LncRNA BRE-AS1 regulates the JAK2/STAT3-mediated inflammatory activation via the miR-30b-5p/SOC3 axis in THP-1 cells

Abstract

Long non-coding RNAs (lncRNAs) have emerged as pivotal regulators in numerous biological processes, including macrophage-mediated inflammatory responses, which play a critical role in the progress of diverse diseases. This study focuses on the regulatory function of lncRNA brain and reproductive organ-expressed protein (BRE) antisense RNA 1 (BRE-AS1) in modulating the inflammatory activation of monocytes/macrophages. Employing the THP-1 cell line as a model, we demonstrate that lipopolysaccharide (LPS) treatment significantly upregulates BRE-AS1 expression. Notably, specific knockdown of BRE-AS1 via siRNA transfection enhances LPS-induced expression of interleukin (IL)-6 and IL-1β, while not affecting tumor necrosis factor (TNF)-α levels. This selective augmentation of pro-inflammatory cytokine production coincides with increased phosphorylation of Janus kinase (JAK)2 and signal transducer and activator of transcription (STAT)3. Furthermore, BRE-AS1 suppression results in the downregulation of suppressor of cytokine signaling (SOCS)3, an established inhibitor of the JAK2/STAT3 pathway. Bioinformatics analysis identified binding sites for miR-30b-5p on both BRE-AS1 and SOCS3 mRNA. Intervention with a miR-30b-5p inhibitor and a synthetic RNA fragment that represents the miR-30b-5p binding site on BRE-AS1 attenuates the pro-inflammatory effects of BRE-AS1 knockdown. Conversely, a miR-30b-5p mimic replicated the BRE-AS1 attenuation outcomes. Our findings elucidate the role of lncRNA BRE-AS1 in modulating inflammatory activation in THP-1 cells via the miR-30b-5p/SOCS3/JAK2/STAT3 signaling pathway, proposing that manipulation of macrophage BRE-AS1 activity may offer a novel therapeutic avenue in diseases characterized by macrophage-driven pathogenesis.

Keywords: JAK2; LncRNA; SOCS3; STAT3; miRNA.

Fig. 1

Upregulation of BRE-AS1 Expression Following LPS Treatment. A, B THP-1 cells were treated with LPS (1000 ng/ml) for specified durations. The expression levels of BRE-AS1 A and TNF-α mRNA B were quantified using real-time PCR. *p < 0.05; ***p < 0.001 (n = 3 and the error bars represent SEM).

Fig. 2

Reduction of BRE-AS1 Enhances IL-6 and IL-1β Expression Without Affecting TNF-α. A The effectiveness of the knockdown was evaluated by real-time PCR 24 h after transfecting THP-1 cells with scramble or BRE-AS1 siRNA (n = 3). B–D Following LPS stimulation (1000 ng/ml), mRNA levels of cytokines were quantified using real-time PCR (n = 3). E, F Cytokine secretion levels were measured by ELISA after LPS stimulation for 24 h at specified concentrations for IL-6 E and at indicated durations for TNF-α with LPS (1000 ng/ml) F (n = 4). **p < 0.01; ***p < 0.001. Error bars represent SEM.

Fig. 3

Inhibition of BRE-AS1 Enhances STAT3 Phosphorylation and IL-6 Expression via the SOCS3/JAK2/STAT3 Pathway. A–E THP-1 cells, after being transfected with BRE-AS1 siRNA, were stimulated with LPS (1000 ng/ml). Protein levels of SOCS3, JAK2, p-JAK2, STAT3, and p-STAT3 were analyzed by Western blot in cell extracts. B Protein expression levels in (A) were quantified using ImageJ software (n = 4). C–E mRNA levels of SOCS3, JAK2, and STAT3 were measured using real-time PCR (n = 4). **p < 0.01; ***p < 0.001. Error bars represent SEM.

Fig. 4

BRE-AS1 Modulation of SOCS3 mRNA via miR-30b-5p Interaction. (A) Identification of complementary binding sites between BRE-AS1, miR-30b-5p, and SOCS3 was performed. (B-F) THP-1 cells were transfected with BRE-AS1 siRNA and a decoy RNA (BRE-AS1 fragment) for 24 h before being stimulated with LPS (1000 ng/ml). (B-D) mRNA expression levels of SOCS3, IL-6, and IL-1β were quantified using real-time PCR (n = 3). (E) IL-6 secretion levels were determined by ELISA (n = 4). (F) Protein expression in the JAK2/STAT3 pathway was analyzed by Western blot. *p < 0.05; **p < 0.01; ***p < 0.001. Error bars represent SEM.

Fig. 5

BRE-AS1 Modulation of SOCS3 mRNA via miR-30b-5p Interaction. A–C THP-1 cells were transfected with a miR-30b-5p inhibitor alongside BRE-AS1 siRNA for 24 h before LPS stimulation (1000 ng/ml). A, B mRNA levels of SOCS3 and IL-6 were quantified using real-time PCR (n = 4). C Protein levels within the SOCS3/JAK2/STAT3 pathway were analyzed by Western blot. D–G THP-1 cells underwent transfection with miR-30b-5p mimic and mimic control for 24 h. D–F The mRNA expression of SOCS3, IL-6, and IL-1β was assessed using real-time PCR (n = 3). (G) Protein levels of the SOCS3/JAK2/STAT3 pathway were evaluated by Western blot. *p < 0.05; **p < 0.01; ***p < 0.001. Error bars represent SEM.

Fig. 6

BRE-AS1 regulates macrophage inflammatory activation via the miR-30b-5p/SOCS3/JAK2/STAT3 Pathway. LncRNA BRE-AS1 modulates SOCS3 expression by serving as a competitive endogenous RNA for miR-30b-5p. SOCS3 subsequently controls the phosphorylation and activation of JAK2 and STAT3, which in turn affects the levels of IL-6 and IL-1β expression.