Ring Finger Protein 43 Research Articles

A DNA repair player, ring finger protein 43, relieves etoposide‐induced topoisomerase II poisoning

Ring finger protein 43 (RNF43) is an E3 ubiquitin ligase which is well-known for its role in negative regulation of the Wnt-signaling pathway. However, the function in DNA double-strand break repairs has not been investigated. In this study, we used a lymphoblast cell line, DT40, and mouse embryonic fibroblast as cellular models to study DNA double-strand break (DSB) repairs. For this purpose, we created RNF43 knockout, RNF43-/- DT40 cell line to investigate DSB repairs. We found that deletion of RNF43 does not interfere with cell proliferation. However, after exposure to various types of DNA-damaging agents, RNF43-/- cells become more sensitive to topoisomerase II inhibitors, etoposide, and ICRF193, than wild type cells. Our results also showed that depletion of RNF43 results in apoptosis upon etoposide-mediated DNA damage. The delay in resolution of γH2AX and 53BP1 foci formation after etoposide treatment, as well as epistasis analysis with DNAPKcs, suggested that RNF43 might participate in DNA repair of etoposide-induced DSB via non-homologous end joining. Disturbed γH2AX foci formation in MEFs following pulse etoposide treatment supported the notion that RNF43 also functions DNA repair in mammalian cells. These findings propose two possible functions of RNF43, either participating in NHEJ or removing the blockage of 5' topo II adducts from DSB ends.

Overexpression of RNF126 Promotes the Development of Colorectal Cancer via Enhancing p53 Ubiquitination and Degradation.

BackgroundRING finger protein 126 (RNF126), as a novel E3 ubiquitin ligase, plays an oncogenic role in several solid cancers. But its potential role in colorectal cancer (CRC) that harbored 50% mutant p53, to our knowledge, is rarely reported.Materials and MethodsWe investigated the clinical significance and relationship of RNF126 and p53 in CRC tissues and cells. Meanwhile, WB, qRT-PCR, co-IP, MTT, and transwell were used to investigate the function and molecular mechanism of RNF126 in regulating malignant biology in vitro.ResultsRNF126 was overexpressed in human CRC specimens, which was tightly associated with tumor size (P=0.021), T stage (P=0.030), lymph node metastasis (P=0.006), TNM stage (P=0.001), and the poor survival (P=0.003) of CRC patients. RNF126 had no association with p53 mutation in CRC specimens, and in p53 mutant Colo-205 and SW620 cells. However, in p53 wildtype HCT116 and HCT-8 cells, RNF126 silencing upregulated p53 and p21 but inhibited Rb phosphorylation at Serine 780 (pRb), which was inhibited by p53siRNA. Conversely, RNF126 overexpression downregulated p53 and p21 but promoted pRb expression, which was reversed by a classic proteasome inhibitor, MG132. However, the mRNA levels of above target genes were unchanged, implying a ubiquitination dependent post-translational modification involving in above regulation. Meanwhile, RNF126 was co-immunoprecipitated with p53 and p21 to form a triple complex. RNF126 silencing and overexpression inhibited and promoted p53 ubiquitination and degradation in vitro, respectively. In addition, p53siRNA reversed RNF126 silencing-inhibited cell proliferation, drug resistance, and cell mobility in HCT116 cells. Conversely, MG132 inhibited RNF126 overexpression-promoted above cell biology in HCT-8 cells.ConclusionOverexpression of RNF126 was remarkably associated with multiple advanced clinical characters of CRC patients independent of mutant p53. RNF126 promotes cell proliferation, mobility, and drug resistance in CRC via enhancing p53 ubiquitination and degradation.

RNF180 mediates STAT3 activity by regulating the expression of RhoC via the proteasomal pathway in gastric cancer cells

Ring finger protein 180 (RNF180) is an important member of the E3 ubiquitin ligase family. As a tumor suppressor gene, RNF180 is significantly associated with the prognosis of patients with gastric cancer (GC) and can inhibit the proliferation, invasion, and migration of GC cells. Signal transducer and activator of transcription 3 (STAT3) are considered one of the most common oncogenes in human cancers with a key role in GC progression. In this study, we explored the molecular signaling pathways by which RNF180 could potentially regulate STAT3 through transcriptomics and proteomics experiments. Here, we found RNF180 overexpression could suppress STAT3 phosphorylation in GC cells. Ubiquitin label-free experiments showed that the ubiquitination level of Ras homolog gene family member C (RhoC) is significantly increased in GC cells transfected with an RNF180 expression vector (RNF180-GFP vector) compared with cells transfected with an empty vector (vehicle vector). We subsequently demonstrated that RNF180 could directly combine with RhoC and promote the ubiquitination and degradation of RhoC protein in GC cells. The phosphorylation level of STAT3 significantly decreased in GC cells after RhoC knockdown using small hairpin RNA (shRNA). Together, these results reveal RNF180 could inhibit GC progression by reducing the phosphorylation of STAT3 via the ubiquitination and degradation of RhoC protein in GC cells. Thus, the protein may be considered a novel therapeutic target for patients with GC.

Hsa_circRNA_100146 Acts as a Sponge of miR-149-5p in Promoting Bladder Cancer Progression via Regulating RNF2.

BackgroundMounting evidence has demonstrated that circular RNAs (circRNAs) play indispensable roles in the progression of bladder cancer. Public database mining showed that hsa_circRNA_100146 (circRNA_100146) was highly expressed in bladder cancer. This study aimed to characterize the biological role of circRNA_100146 and clarify the underlying mechanism in bladder cancer.MethodsWe evaluated the relationship between circRNA_100146 expression and clinicopathological features. Furthermore, gain- and loss-of-function studies were conducted in bladder cancer cells via transfection with gene-carrying plasmids (over-expression) or specific short hairpin RNAs (knockdown). Moreover, computational algorithms and dual-luciferase reporter assays were performed to explore the possible mechanisms of action. Additionally, in vivo xenograft experiments were performed to further analyze the effect of circRNA_100146 on tumor growth.ResultsOur data showed that circRNA_100146 expression was increased in bladder cancer tissues and cell lines, and that high expression of circRNA_100146 was correlated with poor patient prognosis. Upregulation of circRNA_100146 promoted cell proliferation, migration, and invasion, and inhibited cell apoptosis, whereas knockdown of circRNA_100146 displayed opposite effects on bladder cancer cells. Notably, circRNA_100146 could combine with miR-149-5p and promote ring finger protein 2 (RNF2) expression, thereby facilitating the progression of bladder cancer. Furthermore, overexpression of RNF2 reversed the effects of circRNA_100146 knockdown on the biological behaviors of bladder cancer cells. The in vivo experiments revealed that downregulation of circRNA_100146 dramatically delayed tumor growth.ConclusionOur findings indicate that circRNA_100146 functions as a sponge of miR-149-5p in promoting bladder cancer progression by regulating RNF2 expression and that circRNA_100146 may serve as a novel biomarker in human bladder cancer.

Inhibition of RNF6 alleviates traumatic brain injury by suppressing STAT3 signaling in rats.

BackgroundTraumatic brain injury (TBI) has ranked as one of the leading causes of disability and death in the world. The neuroinflammation mediated by signal transducer and activator of transcription 3 (STAT3) signaling during the progression of TBI leads to long‐term neurodegeneration. Ring finger protein 6 (RNF‐6) is an E3 ubiquitin ligase and can regulate the activity of STAT3 signaling pathway by targeting its inhibitors. However, the mechanism underlying this process in TBI remains poorly understood.MethodsIn this research, cortical impact injury was used to construct the TBI rat model. Western blot assay was performed to evaluate the protein levels of RNF6, Src homology 2 domain‐containing protein tyrosine phosphatase 1 (SHP‐1), and STAT3/pSTAT3. QRT‐PCR assay was performed to assess the RNA levels of RNF6 and other cytokines. The neural function of TBI rats was estimated by modified Neurological Severity Scores test.ResultsThe expression of RNF‐6 was up‐regulated in the brain tissues of TBI rats. Down‐regulation of RNF6 alleviated the symptoms and improved the neural recovery postinjury in TBI rats. Inhibition of RNF6 suppressed the cerebral inflammation by up‐regulating the protein level of SHP‐1 and down‐regulating the phosphorylation level of STAT3.ConclusionInhibition of RNF6 alleviated TBI by suppressing the STAT3 signaling in TBI rats.

Abstract CT167: First-in-human clinical trial of SC-006, a RNF43 targeted antibody-drug conjugate, as a monotherapy and in combination with budigalimab in patients with advanced colorectal cancer

Abstract Background: Colorectal cancer (CRC) is the third most common cancer in the United States. Aberrant activation of the Wnt signaling pathway is associated with CRC progression. Ring finger protein 43 (RNF43) is a single-pass type 1 transmembrane protein and a key feedback regulator of Wnt. SC-006 is a RNF43 targeted antibody drug conjugate, linked to a pyrrolobenzodiazepine dimer DNA crosslinking agent. Budigalimab (ABBV-181) is a PD-1 targeting, humanized, recombinant immunoglobulin G1 monoclonal antibody with Fc mutations (L234A, L235A) shown to reduce Fc receptor interactions and limit effector function. This open-label, Phase 1 clinical trial assessed the safety and efficacy of SC-006 as monotherapy and in combination with budigalimab in patients with CRC (NCT03035279). Methods: Evaluable patients had histologically or cytologically confirmed advanced metastatic or unresectable CRC. The trial consisted of 2 parts: part A (SC-006-monotherapy, 2 to 12 µg/kg, IV) and part B (SC-006 in combination with budigalimab at a fixed dose of 375 mg/kg IV every 3 weeks [Q3W]). Efficacy was assessed per RECIST 1.1 by radiographic assessments. Results: As of April 2019, 29 patients were enrolled (n=20 in part A and n=9 in part B). All patients experienced at least one treatment-emergent adverse event (TEAE). The most common TEAEs (>20% of patients) are summarized in Table 1. Two patients (7%) had dose-limiting toxicities of grade 4 thrombocytopenia. All 29 patients discontinued from the study, with study drug being discontinued for 76% of patients due to progressive disease. One death occurred in part A due to gastrointestinal hemorrhage with possible relation to SC-006. Serious TEAEs occurred in 8 patients (28%); these included thrombocytopenia and malignant neoplasm progression in 2 patients (7%) each. Progressive disease occurred in 18 patients (62%), stable disease was reported in 10 patients (34%) and the response for 1 patient was not reported. Conclusion: SC-006 dose escalation was limited by thrombocytopenia observed below the predicted efficacious dose, leading to study termination. Table 1.Summary of the Most Common Treatment-Emergent Adverse EventsPart A: SC-006-monotherapyPart B: SC-006+budigalimabTEAEs (>20% patients), n (%)2 ug/kg4 ug/kg8 ug/kg4 ug/kg8 ug/kg12 ug/kg4 ug/kg Q6W+budigalimab8 ug/kg Q6W+budigalimabOverallQ3WQ3WQ3WQ6WQ6WQ6W(N=1)(N=3)(N=1)(N=2)(N=10)(N=3)(N=4)(N=5)(N=29)Thrombocytopenia01 (33)1 (100)1 (50)4 (40)3 (100)1 (25)4 (80)15 (52)Fatigue0001 (50)3 (30)2 (67)3 (75)3 (60)12 (41)Nausea0001 (50)5 (50)1 (33)2 (50)2 (40)11 (38)Edema peripheral01 (33)01 (50)4 (40)02 (50)1 (20)9 (31)Decreased appetite01 (33)01 (50)4 (40)002 (40)8 (28)Dyspnea00004 (40)3 (100)01 (20)8 (28)Vomiting0001 (50)3 (30)1 (33)2 (50)1 (20)8 (28)Constipation0001 (50)3 (30)01 (25)2 (40)7 (24) Citation Format: Katrina S. Pedersen, Amit Mahipal, Andrea Cercek, Susanna Ulahannan, Michael Rossi, Abraham Fong, Daniel Brickman, Johanna Bendell. First-in-human clinical trial of SC-006, a RNF43 targeted antibody-drug conjugate, as a monotherapy and in combination with budigalimab in patients with advanced colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT167.

MiR-203a suppresses cell proliferation by targeting RING-finger protein 6 in colorectal cancer.

Colorectal cancer (CRC) is one of most common cancers worldwide. Although miR-203a is reported as a tumor suppressor involved in cell progression in some cancers, the role of miR-203a in CRC is still controversial and the underling mechanism of miR-203a in CRC remains unclear. Here, we demonstrated that low expression of miR-203a had poorer survival in CRC patients. miR-203a was down-regulated in most human colon cancer cells. Overexpression of miR-203a could inhibit colon cancer cell proliferation and arrest cell cycle in G1 phase. Bioinformatics and dual luciferase reporter assay confirmed that RING-finger protein 6 (RNF6) was a target gene of miR-203a. Silencing RNF6 inhibited cell proliferation and arrest cell cycle in G1 phase. RNF6 overexpression reversed the effects of miR-203a overexpression in colon cancer cells. Taken together, our data indicate that miR-203a inhibits colon cancer cell proliferation by targeting RNF6, offer novel insights into the regulatory network of miR-203a-modulated cell cycle and proliferation, and suggest that miR-203a a potential therapeutic target in CRC treatment.

The isoflavonoid calycosin inhibits inflammation and enhances beta cell function in gestational diabetes mellitus by suppressing RNF38 expression

Background Gestational diabetes mellitus (GDM) is a medical complication and metabolic disorder associated with pregnancy. Calycosin is a traditional Chinese herbal medicine that is used for the treatment of multiple diseases. This study focused on exploring the effects and underlying mechanisms of Calycosin on GDM. Methods The db/+ diabetic mice model of GDM was used to evaluate the effects of calycosin administration on the symptoms of GDM mice. Blood glucose, cytokine production (interleukin 6, IL-6; tumor necrosis factor-α, TNF-α), and insulin levels were measured by ELISA assay. The expression level of signal transducer and activator of transcription 3 (STAT3), ring finger protein 38 (RNF38), and SH2-containing protein tyrosine phosphatase 1 (SHP-1) were determined by Western Blot assay. Beta cell proliferation was assessed by CCK-8 assay. Results Our data indicated that administration of calycosin significantly improved the GDM symptoms in pregnant db/+ mice as demonstrated by reduced blood glucose, TNF-a, and IL-6 levels as well as increased insulin level, and body weight. Furthermore, we revealed that RNF38/SHP-1/STAT3 signaling should play a critical role in calycosin-promoted beta cell function, and forced expression of RNF38 attenuated the positive effects of calycosin on beta cells. Conclusion Our study implied that calycosin exerts favorable effects on GDM mice via rebalancing insulin sensitivity and inflammatory response.

MiR224-5p Inhibitor Restrains Neuronal Apoptosis by Targeting NR4A1 in the Oxygen-Glucose Deprivation (OGD) Model.

This study was designed to investigate the molecular mechanism of stroke and to explore the effect of miR-224-5p in hypoxic cortical neurons. Firstly, we established a middle cerebral artery occlusion (MCAO) model with Sprague–Dawley rats. Triphenyltetrazolium chloride (TTC) staining showed the brain infarction of an MCAO rat. Longa scores of rats were significantly increased in 12th, 24th, and 48th hours after MCAO. Then, we found that miR-224-5p was increased after MCAO in rats by qRT-PCR. In order to investigate the effect of miR-224-5p in hypoxic neurons, we established an oxygen-glucose deprivation (OGD) model with cortical neurons. MiR-224-5p was also upregulated in neurons after OGD by qRT-PCR. After transfection of the miR-224-5p inhibitor, the number of neurons in the anti-miR-224-5p group significantly increased (P < 0.01) in comparison to the anti-NC group. Furthermore, Tuj1+ (neuronal marker) staining and TUNEL assay (to detect apoptotic cells) were performed in neurons. The survival of neurons in the anti-miR-224-5p group was significantly improved (P < 0.01), while the apoptosis of neurons in the anti-miR-224-5p group was significantly decreased (P < 0.01), when compared with that of the anti-NC group. In addition, we predicted that potential target genes of miR-224-5p were nuclear receptor subfamily 4 group A member 1 (NR4A1), interleukin 1 receptor antagonist (IL1RN), and ring finger protein 38 (RNF38) with bioinformatics databases, such as TargetScan, miRDB, miRmap, and miRanda. The result of qRT-PCR confirmed that NR4A1 was significantly decreased after hypoxic injury (P < 0.01). Meanwhile, luciferase reporter’s assay indicated that NR4A1 was the direct target of miR-224-5p. Compared with the anti-miR-224-5p + siNC group, the number of cortical neurons and the length of the neuron axon in the anti-miR-224-5p + si-NR4A1 group were significantly decreased (P < 0.01), and the number of neuronal apoptosis in the anti-miR-224-5p + si-NR4A1 group was increased (P < 0.01). In conclusion, miR-224-5p played a crucial role in hypoxic neuron injury through NR4A1, which might be an important regulatory mechanism in OGD injury of neurons.

Cell stemness is maintained upon concurrent expression of RB and the mitochondrial ribosomal protein S18-2

Stemness encompasses the capability of a cell for self-renewal and differentiation. The stem cell maintains a balance between proliferation, quiescence, and regeneration via interactions with the microenvironment. Previously, we showed that ectopic expression of the mitochondrial ribosomal protein S18-2 (MRPS18-2) led to immortalization of primary fibroblasts, accompanied by induction of an embryonic stem cell (ESC) phenotype. Moreover, we demonstrated interaction between S18-2 and the retinoblastoma-associated protein (RB) and hypothesized that the simultaneous expression of RB and S18-2 is essential for maintaining cell stemness. Here, we experimentally investigated the role of S18-2 in cell stemness and differentiation. Concurrent expression of RB and S18-2 resulted in immortalization of Rb1-/- primary mouse embryonic fibroblasts and in aggressive tumor growth in severe combined immunodeficiency mice. These cells, which express both RB and S18-2 at high levels, exhibited the potential to differentiate into various lineages in vitro, including osteogenic, chondrogenic, and adipogenic lineages. Mechanistically, S18-2 formed a multimeric protein complex with prohibitin and the ring finger protein 2 (RNF2). This molecular complex increased the monoubiquitination of histone H2ALys119, a characteristic trait of ESCs, by enhanced E3-ligase activity of RNF2. Furthermore, we found enrichment of KLF4 at the S18-2 promoter region and that the S18-2 expression is positively correlated with KLF4 levels. Importantly, knockdown of S18-2 in zebrafish larvae led to embryonic lethality. Collectively, our findings suggest an important role for S18-2 in cell stemness and differentiation and potentially also in cancerogenesis.

RNF8 Dysregulation and Down-regulation During HTLV-1 Infection Promote Genomic Instability in Adult T-Cell Leukemia.

The genomic instability associated with adult T cell leukemia/lymphoma (ATL) is causally linked to Tax, the HTLV-1 viral oncoprotein, but the underlying mechanism is not fully understood. We have previously shown that Tax hijacks and aberrantly activates ring finger protein 8 (RNF8) - a lysine 63 (K63)-specific ubiquitin E3 ligase critical for DNA double-strand break (DSB) repair signaling - to assemble K63-linked polyubiquitin chains (K63-pUbs) in the cytosol. Tax and the cytosolic K63-pUbs, in turn, initiate additional recruitment of linear ubiquitin assembly complex (LUBAC) to produce hybrid K63-M1 pUbs, which trigger a kinase cascade that leads to canonical IKK:NF-κB activation. Here we demonstrate that HTLV-1-infected cells are impaired in DNA damage response (DDR). This impairment correlates with the induction of microscopically visible nuclear speckles by Tax known as the Tax-speckle structures (TSS), which act as pseudo DNA damage signaling scaffolds that sequester DDR factors such as BRCA1, DNA-PK, and MDC1. We show that TSS co-localize with Tax, RNF8 and K63-pUbs, and their formation depends on RNF8. Tax mutants defective or attenuated in inducing K63-pUb assembly are deficient or tempered in TSS induction and DDR impairment. Finally, our results indicate that loss of RNF8 expression reduces HTLV-1 viral gene expression and frequently occurs in ATL cells. Thus, during HTLV-1 infection, Tax activates RNF8 to assemble nuclear K63-pUbs that sequester DDR factors in Tax speckles, disrupting DDR signaling and DSB repair. Down-regulation of RNF8 expression is positively selected during infection and progression to disease, and further exacerbates the genomic instability of ATL.

Clinical significance of ring finger protein 2 high expression in skin squamous cell carcinoma.

Although ring finger protein 2 (RNF2) serves an important role in the occurrence, development and regulation of various types of cancer, RNF2 expression in skin squamous cell carcinoma (SCC) remains unknown. The aim of the present study was to investigate the role of RNF2 expression in SCC and adjacent tissues from patients. The protein and gene expression levels of RNF2 in SCC and adjacent tissues were detected by immunohistochemistry (IHC), western blot analysis and semi-quantitative reverse transcription (RT) PCR. Single factor analysis was used to study the association between RNF2 expression level and the clinicopathological characteristics of patients with SCC. Multifactor Cox survival analysis was used to examine the association between RNF2 expression and the overall survival rate of postoperative patients with SCC. The results from IHC staining demonstrated that the positive expression rate of RNF2 was 84.68% (210/248) and 56.05% (139/248) in SCC and in adjacent tissues, respectively. Furthermore, results from western blot analysis demonstrated that RNF2 protein expression in SCC tissues was significantly higher compared with that in the adjacent tissues (P<0.05). The positive rate of RNF2 mRNA in SCC was 81.05% (201/248), which was significantly higher compared with that in the adjacent tissues 54.44% (135/248; P<0.05). Furthermore, RNF2 protein and gene expression levels were associated with tumor diameter, tumor stage, tumor metastasis and the degree of tumor differentiation in patients with SCC. Patients exhibiting higher RNF2 protein expression in SCC tissues had a significantly shorter disease-specific survival rate compared with patients with low RNF2 expression. In addition, RNF2 protein expression, tumor diameter, tumors site and tumor stage were independent factors affecting the overall survival rate of postoperative patients. High protein and gene expression levels of RNF2 in SCC tissues may be associated with the occurrence and development of SCC and prognosis of patients. The results form this study may serve the development of novel therapeutic options and diagnostic strategies for patients with SCC.

RNF8 promotes high linear energy transfer carbon-ion-induced DNA double-stranded break repair in serum-starved human cells

The cell-killing effect of radiotherapy largely depends on unrepaired DNA double-stranded breaks (DSBs) or lethal chromosome aberrations induced by DSBs. Thus, the capability of DSB repair is critically important for the cancer-cell-killing effect of ionizing radiation. Here, we investigated the involvement of the DNA damage signaling factors ataxia telangiectasia mutated (ATM), ring finger protein 8 (RNF8), and RNF168 in quiescent G0/G1 cells, which are expressed in the majority of cell populations in tumors, after high linear energy transfer (LET) carbon-ion irradiation. Interestingly, ATM inhibition caused a substantial DSB repair defect after high-LET carbon-ion irradiation. Similarly, RNF8 or RNF168 depletion caused a substantial DSB repair defect. ATM inhibition did not exert an additive effect in RNF8-depleted cells, suggesting that ATM and RNF8 function in the same pathway. Importantly, we found that the RNF8 RING mutant showed a similar DSB repair defect, suggesting the requirement of ubiquitin ligase activity in this repair pathway. The RNF8 FHA domain was also required for DSB repair in this axis. Furthermore, the p53-binding protein 1 (53BP1), which is an important downstream factor in RNF8-dependent DSB repair, was also required for this repair. Importantly, either ATM inhibition or RNF8 depletion increased the frequency of chromosomal breaks, but reduced dicentric chromosome formation, demonstrating that ATM/RNF8 is required for the rejoining of DSB ends for the formation of dicentric chromosomes. Finally, we showed that RNF8 depletion augmented radiosensitivity after high-LET carbon-ion irradiation. This study suggests that the inhibition of RNF8 activity or its downstream pathway may augment the efficacy of high-LET carbon-ion therapy.

&lt;p&gt;Identification of High-Frequency Methylation Sites in RNF180 Promoter Region Affecting Expression and Their Relationship with Prognosis of Gastric Cancer&lt;/p&gt;

BackgroundRing finger protein 180 (RNF180) is a tumor suppressor gene regulated by promoter methylation. We previously demonstrated that the RNF180 promoter methylation could be a risk factor for gastric cancer (GC); and eight high-frequency hypermethylated CpG sites were associated with GC. However, it is not clear whether these key sites can affect gene expression and involve in prognosis. The aim of this study was to investigate the effects of above CpG sites on the gene expression and prognosis of GC.Patients and MethodsA total of 164 GC tissues were enrolled and followed up. Tissue samples were used for DNA and RNA isolation. Methylation status of RNF180 was detected using bisulfite sequencing PCR (BSP). Expression levels of RNF180 were detected using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). JASPAR and PROMO databases were used to predict the transcription factors (TFs) binding to the CpG site.ResultsThe methylation in RNF180 promoter region increased and mRNA expression decreased in GC tissue. Correlation analysis revealed that the average methylation rate (AMR) and four CpG sites methylation rate were negatively related to RNF180 expression, including M3(−165)(Chr5:64165942), M5(−148)(Chr5:64,165,959), M7(−133)(Chr5:64,165,974) and M8(−130)(Chr5:64,165,977). Furthermore, the methylation rate of M5(−148)(Chr5:64,165,959) and M27(−26)(Chr5:64,166,081) above 0.3 indicated poor prognosis (PM5 = 0.008, PM27 = 0.003, HRM5(−148) = 2.000 (1.201,3.332), HRM27(−26)=2.389 (1.336,4.271)), which could be independent factors of prognosis.ConclusionBy focusing on the methylation sites in the RNF180 promoter region, we identified two high-frequency methylation sites, M5(−148)(Chr5:64,165,959) and M27(−26)(Chr5:64,166,081), which could affect gene expression and predict the prognosis of GC. In the future, the possible molecular mechanism involved needs to be further studied.

Karyopherin α-2 Mediates MDC1 Nuclear Import through a Functional Nuclear Localization Signal in the tBRCT Domain of MDC1.

Mediator of DNA damage checkpoint protein 1 (MDC1) plays a vital role in DNA damage response (DDR) by coordinating the repair of double strand breaks (DSBs). Here, we identified a novel interaction between MDC1 and karyopherin α-2 (KPNA2), a nucleocytoplasmic transport adaptor, and showed that KPNA2 is necessary for MDC1 nuclear import. Thereafter, we identified a functional nuclear localization signal (NLS) between amino acid residues 1989–1994 of the two Breast Cancer 1 (BRCA1) carboxyl-terminal (tBRCT) domain of MDC1 and demonstrated disruption of this NLS impaired interaction between MDC1 and KPNA2 and reduced nuclear localization of MDC1. In KPNA2-depleted cells, the recruitment of MDC1, along with the downstream signaling p roteins Ring Finger Protein 8 (RNF8), 53BP1-binding protein 1 (53BP1), BRCA1, and Ring Finger Protein 168 (RNF168), to DNA damage sites was abolished. Additionally, KPNA2-depleted cells had a decreased rate of homologous recombination (HR) repair. Our data suggest that KPNA2-mediated MDC1 nuclear import is important for DDR signaling and DSB repair.

Long non-coding RNA SNHG5 promotes human hepatocellular carcinoma progression by regulating miR-363-3p/RNF38 axis.

The involvement of long non-coding RNA (LncRNAs) in HCC development has been widely recognized in recent decades. LncRNA small nucleolar RNA host gene 5 (SNHG5) has been identified to be implicated in the development of many tumors, and this study aimed to explore the role of SNHG5 in HCC tumorigenesis. The expression levels of SNHG5, miR-363-3p, and Ring Finger Protein 38 (RNF38) were measured by using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) or Western blot assay, respectively. Cell proliferation was analyzed by MTT assay. Flow cytometry was used to investigate cell apoptosis. Cell migration and invasion abilities were detected by transwell assay. The relationship among SNHG5, miR-363-3p, and RNF38 was confirmed using bioinformatics analysis and Luciferase reporter assay. The expression of SNHG5 and RNF38 was elevated in HCC tissues and cell lines, and highly expressed SNHG5 and RNF38 could induce apoptosis and repress proliferation, migration, as well as invasion in HCC cells. Further investigations showed that SNHG5 might act as a competing endogenous RNA of miR-26a-5p and thereby cause the derepression of the downstream target RNF38. Moreover, rescue experiments indicated that SNHG5 silence inhibited the progression of HCC cells by regulating miR-363-3p, and the facilitated effects of RNF38 in the progression of HCC cells were regulated by miR-363-3p. LncRNA SNHG5 may promote human HCC progression by regulating the miR-363-3p/RNF38 axis, providing a novel insight into the pathogenesis of HCC and therapeutic strategy for HCC treatment.

RNF126 as a Marker of Prognosis and Proliferation of Gastric Cancer.

Ring finger protein 126 (RNF126) belongs to the family of RING E3 ubiquitin ligases. Although RNF126 has been reported to be overexpressed in several cancers, the role of RNF126 in gastric cancer remains unclear. We investigated the RNF126 expression in 170 primary gastric cancer tissues by immunohistochemistry, and explored its prognostic impact. The effect of the RNF126 expression on the proliferation of cancer cells was evaluated in vitro. The RNF126 expression was significantly associated with tumor depth and presence of venous invasion. The RNF126 status was identified as an independent prognostic factor (p<0.001). RNF126 gene silencing significantly inhibited the proliferation of gastric cancer cells, induced G1 phase arrest and increased the p21 protein level. RNF126 expression has a significant prognostic value in gastric cancer. RNF126 may play an important role in tumor progression of gastric cancer.

Potential Influences of RNF6 on Prognosis and Metastasis of Colorectal Cancer: A Clinical Analysis.

IntroductionRing finger protein 6 (RNF6) locates on the chromatin 13q.12.13, where amplification is frequently occurred in colorectal cancer (CRC). Previous studies have reported the role of RNF6 to accelerate the progression and metastasis of CRC.MethodsIn this paper, we mainly analyzed the potential of RNF6 to predict the prognosis and metastasis of CRC. Based on the cut-off value of RNF6, enrolled CRC patients were assigned into high- and low-level group. Correlation between RNF6 level and survival of CRC patients was assessed.ResultsOur findings revealed that RNF6 was upregulated in CRC tissues. IHC staining demonstrated higher positive expression of RNF6 in CRC tissues. Nearly 61.2% CRC patients had a positive expression of RNF6. Moreover, RNF6 was closely linked to lymphovascular invasion (LV) (P=0.006), invasion depth (P=0.001), metastasis (P<0.001) and TNM staging (P<0.001). In CRC tissues, RNF6 level was negatively correlated to that of E-cadherin (r=−0.7093, P<0.0001). OS (overall survival) and RFS (recurrence-free survival) were worse in CRC patients with high-level RNF6, and tumor cell metastasis was believed to be the major reason.ConclusionTherefore, RNF6 was confirmed to be a hallmark predicting the prognosis and metastasis in CRC patients.

RNF8 promotes efficient DSB repair by inhibiting the pro-apoptotic activity of p53 through regulating the function of Tip60.

ObjectivesRING finger protein 8 (RNF8) is an E3 ligase that plays an essential role in DSB repair. p53 is a well‐established tumour suppressor and cellular gatekeeper of genome stability. This study aimed at investigating the functional correlations between RNF8 and p53 in DSB damage repair.Materials and methodsIn this article, wild‐type, knockout and shRNA‐depleted HCT116 and U2OS cells were stressed, and the roles of RNF8 and p53 were examined. RT‐PCR and Western blot were utilized to investigate the expression of related genes in damaged cells. Cell proliferation, apoptosis and neutral cell comet assays were applied to determine the effects of DSB damage on differently treated cells. DR‐GFP, EJ5‐GFP and LacI‐LacO targeting systems, flow cytometry, mass spectrometry, IP, IF, GST pull‐down assay were used to explore the molecular mechanism of RNF8 and p53 in DSB damage repair.ResultsWe found that RNF8 knockdown increased cellular sensitivity to DSB damage and decreased cell proliferation, which was correlated with high expression of the p53 gene. RNF8 improved the efficiency of DSB repair by inhibiting the pro‐apoptotic function of p53. We also found that RNF8 restrains cell apoptosis by inhibiting over‐activation of ATM and subsequently reducing p53 acetylation at K120 through regulating Tip60.ConclusionsTaken together, these findings suggested that RNF8 promotes efficient DSB repair by inhibiting the pro‐apoptotic activity of p53 through regulating the function of Tip60.

Knockdown of NRAGE Impairs Homologous Recombination Repair and Sensitizes Hepatoblastoma Cells to Ionizing Radiation.

Background: NRAGE (neurotrophin receptor-interacting melanoma antigen-encoding gene homolog) has a complex role and regulates cell growth in different tumor cells. Although NRAGE was been discovered for more than 10 years ago, the function of NRAGE in hepatoblastoma (HB) cells is currently unknown. Materials and Methods: The expression of NRAGE was detected by reverse transcription-quantitative polymerase chain reaction assay or western blotting assay. Cellular apoptosis was analyzed to estimate the effect of NRAGE under radiation. The ability of clonogenic capacity was evaluated to confirm the influence of proliferation for NRAGE by radiation. The immunofluorescence assay was used to further study the expression of NRAGE under radiation. A nude mouse tumor xenograft model was constructed to confirm the effect of NRAGE deficiency under radiation conditions in vivo. Results: The authors determined that deletion of NRAGE significantly inhibited HB cell proliferation in vitro and in vivo, and NRAGE knockdown apparently sensitized HB cells to ionizing radiation (IR). Further mechanistic studies revealed that NRAGE plays a critical role in homologous recombination by inhibiting the expression of RNF8 (ring finger protein 8) and BARD1 (BRCA1 associated RING domain 1) and the recruitment of RAD51. Conclusions: The authors demonstrated that downregulation of NRAGE sensitizes HB cell lines to IR in vitro and in vivo. It provides a promising therapeutic strategy for HB patients by specifically targeting NRAGE.