Effect of Regulating A20 Expression on NF-κB Expression and Biological Characteristics of Jurkat Cells / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of regulating A20 expression on NF-κB and biological characteristics of Jurkat cells with glucocorticoid (GC) resistance.@*METHODS@#CCRF CEM and Jurkat cells were treated with dexamethasone (DEX) at concentrations of 100、10、1、0.1、0.01 and 0.001 μmol/L, and cultured for 24、48 and 72 h. The proliferation inhibition rate of Jurkat cell was detected by CCK-8. A20 plasmid was constructed, A20-siRNA was designed and synthesized, and transfected into Jurkat cells by liposome. CCK-8 was used to detect the proliferation rates of Jurkat cells in different concentrations of DEX group, DEX combined with A20 plasmid group and A20-siRNA group. The mRNA expression level of NF-κB was detected by RT-qPCR, the protein expression level of NF-κB was detected by Western blot, and the apoptosis of Jurkat cells was examined by flow cytometry.@*RESULTS@#The inhibitory effects of DEX at different concentrations on the growth of CCRF CEM cells were time-dependent (r=0.984, P＜0.05) and concentration-dependent (r=0.966, P＜0.05). At the point of 24 hour, the IC approached 1 μmol/L in CCRF CEM cells. Great large differences began to appear between 1 and 10 μmol/L, the proliferation rate of Jurkat cells treated with 1 μmol/L DEX did not show a significant change. Therefore, 1 μmol/L was selected as control group. The cell proliferation rate of A20 plasmid transfection combined with different concentrations of DEX group was lower than that of DEX group and A20-siRNA combined with DEX group. After transfection of A20 plasmid, the expression level of NF-κB was significantly lower than that of control group (P＜0.05), and the apoptotic rate was significantly higher than that of control group (P＜0.05). After transfection of Jurkat cells with A20-siRNA, the expression level of NF-κB was significantly higher than that of control group (P＜0.05). The apoptotic rate of cells in A20-siRNA group was not significantly changed (P＞0.05).@*CONCLUSION@#Jurkat cells are resistant to DEX. A20 overexpression combined with DEX can increase sensitivity of Jurkat cells with GC resistance and decrease the proliferation rate of Jurkat cells, down-regulate the expression level of NF-κB and promote the apoptosis of Jurkat cells.

Correlation of EBV Infection with Expression of TNF-α-Inducing Protein 3 Gene and A20 Protein in Hodgkin's Lmphoma / 中国实验血液学杂志

OBJECTIVE@#To analyze the correlation of EBV infection with expression of TNF-α-inducing protein 3 gene and A20 protein in Hodgkin lmphoma.@*METHODS@#The clinical data and pathological specimens of 65 cases of Hodgkin's lymphoma in our hospital were analyzed retrospectively, and the tissue chips were made for the rich area of the tumor cells. The latent membrane protein 1 encoded by EBV was measured by immunohistochemical staining, and the RNA encoded by EBV was measured by in situ hybridization to analyze the infection state. The gene expression of tumor necrosis factor.α-induced protein 3 was detected by fluorescence in situ hybridization, and the expression of A20 protein encoded by EBV was detected by immunohistochemical staining. The obtained data were processed by SPSS 23.0 version statistical software.@*RESULTS@#The positive rate of latent membrane protein 1 was 26.15% (17/65), the positive rate of EBV encoded RNA was 26.15% (17/65), and the coincidence rate was 100.00%. In 65 patients, A20 protein expression was lost in 18 cases (27.69%), and 14 cases (21.54%) showed homozygous or heterozygous deletion of tumor necrosis factorα protein 3 gene. Only 1 case showed A20 loss combined with homozygous deletion of TNFα inducible protein 3. Correlation analysis showed that EBV infection did not significantly relate with expression loss of A20 protein and the gene deletion of TNF-α inducing protein 3 (P>0.05).@*CONCLUSION@#The expression loss of A20 protein and gene detection of TNFα inducing protein 3 are found in both EBV negative and positive patients with Hodgkin's lymphoma, however the results of immunohistochemical staining and fluorescence in situ hybridization are not complete consistant, the reason may closely relate with the technical factors.

Expressions of Tumor Necrosis Factor alpha-induced Protein 3 and Mammary Serine Protease Inhibitor in Radiotherapy of Nasopharyngeal Carcinoma / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the expressions of tumor necrosis factor alpha-induced protein 3(TNFAIP3) and mammary serine protease inhibitor (Maspin) in the radiotherapy of nasopharyngeal carcinoma and explore the differences in radiosensitivity and radioresistance,the relation with the occurrence and development of radioresistance.</p><p><b>METHODS</b>The TNFAIP3 and Maspin mRNA expressions were detected by using TNFAIP3 and Maspin multi-point labeled DIG probes in situ hybridization.</p><p><b>RESULTS</b>In radiosensitivity and radioresistance of nasopharyngeal carcinoma,the moderately and strongly positive TNFAIP3 mRNA expression rates were 27.50% and 48.33% (P=0.037), and the moderately and strongly positive Maspin mRNA expression rates were 67.50% and 46.67% (P=0.040). In the radioresistance of nasopharyngeal carcinoma,TNFAIP3 mRNA moderately and strongly positive expressions were positively correlated with TNM stage (P=0.005). In distant metastasis and no distant metastasis (70.00% and 37.50%, P=0.018), the expression rates had statistical significance. The Maspin mRNA moderately and strongly positive expressions were positively correlated with TNM stage (P=0.039) and T stage (P=0.021). In distant metastasis and no distant metastasis (65.00% and 37.50%, P=0.044), the expression rates had statistical significance.</p><p><b>CONCLUSION</b>TNFAIP3 may be involved in the development of radioresistant nasopharyngeal carcinoma,and Maspin may be related with the invasion and metastasis of radioresistant nasopharyngeal carcinoma.</p>

Protective effect of cotransfection of A20 and HO-1 gene against the apoptosis induced by TNF-α in rat islets in vitro / 中华儿科杂志

<p><b>OBJECTIVE</b>To establish the method for cotransferring human A20 gene and human heme oxygenase-1 (HO-1) gene into the isolated rat islets using lentiviral transfection system, and to study the protective effect of A20 and HO-1 protein against the apoptosis induced by cycloheximide (CHX) and TNF-α, and finally to explore the underlying mechanism.</p><p><b>METHOD</b>The A20 gene and HO-1 gene were cloned and inserted into the lentiviral transfection system. The efficacy of gene transfer was measured by the intensity of the enhanced green fluorescent protein (EGFP) fluorescence-positive islets. Western blot was applied to verify the expression of the A20 and HO-1 genes. To induce apoptosis in vitro, the isolated islets were treated with CHX+TNF-α, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and the fluorescence-activated cell sorting (FACS) methods were used to evaluate the apoptosis of the islet cells and Western blot was used to detect caspase-3 activation.</p><p><b>RESULT</b>(1) A20 and HO-1 genes were introduced into the isolated islets by lentiviral transfection, both of the genes were highly expressed in the islets after 96 hours culture detected by Western blot method. (2) The insulin levels in the cell culture medium from A20 and/or HO-1 transgenic islets were significantly higher than that in non-transgenic controls (P < 0.01). (3)After CHX + TNF-alpha treatment, the cell culture medium insulin concentration in the A20 gene transfected group [(93.58 ± 4.12)µg/ml], HO-1 gene transfected group [(88.98 ± 4.77) µg/ml ] and A20/HO-1 co-transfected group [(103.33 ± 3.16) µg/ml] were significantly higher than that in the EGFP group [(9.03 ± 0.65) µg/ml ] and the control group [(8.86 ± 0.38) µg/ml] (P < 0.001). Minimum expression level of the activated caspase-3 was found in the A20/HO-1 co-transfected group.</p><p><b>CONCLUSION</b>The lentiviral gene transfer system was an efficient and stable gene transfer vector, the over-expressed A20 and HO-1 protein delivered via lentivirus could preserve rats' islets function and act against the apoptosis induced by CHX and TNF-α.</p>

Variants of tumor necrosis factor-induced protein 3 gene are associated with left ventricular hypertrophy in hypertensive patients / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Tumor necrosis factor-induced protein 3 (TNFAIP3) gene has been shown important in cardiac remodeling. The aim of the present study was to investigate whether the variants of TNFAIP3 gene are associated with left ventricular hypertrophy (LVH) in hypertensive patients.</p><p><b>METHODS</b>Four representatives of all the other single nucleotide polymorphisms (SNPs) in TNFAIP3 gene were tested for association with hypertrophy in two independent hypertensive populations (n = 2120 and n = 324).</p><p><b>RESULTS</b>We found that only the tag SNP (rs5029939) was consistently lower in the hypertensives with cardiac hypertrophy than in those without cardiac hypertrophy in the two study populations, indicating a protective effect on LVH (odds ratio (OR) (95% confidence interval (CI)) 0.58 (0.358 - 0.863), P = 0.035; OR (95%CI) = 0.477 (0.225 - 0.815), P < 0.05, respectively). Multiple regression analyses confirmed that the patients with G allele of rs5029939 had less thickness in inter-ventricular septum, left ventricular posterior wall, relative wall thickness and left ventricular mass index than did those with CC allele in the hypertensive patients in both study populations (all P < 0.01).</p><p><b>CONCLUSION</b>These findings indicate that the SNP (rs5029939) in the TNFAIP3 gene may serve as a novel protective genetic marker for the development of LVH in patients with hypertension.</p>

Immune regulation role of A20 and its clinical significance / 中国实验血液学杂志

A20 was originally identified as a TNFα-induced protein 3 (TNFAIP3), a key regulator of inflammation signalling pathways, as well as a NF-κB inhibitor. It plays a critical role in regulation of innate and adoptive immunity. Recently, A20 has also been proposed to function as a tumor suppressor. Lacking A20 gene is involved in inflammation-mediated autoimmune disease and tumorigenesis in several human B-cell lymphomas. Current advance concerning the feature of A20 expression in immune cells, the biological function, the immune regulated function in native immunity, humoral and cellular immunity, the inactivation of A20 in lymphocytic malignancies and the polymorphism and abnormal expression of A20 in autoimmune disease indicate that the clinical significance of A20 should be worthy to recognize and to be further employed in induction of immune tolerance, antitumor immuno-regulated therapy and antiviral immunotherapy and so on.

A20 inhibits human salivary adenoid cystic carcinoma cells invasion via blocking nuclear factor-kappaB activation / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>A20, also known as tumor necrosis factor alpha induced protein 3 (TNFaip3), is a cytoplasmic zinc finger protein that inhibits nuclear factor kappa-B (NF-kappaB) activity and prevents tumor necrosis factor (TNF)-mediated programmed cell death. NF-kappaB is a transcription factor that regulates expression of genes involved in cell proliferation, cell survival and anti-apoptosis. Several studies have implicated that the NF-kappaB signal pathway is associated with angiogenesis and clinico-pathological process of adenoid cystic carcinoma (ACC) of the salivary glands.</p><p><b>METHODS</b>The ability of overexpression of A20 to influence the biological behavior and invasion of ACC cells was examined. The cells were stably transfected with full-length A20 cDNA. Stable gene transfer was verified by realtime-polymerase chain reaction (PCR) and Western blot analysis. The change of cell biological behavior was examined by methyl thiazolyl tetrazolium (MTT) and NF-kappaB luciferase reporter assay and the invasion of the cells was examined by a Matrigel invasion chamber.</p><p><b>RESULTS</b>pEGPFN3-A20 gene was stably transferred into ACC-2 cells and overexpressed. When cells were treated with TNFalpha, the NF-kappaB activity of ACC-2-A20 cells could be down-regulated about 46.32% in contrast to ACC-2-GFP cells (P < 0.05). A20 potently inhibited growth of A20 transfectant ACC-2-A20 compared with control vector transfected groups and the ACC-2 empty control group (P < 0.05). The ACC-2-A20 cells showed significantly reduced ability to invade through Matrigei-coated filters compared to ACC-2-GFP and ACC-2 cells. The inhibition rate was up to 71.05% (P < 0.05).</p><p><b>CONCLUSIONS</b>A20 gene transfer is associated with decreased tumor invasion, in part via the down-regulation of NF-kappaB expression, providing evidence for a potential application of A20 in designing a treatment modality for salivary gland cancers such as ACC.</p>