas circ-ITCH knockdown appeared an opposite effect. Bioinformatic analysis and luciferase reporter assay confirmed that circ-ITCH acted as miR-199a-5p sponge and increased the level of Klotho. The expression level of miR-199-5p was up-regulated in GC tissues and negatively correlated with that of circ-ITCH. MiR-199a-5p mimics reversed the effects on inhibiting metastasis induced by circ-ITCH overexpression and decreased the level of Klotho in GC cells. Our findings indicate that circ-ITCH suppresses metastasis of GC by acting as the sponge of miR-199a-5p and increasing Klotho expression, which serves as a potential biomarker and targets for the diagnosis and therapy of GC. Abbreviations: CircRNAs: circular RNAs; GC: gastric cancer; circ-ITCH: circular RNA Itchy E3 ubiquitin protein ligase; ceRNA: competitive endogenous RNA; EMT: Epithelial–mesenchymal transition; siRNA: Small interfering RNA; TEM: transmission electron microscope; NTA: nanoparticle tracking analysis.

Lidocaine Inhibits Hepatocellular Carcinoma Development by Modulating circ_ITCH/miR-421/CPEB3 Axis.

Lidocaine plays an anticancer role in hepatocellular carcinoma. Nevertheless, the mechanism of lidocaine in hepatocellular carcinoma remains largely unclear. This study aims to assess the function of lidocaine and explore the potential regulatory mechanism. Hepatocellular carcinoma cells were challenged via lidocaine. Cell proliferation, apoptosis, migration, and invasion were detected via colony formation, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, flow cytometry, Western blot, and transwell analyses. Circular RNA itchy E3 ubiquitin protein ligase (circ_ITCH), microRNA-421 (miR-421), and cytoplasmic polyadenylation element-binding protein 3 (CPEB3) abundances were detected via quantitative reverse transcription polymerase chain reaction or Western blot. The relationship between miR-421 and circ_ITCH or CPEB3 was tested via dual-luciferase reporter analysis. The role of circ_ITCH in lidocaine-challenged cell growth in vivo was assessed via xenograft model. Lidocaine inhibited hepatocellular carcinoma cell proliferation by decreasing colony formation and cell viability. Lidocaine suppressed hepatocellular carcinoma cell migration and invasion and promoted apoptosis. circ_ITCH and CPEB3 levels were decreased in hepatocellular carcinoma tissues and cells, and were restored in cells via lidocaine treatment. circ_ITCH knockdown weakened the suppressive effect of lidocaine on hepatocellular carcinoma development, which was abolished via CPEB3 overexpression. circ_ITCH could modulate CPEB3 by competitively binding with miR-421. miR-421 knockdown mitigated the effect of circ_ITCH silence in lidocaine-challenged cells. circ_ITCH knockdown increased xenograft tumor growth. Lidocaine represses hepatocellular carcinoma cell proliferation, migration, and invasion and promotes apoptosis via regulating circ_ITCH/miR-421/CPEB3 axis, indicating a new insight into the mechanism of lidocaine in hepatocellular carcinoma.

CircRNA ITCH increases bortezomib sensitivity through regulating the miR-615-3p/PRKCD axis in multiple myeloma

AimsBortezomib (BTZ) is described as the first-line agent for multiple myeloma (MM) chemotherapy, but the emergence of BTZ resistance usually results in the failure of chemotherapy in MM. Circular RNA (circRNA) itchy E3 ubiquitin protein ligase (circITCH) is a novel identified circRNA that plays a vital role in the development of human cancers. However, the role of circITCH in the development of BTZ resistance in MM remains elusive. Materials and methodsThe expression of circITCH, miR-615-3p, and protein kinase C, delta (PRKCD) was detected with quantitative reverse transcription PCR and western blot. The effects of circITCH on the sensitivity of MM cells to BTZ were assessed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, flow cytometry, and xenograft tumor assay. The interaction of circITCH, microRNA-615-3p, and PRKCD was explored using luciferase reporter assay and RNA immunoprecipitation assay. Key findingscircITCH was downregulated in MM bone marrow specimens and cell lines, as well as BTZ-resistant MM cells. Reduced expression of circITCH was indicative of poor prognosis in MM patients. Upregulation of circITCH enhanced the sensitivity of BTZ-resistant MM cells to BTZ in vitro and in vivo. Furthermore, circITCH was identified as a sponge for miR-615-3p, and PRKCD is confirmed as a direct target of miR-615-3p. Besides, circITCH overexpression enhanced the sensitivity of MM cells to BTZ through miR-615-3p/PRKCD axis. SignificancecircITCH overexpression enhanced the sensitivity of MM cells to BTZ through miR-615-3p/PRKCD axis, providing a novel potential target for combating BTZ resistance in patients with MM.

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.

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.

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.