Long Non-Coding RNA GAS5 and Intestinal MMP2 and MMP9 Expression: A Translational Study in Pediatric Patients with IBD

Abstract

Background: The long non-coding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) seems to be involved in the regulation of mediators of tissue injury, in particular matrix metalloproteinases (MMPs), implicated in the pathogenesis of inflammatory bowel disease (IBD). We investigated the role of GAS5 in regulating MMP2 and MMP9 expression in pediatric patients with IBD and in vitro.

Methods: In total, 25 IBD patients were enrolled: For each patient paired inflamed and non-inflamed biopsies were collected. RNA was extracted and GAS5, MMP2, and MMP9 were quantified by TaqMan assay. The expression of GAS5 and MMPs was also determined in the human monocytic THP1 cells differentiated into macrophages and stimulated with lipopolysaccharide (LPS). The function of GAS5 was assessed by overexpressing the lncRNA and evaluating the MMPs levels.

Results: Real-time PCR results demonstrated a downregulation of GAS5 and an upregulation of both MMPs in inflamed tissues. In vitro data confirmed the trend observed in patients for the three genes: The stimulation with LPS promoted a downregulation of GAS5 while an increase of MMPs was observed. Overexpression experiments showed that higher levels of GAS5 lead to a decrease of both enzymes.

Conclusion: These results provide new information about the role of GAS5 in IBD: The lncRNA could mediate tissue damage by modulating the expression of MMPs.

Keywords: GAS5; MMP2; MMP9; inflammatory bowel disease; long non-coding-RNA.

Figure 1

Growth arrest–specific transcript 5 (GAS5) levels in colon biopsies of inflammatory bowel disease (IBD) pediatric patients. Expression was evaluated in inflamed (INF) and non-inflamed (NON-INF) tissues for each patient. GAS5 expression was normalized using RPLP0 gene. Relative expression (RE) values are expressed as Log₂ 2^−ΔCt. Paired t test * p < 0.05.

Figure 2

Correlation between GAS5 levels evaluated in inflamed tissue and the clinical score; Spearman test p = 0.048; r = −0.40.

Figure 3

MMP (matrix metalloproteinase) levels in colon biopsies of IBD pediatric patients. MMP2 (A) and MMP9 (B) expression was evaluated in inflamed (INF) and non-inflamed (NON-INF) tissues for each patient and normalized using RPLP0 gene. Relative expression (RE) values are expressed as Log₂ 2^−ΔCt. Paired t test * p < 0.05, ** p < 0.01.

Figure 4

MMP2 (74 kDa; A and B) and MMP9 (92 kDa; A and C) protein expression in colon biopsies of IBD pediatric patients analyzed by immunoblotting. Protein lysates were obtained from inflamed (INF) and non-inflamed (NON-INF) tissues. Actin (42 kDa) was used as internal loading control. Representative western blot reflecting MMP2, MMP9 and actin protein levels. Paired t test * p < 0.05.

Figure 5

Relative expression (RE, values are expressed as 2^−ΔCt) of GAS5 (A), MMP2 (B), and MMP9 (C) in THP1 cells exposed to LPS (lipopolysaccharide), PMA (phorbol-12-myristate 13-acetate), and PMA + LPS. GAS5, MMP2, and MMP9 expression was normalized using 18S gene. One-way ANOVA GAS5 p = 0.003, MMP2 p = 0.018, MMP9 p < 0.0001 Bonferroni’s multiple comparison post-test * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Evaluation of GAS5 levels after transfection with pcDNA-GAS5 plasmid in monocytes (GAS5_MONO) and macrophages (GAS5_MACRO). GAS5 expression was normalized using GAPDH gene and relative expression (RE) values are expressed as 2^−ΔCt. One-way ANOVA GAS5 p = 0.0005, Bonferroni’s multiple comparison post-test ** p < 0.01, *** p < 0.001.

Figure 7

Levels of MMP2 (A) and MMP9 (B) after overexpression of GAS5 (GAS5) in monocyte stimulated with LPS (LPS) and macrophages stimulated with LPS (PMA + LPS) and in controls (EMPTY). MMP2 and MMP9 expression were normalized using GAPDH gene and relative expression (RE) values are expressed as 2^−ΔΔCt. Two-way ANOVA: EMPTY vs. GAS5 MMP2 p = 0.002, MMP9 p < 0.0001 Bonferroni’s multiple comparison post-test * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 8

Evaluation of levels of MMP2 (A) and MMP9 (B) after overexpression of GAS5 (GAS5) in monocyte stimulated with LPS (LPS) and macrophages stimulated with LPS (PMA + LPS) and control (EMPTY). Two-way ANOVA: EMPTY vs. GAS5 MMP2 p = 0.02, MMP9 p < 0.0001 Bonferroni’s multiple comparison post-test * p < 0.05, ** p < 0.01; ns = not significant.