Introduction: Fibroblast-like synoviocytes (FLS) play an important role in the pathogenesis of rheumatoid arthritis (RA). This study aimed to investigate the role of glucose 6-phosphate isomerase (GPI) in the proliferation of RA-FLS. Methods: The distribution of GPI in synovial tissues from RA and osteoarthritis (OA) patients was examined by immunohistochemical analysis. FLS were isolated and cultured, cellular GPI level was detected by real-time polymerase chain reaction (PCR) and Western blot analysis, and secreted GPI was detected by Western blot and enzyme-linked immunosorbent assay (ELISA). Doxorubicin (Adriamycin, ADR) was used to induce apoptosis. Cell proliferation was determined by MTS assay. Flow cytometry was used to detect cell cycle and apoptosis. Secreted pro-inflammatory cytokines were measured by ELISA. Results: GPI was abundant in RA-FLS and was an autocrine factor of FLS. The proliferation of both RA and OA FLS was increased after GPI overexpression, but was decreased after GPI knockdown. Meanwhile, exogenous GPI stimulated, while GPI antibody inhibited, FLS proliferation. GPI positively regulated its receptor glycoprotein 78 and promoted G1/S phase transition via extracellular regulated protein kinases activation and Cyclin D1 upregulation. GPI inhibited ADR-induced apoptosis accompanied by decreased Fas and increased Survivin in RA FLS. Furthermore, GPI increased the secretion of tumor necrosis factor-α and interleukin-1β by FLS. Conclusions: GPI plays a pathophysiologic role in RA by stimulating the proliferation, inhibiting the apoptosis, and increasing pro-inflammatory cytokine secretion of FLS.

The higher level of Glucose-6-phosphate isomerase (G6PI) has been found in both synovial tissue and synovial fluid of rheumatoid arthritis (RA) patients, while the function of G6PI in RA remains unclear. Herein we found the enrichment of G6PI in microvascular endothelial cells of synovial tissue in RA patients, where a 3% O₂ hypoxia environment has been identified. In order to determine the correlation between the high G6PI level and the low oxygen concentration in RA, a hypoxia condition (~3% O₂) in vitro was applied to mimic the RA environment in vivo. Hypoxia promoted cellular proliferation of rheumatoid arthritis synovial fibroblasts (RASFs), and induced cell migration and angiogenic tube formation of human dermal microvascular endothelial cells (HDMECs), which were accompanied with the increased expression of G6PI and HIF-1α. Through application of G6PI loss-of-function assays, we confirmed the requirement of G6PI expression for those hypoxia-induced phenotype in RA. In addition, we demonstrated for the first time that G6PI plays key roles in regulating VEGF secretion from RASFs to regulate the hypoxia-induced angiogenesis in RA. Taken together, we demonstrated a novel pathway regulating hypoxia-induced angiogenesis in RA mediated by G6PI.

Chronic kidney disease (CKD) affects more than 10% of the global population and is associated with significant morbidity and mortality. In most cases, this disease is developed silently, and it can progress to the end-stage renal failure. Therefore, early detection becomes critical for initiating effective interventions. Routine diagnosis of CKD requires both blood test and urinalyses in a clinical laboratory, which are time-consuming and have low sensitivity and specificity. Surface-enhanced Raman scattering (SERS) is an emerging method for rapidly assessing kidney function or injury. This study was designed to compare the differences between the SERS properties of the serum and urine for easy and simple detection of CKD. Enrolled for this study were 126 CKD patients (Stages 2–5) and 97 healthy individuals. SERS spectra of both the serum and urine samples were acquired using a Raman spectrometer (785 nm excitation). The correlation of chemical parameters of kidney function with the spectra was examined using prinicpal component analysis (PCA) combined with linear discriminant analysis (LDA) and partial least squares (PLS) analysis. Here, we showed that CKD was discriminated from non-CKD controls using PCA–LDA with a sensitivity of 74.6% and a specificity of 93.8% for the serum spectra, and 78.0% and 86.0 % for the urine spectra. The integration area under the receiver operating characteristic curve was 0.937 ± 0.015 ( p < 0.0001) for the serum and 0.886 ± 0.025 ( p < 0.0001) for the urine. The different stages of CKD were separated with the accuracy of 78.0% and 75.4% by the serum and urine spectra, respectively. PLS prediction (R ² ) of the serum spectra was 0.8540 for the serum urea ( p < 0.001), 0.8536 for the serum creatinine ( p < 0.001), 0.7500 for the estimated glomerular filtration rate (eGFR) ( p < 0.001), whereas the prediction (R ² ) of urine spectra was 0.7335 for the urine urea ( p < 0.001), 0.7901 for the urine creatinine ( p < 0.001), 0.4644 for the eGFR ( p < 0.001) and 0.6579 for the urine microalbumin ( p < 0.001). In conclusion, the accuracy of associations between SERS findings of the serum and urine samples with clinical conclusions of CKD diagnosis in this limited number of patients is similar, suggesting that SERS may be used as a rapid and easy-to-use method for early screening of CKD, which however needs further evaluation in a large cohort study.

INTRODUCTION: Emerging evidence has demonstrated the potential of the circulating tumor DNA (ctDNA) methylation in the application of cancer diagnosis. METHODS: Three genes including Septin9, Syndecan-2 (SDC2), and branched-chain amino acid transaminase 1 (BCAT1), which have been well demonstrated to have aberrant expression in colorectal cancer (CRC) as tumor suppressors, were selected for detection. A total of 234 peripheral plasma samples from 104 patients with CRC and 130 patients with colorectal polyps, and 60 plasma samples from healthy controls, were collected before any treatment. A real-time polymerase chain reaction-based gene panel was used to detect the methylation of Septin9, SDC2, and BCAT1. The composite score (P) was calculated according to the cycle threshold values of the 3 methylated genes using the logistic regression equation. RESULTS: The ctDNA methylation of the 3 genes had a significantly higher level in patients with CRC, compared with patients with colorectal polyps and healthy controls. The composite score (P) showed association with tumor stages in CRC but not with the tumor location (colon or rectum). In addition, BCAT1 and Septin9 showed better performance for CRC diagnosis, by which CRC was able to distinguish from polyps with sensitivity of 83.7%, specificity of 93.9%, and area under the curve of 0.908. The diagnostic efficiency was significantly improved by combining composite score (P), carcinoembryonic antigen, and fecal immunochemical test for hemoglobin (area under the curve = 0.962). DISCUSSION: The composite score (P) derived from the ctDNA methylation levels of Septin9, SDC2, and BCAT1 can be used for CRC diagnosis with high sensitivity and high specificity. A combination of ctDNA methylation, carcinoembryonic antigen, and fecal immunochemical test for hemoglobin was proved to be the most effective approach to diagnose CRC.

Hypoxia plays an important role in the proliferation of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS), leading to pathology of RA. This study was conducted to evaluate hypoxia-induced microRNAs (hypoxamiR) in RA-FLS and its role in the function of RA-FLS. RA-FLS were cultured under normoxia (21% O2) or hypoxia (3% O2) condition, followed by a microRNA (miRNA) array analysis. The upregulation of miR-191 by hypoxia was confirmed in RA-FLS and FLS from osteoarthritis (OA) patients by quantitative real-time polymerase chain reaction (RT-PCR). Transfection of miR-191 mimic and inhibitor was used to investigate the function of miR-191 in RA-FLS. The functional targets of miR-191 were predicted by bioinfomatics and then validated by reporter gene assay. A subset of miRNAs was identified to be induced by hypoxia including miR-191. The upregulation of miR-191 was found to be specific in hypoxic RA-FLS, compared to hypoxic OA-FLS. We observed that miR-191 in RA-FLS increased cellular proliferation via promoting G1/S transition of the cell cycle and suppressed cell apoptosis induced by cell starvation. Bioinformatical analysis and experimental assays identified CCAAT/enhancer binding protein β (C/EBPβ) as a target gene of miR-191 in RA-FLS. Enforced expression of C/EBPβ rescued the cellular phenotypes induced by miR-191. In addition, an inverse correlation between the C/EBPβ level and hypoxia stimulation was found in RA-FLS, and overexpression of C/EBPβ could partly rescue the hypoxia-induced cell proliferation. We demonstrated the miR-191-C/EBPβ signaling pathway mediating the hypoxia-induced cell proliferation in RA.