Deregulated miR-146a-3p alleviates disease progression in atherosclerosis through inactivating NF-κB: An experimental study

Abstract

Background: Atherosclerosis (AS), as a complex chronic inflammatory disease, is 1 of the main causes of cardiovascular and cerebrovascular diseases. This study aimed to confirm the direct interaction between miR-146a-3p and NF-κB, and explore the role of miR-146a-3p/NF-κB in the regulation of inflammation in AS.

Methods: Bioinformatic prediction and dual-luciferase reporter assay were used to confirm the interaction between miR-146a-3p and NF-κB. Lipopolysaccharides stimulation was performed to establish AS inflammatory cell model, and the levels of pro-inflammatory cytokines were estimated using an enzyme-linked immunosorbent assay. miR-146a-3p and NF-κB expression were evaluated using reverse transcription quantitative PCR, and their clinical value was examined using a receiver operating characteristic curve.

Results: Inflammatory cell model showed increased IL-1β, IL-6, and TNF-α. NF-κB was a target gene of miR-146a-3p, and mediated the inhibitory effects of miR-146a-3p on inflammatory responses in the cell model. In patients with AS, miR-146a-3p/NF-κB was associated with patients' clinical data and inflammatory cytokine levels, and aberrant miR-146a-3p and NF-κB showed diagnostic accuracy to distinguish AS patients from healthy populations.

Conclusion: miR-146a-3p might inhibit inflammation by targeting NF-κB in AS progression, and miR-146a-3p/ NF-κB might provide novel biomarkers and therapeutic targets for the prevention of AS and related vascular events.

Figure 1.

NF-κB was a direct target of miR-146a-3p. (A) Complementary sequences of miR-146a-3p in the 3’-UTR of NF-κB. (B) Results of dual-luciferase reporter assay. (C) miR-146a-3p expression was successfully regulated by cell transfection. (D) NF-κB expression was inhibited by miR-146a-3p overexpression but was promoted by miR-146a-3p inhibition. **P < .01, ***P < .001 compared to untreated or controls. 3’-UTR = 3’-untranslated region, miR-146 = microRNA-146, NF-κB = NF-activated B cell Kappa-light-chain enhancer.

Figure 2.

miR-146a-3p inhibited inflammation by targeting NF-κB in the cell model. (A) LPS inhibited miR-146a-3p expression, but miR-146a-3p mimic promoted miR-146a-3p expression. (B) LPS promoted NF-κB expression, but miR-146a-3p overexpression inhibited NF-κB expression, while this inhibition was rescued by pcDNA3.1- NF-κB. (C–E) LPS led to increased IL-1β, IL-6, and TNF-α, but this effect was reduced by miR-146a-3p overexpression and enhanced by NF-κB overexpression. ***P < .001 compared to controls; ^###P < .001 compared to LPS; ^&&&P < .001 compared to LPS + miR-146a-3p mimic. miR-146 = microRNA-146, IL = interleukin, LPS = lipopolysaccharides, NF-κB = NF-activated B cell Kappa-light-chain enhancer, TNF = tumor necrosis factor.

Figure 3.

Expression of miR-146a-3p and NF-κB in the serum of AS patients. (A) Serum miR-146a-3p was decreased in AS patients. (B) Serum NF-κB was increased in AS patients. (C) Serum miR-146a-3p levels were negatively correlated with NF-κB in AS patients. ***P < .001. AS = atherosclerosis, miR-146 = microRNA-146, NF-κB = NF-activated B cell Kappa-light-chain enhancer.

Figure 4.

ROC curves based on serum miR-146a-3p (A) and NF-κB (B). miR-146 = microRNA-146, NF-κB = NF-activated B cell Kappa-light-chain enhancer, ROC = receiver operating characteristic curve.