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Synovial fluid (SF) accumulates extensively in joints of individuals with rheumatoid arthritis (RA), which reflects the pathological state of the synovium and disease activity. This study applied quasi-targeted liquid chromatography-mass spectrometry/mass spectrometry, an advanced metabolomics technique, to find characteristic metabolisms in RA.SF samples from the patients (n=20) were collected and examined using the metabolomic technique. SF samples from patients with osteoarthritis (OA) (n=20) were used as controls.Four hundred and seventy-nine variable metabolites were detected, and 250 of these metabolites were identified by searching the Human Metabolome Database (HMDB) and a self-constructed information list of possible metabolites. S-plot and volcano plot analysis detected 22 metabolites with differential levels in RA SF compared with those in OA SF. With these 22 candidate metabolites, pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database detected upregulation of pyrimidine metabolism and purine metabolism, and downregulation of fatty acid biosynthesis and unsaturated fatty acid biosynthesis in RA SF. Receiver operating characteristic (ROC) analysis and logistic regression models detected increased levels of guaiacol, naringenin, phenylpropanolamine and vanillylmandelic acid in RA SF. Furthermore, the naringenin level showed positive correlation with rheumatic factor (RF) and anti-cyclic citrillinated peptides (anti-CCP) levels.Our study suggests disturbed pyrimidine metabolism, purine metabolism, fatty acid biosynthesis and unsaturated fatty acid biosynthesis, as well as increased naringenin level, are characteristic metabolisms in RA.

Decreased nicotinamide adenine dinucleotide (NAD+) levels accompany aging. CD38 is the main cellular NADase. Cyanidin-3-O-glucoside (C3G), a natural inhibitor of CD38, is a well-known drug that extends the human lifespan. We investigated mechanisms of CD38 in cell senescence and C3G in antiaging. Myocardial H9c2 cells were induced to senescence with D-gal. CD38 siRNA, C3G and UBCS039 (a chemical activator of Sirt6) inhibited D-gal-induced senescence by reducing reactive oxygen species, hexokinase 2 and SA

Many studies have found that CD38 expression is increased in rheumatoid arthritis (RA), an autoimmune disease in which immune tolerance is dysregulated. Inhibition of CD38 expression or activity can significantly alleviate collagen-induced arthritis (CIA), a well-known animal model used for the study of RA. This study aimed to confirm the therapeutic effect of 78c, a specific inhibitor of CD38, and the role of CD38+ NK cells in immune imbalance in RA.CIA mice were injected with 78c to observe the therapeutic effect. CD38+ NK cells were extracted from human peripheral blood and treated with 78c. The pretreated NK cells were co-cultured with CD4+ T cells.We found that 78c significantly suppressed joint inflammation; reduced the levels of B cells, IL-6 and TNF-α; and increased the levels of IL-10, energy metabolism and spontaneous movement in CIA mice. 78c treatment also increased Treg cell numbers and decreased the Th1/Th2 ratio in the CIA model animals. Moreover, the proportion of CD38+ NK cells was increased in the CIA mice and significantly decreased following 78c treatment. Human CD4+ T cells that were co-cultured with 78c-pretreated CD38+ NK cells differentiated into more Treg cells and had lower Th17/Treg and Th1/Th2 ratios than CD4+ T cells co-cultured with CD38+ NK cells without the pretreatment. Transcriptomic analyses demonstrated that 78c changed expression pro les in CD38+ NK cells.These results suggested that 78c could be used for the treatment of RA and CIA as it alleviates the inhibitory effect of CD38+ NK cells on CD4+ T cell differentiation to Treg cells to restore immune balance.

PADI4, an enzyme catalyzing arginine residues to citrulline residues, is highly expressed in malignant tumors. This study prepared a monoclonal anti-human PADI4 antibody and investigated the therapeutic effect of the antibody on breast tumors and the functional mechanism.After treatment with PADI4 antibody, the changes in tumor-bearing mice were examined by PET-CT, pathological assays, biochemical tests, routine blood tests, cytokine assays and metabolic assays. We used PADI4 recombinant protein to catalyze fibronectin (Fn) and then used citrullinated fibronectin (Cit-Fn) to culture MDA-MB-231 cells. We also treated Cit-Fn cultured cells with PADI4 antibody. The cultured cells were examined using cell proliferation, apoptosis, colony formation, migration and glycolic ATP production. Citrullination in the tumor tissues and peripheral blood was measured using Western blotting and ELISA, respectively.Following PADI4 antibody treatment, tumor growth was significantly suppressed, and the number of apoptotic cells in tumor tissues was increased. The citrullination level in peripheral blood and tumor tissues was decreased, EMT-related gene expression in tumors was also decreased, and the spontaneous movement of tumor-bearing mice was increased following treatment. Following antibody treatment, the serum concentrations of IL-10, IL-12p70, IL-23, ALT and AST were significantly decreased. MDA-MB-231 cells treated with Cit-Fn showed increased cell proliferation, cell migration, colony formation and glycolytic ATP production and decreased apoptosis. The growth and migration of MDA-MB-231 cells were reduced following PADI4 antibody treatment, and PADI4 antibody inhibited the citrullination of fibronectin in vitro.The PADI4 antibody had a therapeutic effect on breast tumors by inhibiting the citrullination of fibronectin to change the tumor tissue microenvironment. PADI4 antibody is a potential means for tumor treatment.

Background. Rho guanine nucleotide exchange factor 10-like protein (ARHGEF10L) is a member of the guanine nucleotide exchange factor family, which regulates Rho GTPase activities, thus contributing to tumorigenesis. Our previous study demonstrated a strong association between the ARHGEF10L gene and the risk of cervical carcinoma. This study investigated the pathogenic role and mechanism of ARHGEF10L in cervical tumors. Methods. The HeLa cell line, which was derived from cervical carcinoma, was transfected with ARHGEF10L-overexpressing plasmids or anti-ARHGEF10L siRNA. Cell counting kit-8 assays, wound-healing assays, and cell apoptosis assays were performed to investigate the effects of ARHGEF10L on cell activities. A Rho pull-down assay and RNA-sequencing analysis were performed to investigate the pathogenic pathway of ARHGEF10L involvement in cervical tumors. Results. ARHGEF10L overexpression promoted cell proliferation and migration, reduced cell apoptosis, and induced epithelial-to-mesenchymal transition (EMT) via downregulation of E-cadherin and upregulation of N-cadherin and Slug in transfected HeLa cells. The overexpression of ARHGEF10L also upregulated GTP-RhoA, ROCK1, and phospho-ezrin/radixin/moesin (ERM) expression in HeLa cells. RNA-sequencing analysis detected altered transcription of 31 genes in HeLa cells with ARHGEF10L overexpression. Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) pathway analyses identified significant differences in cyclin-dependent protein serine/threonine kinase activity, cell responses to vitamin A, and Toll-like receptor signaling pathways. Both real-time PCR and Western blotting verified the increased expression of heat shock 70 kDa protein 6 (HSPA6) in ARHGEF10L-overexpressing HeLa cells. Since we reported that ARHGEF10L played a role through RhoA-ROCK1-ERM signaling, an important pathway in tumorigenesis, and stimulated EMT and HSPA6 expression in liver tumors and gastric tumor cells, we suggest that ARHGEF10L is a novel oncogene in many tumors.

Background: CD38+ NK (CD3− CD16+ CD38+ CD56+) cells were increased in rheumatoid arthritis (RA), which suppressed Treg cell differentiation. This study explored how CD38+ NK cells regulated CD4+ T-cell differentiation into Treg cells in RA. Methods: Proportions of CD38+ NK cells and their counterpart CD38+ NK-like T (CD3+ CD16+ CD38+ CD56+) cells were measured in RA and rats with collagen-induced arthritis (CIA). CD38+ NK cells and CD38+ NK-like T cells were cocultured with CD4+ T cells, respectively. Results: A significantly increased proportion of CD38+ NK cells and a decreased proportion of CD38+ NK-like T cells were detected in RA and CIA blood and synovial fluids. When CD4+ T cells were cocultured with CD38+ NK cells, mammalian target of rapamycin (mTOR) signaling was activated, and Th1/Th2 and Th17/Treg ratios were increased. When CD38+ NK cells were pretreated with anti-CD38 antibody, Treg cell proportion was increased, and Th1/Th2 and Th17/Treg ratios were decreased. CD38+ NK-like T cells showed the opposite results. CD38+ NK cells and CD38+ NK-like-T cells activated differential gene expressions and pathways in CD4+ T cells and initiated Th1 and Th2 cell differentiation by differential gene nodes. Conclusions: This study suggest that the high CD38+ NK cell proportion and low CD38+ NK-like T cell proportion in RA suppress Treg cell differentiation by stimulating mTOR signaling in CD4+ T cells, which consequentially disturbs the immune tolerance.

Background Increased CD38 expression and CD38+ cell proportion as well as their importance had been reported in rheumatoid arthritis (RA).Methods The proportion of lymphocyte subtypes in RA patients and rats with collagen-induced arthritis (CIA) was examined using flow cytometry. CD38+ NK cells, CD38+ NKT cells and CD4+ T cells as well as mononuclear cells (MNCs) depleted of CD38+ cells were isolated from RA synovial fluid using flow cytometry and cocultured in transwell apparatus.Results This study detected a significantly increased CD38+ NK cell proportion and a decreased CD38+ NKT cell proportion in RA peripheral blood and synovial fluid. The CD38+ NK/CD38+ NKT ratio was positively correlated with the disease activity. A similar result was observed in CIA rats. When CD38+ NK cells were cocultured with MNCs, the Treg cell proportion in MNCs and IL-10 level significantly decreased, and Th17 cell proportion and IFN-γ level increased. When the CD38+ NK cells were pretreated with monoclonal anti-CD38 antibody, Treg cell proportion and IL-10 level significantly increased, and the Th17 cell proportion and IFN-γ and IL-6 level decreased. When CD38+ NK cells were cocultured with CD4+ T cells, the Th1/Th2 and Th17/Treg ratios significantly increased, and mTOR signaling was activated in the cells. When the CD38+ NK cells were pretreated with the anti-CD38 antibody, the opposite result was obtained. Coculturing CD38+ NKT cells with MNCs or CD4+T cells showed opposite results. The anti-CD38 antibody also significantly increased TGF-β expression in the CD38+ NK cells.Conclusions Our results suggest that a high CD38+ NK and low CD38+ NKT proportion in RA elevates Th1/Th2 and Th17/Treg ratio to contribute to the pathogenesis.

Objective. Regulator of chromosome condensation 2 (RCC2) has been reported to be involved in the regulation of cell cleavage. This study investigated the effect of RCC2 expression on breast tumorigenesis. Methods. MCF-7 cells originating from estrogen receptor-positive (ER+) breast cancer were transfected with anti-RCC2 siRNA or RCC2-expressing plasmids. Cell proliferation, apoptosis, migration, and cytokine production in the transfected cells were examined using the CCK-8 assay, wound healing assay, and flow cytometry, respectively. PCR array was used to investigate the tumorigenic pathway of RCC2 in MCF-7 cells transfected with the anti-RCC2 siRNA. MCF-7 cells were also transfected with lentivirus-containing anti-RCC2 short hairpin RNA and were injected into BALB/c nude mice to generate tumor-bearing mice. Tumor growth in the mouse model was examined using magnetic resonance imaging by diffusion-weighted imaging analysis. Results. Western blotting and immunohistochemistry detected significantly increased expression of RCC2 in ER + breast tumor tissues compared with breast fibroadenoma samples. Inhibiting RCC2 expression decreased cell migration and stimulated apoptosis in MCF-7 cells, while overexpressing RCC2 stimulated cell migration and inhibited apoptosis. The inhibition of RCC2 expression significantly decreased breast tumor growth and IL-6 levels in the tumor-bearing mice. PCR array demonstrated that inhibiting RCC2 expression significantly decreased the expression of IGF1 and TWIST1, two well-known tumor-enhancing genes, in MCF-7 cells; conversely, overexpressing RCC2 increased the expression levels of these two genes in the transfected cells. This result was verified in the mouse model following inhibition of RCC2 expression in MCF-7 cells. Additionally, estradiol-17β suppressed MCF-7 cell apoptosis, stimulated cell proliferation and cell migration, and increased RCC2, IGF1, and TWIST1 expression. The siRNA-mediated inhibition of RCC2 expression alleviated the inhibitory effects of estrogen on apoptosis in MCF-7 cells, while overexpressing RCC2 enhanced the estrogen-driven inhibition of apoptosis. Modifying RCC2 expression had no impact on MCF-7 cell proliferation in the presence or absence of estradiol-17β. Conclusions. Our results suggest that estrogen-induced RCC2 expression prompts IGF1, TWIST1, and IL-6 expression, stimulates cell migration, and inhibits apoptosis to contribute to ER + breast tumorigenesis.

PADI4 post-translationally converts peptidylarginine to citrulline. PADI4 can disrupt the apoptotic process via the citrullination of histone H3 in the promoter of p53-target genes. The current study focused on PADI4 expression in various subtypes of oesophageal carcinoma (EC) by immunohistochemistry, western blotting and real time PCR. The study also investigated the effect of bile acid deoxycholate (DCA) on PADI4 expression in Eca-109 cells that originated from EC. Apoptosis and DCA-induced toxicity were analyzed by TUNEL, MTT assay and flow cytometry. Additionally, the present study investigated the correlation between single nucleotide polymorphism (SNP) in PADI4 gene and EC risk in Chinese population using Illumina GoldenGate assay. Compared with paraneoplastic tissues, the transcriptional and translational levels of PADI4 were significantly elevated in oesophageal squamous cell carcinoma (ESCC, n=9) and oesophageal adenocarcinoma (EAC, n=5) tissues. Immunolabeling detected expression of PADI4 in ESCC tissues (98.56%, n=139), EAC samples (87.5%, n=16) and oesophageal small cell undifferentiated carcinoma (91.7%, n=12) but not in normal tissues (0%, n=16). Furthermore, PADI4 levels is positively correlated with the pathological classification of ESCC (p=0.009). PADI4 expression levels were consistent with the number of apoptotic cells in the induced Eca-109 cells. rs10437048 [OR= 0.012831; 95% CI, 0.001746~0.094278; p=1.556×10-12] were significantly associated with decreased risk of EC, whereas rs41265997 [OR=12.7; 95% CI, 0.857077~33.207214; p=3.896×10-8] were significantly associated with increased risk of EC. rs41265997 in exon 3 of PADI4 gene is non-synonymous and converts ACG to ATG resulting in a threonine /methionine conversion at position 274 of the protein. Haplotypes GC that carries the variant alleles for rs2501796 and rs2477134 was significantly associated with increased risk of EC (frequency=0.085, p=0.0256, OR=2.7). The results suggest that PADI4 expression is related to the tumorigenic process of EC and the DCA-induced apoptosis. The PADI4 gene may be a valid EC susceptibility gene.

PADI4 post-translationally converts peptidylarginine to citrulline. PADI4 can disrupt the apoptotic process via the citrullination of histone H3 in the promoter of p53-target genes. The current study focused on PADI4 expression in various subtypes of oesophageal carcinoma (EC) by immunohistochemistry, western blotting and real time PCR. The study also investigated the effect of bile acid deoxycholate (DCA) on PADI4 expression in Eca-109 cells that originated from EC. Apoptosis and DCA-induced toxicity were analyzed by TUNEL, MTT assay and flow cytometry. Additionally, the present study investigated the correlation between single nucleotide polymorphism (SNP) in PADI4 gene and EC risk in Chinese population using Illumina GoldenGate assay. Compared with paraneoplastic tissues, the transcriptional and translational levels of PADI4 were significantly elevated in oesophageal squamous cell carcinoma (ESCC, n=9) and oesophageal adenocarcinoma (EAC, n=5) tissues. Immunolabeling detected expression of PADI4 in ESCC tissues (98.56%, n=139), EAC samples (87.5%, n=16) and oesophageal small cell undifferentiated carcinoma (91.7%, n=12) but not in normal tissues (0%, n=16). Furthermore, PADI4 levels is positively correlated with the pathological classification of ESCC (p=0.009). PADI4 expression levels were consistent with the number of apoptotic cells in the induced Eca-109 cells. rs10437048 [OR= 0.012831; 95% CI, 0.001746~0.094278; p=1.556×10(-12)] were significantly associated with decreased risk of EC, whereas rs41265997 [OR=12.7; 95% CI, 0.857077~33.207214; p=3.896×10(-8)] were significantly associated with increased risk of EC. rs41265997 in exon 3 of PADI4 gene is non-synonymous and converts ACG to ATG resulting in a threonine /methionine conversion at position 274 of the protein. Haplotypes GC that carries the variant alleles for rs2501796 and rs2477134 was significantly associated with increased risk of EC (frequency=0.085, p=0.0256, OR=2.7). The results suggest that PADI4 expression is related to the tumorigenic process of EC and the DCA-induced apoptosis. The PADI4 gene may be a valid EC susceptibility gene.

This study used a proteomic approach to screen the proteins with decreased expression in the synovial tissues of rheumatoid arthritis (RA) patients by comparing their expression profiles to that of osteoarthritis (OA) and ankylosing spondylitis (AS) patients. The result was complemented by a SNP analysis.Proteins extracted from the synovial membranes (n=10 for each disease) were separated by 2-D electrophoresis. The proteins with significantly decreased expression in the RA samples were subjected to MALDI-TOF/TOF MS. The result was verified using western blotting. Tag SNPs located in the targeted gene were assessed using the Taqman assay in a cohort of 267 Chinese patients with RA, 51 patients with AS and 160 healthy controls. The genotyping result was confirmed in a large cohort of 389 patients with RA, 200 patients with AS and 371 healthy controls.The proteomic approach detected significantly decreased expression of vitamin D-binding protein (VDBP) in the synovial membranes from patients with RA, which was confirmed with western blot analysis. rs2282679 was significantly associated with RA and AS (p=0.026794 and 0.007566, respectively). The result was confirmed in a large cohort of RA (OR=0.678639, 95%CI=[0.541113~0.851118], p=0.000776) and AS (OR=0.564053, 95%CI=[0.433716~0.733558], p=1.79e-005).1,25-dihydroxyvitamin D3 inhibits cell proliferation, immunoglobulin production and the release of cytokines through binding to VDBP. VDBP also mediates bone resorption by activating osteoclasts. The decreased expression and the genetic effect of VDBP in RA suggest a novel pathogenic pathway that vitamin D contributes to the arthritic process of the disease.

Introduction Expression of TXNDC5, which is induced by hypoxia, stimulates cell proliferation and angiogenesis. Our previous study detected increased TXNDC5 expression in the synovial tissues of rheumatoid arthritis (RA) patients using proteomic methods. The current study investigated a pathogenic role for TXNDC5 in RA. Method Expression of TXNDC5 in synovial membranes was quantitatively analyzed by immunohistochemistry, Western blotting and real-time polymerase chain reaction (PCR). Serum TXNDC5 levels and serum anti-TXNDC5 antibody levels were determined using sandwich enzyme-linked immunosorbent assay (ELISA). A total of 96 single nucleotide polymorphisms (SNPs) in or near the TXNDC5 gene were genotyped using custom-designed Illumina 96-SNP VeraCode microassay. Allele frequencies and genotype frequencies of SNPs were assessed using a case-control design in a cohort of 267 Chinese patients with RA, 51 patients with ankylosing spondylitis (AS) and 160 healthy controls. Additional genotyping of 951 patients with RA and 898 healthy controls was performed for four SNPs (rs2277105, rs369086, rs443861 and rs11962800) using the TaqMan method. Results Real-time PCR, Western blotting and immunohistochemistry detected significantly higher TXNDC5 expression in the synovial tissues of RA patients compared to samples from patients with osteoarthritis (OA) or AS. ELISA detected significantly higher levels of TXNDC5 in the blood of RA patients compared to OA, AS and systemic lupus erythematosus patients, and healthy controls. ELISA did not detect significantly different levels of anti-TXNDC5 antibody in the blood of RA, OA and AS patients and healthy controls. A total of 9 SNPs (rs9505298, rs41302895, rs1225936, rs1225938, rs372578, rs443861, rs408014, rs9392189 and rs2743992) showed significant association with RA, while 16 SNPs (rs1044104, rs1225937, rs1225938, rs372578, rs89715, rs378963, rs1225944, rs1225947, rs1238994, rs369086, rs408014, rs368074, rs1225954, rs1225955, rs13209404 and rs3812162) showed significant association with AS. Taqman SNP assay demonstrated that rs443861 has an association with RA, which correlates with the microassay results. Conclusions TXNDC5 is up-regulated in synovial tissues of RA patients. TXNDC5 has a genetic effect on the risk of RA and AS.

PADI4 post-translationally converts peptidylarginine to citrulline, a process called citrullination. Studies have demonstrated the high expression of PADI4 in various malignant tumour tissues. PADI4 is also expressed at high levels in the blood of patients with some malignant tumours. Thus far, citrullination of histone, cytokeratin, antithrombin and fibronectin have been confirmed to be involved in abnormal apoptosis, high coagulation, and disordered cell proliferation and differentiation, all of which are main features of malignant tumours. PADI4 is expressed in CD34+ stem cells in normal tissues, and many more CD34+ cells expressing PADI4 are present in tumour tissues. These findings suggest that PADI4 may play an important role in tumourigenesis.