Cholinergic dysfunction-induced insufficient activation of alpha7 nicotinic acetylcholine receptor drives the development of rheumatoid arthritis through promoting protein citrullination via the SP3/PAD4 pathway

Both cholinergic dysfunction and protein citrullination are the hallmarks of rheumatoid arthritis (RA), but the relationship between the two phenomena remains unclear. We explored whether and how cholinergic dysfunction accelerates protein citrullination and consequently drives the development of RA. Cholinergic function and protein citrullination levels in patients with RA and collagen-induced arthritis (CIA) mice were collected. In both neuron-macrophage coculture system and CIA mice, the effect of cholinergic dysfunction on protein citrullination and expression of peptidylarginine deiminases (PADs) was assessed by immunofluorescence. The key transcription factors for PAD4 expression were predicted and validated. Cholinergic dysfunction in the patients with RA and CIA mice negatively correlated with the degree of protein citrullination in synovial tissues. The cholinergic or alpha7 nicotinic acetylcholine receptor (α7nAChR) deactivation and activation resulted in the promotion and reduction of protein citrullination in vitro and in vivo, respectively. Especially, the activation deficiency of α7nAChR induced the earlier onset and aggravation of CIA. Furthermore, deactivation of α7nAChR increased the expression of PAD4 and specificity protein-3 (SP3) in vitro and in vivo. Our results suggest that cholinergic dysfunction-induced deficient α7nAChR activation, which induces the expression of SP3 and its downstream molecule PAD4, accelerating protein citrullination and the development of RA.

Roles of TSA upregulation miR-4298 targeting inhibition of PADI4 expression in inducing U251 cells apoptosis / 国际肿瘤学杂志

Objective:To investigate the mechanism of microRNA-4298 (miR-4298) targeting inhibition of peptidylarginine deiminase 4 (PADI4) expression in U251 cells apoptosis induced by histone deacetylase inhibitor trichostatin A (TSA).Methods:The experiment was divided into TSA group (0.2 μmol/L TSA) and contral group. CCK8 method was used to detect the effect of TSA on the proliferation of U251 cells. Flow cytometry was used to detect the apoptosis level of U251 cells after drug action. Reverse transcription PCR and Western blotting experiments were used to determine the changes of PADI4 gene and protein expression after miR-4298 interference. Luciferase assay was used to determine the effects of miR-4298 on targeted binding and luciferase activity in the 3′UTR region of PADI4. U251 cells were transfected with PADI4 to observe the rescue effect of miR-4298 on apoptosis. Each experiment was divided into 3 groups, NC group, miR-4298 mimic group, TSA+ miR-4298 mimic group and NC group, miR-4298 inhibitor group, TSA+ miR-4298 inhibitor group.Results:U251 cells were treated with 0.2 μmol/L TSA for 4 days, the Absorbancy ( A) values of the control group was 1.168±0.148, which was higher than those of the TSA group (0.737±0.007), with statistically significant difference ( t=4.948, P=0.008). Compared with the control group, after treatment with 0.2 μmol/L TSA for 48 h, U251 cells showed obvious apoptosis (27.62%±3.49% vs. 4.99%±0.13%, t=11.190, P<0.001). Compared with those before the drug treatment, the PADI4 gene expression (0.386±0.020 vs. 0.903±0.021) and protein expression (0.276±0.041 vs. 0.777±0.031) after TSA treatment for 48 h both were decreased, with statistically significant differences ( t=30.400, P<0.001; t=16.770, P<0.001). The expression of miR-4298 during the process of TSA-induced U251 cell apoptosis increased (2.573±0.289 vs. 1.003±0.136; t=8.487, P=0.001). There was a negative correlation between the miR-4298 expression and PADI4 expression ( r=-0.877, P=0.002). Luciferase experiment confirmed that miR-4298 has targeted binding and luciferase inhibitory effects on the 3′UTR region of wild-type PADI4. Compared with NC group (0.920±0.026), the relative expression levels of PADI4 gene of miR-4298 mimic group (0.413±0.049) and TSA+ miR-4298 mimic group (0.213±0.035) were decreased, with statistically significant differences (all P<0.001). The PADI4 protein expression level change was consistent with the gene change trend. Compared with NC group (3.78%±0.68%), the apoptosis levels of miR-4298 mimic group (7.96%±1.10%) and TSA+ miR-4298 mimic group (13.74%±1.26%) were increased, with statistically significant differences ( P=0.005; P<0.001). Compared with NC group (0.183±0.025), the PADI4 gene expression of miR-4298 inhibitor group (0.483±0.032) and TSA+ miR-4298 inhibitor group (0.386±0.025) were increased, with statistically significant differences ( P<0.001; P=0.015). The PADI4 protein expression level change was consistent with the gene change trend. Compared with NC group (4.96%±0.59%), the apoptosis levels of miR-4298 inhibitor group (23.83%±2.20%) and TSA+ miR-4298 inhibitor group (9.55%±1.49%) were increased, with statistically significant differences (all P<0.001). In the rescue experiment, the expression level of PADI4 in miR-4298 mimic group was significantly increased ( P<0.001), while the expression level of PADI4 in miR-4298 mimic+ PADI4 group was relatively reversed ( P=0.002). Conclusion:miR-4298 can participate in the process of U251 cell apoptosis by targeting the expression of PADI4 gene, and miR-4298 may be the target of targeted intervention therapy for glioma.

Meta-analysis of the association between PADI4 -92C/G polymorphism and rheumatoid arthritis in the Chinese population

Many studies have evaluated the correlation between peptidylarginine deiminase 4 (PADI4) -92C/G polymorphism and rheumatoid arthritis (RA), but the results remain inconclusive. Therefore, we performed a meta-analysis in the Chinese population to provide comprehensive data on the association between PADI4 -92C/G polymorphism and RA. Eligible studies published before May 2016 were identified in PubMed and Chinese databases. The strengths of these associations were assessed by pooled odds ratios (OR) and 95% confidence interval (CI). Eight studies documenting a total of 1351 RA cases and 1585 controls were included in this meta-analysis. In the overall analysis, a significant association between the PADI4 -92C/G polymorphism and RA was found in the Chinese population (G vs C: OR=1.32, 95%CI=1.02-1.71; GG+CG vs CC: OR=1.75, 95%CI=1.20-2.53). The subgroup analyses stratified by geographic area(s) and source of controls revealed significant results in South China, in hospital-based studies and population-based studies. In summary, this meta-analysis suggested that PADI4 -92C/G polymorphism may be associated with the RA incidence in the Chinese population, especially for South China. Further studies conducted on other ethnic groups are required for definite conclusions.

Peptidylarginine deiminase 4 probably play an important role in rheumatoid arthritis by involving in the formation of the neutrophil extracellular traps / 中华风湿病学杂志

Objective To detect the levels of peptidylarginine deiminase 4 (PAD4) mRNA and neutrophil extracellular traps (NETs) of the peripheral blood neutrophils in rheumatoid arthritis (RA) patients and to study the association between PAD4 and NETs in RA.The anti-cyclic citrullinated peptide (anti CCP) antibodies,disease activity score (DAS28) score,erythrocyte sedimentation rate (ESR) were recorded.Its value in the pathogenesis of RA was explored.T test,rank-sum test,Pearson and Spearman correlative analysis were used for statical analysis.Methods The serum double-stranded DNA (dsDNA)/NETs in 43 RA patients and 20 healthy controls were detected by PicoGreen dsDNA Quantitation Kits (Invitrogen).Real-time quantitative polymerase chain reaction (RT-PCR) was used to determine the expression of PAD4 mRNA in neutrophils,and the relationship between dsDNA/NETs and their clinical indexes were analyzed.Results ① The level of peripheral blood neutrophils PAD4 mRNA in RA was significantly higher than healthy controls [1.493(0.831,2.607)] vs [0.631(0.358,1.489)],(Z=-2.07,P=0.039).The levels of serum PAD4 mRNA in RA treated with disease-modifying antirheumatic drugs (DMARDs) were lower than untreated with DMARDs but no significant difference was found (Z=-1.19,P=0.234).② The levels of serum dsDNA/NETs in RA patients were significantly higher than in healthy controls (t=3.4,P=0.001).③ The levels of serum dsDNA/NETs in RA were positively correlated with DAS28 score,ESR,CRP and PAD4 mRNA (r=0.36,P=0.036;r=0.345,P=0.042;r=0.36,P=0.043;r=0.42,P=0.017) but not with anti-CCP antibodies (r=0.277,P=0.154).Conclusion ① PAD4 may play an important role in the pathogenesis of RA.② NETs maybe associated with disease activity and play an important role in RA pathogenesis,inhibit NETs generation or improve the ability to clear NETs,perhaps can treat RA.③ PAD4 probably play an important role in rheumatoid arthritis by influencing the formation of the NETs.

Expressions and clinicopathological significances of BMI-1 and PADI4 in esophageal squamous cell car-cinoma / 国际肿瘤学杂志

Objective To evaluate the expression levels of peptidylarginine deiminase 4 (PADI4)and B-cells pecific Moloney leukemia virus insert site-1 (BMI-1 )in esophageal squamous cell carcinoma (ESCC) tissues and pericarcinous tissues.To explore the function and clinical significance in the development of ESCC and their association.Methods The expression levels of PADI4 and BMI-1 were measured by immunohisto-chemistry,Western blotting and quantitative real time PCR in ESCC tissues and pericarcinous tissues from 86 patients.The relationships between the expressions of PADI4 and BMI-1 and the clinicopathologic characte-ristics were analyzed.Results The immunohistochemistry showed that the expressions of PADI4 and BMI-1 in ESCC tissues (68.6% and 73.3%)were significantly higher than those in pericarcinous tissues (37.2% and 30.2%,χ2 =1 7.01 1 ,P =0.000;χ2 =31 .876,P =0.000).Western blotting indicated that the levels of PADI4 and BMI-1 were higher than those in pericarcinous tissues (0.91 9 ±0.098 vs.0.71 8 ±0.1 03,t =2.462,P =0.021 ;0.975 ±0.074 vs.0.71 7 ±0.071 ,t =2.640,P =0.01 4).The expressions of BMI-1 and PADI4 mRNA in ESCC tissues were higher than those in pericarcinous tissues,but the differences were not sta-tistically significant (0.091 ±0.005 vs.0.038 ±0.002,t =1 .701 ,P =0.1 01 ;0.1 1 4 ±0.075 vs.0.048 ± 0.003,t =1 .499,P =0.1 46)by the quantitative real time PCR.The expression of PADI4 was correlated with lymph node metastasis (χ2 =5.771 ,P =0.01 6),depth of invasion (χ2 =6.672,P =0.01 0)and clinical stage (χ2 =5.771 ,P =0.01 6).The BMI-1 gene expression had a correlation with lymph node metastasis (χ2 =7.1 76,P =0.007),the differentiation degree (χ2 =1 3.787,P =0.001 )and clinical stage (χ2 =7.1 76,P =0.007).In addition,there was a positive correlation between PADI4 and BMI-1 expression in ESCC by immunohistochemistry and quantitative real time PCR (r =0.21 4,P =0.047;r =0.534,P =0.005).Conclusion The expression levels of PADI4 and BMI-1 are significantly higher in ESCC compared to pericarcinous tissues.PADI4 and BMI-1 are positively correlated and may contribute to the diagnosis and prog-nosis of the ESCC.

Correlation between Anti Vraf Murine Sarcomaviral Oncogene Homologue B1 Antibodies and Anti Pepti-dylarginine Deiminase 4 Antibodies with Rheumatoid Arthritis / 沈阳医学院学报

Rheumatoid arthritis(RA)is a joint synovial inflammation which is a prominent feature of chronic,erosive autoimmune disease. Early clinical symptoms are occult and atypical. If not for the early treatment,an illness will break out repeatedly,ultimately lead to the destruction of the joints,can be deformed or even lose labor ability,seriously affect the patients quality of life. RA patho?genesis is not clear, the antigen?antibody reaction plays an important role in the process of its development. Autoantibodies with higher specificity and sensitivity detected in the early RA disease for the diagnosis of RA is very necessary,can also be used for RA prognosis and monitoring of RA disease activity.

Effect of siRNA against peptidylarginine deiminase 4 gene on the apoptosis of fibroblast-like synoviocytes from synovium of rheumatoid arthritis patients / 中华风湿病学杂志

Objective To evaluate the effects of small interfering RNA (siRNA) against peptidylarginine deiminase 4 (PADI4) gene on apoptosis of fibroblast-like synoviocytes (FLS) from synovium of rheumatoid arthritis (RA).Methods The siRNA targeting PADI4 was constructed and transfected into FLS cells in RA via LipofectamineTM 2000.The expression level of PDAI4 mRNA was detected by using real-time quantitative polymerase chain reaction (real-time PCR).The protein expression of PADI4,CyclinB1 and P21 was detected by Western blotting.The apoptosis of FLS cells in RA was examined by flow cytometry.The levels of IL-1β were detected by ELISA.T-test was used for statistical analysis.Results siRNA-PADI4 efficiently down-regulated the PADI4 expression compared with control group,1.00±0.20 vs 0.38±0.20 (t=9.607,P＜0.01),0.39±0.23(t=8.394,P＜0.01).FCM analysis showed that the percentage of apoptosis cells in PADI4 siRNA group in FLS was (5.4±0.6)％ (t=-19.223,P＜0.01) and (6.1±0.6)％ respectively (t=-24.229,P＜0.01),which was significantly higher than that in the control group in FLS (1.6±0.3)％.The expression of CyclinB1 protein was decreased,and P21 increased.The concentrations of IL-1β in culture medium of the transfected group were (26.8±0.7) ng/ml (t=-10.747,P＜0.01) and (27.7±0.7) ng/ml (t=-10.967,P＜0.01),higher than the control group [(23.9±0.7) ng/ml].Conclusion After being transfected with PADI4 siRNA,the apoptosis of FLS cells in RA is increased.Our results have demonstrated the potential role of CyclinB1 and P21 in PADI4 signaling.

Study on the single nucleotide polymorphisms of peptidylarginine deiminase 4 in patients with rheumatoid arthritis in Chinese Han population in Hebei province / 中华风湿病学杂志

Objective To elucidate the association of the susceptibility for rheumatoid arthritis ( RA )and peptidylarginine deiminase 4(PADI4) genetic single nucleotide polymorphism (SNP) in Han population in Hebei province.Methods This hospital-based ease-control study included 105 untreated RA patients and 96 healthy controls.The genotypes and allele frequencies of padi4_92 gene polymorphisms were analyzed by PCR-RFLP method,and the analysis of linkage disequilibrium and haplotype construction were performed for padi4_92,padi4_94 and padi4_104 SNPs.The Peason Chi-square test and Woolf statistic method were used to analyze the odds ration (OR) and 95％ confidence interval (95％CI).Results Significant differences in the frequency of PADI4 alleles and genotypes between the cases and controls were observed.The combined effect of padi4_92,padi4_94 and padi4_104 SNPs was analyzed by SHEsis snd Genehunter software,and got three haplotypes,CCC,GTT and GCC.There was significant difference in haplotypes distribution of 3 SNPs of padi4 between the two groups.This analysis of haplotypes revealed that haplotype of PADI4 was a risk factor for RA The ORs for these three haplotypes for RA susceptibility were 0.634 (95％CI=0.425-0.946),1.306 (95％CI=0.864-1.975),4.286 (95％CI=1.274-14.424),respectively.Conclnsion The SNPs of PADI4 may contribute to genetic susceptibility to RA in Han population in Hebei Province.

The significance of serum peptidylarginine deiminase 4 in rheumatoid arthritis / 中华内科杂志

Objective To detect the levels of serum peptidylarginine deiminase (PADI)4 and explore its significance in rheumatoid arthritis ( RA ). Methods The presence of PADI4, anti-PADI4antibodies and anti-cyclic citrullinated peptide (CCP) antibodies levels were examined by enzyme-linked immunosorbent assay (ELISA). Serum samples were obtained from 100 patients with RA, 23 patients with other rheumatic diseases, and 24 healthy controls. The associations between PADI4 and the disease activity score using 28 joint counts ( DAS28 ) score, anti-CCP antibodies, erythrocyte sedimentation rate ( ESR ),C-reactive protein ( CRP), PADI4-94 ( rs 2240340 ) and PADI-104 ( rs 1748033 ) gene polymorphisms and other indexes were analyzed in RA. Results The levels of PADI4 in RA patients were significantly higher than in other rheumatic diseases and healthy controls ( F = 8. 75, P ＜ 0. 001 ). There was no significantly difference in levels of PADI4 among ADI4-94 ( rs 2240340)/PADI-104 ( rs 1748033 ) genotypes. Conclusions PADI4 is associated with disease activity involved in the RA pathogenesis and may be a RA pathogenic antigen.

Association of polymorphism of peptidylarginine deaminase-4 gene and rheumatoid arthritis / 中华风湿病学杂志

Objective To investigate the distribution characteristics of single nucleotide polymorphisms (SNPs) of peptide arginine deaminase4 (PADI4)-94 and PADI4-104 with rheumatoid arthritis(RA)and study the association between PADI4-94/PADI4-104 SNPs and RA susceptibility.Methods PCR-LDR was used to identify the gene and genotype of 116 rheumatoid arthritis patients and 100 healthy controls and calculated the frequencies.Chi-square test was used to analyze the statistical significance.The presence of anti-PADI4 and PADI4 protein in the peripheral blood of patients with RA was examined using enzyme-linked three kinds of genotypes (A/A,G/G and A,G),PADI4-94 allele A,G and A/A,C/G and A/G genotype frequencies in RA group and healthy controls were 0.460/0.392,0.540/0.608,0.204/0.186,0.283/0.402 and 0.513/0.412,respectively.There was no significant difference between the two groups (X2=1.996,P=0.157;X2=3.407,P=0.182);PADI4-104 allele A,G and A/A,G/G and A/G genotype frequencies in RA group and healthy controls were 0.412/0.345,0.588/0.655,0.150/0.144,0.327/0.454 and 0.522/0.402,respectively.There no significant differences between PADI4-94/PADI4-104 genotypes and ESR,DAS28 score,CRP,anti-CCP antibodies,PADI4 protein,and anti-PADI4 antibodies(P values were 0.46/0.67,0.62/0.57,0.12/0.23,0.81/0.43,0.78/0.75,0.38/0.31).Conclusion The SNPs of PADI4-94 and PADI4-104 in Chinese RA population can be identified,but they may not be the susceptibility genotypes of RA.

The association between rheumatoid arthritis and single nucleotide polymorphisms of peptidyiarginine deiminase 4 in Han population of Hebei province / 中华风湿病学杂志

Objective To study the association of peptidylarginine deiminase 4 (PADI4) (padi4_94 and padi4_104) genetic single nucleotide polymorphism (SNP) and rheumatoid arthritis (RA) susceptibility in Han population of Hebei province. Methods This hospital-based case-control study included 115 RA patients and 106 healthy controls. All the individuals were recruited from Han Hebei residents and were randomly selected. The genotype and allele frequencies of PADI4 gene polymorphisms (padi4_94 and padi4_104) were analyzed by PCR-DNA sequencing method. Results The distribution of padi4_94 and padi4_104 genotypes between the two groups was not significantly deviated from that expected by Hardy-Weinberg equilibrium (P>0.05). The combined effect of padi4_94 and padi4_104 SNPs was analyzed and three haplotypos (AA, AG and GG) were found but without GA haplotype. The genotype and allele distribution of padi4_94 and padi4_104 in the patients with RA was not significantly different from that in healthy controls,and the analysis of haplotypes revealed that no haplotypes were risk factors for RA. Conclusion In Han population of Hebei province, the SNPs of PADI4 (padi4_94 or/and padi4_104) is no associated with RA susceptibility. Therefore, it should not be regarded as a genetic risk factor for RA.