LncRNAs Landscape in the patients of primary gout by microarray analysis

Abstract

To determine the expression profile and clinical significance of long non-coding RNAs (lncRNAs) in peripheral blood mononuclear cells (PBMCs) of patients with primary gout and healthy control subjects. Human lncRNA microarrays were used to identify the differentially expressed lncRNAs and mRNAs in primary gout patients (n = 6) and healthy control subjects (n = 6). Bioinformatics analyses were performed to predict the roles of differently expressed lncRNAs and mRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression levels of 8 lnRNAs in 64 primary gout patients and 32 healthy control subjects. Spearman's correlation was used to analyze the correlation between these eight lncRNAs and the laboratory values of gout patients. A receiver operating characteristic (ROC) curve was constructed to evaluate the diagnostic value of the lncRNAs identified in gout. The microarray analysis identified 1479 differentially expressed lncRNAs (879 more highly expressed and 600 more lowly expressed), 862 differentially expressed mRNAs (390 more highly expressed and 472 more lowly expressed) in primary gout (fold change > 2, P < 0.05), respectively. The bioinformatic analysis indicated that the differentially expressed lncRNAs regulated the abnormally expressed mRNAs, which were involved in the pathogenesis of gout through several different pathways. The expression levels of TCONS_00004393 and ENST00000566457 were significantly increased in the acute gout flare group than those in the intercritical gout group or healthy subjects (P<0.01). Moreover, inflammation indicators were positive correlated with TCONS_00004393 and ENST00000566457 expression levels. The areas under the ROC curve of ENST00000566457 and NR-026756 were 0.868 and 0.948, respectively. Our results provide novel insight into the mechanisms of primary gout, and reveal that TCONS_00004393 and ENST00000566457 might be as candidate targets for the treatment of gout flare; ENST00000566457 and NR-026756 could effectively discriminate between the gout and the healthy control groups.

Fig 1. Expression profiles of lncRNAs or mRNAs in gout patients compared with healthy controls.

(A) Volcano plot of differentially expressed lncRNAs. (B) Volcano plot of differentially expressed mRNAs. The horizontal green line represents a P-value of 0.05, and vertical green lines represent 2.0-fold changes up and down. X-axes is the fold change values (log2 scaled), and Y-axes is the P-values (log10 scaled). (C) Heatmap of 32 significantly differentially expressed lncRNAs. AG (acute gout flare) patients group: AG1, AG2, AG3; IG (intercritical gout) patients group: IG1, IG2, IG3; HC (healthy control) group: HC1, HC2, HC3. Red and green colors represent more highly expressed and more lowly expressed genes, respectively. (D) Relative expression levels of 8 lncRNAs in 6 gout patients and 6 healthy control subjects. The Y-axis is the ratio of the relative expression level of lncRNA in the gout group to the healthy control group.

Fig 2. Bioinformatics analysis.

(A) Gene ontology analyses of differentially expressed mRNAs according to the biological process (BP). (B) KEGG pathways analysis of differentially expressed mRNAs. The top 10 enriched GO or KEGG terms of more highly expressed mRNAs (A1, B1) and more lowly expressed mRNAs (A2, B2) are shown. The y-axis represents the most significantly enriched pathways, and the x-axis represents their scores (negative logarithm of P-value). (C) IncRNA-mRNA co-expression network. (C1) Ninety lncRNAs interacted with nine mRNAs in the meaningful “TOLL-like receptor” signaling pathway. (C2) One hundred and eighty lncRNAs interacted with seven mRNAs in the “NOD-like receptor” signaling pathway.

Fig 3. Expression levels of eight lncRNAs in gout patients and healthy control subjects.

(A) Relative expression levels of 8 lncRNAs in 64 gout patients and 6 healthy control subjects. (B) Relative expression levels of 8 lncRNAs in 32 acute gout patients, 32 intercritical gout patients and 32 healthy control subjects. qRT-PCR indicates quantitative real-time polymerase chain reaction. Gout: primary gout, including acute gout and intercritical gout, AG: acute gout flare, IG: intercritical gout, HC: healthy control subjects, P < 0.05, * * P < 0.01, * * * P < 0.001.

Fig 4. Correlations between the expression level of lncRNAs and clinical characteristics of gout analyzed with Spearman’s coefficient.

(A) The expression level of lncRNAs were related to laboratory inflammation indicators. (B) The expression level of lncRNAs were related to laboratory metabolic indicators.

Fig 5. Assessment of the diagnostic value of the lncRNAs for gout.

Receiver operating characteristic (ROC) curve analysis of five differently expressed lncRNAs risk scores in gout and healthy control subjects. AUC = area under the curve.