LncRNA MALAT1 Regulating Lung Carcinoma Progression via the miR-491-5p/UBE2C Axis.

Long noncoding RNAs (lncRNAs) play a critical role in the development of lung carcinoma. The mechanism of MALAT1 in lung carcinoma development is not understood very well. This study aimed to investigate the role of MALAT1 in lung carcinoma progression and the mechanism underlying the role of miR-491-5p in the MALAT1 mediated regulation of UBE2C expression. The results indicated that the expression of MALAT1 was often augmented in lung carcinoma cells. Suppression of MALAT1 blocked the proliferation, invasion and migration ability of cancer cells and inhibited the expression of UBE2C. UBE2C restoration attenuated the MALAT1 knockdown-induced anti-cancer effects. Moreover, UBE2C and MALAT1 were indicated as targets of miR-491-5p and inhibition of miR-491-5p restored the MALAT1 knockdown-induced inhibition of the progression of lung carcinoma. Furthermore, MALAT1 sponged miR-491-5p to upregulate UBE2C expression, causing it to act as a competing endogenous RNA. Collectively, MALAT1 downregulation suppressed lung carcinoma progression by regulating the miR-491-5p/UBE2C axis. These results indicate that MALAT1 could be a molecular target for lung carcinoma treatment and prognosis.

UBE2V2 Positively Correlates With PD-L1 Expression and Confers Poor Patient Survival in Lung Adenocarcinoma.

This research aims to explore the diagnostic and prognostic value of ubiquitin-conjugating enzyme E2 variant 2 (UBE2V2) in lung adenocarcinoma (LUAD). The expression of UBE2V2 in clinical specimens was evaluated by bioinformatics analyses and immunohistochemistry. Bioinformatics analyses relying on the The Cancer Genome Atlas (TCGA) database suggested the elevated UBE2V2 mRNA levels in LUAD in comparison to adjacent normal tissues. Gene set enrichment analyses and gene ontology term enrichment analyses further showed the involvement of UBE2V2 in the modulation of cell cycle and immune associated signaling. The correlation analyses in TCGA LUAD data set revealed the positive correlation between UBE2V2 and CCNE1, CCNE2, CCNA2, CCNB1, CCNB2, cyclin-dependent kinase (CDK)2, CDK4, and CDK1 at the mRNA level. Moreover, UBE2V2 mRNA levels were positively correlated with PD-L1 mRNA levels, the T classification, and poor survival of LUAD patients, and were negatively correlated with type II interferon response. Consistent with the results obtained from TCGA data mining, immunohistochemistry demonstrated that UBE2V2 protein levels were upregulated in LUAD in comparison to normal tissues and were positively associated with T classification. Intriguingly, a positive correlation between UBE2V2 protein levels and PD-L1 expression was also elucidated in clinical samples. Besides, UBE2V2 expression indicated a poor prognosis in LUAD patients. Our study found that UBE2V2 was identified as an independent prognostic indicator for LUAD and might serve as an alternative target for LUAD treatment.

Knockdown of long non-coding RNA RMRP protects cerebral ischemia-reperfusion injury via the microRNA-613/ATG3 axis and the JAK2/STAT3 pathway.

Cerebral ischemia-reperfusion (I/R) injury can induce the mitophagy of neurons in the ischemic brain. Long non-coding RNAs (lncRNAs) play an important role in the pathogenesis of various injuries, especially in cerebral I/R injury. The purpose of this study is to investigate the molecular mechanism of lncRNA RNA component of mitochondrial RNA processing endoribonuclease (RMRP) in cerebral I/R injury. The middle cerebral artery occlusion (MCAO) mouse model was established. Neurological deficit score, pathological structure, infarcted area, neuron number, cell apoptosis, and coagulation ability of MCAO mice were evaluated. The expressions of RMRP, microRNA (miR)-613, and ATG3 in MCAO mice were detected. The binding relationships among miR-613, RMRP, and ATG3 were predicted and verified. Neuro 2A (N2a) cells were treated with oxygen-glucose deprivation/reperfusion (OGD/R) to simulate I/R injury. Cell viability and apoptosis assays were performed. The effects of miR-613, ATG3, and RMRP on I/R injury were verified by functional rescue experiments. JAK2/STAT3 phosphorylation level was detected. We found significantly upregulated RMRP and ATG3, and downregulated miR-613 expressions in MCAO mice. RMRP could escalate ATG3 mRNA expression through miR-613. RMRP knockdown promoted viability and inhibited apoptosis of OGD/R-treated N2a cells, which could be reversed by miR-613 inhibition or ATG3 overexpression. RMRP overexpression inhibited the activation of JAK2/STAT3 signaling pathway. We demonstrated that lncRNA RMRP competitively bound to miR-613, leading to the increase of ATG3 expression and the inhibition the JAK2/STAT3 pathway, thus promoting cerebral I/R injury in mice.

miR-708 and miR-335-3p Inhibit the Apoptosis of Retinal Ganglion Cells Through Suppressing Autophagy.

This study aimed to clarify the regulation role of miR-708 and miR-335-3p in retinal ganglion cell (RGC) autophagy and apoptosis in glaucoma. Chronic glaucoma mice were established by laser photocoagulation. RGCs were isolated and transfected with a series of plasmids and the cultured in 60 mmHg pressure. miR-335-3p, miR-708, and ATG3 mRNA expressions were detected by qRT-PCR. Protein levels of ATG3, autophagy-related protein LC3, and p62 were detected by Western blot. The apoptosis of RGCs was detected by flow cytometry. The regulation role of miR-335-3p/miR-708 in ATG3 was confirmed by the dual-luciferase reporter gene. The expressions of several miRNAs were measured in retinal tissues from chronic glaucoma mice and RGCs under pressure conditions, and results showed that both miR-335-3p and miR-708 were down-regulated. Besides, the inhibition of miR-708 and miR-335-3p induced the apoptosis of RGCs through promoting autophagy. Also, miR-708 and miR-335-3p could bind to ATG3 and targeted regulated ATG3. Furthermore, the interference with miR-708/miR-335-3p induced RGC apoptosis by up-regulating ATG3 to promote autophagy. In general, the down-regulation of miR-708 and miR-335-3p contributed to the apoptosis of RGCs through promoting autophagy in glaucoma.

Induction of NEDD8-conjugating enzyme E2 UBE2F by platinum protects lung cancer cells from apoptosis and confers to platinum-insensitivity.

Platinum is a widely used first-line chemotherapy in treating non-small cell lung cancer of adenocarcinoma. Unfortunately, platinum resistance leads to relapse and therapeutic failure, enabling the development of platinum-sensitization strategies to be of great clinical significance. Here, we report that the upregulation of the NEDD8-conjugating enzyme UBE2F is an important way for lung cancer cells to escape platinum-induced cell apoptosis, which confers to insensitivity to platinum-based chemotherapy. Mechanistically, platinum treatment impairs the complex formation for proteasome-mediated UBE2F degradation, evidenced by the weaker association between UBE2F and Ring-box protein 1 (RBX1), an essential component of Cullin-Ring E3 ligases (CRLs), thus leading to the accumulation of UBE2F. The accumulated UBE2F promotes the neddylation levels and activity of Cullin5, in accord with the lower expression of pro-apoptotic protein NOXA, a well-known substrate of Cullin-Ring E3 ligase 5 (CRL5). Additionally, knockout of UBE2F significantly sensitizes lung cancer cells to platinum treatment by enhancing the protein levels of NOXA and subsequently promoting cell apoptosis. Our observations uncover a previously unknown regulatory mechanism of UBE2F stability upon platinum chemotherapy and suggest that UBE2F might be a novel therapy target for sensitizing lung cancer cells to platinum-based chemotherapy.

Elevated TOP2A and UBE2C expressions correlate with poor prognosis in patients with surgically resected lung adenocarcinoma: a study based on immunohistochemical analysis and bioinformatics.

PURPOSE: Lung cancer has the highest morbidity and mortality among all cancer types. Reliable prognostic biomarkers are needed to identify high-risk patients apart from TNM system for precision medicine. The present study is designed to identify robust prognostic biomarkers in lung adenocarcinoma (LUAD) based on integration of multiple GEO datasets, The Cancer Genome Atlas (TCGA) database and Clinical Proteomic Tumor Analysis Consortium (CPTAC) database. METHODS: Four LUAD GEO datasets (GSE10072, GSE2514, GSE43458, and GSE32863) and TCGA database were implemented to analyze the differently expressed genes (DEGs). Gene ontology, KEGG pathway, and protein-protein interaction network (PPI) were conducted based on the above DEGs. Hub genes were selected based on connectivity degree in the PPI network. Expression analysis and Kaplan-Meier survival analysis were conducted in CPTAC lung adenocarcinomas cohort. Kaplan-Meier survival analysis and Cox proportional hazards regression were performed on these hub genes using TCGA and our own cohort. RESULTS: A total of 430 shared genes in all five datasets were identified as DEGs. Based on their PPI network, nine hub genes were selected and all of them were significantly associated with overall survival using GEPIA analysis. Two hub genes, TOP2A and UBE2C, were further combined and showed poorer prognosis in both TCGA dataset and our validated cohort. Analysis in CPTAC revealed that TOP2A and UBE2C were significantly highly expressed in tumor sample. Multivariable analysis suggested TOP2A and UBE2C as independent prognostic factors in LUAD. CONCLUSION: Using data mining approach, we identified TOP2A and UBE2C as two robust prognostic factors in LUAD. We also demonstrated the TOP2A/UBE2C co-expression status in LUAD, and TOP2A/UBE2C co-expression correlated with poorer prognosis. More in-depth research is needed for transforming this result into clinical setting.

Expression of SUMO enzymes is fiber type dependent in skeletal muscles and is dysregulated in muscle disuse.

SUMOylation is a dynamic, reversible, enzymatic drug-targetable post-translational modification (PTM) reaction where the Small Ubiquitin-like Modifier (SUMO) moieties are attached to proteins. This reaction regulates various biological functions like cell growth, differentiation, and it is crucial for maintaining organ homeostasis. However, the actions of SUMO in skeletal muscle pathophysiology are still not investigated. In this study, we quantified the abundance of the SUMO enzymes and determined the distribution of SUMOylated proteins along the fibers of nine different muscles. We find that skeletal muscles contain a distinctive group of SUMO enzymes and SUMOylated proteins in relation to their different metabolism, functions, and fiber type composition. In addition, before the activation of protein degradation pathways, this unique set is quickly altered in response to muscle sedentariness. Finally, we demonstrated that PAX6 acts as an upstream regulator of the SUMO conjugation reaction, which can become a potential therapeutic marker to prevent muscle diseases generated by inactivity.

High ubiquitin conjugating enzyme E2 T mRNA expression and its prognostic significance in lung adenocarcinoma: A study based on the TCGA database.

Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer with a high mortality disease which has been positioned the first and second cancer morbidity of men and women in China, separately. Our study was to assess the prognostic meaningful of ubiquitin conjugating enzyme E2 T (UBE2T) expression in LUAD dependent on data acquired from The Cancer Genome Atlas (TCGA) and so as to increase further knowledge into the biological pathways involved in LUAD pathogenesis related to UBE2T.Information on gene expression and comparing clinical data were recognized and downloaded from TCGA. Gene set enrichment analysis (GSEA) created an arranged list of all genes s indicated by their connection with UBE2T expression.Our study cohort included 265 (54.5%) female and 221 (36.0%) male patients. The scatter plot and paired plot showed the difference of UBE2T expression between normal and tumor samples (P < .01). Overall survival (OS) analysis demonstrated that LUAD with UBE2T-high had a more terrible prognosis than that with UBE2T-low (P < .01). Multivariate analysis with the cox proportional hazards model indicated that the expression of UBE2T (hazard ratio [HR]: 1.28; 95% Confidence Interval (CI): 1.06-1.56; P = .011) and stage (HR: 2.02; 95% CI: 1.27-3.21; P = .003) were independent prognostic factors for patients with LUAD. The GSEA results showed that cell cycle, DNA replication, RNA degradation, oxidative phosphorylation, pathogenic Escherichia coli infection, citrate cycle tricarboxylic acid cycle, Alzheimer's disease, P53 signaling pathway, and purine metabolism are differentially enriched in UBE2T high expression phenotype.Our study found that the expression of UBE2T was significantly increased in LUAD patients and associated with several clinical features. UBE2T may be a potentially useful prognostic molecular biomarker of bad survival in LUAD, while further experimental ought to be performed to demonstrate the biologic effect of UBE2T.

The expression of ubiquitin-conjugating enzyme E2C and KAI1 in ovarian carcinoma and their clinical significance.

Ubiquitin-conjugating enzyme E2C (UBE2C) is considered to play an important role in the tumorigenesis of many cancers and promote cell cycle progression. Kangai 1 (KAI1) is considered as a suppressor gene of tumor metastasis. However, the clinicopathological significance and their each relationship of UBE2C and KAI1 in epithelial ovarian carcinoma (EOC) are not widely reported. The purpose of this study is to detect the expression of UBE2C and KAI1 in EOC and their clinical significance.The expression of UBE2C and KAI1 in 180 cases of EOC tissues, 60 cases of normal ovarian epithelial tissues, and 60 cases of ovarian benign tumor tissues were detected by immunohistochemistry. Patients data were also collected.Positive expression of UBE2C in EOC (38.9%) was significantly higher than that both in the normal group (0%) and benign tumors group (10.0%). Furthermore, the expression of UBE2C was positively associated with grades of differentiation, implants, lymph node metastasis (LNM), as well as the International Federation of Gynecology and Obstetrics (FIGO) stages. Positive expression of KAI1 in EOC (25.0%) was significantly lower than that both in the normal group (100%) and benign tumors group (75.0%). And the expression of KAI1 was inversely associated with grades of differentiation, implants, LNM, and FIGO stages. Kaplan-Meier survival analyses demonstrated that UBE2C positive expression for patients with EOC had unfavorably overall survival (OS) time when compared with negative UBE2C for patients. And KAI1 positive expression for patients had favorably OS time when compared with negative KAI1 for patients. Multivariate analysis showed that positive expression of UBE2C and KAI1, implants, and FIGO stages were considered as independently prognostic factors for OS in patients with EOC. Moreover, UBE2C expression was significantly higher in high grade serous adenocarcinoma (SA) when compared with low grade SA; and KAI1 expression was significantly lower in high grade SA when compared with low grade SA. High grade SA patients had higher rates of implants, LNM, and high FIGO stages when compared with low grade SA. High grade SA patients had unfavorably OS time when compared with low grade SA.UBE2C and KAI1 should be considered as potential biomarkers of EOC prognosis.

Immunohistochemical expression pattern of RIP5, FGFR1, FGFR2 and HIP2 in the normal human kidney development.

AIM: Present study analyses the co-localisation of RIP5 with FGFR1, FGFR2 and HIP2 in the developing kidney, as RIP5 is a major determinant of urinary tract development, downstream of FGF-signaling. METHODS: Paraffin embedded human kidney tissues of 16 conceptuses between the 6th-22th developmental week were analysed using double-immunofluorescence method with RIP5/FGFR1/FGFR2 and HIP2 markers. Quantification of positive cells were performed using Kruskal-Wallis test. RESULTS: In the 6th week of kidney development RIP5 (89.6%) and HIP2 (39.6%) are strongly expressed in the metanephric mesenchyme. FGFR1 shows moderate/strong expression in the developing nephrons (87.3%) and collecting ducts (70.5%) (p < 0.05). RIP5/FGFR1 co-localized at the marginal zone and the ureteric bud with predominant FGFR1 expression. FGFR2 (26.1%) shows similar expression pattern as FGFR1 (70.5%) in the same kidney structures. RIP5/FGFR2 co-localized at the marginal zone and the collecting ducts (predominant expression of FGFR2). HIP2 is strongly expressed in collecting ducts (96.7%), and co-localized with RIP5. In 10th week, RIP5 expression decrease (74.2%), while the pattern of expression of RIP5 and FGFR1 in collecting ducts (33.4% and 91.9%) and developing nephrons (21.9% and 32.4%) (p < 0.05) is similar to that in the 6th developmental week. Ureter is moderately expressing RIP5 while FGFR1 is strongly expressed in the ureteric wall. FGFR2 is strongly expressed in the collecting ducts (84.3%) and ureter. HIP2 have 81.1% positive cells in the collecting duct. RIP5/FGFR1 co-localize in collecting ducts and Henley's loop. CONCLUSIONS: The expression pattern of RIP5, FGFR1, FGFR2 and HIP2 in the human kidney development might indicate their important roles in metanephric development and ureteric muscle layer differentiation through FGF signaling pathways.

Deregulation of LRSAM1 expression impairs the levels of TSG101, UBE2N, VPS28, MDM2 and EGFR.

CMT is the most common hereditary neuromuscular disorder of the peripheral nervous system with a prevalence of 1/2500 individuals and it is caused by mutations in more than 80 genes. LRSAM1, a RING finger ubiquitin ligase also known as TSG101-associated ligase (TAL), has been associated with Charcot-Marie-Tooth disease type 2P (CMT2P) and to date eight causative mutations have been identified. Little is currently known on the pathogenetic mechanisms that lead to the disease. We investigated the effect of LRSAM1 deregulation on possible LRSAM1 interacting molecules in cell based models. Possible LRSAM1 interacting molecules were identified using protein-protein interaction databases and literature data. Expression analysis of these molecules was performed in both CMT2P patient and control lymphoblastoid cell lines as well as in LRSAM1 and TSG101 downregulated SH-SY5Y cells.TSG101, UBE2N, VPS28, EGFR and MDM2 levels were significantly decreased in the CMT2P patient lymphoblastoid cell line as well as in LRSAM1 downregulated cells. TSG101 downregulation had a significant effect only on the expression of VPS28 and MDM2 and it did not affect the levels of LRSAM1. This study confirms that LRSAM1 is a regulator of TSG101 expression. Furthermore, deregulation of LRSAM1 significantly affects the levels of UBE2N, VPS28, EGFR and MDM2.

Disturbed Flow Increases UBE2C (Ubiquitin E2 Ligase C) via Loss of miR-483-3p, Inducing Aortic Valve Calcification by the pVHL (von Hippel-Lindau Protein) and HIF-1α (Hypoxia-Inducible Factor-1α) Pathway in Endothelial Cells.

Objective- Calcific aortic valve (AV) disease, characterized by AV sclerosis and calcification, is a major cause of death in the aging population; however, there are no effective medical therapies other than valve replacement. AV calcification preferentially occurs on the fibrosa side, exposed to disturbed flow (d-flow), whereas the ventricularis side exposed to predominantly stable flow remains protected by unclear mechanisms. Here, we tested the role of novel flow-sensitive UBE2C (ubiquitin E2 ligase C) and microRNA-483-3p (miR-483) in flow-dependent AV endothelial function and AV calcification. Approach and Results- Human AV endothelial cells and fresh porcine AV leaflets were exposed to stable flow or d-flow. We found that UBE2C was upregulated by d-flow in human AV endothelial cells in the miR-483-dependent manner. UBE2C mediated OS-induced endothelial inflammation and endothelial-mesenchymal transition by increasing the HIF-1α (hypoxia-inducible factor-1α) level. UBE2C increased HIF-1α by ubiquitinating and degrading its upstream regulator pVHL (von Hippel-Lindau protein). These in vitro findings were corroborated by immunostaining studies using diseased human AV leaflets. In addition, we found that reduction of miR-483 by d-flow led to increased UBE2C expression in human AV endothelial cells. The miR-483 mimic protected against endothelial inflammation and endothelial-mesenchymal transition in human AV endothelial cells and calcification of porcine AV leaflets by downregulating UBE2C. Moreover, treatment with the HIF-1α inhibitor (PX478) significantly reduced porcine AV calcification in static and d-flow conditions. Conclusions- These results suggest that miR-483 and UBE2C and pVHL are novel flow-sensitive anti- and pro-calcific AV disease molecules, respectively, that regulate the HIF-1α pathway in AV. The miR-483 mimic and HIF-1α pathway inhibitors may serve as potential therapeutics of calcific AV disease.

Gain of UBE2D1 facilitates hepatocellular carcinoma progression and is associated with DNA damage caused by continuous IL-6.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common type of liver cancer with increasing incidence and poor prognosis. Ubiquitination regulators are reported to play crucial roles in HCC carcinogenesis. UBE2D1, one of family member of E2 ubiquitin conjugating enzyme, mediates the ubiquitination and degradation of tumor suppressor protein p53. However, the expression and functional roles of UBE2D1 in HCC was unknown. METHODS: Immunohistochemistry (IHC), western blotting, and real-time PCR were used to detect the protein, transcription and genomic levels of UBE2D1 in HCC tissues with paired nontumor tissues, precancerous lesions and hepatitis liver tissues. Four HCC cell lines and two immortalized hepatic cell lines were used to evaluate the functional roles and underlying mechanisms of UBE2D1 in HCC initiation and progression in vitro and in vivo. The contributors to UBE2D1 genomic amplification were first evaluated by performing a correlation analysis between UBE2D1 genomic levels with clinical data of HCC patients, and then evaluated in HCC and hepatic cell lines. RESULTS: Expression of UBE2D1 was significantly increased in HCC tissues and precancerous lesions and was associated with reduced survival of HCC patients. Upregulation of UBE2D1 promoted HCC growth in vitro and in vivo by decreasing the p53 in ubiquitination-dependent pathway. High expression of UBE2D1 was attributed to the recurrent genomic copy number gain, which was associated with high serum IL-6 level of HCC patients. Further experiments showed that continuous IL-6 activated the DNA damage response and genomic instability by repressing DNA damage checkpoint protein RAD51B. Moreover, continuous IL-6 could significantly facilitate the HCC growth especially with the genomic gain of UBE2D1. CONCLUSIONS: Our findings showed that UBE2D1 played a crucial role in HCC progression, and suggested a novel pattern of continuous IL-6 to promote cancers by inducing the genomic alterations of specific oncogenes.

Differential Expression of Sumoylation Enzymes SAE1, U BA2, UBC9, PIAS1 and RanBP2 in Major Ocular Tissues of Mouse Eye.

Background: It is now well established that protein sumoylation is an important mechanism to regulate multiple cellular processes including gene transcription, chromatin structure, cell proliferation and differentiation, as well as pathogenesis. Objective: In the vertebrate eye, we and others have previously shown that sumoylation can regulate differentiation of major ocular tissues including retina and lens. However, the expression patterns of the three types of sumoylation enzymes, the activating enzymes SAE1 and UBA2, the conjugating enzyme UBC9, and the ligating enzymes such as RanBP2 and PIAS1 have not been well studied in the ocular tissues. Conclusion: In the present study, using QRT-PCR and western blot analysis, we have determined the differentiatial expression patterns of the above three types of enzymes, and the obtained results lay down a foundation for further exploration of sumoylation functions in vertebrate eye.

UBE2M Is a Stress-Inducible Dual E2 for Neddylation and Ubiquitylation that Promotes Targeted Degradation of UBE2F.

UBE2M and UBE2F are two family members of neddylation E2 conjugating enzyme that, together with E3s, activate CRLs (Cullin-RING Ligases) by catalyzing cullin neddylation. However, whether and how two E2s cross-talk with each other are largely unknown. Here, we report that UBE2M is a stress-inducible gene subjected to cis-transactivation by HIF-1 and AP1, and MLN4924, a small molecule inhibitor of E1 NEDD8-activating enzyme (NAE), upregulates UBE2M via blocking degradation of HIF-1α and c-JUN. UBE2M is a dual E2 for targeted ubiquitylation and degradation of UBE2F, acting as a neddylation E2 to activate CUL3-Keap1 E3 under physiological conditions but as a ubiquitylation E2 for Parkin-DJ-1 E3 under stressed conditions. UBE2M-induced UBE2F degradation leads to CRL5 inactivation and subsequent NOXA accumulation to suppress the growth of lung cancer cells. Collectively, our study establishes a negative regulatory axis between two neddylation E2s with UBE2M ubiquitylating UBE2F, and two CRLs with CRL3 inactivating CRL5.

SUMOylation regulates cytochrome P450 2E1 expression and activity in alcoholic liver disease.

Alcohol acts through numerous pathways leading to alcoholic liver disease (ALD). Cytochrome P450 (CYP2E1), an ethanol-inducible enzyme, metabolizes ethanol-producing toxic reactive oxygen species (ROS) and is regulated at the posttranslational level. Small ubiquitin-like modifier (SUMO)ylation is a posttranslational modification that involves the addition of SUMOs, which modulate protein stability, activity, and localization. We demonstrated that ubiquitin-conjugation enzyme 9, the SUMO-conjugating enzyme, is induced in the livers of an intragastric ethanol mouse model. Our aim is to examine whether SUMOylation could regulate ethanol-induced CYP2E1 expression in ALD and to elucidate the molecular mechanism(s). CYP2E1 and UBC9 expression in vitro and in vivo was detected by real-time PCR and immunoblotting/immunostaining. SUMOylation was assayed by mass spectrometry and coimmunoprecipitation. Ubc9 expression was induced in ethanol-fed mouse livers, and silencing inhibited ethanol-mediated CYP2E1 microsomal retention and enzymatic activity. CYP2E1 SUMOylation was found to be induced by ethanol in vitro and in vivo. Ubc9 silencing prevents ethanol-induced lipid accumulation and ROS production. UBC9 was highly expressed in human ALD livers. Finally, we found that lysine 410 is a key SUMOylated residue contributing to CYP2E1 protein stability and activity preventing CYP2E1 SUMOylation. Ethanol-mediated up-regulation of CYP2E1 via SUMOylation enhancing its protein stability and activity and may have important implications in ALD.-Tomasi, M. L., Ramani, K., Ryoo, M., Cossu, C., Floris, A., Murray, B. J., Iglesias-Ara, A., Spissu, Y., Mavila, N. SUMOylation regulates cytochrome P450 2E1 expression and activity in alcoholic liver disease.

Sodium Iodate-Induced Mouse Model of Age-Related Macular Degeneration Displayed Altered Expression Patterns of Sumoylation Enzymes E1, E2 and E3.

PURPOSE: Protein sumoylation is a highly dynamic and reversible post-translational modification, involving covalently conjugation of the small ubiquitin-like modifier (SUMO) to the lysine residue of the target protein. Similar to ubiquitination, sumoylation is catalyzed by E1, E2 and several E3 ligases. However, sumoylation usually does not cause protein degradation but alter the target function through diverse mechanisms. Increasing evidences have shown that sumoylation plays pivotal roles in the pathogenesis of human diseases, including neuron degeneration, cancer and heart disease, etc. We and others have shown that sumoylation is critically implicated in mouse eye development. However, the expression of sumoylation machinery has not been characterized in normal and pathogenic retina. Worldwide, age-related macular degeneration (AMD) is the leading cause of irreversible blindness in aged person. In the present study, we investigated the expression of the major sumoylation enzymes in normal mice and sodium iodateinduced AMD mouse model. METHODS: Four-week-old C57BL/6J mice were used in our experiment. A sterile 1% NaIO3 solution was freshly prepared in PBS from solid NaIO3. Experimental mice were injected with 70 mg/kg NaIO3, and similar volumes of PBS as control. Eyes were enucleated and immersion in FAA fixation overnight and processed for eye cross-sections. After fixation, cross sections eyes were dehydrated, embedded in paraffin, and 6 mm transverse sections were cut using the rotary microtome. Then paraffin sections were stained with hematoxylin and eosin (H&E), and mouse retinal thickness was observed to assess the histopathologic changes. RESULTS: Significantly declined RNA levels of E1, E2 and E3 ligase PIAS1 in NaIO3-injected mouse RPE one day-post treatment. Consistently, the protein level of PIAS1 was also decreased at this time point. At the late stage of treatment (three days post-injection), significantly reduced expression of E1 enzyme SAE1/UBA2 was detected in NaIO3-injected mouse retinas. In the contrary, dramatically increased E3 ligase RanBP2 was found in the injected-retinas. CONCLUSION: Together, our results demonstrated for the first time the dynamic expression of sumoylation pathway enzymes during the progression of retina degeneration induced by oxidative stress. Dynamic expression of E1, E2 and E3 enzymes were found during the time course of RPE and retina degeneration, which revealed the potential regulatory roles of sumoylation in AMD pathogenesis.

UBE2C is involved in the functions of ECRG4 on esophageal squamous cell carcinoma.

BACKGROUND: Esophageal cancerrelated gene 4 (ECRG4) is down-regulated in esophageal squamous-cell carcinoma (ESCC) and inhibits the tumorigenicity of ESCC cells. Ubiquitin conjugating enzyme E2 (UBE2C), an E2 ubiquitin-conjugating enzyme, is upregulated in numerous human cancers, including ESCC. METHODS: mRNA and protein expression was determined by real-time PCR and western blotting analysis, respectively. Cell apoptosis was assessed by Annexin V-fluorescein isothiocyanate staining and flow cytometry analysis. RESULTS: By analyzing previous quantitative proteomics data on EC9706 cells, we found that UBE2C was significantly down-regulated in ECRG4 overexpressed cells. Western blotting analysis validated the proteomics results in both EC9706 and EC-18 cells. In addition, Pearson's correlation analysis demonstrated a negative correlation between the mRNA levels of ECRG4 and UBE2C in ESCC tissues. Then, we found that Nuclear Factor-κB (NF-κB) inhibitor, pyrriolidine-dithiocarbamate (PDTC), could inhibit NF-κB p65 nuclear translocation and UBE2C expression, which was partially reversed by ECRG4 silence. More importantly, UBE2C knockdown in TE-1 cells significantly inhibited cell proliferation and induced cell apoptosis, which was partially reversed by ECRG4 knockdown. CONCLUSIONS: ECRG4 down-regulated UBE2C expression in ESCC cells via NF-κB signaling. UBE2C was involved in the anti-proliferative and pro-apoptotic functions of ECRG4 in ESCC cells.

Differential UBE2C and HOXA1 expression in melanocytic nevi and melanoma.

BACKGROUND: Recent molecular advances suggest that Spitz nevi and other spitzoid neoplasms are biologically distinct from melanoma and conventional nevi. The ubiquitin ligase UBE2C and the homeobox transcription factor HOXA1 are candidate oncogenes in melanoma. METHODS: Using RNA expression analysis and immunohistochemistry, we evaluated these biomarkers in Spitz nevi (n = 20), melanoma (n = 20), and by immunohistochemistry in conventional nevi (n = 20). RESULTS: RNA analysis with branched DNA multiplex assay identified upregulation of UBE2C in melanomas vs Spitz nevi (P = .003), whereas HOXA1 was downregulated in melanoma (P < .0001). Immunohistochemical analysis confirmed increased nuclear expression of UBE2C in melanoma (mean = 18% of cells; range 3%-44%) when compared with Spitz nevi (mean = 9%; range 2%-28%; P = .001) and conventional nevi (mean = 1.5%; range 0-9%; P < .0001). Strong UBE2C staining was identified in cells undergoing mitosis. UBE2C RNA and protein detection correlated with mitotic rate (P < .0001). On the other hand, HOXA1 nuclear staining was low in melanoma (mean = 69%; range 5%-100%) when compared with Spitz nevi (mean = 94%; range 66%-100%; P = .0024) and conventional nevi (mean = 94%; range 83%-99%; P = .009). CONCLUSIONS: UBE2C and HOXA1 RNA and protein are differentially expressed in conventional and Spitz nevi and melanoma.