BACKGROUND: Cyclooxygenase-2 (COX-2) is an enzyme that promotes proliferation of tumor cells. HuR is a member of the family of embryonic lethal abnormal vision-like proteins. Recent studies show that cytoplasmic HuR stabilizes the mRNA of COX-2 and regulates the expression of COX-2. Moreover, cytoplasmic HuR expression is associated with a poorer prognosis for patients with some cancers. The aim of this study was to investigate the expression patterns of and the relationship between COX-2 and HuR in gallbladder carcinoma. METHODS: We analyzed COX-2 and HuR expression by immunohistochemical staining of 108 gallbladder carcinomas. RESULTS: COX-2 expression and nuclear and cytoplasmic HuR expression were seen in, respectively, 61 (56.5%), 77 (71.3%), and 4 (3.7%) cases. COX-2 and nuclear HuR were simultaneously expressed in 44 of the 108 samples without any quantitative association between the levels of each. COX-2 expression correlated with tumor stage, differentiation (based on histology), lymph node metastasis, perineural invasion, and survival. Nuclear and cytological expression of HuR did not correlate with any clinical parameters. CONCLUSIONS: COX-2 expression but not HuR may play an important role in the prognosis of patients with gallbladder carcinoma.
Cyclooxygenase 2 ; Cytoplasm ; Gallbladder ; Gallbladder Neoplasms ; ELAV Proteins ; ELAV-Like Protein 1 ; Humans ; Lymph Nodes ; Neoplasm Metastasis ; Prognosis ; Proteins ; RNA, Messenger

OBJECTIVE: To investigate the regulatory role of the long non-coding RNA LINC00926 in pyroptosis of hypoxia-induced human umbilical vein vascular endothelial cells (HUVECs) and explore the molecular mechanism. METHODS: HUVECs were transfected with a LINC00926-overexpressing plasmid (OE-LINC00926), a siRNA targeting ELAVL1, or both, followed by exposure to hypoxia (5% O2) or normoxia. The expression of LINC00926 and ELAVL1 in hypoxia-treated HUVECs was detected using real-time quantitative PCR (RT-qPCR) and Western blotting. Cell proliferation was detected using Cell Counting Kit-8 (CCK-8), and the levels of IL-1β in the cell cultures was determined with ELISA. The protein expression levels of pyroptosis-related proteins (caspase-1, cleaved caspase-1 and NLRP3) in the treated cells were analyzed using Western blotting, and the binding between LINC00926 and ELAVL1 was verified with RNA immunoprecipitation (RIP) assay. RESULTS: Exposure to hypoxia obviously up-regulated the mRNA expression of LINC00926 and the protein expression of ELAVL1 in HUVECs, but did not affect the mRNA expression of ELAVL1. LINC00926 overexpression in the cells significantly inhibited cell proliferation, increased IL-1β level and enhanced the expressions of pyroptosis-related proteins (all P < 0.05). LINC00926 overexpression further up-regulated the protein expression of ELAVL1 in hypoxia-exposed HUVECs. The results of RIP assay confirmed the binding between LINC00926 and ELAVL1. ELAVL1 knockdown significantly decreased IL-1β level and the expressions of pyroptosis-related proteins in hypoxia-exposed HUVECs (P < 0.05), while LINC00926 overexpression partially reversed the effects of ELAVL1 knockdown. CONCLUSION LINC00926 promotes pyroptosis of hypoxia-induced HUVECs by recruiting ELAVL1.
Humans ; Caspase 1 ; ELAV-Like Protein 1 ; Human Umbilical Vein Endothelial Cells ; Pyroptosis ; RNA, Messenger ; RNA, Long Noncoding/genetics* ; Cell Hypoxia

BACKGROUND: Hu syndrome, a neurological disorder, is characterized by the remote effect of small cell lung cancer on the neural degeneration. The suspicious effectors for this disease are anti-Hu autoantibodies or Hu-related CD8+ T lymphocytes. Interestingly, the same effectors have been suggested to act against tumor growth and this phenomenon may represent natural tumor immunity. For these diagnostic and therapeutic reasons, the demand for antibodies against Hu protein is rapidly growing. METHODS: Polyclonal and monoclonal antibodies were generated using recombinant HuR protein. Western blot analyses were performed to check the specificity of generated antibodies using various recombinant proteins and cell lysates. Extracellular stimuli for HuR expression had been searched and HuR-associated proteins were isolated from polysome lysates and then separated in a 2-dimensional gel. RESULTS: Polyclonal and monoclonal antibodies against HuR protein were generated and these antibodies showed HuR specificity. Antibodies were also useful to detect and immunoprecipitate endogenous HuR protein in Jurkat and BJAB. This report also revealed that TNF-alphatreatment in BJAB up-regulated HuR expression. Lastly, protein profile in HuR-associated mRNA- protein complexes was mapped by 2-dimensional gel electrophoresis. CONCLUSION: This study reported that new antibodies against HuR protein were successfully generated. Currently, project to develop a diagnostic kit is in process. Also, this report showed that TNF-alphaup-regulated HuR expression in BJAB and protein profile associated with HuR protein was mapped.
Antibodies ; Antibodies, Monoclonal ; Autoantibodies ; Blotting, Western ; Electrophoresis ; ELAV Proteins ; ELAV-Like Protein 1 ; Nervous System Diseases ; Ovarian Neoplasms* ; Polyribosomes ; Recombinant Proteins ; Sensitivity and Specificity ; Small Cell Lung Carcinoma ; T-Lymphocytes ; Tumor Necrosis Factor-alpha

OBJECTIVE: To investigate the role of miR-519 in regulating the cell activity of gastric cancer. 
 METHODS: The expression of miR-519 was measured by qPCR. The protein expression of HuR was detected by Western blot. MTT assay was used to detect the cell activity of gastric cancer cells.
 RESULTS: The protein level of HuR in the gastric cancer cells was higher than that in the control cells. Over-expression of HuR resulted in the increase in the cell activity of gastric cancer (P<0.001). Suppression of HuR decreased the cell activity of gastric cancer (P=0.001). The miR-519 level in the gastric cancer cells was lower than that in the control cells (P=0.001). Over-expression of miR-519 decreased the protein level of HuR and the activity of gastric cancer cells.
 CONCLUSION MiR-may inhibit the gastric cancer cell activity through suppression of HuR expression.
Cell Line, Tumor ; Cell Proliferation ; ELAV-Like Protein 1 ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; MicroRNAs ; genetics ; metabolism ; Stomach Neoplasms ; genetics ; metabolism

OBJECTIVE: To investigate whether circular RNA circRSF1 regulates radiation-induced inflammatory phenotype of hepatic stellate cells (HSCs) by binding to HuR protein and repressing its function. METHODS: Human HSC cell line LX2 with HuR overexpression or knockdown was exposed to 8 Gy X-ray irradiation, and the changes in the expression of inflammatory factors (IL-1β, IL-6 and TNF-α) were detected by qRT-PCR. The expressions of IκBα and phosphorylation of NF-κB were detected with Western blotting. The binding of circRSF1 to HuR was verified by RNA pull-down assay and RNA-binding protein immunoprecipitation (RIP). The expressions of inflammatory factors, IκBα and the phosphorylation of NF-κB were detected after modifying the interaction between circRSF1 and HuR. RESULTS: Knockdown of HuR significantly up- regulated the expressions of IL-1β, IL-6 and TNF-α, decreased IκBα expression and promoted NF-κB phosphorylation in irradiated LX2 cells, whereas overexpression of HuR produced the opposite changes (P < 0.05). Overexpression or knockdown of circRSF1 did not significantly affect the expression of HuR. RNA pull-down and RIP experiments confirmed the binding between circRSF1 and HuR. Overexpression of circRSF1 significantly reduced the binding of HuR to IκBα and down-regulated the expression of IκBα (P < 0.05). Overexpression of circRSF1 combined with HuR overexpression partially reversed the up-regulation of the inflammatory factors, down-regulated IκBα expression and increased phosphorylation of NFκB in LX2 cells, while the opposite effects were observed in cells with knockdown of both circRSF1 and HuR (P < 0.05). CONCLUSION circRSF1 reduces IκBα expression by binding to HuR to promote the activation of NF-κB pathway, thereby enhancing radiation- induced inflammatory phenotype of HSCs.
Humans ; Hepatic Stellate Cells/radiation effects* ; Interleukin-6 ; NF-kappa B ; NF-KappaB Inhibitor alpha ; Phenotype ; RNA ; RNA, Circular/metabolism* ; Tumor Necrosis Factor-alpha ; ELAV-Like Protein 1/metabolism*