RABC: Rheumatoid Arthritis Bioinformatics Center.

Advances in sequencing technologies have led to the rapid growth of multi-omics data on rheumatoid arthritis (RA). However, a comprehensive database that systematically collects and classifies the scattered data is still lacking. Here, we developed the Rheumatoid Arthritis Bioinformatics Center (RABC, http://www.onethird-lab.com/RABC/), the first multi-omics data resource platform (data hub) for RA. There are four categories of data in RABC: (i) 175 multi-omics sample sets covering transcriptome, epigenome, genome, and proteome; (ii) 175 209 differentially expressed genes (DEGs), 105 differentially expressed microRNAs (DEMs), 18 464 differentially DNA methylated (DNAm) genes, 1 764 KEGG pathways, 30 488 GO terms, 74 334 SNPs, 242 779 eQTLs, 105 m6A-SNPs and 18 491 669 meta-mQTLs; (iii) prior knowledge on seven types of RA molecular markers from nine public and credible databases; (iv) 127 073 literature information from PubMed (from 1972 to March 2022). RABC provides a user-friendly interface for browsing, searching and downloading these data. In addition, a visualization module also supports users to generate graphs of analysis results by inputting personalized parameters. We believe that RABC will become a valuable resource and make a significant contribution to the study of RA.

Ten Years of EWAS.

Epigenome-wide association study (EWAS) has been applied to analyze DNA methylation variation in complex diseases for a decade, and epigenome as a research target has gradually become a hot topic of current studies. The DNA methylation microarrays, next-generation, and third-generation sequencing technologies have prepared a high-quality platform for EWAS. Here, the progress of EWAS research is reviewed, its contributions to clinical applications, and mainly describe the achievements of four typical diseases. Finally, the challenges encountered by EWAS and make bold predictions for its future development are presented.

Ellagic acid inhibits high glucose-induced injury in rat mesangial cells via the PI3K/Akt/FOXO3a signaling pathway.

The pathological damage of mesangial cells serves an important role in the occurrence and development of diabetic nephropathy. Ellagic acid has been reported to possess antioxidant, antitumor, antiviral and anti-inflammatory properties in several diseases, but the roles of ellagic acid in diabetic nephropathy are unclear. The main aim of the present study was to investigate the effect of ellagic acid on high glucose-induced mesangial cell damage. The results revealed that high glucose could induce the hyperproliferation of mesangial cells, decrease the activity of superoxide dismutase, increase the malondialdehyde content, the level of reactive oxygen species, the secretion of inflammatory factors (TNF-α, IL-1ß and IL-6) and the synthesis of extracellular matrix (Fibronectin, MMP-9 and TIMP-1) and activate the PI3K/Akt/FOXO3a signaling pathway. Ellagic acid could attenuate the injury of mesangial cells induced by high glucose in a concentration-dependent manner and its effect was consistent with that of a PI3K inhibitor (LY294002). Moreover, a PI3K agonist (740Y-P) reversed the protective effect of ellagic acid on mesangial cells induced by high glucose. In conclusion, ellagic acid protected mesangial cells from high glucose-induced injury in a concentration-dependent manner. The mechanism may be associated with ellagic acid inhibiting the activation of the PI3K/Akt signaling pathway and reducing the expression levels of downstream transcription factor FOXO3a.

The role of TRPC6 in α1-AR activation-induced calcium signal changes in human podocytes.

BACKGROUND: An accumulating amount of evidence has suggested that there is a contributive role of sympathetic nervous hyperactivity in the pathogenesis of chronic kidney disease (CKD). α1-AR promotes an increase in calcium levels in podocytes and adjusts podocyte contraction. Changes in TRPC6 expression and function can directly affect the podocyte cytoskeleton, which is a key component in podocyte injury. This study proposed to clarify the correlation between α1-AR activation-induced signal cascade reaction and TRPC6 in human podocytes. METHODS: Human podocytes were incubated with the calcium probe Fluo-3/AM. Next, the effects of the α1-AR agonists or antagonists and nonselective TRPC6 blockers on intracellular calcium were observed under laser confocal microscopy. FITC-phalloidin was employed to stain podocytes, and the change of F-actin under the α1-AR activation condition was observed. RESULTS: The α1-AR agonist PE (phenylephrine hydrochloride) induced an increase in intracellular Ca2+ ([Ca2+]i) in human podocytes. Moreover, the downregulation of TRPC6 by siRNA or TRPC blocker could attenuate the PE-induced [Ca2+]i elevation in a phospholipase C (PLC)-dependent pattern. When podocytes were stimulated to the PE, their F-actin fiber cytoskeletal structure was lost. PE subsequently increased the expression of RhoA, and the TRPC6-dependent Ca2+ influx was involved in this process. The abnormal activation of RhoA could result in disturbance of the podocyte skeleton structure, thus leading to podocyte injury. CONCLUSIONS: We concluded that TRPC6 is involved in α1-AR activation-induced calcium signal changes in podocytes. Meanwhile, the α1-AR agonists can destroy the cell's cytoskeletal structure, which is mediated by TRPC6 via the RhoA/ROCK pathway.

Genetic reduction of cilium length by targeting intraflagellar transport 88 protein impedes kidney and liver cyst formation in mouse models of autosomal polycystic kidney disease.

Polycystin-1 (PC1) and -2 (PC2), products of the PKD1 and PKD2 genes, are mutated in autosomal dominant polycystic kidney disease (ADPKD). They localize to the primary cilia; however, their ciliary function is in dispute. Loss of either the primary cilia or PC1 or PC2 causes cyst formation. However, loss of both cilia and PC1 or PC2 inhibits cyst growth via an unknown pathway. To help define a pathway, we studied cilium length in human and mouse kidneys. We found cilia are elongated in kidneys from patients with ADPKD and from both Pkd1 and Pkd2 knockout mice. Cilia elongate following polycystin inactivation. The role of intraflagellar transport proteins in Pkd1-deficient mice is also unknown. We found that inactivation of Ift88 (a gene expressing a core component of intraflagellar transport) in Pkd1 knockout mice, as well as in a new Pkd2 knockout mouse, shortened the elongated cilia, impeded kidney and liver cystogenesis, and reduced cell proliferation. Multi-stage in vivo analysis of signaling pathways revealed ß-catenin activation as a prominent, early, and sustained event in disease onset and progression in Pkd2 single knockout but not in Pkd2.Ift88 double knockout mouse kidneys. Additionally, AMPK, mTOR and ERK pathways were altered in Pkd2 single knockout mice but only AMPK and mTOR pathway alteration were rescued in Pkd2.Ift88 double knockout mice. Thus, our findings advocate an essential role of polycystins in the structure and function of the primary cilia and implicate ß-catenin as a key inducer of cystogenesis downstream of the primary cilia. Our data suggest that modulating cilium length and/or its associated signaling events may offer novel therapeutic approaches for ADPKD.

miR-25-3p promotes proliferation and inhibits autophagy of renal cells in polycystic kidney mice by regulating ATG14-Beclin 1.

MicroRNAs are involved in the regulation of the autophagy and proliferation in several diseases. This study aims to verify the role of miR-25-3p in the proliferation and autophagy of renal cells in polycystic kidney disease (PKD). We found that kidney to body weight and blood urea content were increased in PKD mice. Cystic dilations were increased in kidney tissue from PKD mice, and autophagy-related protein ULK1 and the ratio of LC3-II/LC3-I were decreased, indicating autophagy was inhibited in PKD mice. In addition, miR-25-3p was upregulated in PKD mice, and inhibition of miR-25-3p decreased cystic dilations in kidney tissues, increased ULK1 expression and the ratio of LC3-II/LC3-I, indicating inhibition of miR-25-3p enhanced the autophagy in PKD. Besides, inhibition of miR-25-3p suppressed the proliferation of renal cells and downregulated E2F-1 and PCNA expressions. Importantly, miR-25-3p targetedly suppressed ATG14 expression in PKD cells. Finally, silencing ATG14 abolished the inhibition effect of miR-25-3p inhibitor on renal cell proliferation, and reversed the inhibition effect of miR-25-3p inhibitor on E2F-1 and PCNA expressions in in vitro and in vivo experiments, which suggested that ATG14 was involved in the regulation of miR-25-3p-mediated kidney cell proliferation. Therefore, inhibition of miR-25-3p promoted cell autophagy and suppressed cell proliferation in PKD mice through regulating ATG14.

Ginsenoside Rg3 Reduces Epithelial-Mesenchymal Transition Induced by Transforming Growth Factor-ß1 by Inactivation of AKT in HMrSV5 Peritoneal Mesothelial Cells.

BACKGROUND Ginsenosides, including ginsenoside Rg3, are components of Panax ginseng C.A. Meyer (Araliaceae) used in traditional Chinese medicine. Long-term peritoneal dialysis induces peritoneal fibrosis that impairs ultrafiltration and is associated with epithelial-mesenchymal transition (EMT) of peritoneal cells. This study aimed to investigate the effects of ginsenoside Rg3 on EMT induced by transforming growth factor-ß1 (TGF-ß1) in HMrSV5 human peritoneal mesothelial cells. MATERIAL AND METHODS The cell counting kit-8 (CCK-8) assay measured HMrSV5 cell viability. The expression of EMT markers, E-cadherin, vimentin, and alpha-smooth muscle actin (alpha-SMA) were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The wound-healing assay determined cell migration. The S-phase of the cell cycle was assessed by 5-ethynyl-2'-deoxyuridine (EdU) labeling, and expression of phosphorylated AKT was measured by Western blot. The effect of ginsenoside Rg3 and the AKT activator SC79 on the TGF-ß1-induced EMT of HMrSV5 cells were evaluated. RESULTS Low concentration of ginsenoside Rg3 did not effect cell viability of HMrSV5 cells. TGF-ß1 treatment decreased the expression of E-cadherin, and increased the expression of vimentin and alpha-SMA and promoted cell migration of HMrSV5 cells. However, co-treatment of ginsenoside Rg3 and TGF-ß1 significantly reduced TGF-ß1-induced EMT in HMrSV5 cells. TGF-ß1 increased the phosphorylation of AKT and increased the expression of Smurf2. Ginsenoside Rg3 reduced TGF-ß1-induced activation of AKT and Smurf2. SC79 reversed the effects of ginsenoside Rg3 on TGF-ß1-induced EMT in HMrSV5 cells. CONCLUSIONS Ginsenoside Rg3 inhibited EMT induced by TGF-ß1 in HMrSV5 human peritoneal mesothelial cells by inhibiting the activation of AKT.

microRNA-612 suppresses the malignant development of non-small-cell lung cancer by directly targeting bromodomain-containing protein 4.

Background: Aberrant expression of microRNAs (miRNAs) in non-small-cell lung cancer (NSCLC) has been reported. Dysregulation of miRNAs exerts tumor-suppressing or tumor-promoting actions on the pathology and biological behaviors of NSCLC. miR-612 is associated with many types of human cancer; however, the expression, potential roles, and regulatory mechanisms of miR-612 in NSCLC remain unclear. Material and methods: Here, the expression level of miR-612 in NSCLC tissue specimens and a panel of cell lines were evaluated by RT-qPCR. Cell-Counting Kit 8, flow cytometry, Transwell migration and invasion, and in vivo tumor growth assays were performed to determine the functional role of miR-612 in malignant phenotypes of NSCLC cells. The molecular mechanism underlying the tumor-suppressive roles of miR-612 in NSCLC was investigated. Results: miR-612 was expressed at low levels in NSCLC, and low miR-612 expression was significantly correlated with TNM stage and lymph node metastasis. NSCLC patients with low miR-612 expression had shorter overall survival rate than those with high levels. Exogenous miR-612 expression decreased proliferation, migration, and invasion, and promoted apoptosis of NSCLC cells in vitro. miR-612 upregulation hindered NSCLC tumor growth in vivo. Bromodomain-containing protein 4 (BRD4) was confirmed as a direct target gene of miR-612 in NSCLC cells. BRD4 was obviously overexpressed in human NSCLC tissues and inverse correlated with miR-612 expression. Inhibition of BRD4 expression simulated the tumor-suppressive functions of miR-612 overexpression in NSCLC cells. Reintroduction of miR-612 expression abrogated the miR-612-mediated suppressive effects on NSCLC cells. BRD4 upregulation inhibited activation of the PI3K/Akt pathway in NSCLC cells in vitro and in vivo. Conclusion: This study supports the first evidence that miR-612 exerts tumor-suppressive roles in the aggressive behaviors of NSCLC cells in vitro and in vivo through direct targeting BRD4 and deactivating the PI3K/Akt pathway. Thus, miR-612 might be a promising target for anticancer therapies in patients with NSCLC.

LncRNA MIR210HG promotes proliferation and invasion of non-small cell lung cancer by upregulating methylation of CACNA2D2 promoter via binding to DNMT1.

Background: In recent years, a large number of studies have shown that differentially expressed lncRNAs are capable of promoting the occurrence and development of tumors by regulating cell proliferation and differentiation. However, the biological effects of lncRNAs in non-small cell lung cancer (NSCLC) are still needed to be further investigated. Methods: The differentially expressed lncRNAs in NSCLC tissues in the downloaded profiles from GEO database were analyzed and further verified in 100 pairs of NSCLC samples collected in our hospital. After identification of the target gene MIR210HG, the relationship between MIR210HG expression and clinical data of NSCLC patients was analyzed. Regulatory effects of MIR210HG on proliferation, migration, and invasion of NSCLC cells were detected by CCK-8, colony formation, and transwell assay, respectively. The binding condition of MIR210HG and DNA methyltransferase 1 (DNMT1) was detected by RNA binding protein immunoprecipitation. Subsequently, chromatin immunoprecipitation assay assessed the promoter binding of DNMT1 to CACNA2D2. Rescue experiments were conducted to assess whether CACNA2D2 can reverse the function of MIR210HG. Results: MIR210HG was highly expressed in NSCLC tissues not only in GSE30219 dataset but also in our collected NSCLC tissues. MIR210HG expression was correlated to tumor stage and lymph node metastasis of NSCLC patients. Besides, lower disease-free survival (DFS) and overall survival (OS) were found in NSCLC patients with high-level MIR210HG compared with those with low-level MIR210HG. Regression analysis indicated that MIR210HG was the independent risk factor for DFS and OS of NSCLC patients. In vitro experiments demonstrated that MIR210HG knockdown remarkably inhibited proliferation and migration of NSCLC cells. MIR210HG could recruit DNMT1, thereafter promoting methylation of CACNA2D2 promoter region. CACNA2D2 overexpression remarkably inhibited cell proliferation. Moreover, inhibited proliferation induced by MIR210HG knockdown was reversed by CACNA2D2 knockdown. Conclusion: MIR210HG can promote the tumorigenesis of NSCLC by inhibiting the expression of CACNA2D2. Our findings provide new therapeutic strategies for the future treatment of NSCLC.

EWASdb: epigenome-wide association study database.

DNA methylation, the most intensively studied epigenetic modification, plays an important role in understanding the molecular basis of diseases. Furthermore, epigenome-wide association study (EWAS) provides a systematic approach to identify epigenetic variants underlying common diseases/phenotypes. However, there is no comprehensive database to archive the results of EWASs. To fill this gap, we developed the EWASdb, which is a part of 'The EWAS Project', to store the epigenetic association results of DNA methylation from EWASs. In its current version (v 1.0, up to July 2018), the EWASdb has curated 1319 EWASs associated with 302 diseases/phenotypes. There are three types of EWAS results curated in this database: (i) EWAS for single marker; (ii) EWAS for KEGG pathway and (iii) EWAS for GO (Gene Ontology) category. As the first comprehensive EWAS database, EWASdb has been searched or downloaded by researchers from 43 countries to date. We believe that EWASdb will become a valuable resource and significantly contribute to the epigenetic research of diseases/phenotypes and have potential clinical applications. EWASdb is freely available at http://www.ewas.org.cn/ewasdb or http://www.bioapp.org/ewasdb.

EWAS: epigenome-wide association study software 2.0.

Motivation: With the development of biotechnology, DNA methylation data showed exponential growth. Epigenome-wide association study (EWAS) provide a systematic approach to uncovering epigenetic variants underlying common diseases/phenotypes. But the EWAS software has lagged behind compared with genome-wide association study (GWAS). To meet the requirements of users, we developed a convenient and useful software, EWAS2.0. Results: EWAS2.0 can analyze EWAS data and identify the association between epigenetic variations and disease/phenotype. On the basis of EWAS1.0, we have added more distinctive features. EWAS2.0 software was developed based on our 'population epigenetic framework' and can perform: (i) epigenome-wide single marker association study; (ii) epigenome-wide methylation haplotype (meplotype) association study and (iii) epigenome-wide association meta-analysis. Users can use EWAS2.0 to execute chi-square test, t-test, linear regression analysis, logistic regression analysis, identify the association between epi-alleles, identify the methylation disequilibrium (MD) blocks, calculate the MD coefficient, the frequency of meplotype and Pearson's correlation coefficients and carry out meta-analysis and so on. Finally, we expect EWAS2.0 to become a popular software and be widely used in epigenome-wide associated studies in the future. Availability and implementation: The EWAS software is freely available at http://www.ewas.org.cn or http://www.bioapp.org/ewas.

A Comparison of Co-methylation Relationships Between Rheumatoid Arthritis and Parkinson's Disease.

Rheumatoid arthritis (RA) is a complex autoimmune disease. Recent studies have identified the DNA methylation loci associated with RA and found that DNA methylation was a potential mediator of genetic risk. Parkinson's disease (PD) is a common neurodegenerative disease. Several studies have indicated that DNA methylation levels are linked to PD, and genes related to the immune system are significantly enriched in PD-related methylation modules. Although recent studies have provided profound insights into the DNA methylation of both RA and PD, no shared co-methylation relationships have been identified to date. Therefore, we sought to identify shared co-methylation relationships linked to RA and PD. Here, we calculated the Pearson's correlation coefficient (PCC) of 225,239,700 gene pairs and determined the differences and similarities between the two diseases. The global co-methylation change between in PD cases and controls was larger than that between RA cases and controls. We found 337 gene pairs with large changes that were shared between RA and PD. This co-methylation relationship study represents a new area of study for both RA and PD and provides new ideas for further study of the shared biological mechanisms of RA and PD.

EWAS: epigenome-wide association studies software 1.0 - identifying the association between combinations of methylation levels and diseases.

Similar to the SNP (single nucleotide polymorphism) data, there is non-random association of the DNA methylation level (we call it methylation disequilibrium, MD) between neighboring methylation loci. For the case-control study of complex diseases, it is important to identify the association between methylation levels combination types (we call it methylecomtype) and diseases/phenotypes. We extended the classical framework of SNP haplotype-based association study in population genetics to DNA methylation level data, and developed a software EWAS to identify the disease-related methylecomtypes. EWAS can provide the following basic functions: (1) calculating the DNA methylation disequilibrium coefficient between two CpG loci; (2) identifying the MD blocks across the whole genome; (3) carrying out case-control association study of methylecomtypes and identifying the disease-related methylecomtypes. For a DNA methylation level data set including 689 samples (354 cases and 335 controls) and 473864 CpG loci, it takes only about 25 min to complete the full scan. EWAS v1.0 can rapidly identify the association between combinations of methylation levels (methylecomtypes) and diseases. EWAS v1.0 is freely available at: http://www.ewas.org.cn or http://www.bioapp.org/ewas.

Alpha1-Adrenergic Receptor Activation Stimulates Calcium Entry and Proliferation via TRPC6 Channels in Cultured Human Mesangial Cells.

BACKGROUND AND AIMS: There is accumulating evidence that sympathetic nervous hyperactivity contributes to the pathogenesis of glomerular sclerosis independent of blood pressure effects. A previous study showed that α1-adrenoceptor (α1-AR) antagonists inhibit mesangial cell (MC) proliferation. However, the underlying mechanism remains unclear. METHODS AND RESULTS: We found that α1-AR is expressed in a human mesangial cell line. The α1-AR agonist phenylephrine (PE) induced Ca(2+) influx as well as release from intracellular Ca(2+) stores. Blockade of TRPC6 with siRNA, anti-TRPC6 antibodies and a TRPC blocker attenuated the PE-induced [Ca(2+)]i increase. Additionally, the PE-induced [Ca(2+)]i increase was phospholipase C dependent. Furthermore, PE induced a [Ca(2+)]i increase even when the intracellular Ca(2+) stores were already depleted. This effect was mimicked by an analog of diacylglycerol. These results suggested that, upon α1-AR stimulation, TRPC6 mediates Ca(2+) influx via a receptor-operated Ca(2+) entry mechanism. Finally, TRPC6 contributes to the PE-induced MC proliferation. The mechanisms are associated with the extracellular signal-regulated kinase (ERK) signaling pathway because blockade of TRPC6 and chelation of extracellular Ca(2+) abrogated PE-induced ERK1/2 abrogated PE-induced ERK1/2 phosphorylation. CONCLUSION: TRPC6 channels are involved in α1-AR activation-induced Ca(2+) entry, which mediates proliferation via ERK signaling in human MCs.

High glucose promotes tumor invasion and increases metastasis-associated protein expression in human lung epithelial cells by upregulating heme oxygenase-1 via reactive oxygen species or the TGF-ß1/PI3K/Akt signaling pathway.

BACKGROUND: Growing evidence indicates that heme oxygenase-1 (HO-1) is up-regulated in malignancies and subsequently alters tumor aggressiveness and various cancer-related factors, such as high glucose (HG) levels. HO-1 expression can be induced when glucose concentrations are above 25 mM; however, the role of HO-1 in lung cancer patients with diabetes remains unknown. Therefore, in this study we investigated the promotion of tumor cell invasion and the expression of metastasis-associated proteins by inducing the up-regulation of HO-1 expression by HG treatment in A549 human lung epithelial cells. METHODS: The expression of HO-1and metastasis-associated protein expression was explored by western blot analysis. HO-1 enzymatic activity, reactive oxygen species (ROS) production and TGF-ß1 production were examined by ELISA. Invasiveness was analyzed using a Transwell chamber. RESULTS: HG treatment of A549 cells induced an increase in HO-1 expression, which was mediated by the HG-induced generation of reactive oxygen species (ROS) and transforming growth factor-ß1 (TGF-ß1) in a concentration- and time-dependent manner. Following the increase in HO-1 expression, the enzymatic activity of HO-1 also increased in HG-treated cells. Pretreatment with N-acetyl-L-cysteine (NAC) or with phosphatidylinositol 3-kinase (PI3K)/Akt inhibitors attenuated the HG-induced increase in HO-1 expression. HG treatment of A549 cells enhanced the invasion potential of these cells, as shown with a Transwell assay, and increased metastasis-associated protein expression. However, HO-1 siRNA transfection significantly decreased these capabilities. CONCLUSION: this study is the first to demonstrate that HG treatment of A549 human lung epithelial cells promotes tumor cell invasion and increases metastasis-associated protein expression by up-regulating HO-1 expression via ROS or the TGF-ß1/PI3K/Akt signaling pathway.

MCPerm: a Monte Carlo permutation method for accurately correcting the multiple testing in a meta-analysis of genetic association studies.

Traditional permutation (TradPerm) tests are usually considered the gold standard for multiple testing corrections. However, they can be difficult to complete for the meta-analyses of genetic association studies based on multiple single nucleotide polymorphism loci as they depend on individual-level genotype and phenotype data to perform random shuffles, which are not easy to obtain. Most meta-analyses have therefore been performed using summary statistics from previously published studies. To carry out a permutation using only genotype counts without changing the size of the TradPerm P-value, we developed a Monte Carlo permutation (MCPerm) method. First, for each study included in the meta-analysis, we used a two-step hypergeometric distribution to generate a random number of genotypes in cases and controls. We then carried out a meta-analysis using these random genotype data. Finally, we obtained the corrected permutation P-value of the meta-analysis by repeating the entire process N times. We used five real datasets and five simulation datasets to evaluate the MCPerm method and our results showed the following: (1) MCPerm requires only the summary statistics of the genotype, without the need for individual-level data; (2) Genotype counts generated by our two-step hypergeometric distributions had the same distributions as genotype counts generated by shuffling; (3) MCPerm had almost exactly the same permutation P-values as TradPerm (r = 0.999; P<2.2e-16); (4) The calculation speed of MCPerm is much faster than that of TradPerm. In summary, MCPerm appears to be a viable alternative to TradPerm, and we have developed it as a freely available R package at CRAN: http://cran.r-project.org/web/packages/MCPerm/index.html.