A total of 34 patients with RA (29 females and 5 males; mean age, 53.7 years) admitted to the Department of Rheumatology and Immunology, The First Affiliated Hospital of Nanchang University (Nanchang, China) between October 2014 and April 2016, were enrolled in the present study. The diagnosis of RA was established by the American College of Rheumatology criteria (21). The baseline characteristics of 10 of these patients that were used in microarray analysis are summarized in Table I. For RA patients, Disease activity was calculated using the disease activity score 28 system (22), anti-cyclic citrullinated peptide (CCP) antibody was detected by Anti-CCP Enzyme Immunoassay Test kit (cat. no. EC170502; Shanghai Kexin Biotech Co., Ltd., Shanghai, China) and values >25 RU/ml were considered positive. Erythrocyte sedimentation rate was examined by the Westergren method (23), and values ≤15 mm/h for males and ≤20 mm/h for females were considered normal. Serum C-reactive protein (CRP) and rheumatoid factor (RF) were measured by nephelometry. Values >8 mg/l for CRP and >20 IU/ml were considered positive for RF. A total of 34 healthy controls (29 females and 5 males; mean age, 50.2 years) were selected based on no history of autoimmune disease. Among the patients and controls, 10 patients with RA (Table I) and 10 age-matched and sex-matched normal controls were recruited for isolating peripheral blood mononuclear cells (PBMCs) for microarray analysis. The remaining 24 healthy controls and 24 patients with RA were used to validate the reliability of microarray results by RT-qPCR analysis.

The present study was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University (approval no. 019) and was conducted in compliance with the Declaration of Helsinki. Informed consent was obtained from all participants, including patients and normal controls, prior to initiation of the study.

Blood samples were collected in vacutainer tubes containing EDTA, and PBMCs were isolated by centrifugation against a density. Gradient (1,000 × g, 20 min, 22°C). Total RNA was isolated from freshly obtained PBMCs using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. The integrity of the RNA was assessed by electrophoresis on a 2% denaturing agarose gel and visualized using ethidium bromide. The concentration and quality of the RNA were assessed by absorbance spectrometry measuring absorbance ratios of A260/A280 and A260/A230 using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc.).

Arraystar Human LncRNA Microarray V3.0 (8×60 K; Arraystar, Inc., Rockville, MD, USA) is designed for the global profiling of human lncRNA and mRNA, and is updated from the previous Microarray V2.0. The sample preparation and microarray hybridization were performed according to the manufacturer's protocol. Briefly, ribosomal RNA was removed from total RNA and subsequently mRNA was obtained using an mRNA-ONLY™ Eukaryotic mRNA Isolation kit (Epicentre; Illumina, Inc., San Diego, CA, USA). The random priming method (24) was utilized to amplify each sample according to the manufacturer's protocol (Quick Amp Labeling kit; 5190-0442 Agilent Technologies, Inc., Santa Clara, CA, USA; amplification assay was set at 40°C for 2 h, followed by 65°C for 15 min and 0°C for 5 min), and mRNA was transcribed into fluorescent cRNA without 3′ bias. Labeled cRNA were hybridized to the Human LncRNA Microarray, designed for 30,586 lncRNA and 26,109 mRNA. After washing with Gene Expression Wash Buffer 1 (Agilent Technologies, Inc.), and Gene Expression Wash Buffer 2 (Agilent Technologies, Inc.), the Agilent Scanner G2505C and Agilent feature extraction software (version 11.0.1.1; Agilent Technologies, Inc.) were used to scan and analyze the arrays. Quantile normalization and subsequent data processing were executed using the GeneSpring GX v12.0 software package (Agilent Technologies, Inc.). The microarray work was performed by Shanghai Kangchen Bio-tech, Inc., (Shanghai, China).

The expression levels of differentially expressed lncRNA and mRNA were confirmed by RT-qPCR. Briefly, 5 µg of total RNA from each sample was used for the synthesis of first strand cDNA using a PrimeScript™ RT Reagent Kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol. RT reaction was carried out in a 10 µl reaction volume containing 5X PrimeScript™ Buffer, 1 µl RT specific primer, 0.5 µl PrimeScript™ RT Enzyme Mix, 5 µg of total RNA. RT assay was set at an initial denaturation step at 37°C for 15 min, followed by 85°C for 5 sec. Following first strand cDNA synthesis, PCR reaction was performed in a 10 µl reaction volume containing 1X SYBR Green PCR Master mix (Takara Biotechnology Co., Ltd.), 0.4 µM of each specific forward and reverse primer, 0.5 µl of cDNA template. PCR assay was set at an initial denaturation step at 95°C for 5 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min with an ABI 7500 Real-time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers were as shown in Table II. The gene expression levels were normalized to that of GAPDH in cDNA samples, and all samples were measured in triplicate. The relative expression levels of the genes were calculated using the ∆Ct method where ∆Ct=Ct median gene-Ct median GAPDH (25,26).

Target genes were predicted for differentially expressed lncRNAs. LncRNAs originate in complex transcriptional loci and regulate gene expression through epigenetic regulation of chromatin modification, and transcriptional and post-transcriptional processing (27). In order to determine the underlying roles of lncRNA in RA and shed light on the potential mechanisms of RA, the present study mainly focused on unveiling the relationship between lncRNA and mRNA. Abnormal lncRNA (fold change, ≥3.0) were subjected to bioinformatics analysis for target gene prediction. To increase the accuracy of target prediction, abnormal mRNA (fold change, ≥3.0) were integrated with the predicted lncRNA targets.

GO (www.geneontology.org) and KEGG (www.genome.ad.jp/kegg) databases were utilized to analyze biological functions and signaling pathways affected by abnormally expressed mRNA (28,29). P<0.05 was considered to indicate a statistically significant difference.

Statistical analysis and graphic presentation were performed using GraphPad Prism version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). A Student's t-test was used where the normality test passed; otherwise, the nonparametric Mann-Whitney U test was used to analyze the data. P<0.05 was considered to indicate a statistically significant difference.

To profile differentially expressed lncRNA in RA, a microarray analysis of lncRNA and mRNA expression in the RA group and healthy group was performed (Figs. 1 and 2). The results revealed that 139 lncRNA (88 up and 51 down) and 92 mRNA (67 up and 25 down) were significantly differentially expressed between the RA group and healthy group (fold change ≥10.0; P<0.05). Additionally, 5,045 lncRNA (2,410 up and 2,635 down) and 3,289 mRNA (1,403 up and 1,886 down) were differentially expressed with a fold change >2 (P<0.05). The most significantly deregulated lncRNA were ENST00000583574 (up fold change, 257.7358) and ENST00000559539 (down fold change, 4726.8003). The most significantly deregulated mRNA were ALAS2 (up fold change, 52.16745) and KCNMB3 (down fold change, 4999.96853) (data not shown).

To validate the reliability of the microarray results, three differentially expressed lncRNA and three differentially expressed mRNA were randomly selected and analyzed using RT-qPCR. For lncRNA, as demonstrated in Fig. 3, the results demonstrated that ENST00000445339 was significantly downregulated (P=0.042) and ENST00000506982 was significantly upregulated (P=0.019) in the 24 patients with RA compared to the levels in the healthy group (Fig. 3A and C, respectively). No significant difference was observed in the expression of ENST00000569920 between RA patients and healthy controls (P=0.57; Fig. 3B). For mRNA, the expression of PGLYRP1 was significantly downregulated (P<0.0001), LRRN3 was significantly upregulated (P=0.0003) and CX3CR1 was significantly upregulated (P=0.048) in patients with RA compared with the levels in healthy controls (Fig. 3D-F). This demonstrated that RT-qPCR results were consistent with those of the microarray analysis.

Evidence from previous reports has indicated that lncRNA may be classified into lncRNA with enhancer-like functions, antisense lncRNA and lincRNA (26,30). The results of the present study indicated that 610 intergenic lncRNA (304 upregulated and 306 downregulated), 251 enhancer-like lncRNA (138 upregulated and 113 downregulated) and 134 antisense lncRNA (57 upregulated and 77 downregulated) were differentially expressed (fold change ≥2.0; P<0.05) between the RA group and healthy group. Some nearby coding genes that may be regulated by these differentially expressed lncRNA were also identified (data not shown).

The present results revealed that the majority of differentially expressed lncRNA were from intergenic regions (42%), natural antisense to protein-coding loci (19%), or intronic antisense to protein-coding loci (15%), with the others representing intronic sense-overlapping, exon sense-overlapping, and bidirectional regions. In addition, 2,859 of 5,045 differentially expressed lncRNA were oriented in or around a known protein-coding region (i.e. not intergenic). Therefore, differentially expressed lncRNA (fold change, ≥3.0) were integrated with predicted mRNA (fold change, ≥3.0). The result demonstrated that 109 differentially expressed protein-coding genes were markedly associated with each of the 85 differentially expressed lncRNA to generate 135 lncRNA-mRNA target pairs. Among them, 81 pairs were differentially expressed unidirectionally (upregulated or downregulated), while 54 pairs were differentially expressed bidirectionally (data not shown).

Through GO analysis, it was demonstrated that these abnormal transcripts of lncRNA were associated with biological process, cellular components and molecular function (Fig. 4). The analysis indicated that the differentially expressed mRNA between the RA group and healthy group were significantly enriched (P<0.05) in response to wounding, DNA packaging complex, chemokine activity, RNA metabolic process, nuclear lumen and DNA binding. Pathway analysis indicated that 23 upregulated pathways were identified, including systemic lupus erythematosus and alcoholism pathways. A total of 23 downregulated pathways were identified, including bile secretion and T cell receptor signaling pathway (Fig. 5).