Cardiovascular disease has garnered increased interest and became the pre-eminent health problem worldwide as the leading cause of death and illness[1, 3]. Atherosclerosis constitutes the single most crucial contributor to the outcome of cardiovascular diseases. Recently, numerous animal and cell experiments have focused on the miRNA profile in atherosclerotic processes, and an obvious up-regulation of miR-21 has been demonstrated in atherosclerotic plaques[23, 30]. However, its function on the developmental progress of atherosclerotic plaques remains unclear. In this study, our results have manifested that miR-21 levels were significantly increased, and can negatively regulate lipid accumulation and inflammation cytokine secretion in LPS-stimulated macrophages by TLR-4-dependent signaling.
LPS, as one of the best studied immunostimulatory components of bacteria, has been proven to enhance lipid deposition and lipid-derived macrophage foam cell formation, as well as inflammatory cytokines release. All of these are the characterizations of atherosclerosis, and can regulate the pathological process of atherosclerosis and its complications. In this study, LPS dose- and time-dependently induced the expression of miR-21 mRNA. To clarify the roles of miR-21 in atherosclerosis, we analyzed the effect of miR-21 in LPS-induced lipid accumulation and inflammatory response in macrophages. After transfection with miR-21 mimics, the overexpression of miR-21 was induced in macrophages, and the corresponding down-regulation of it was also performed by anti-miR-21 inhibitor transfection. Lipid-laden foam cell formation is a critical trigger for the development of atherosclerosis. In this study, overexpression of miR-21 dramatically attenuated the ratio of CE/TC, indicating an obvious decrease in lipid accumulation in LPS-stimulated macrophages. Simultaneously, blocking of miR-21 expression accelerated LPS-induced lipid deposition in macrophages. Further analysis suggested that a notable reduction in foam cell formation was observed when overexpression of miR-21 in macrophages exposed to LPS. While in contrast to the control group, inhibiting miR-21 expression induced dramatically lipid droplets formation in macrophages stimulated with LPS. Together, our results suggested that miR-21 negatively regulated LPS-induced lipid accumulation in macrophages.
Toll-like receptors (TLRs) exerts multiple roles in atherosclerosis, and is highly expressed in atherosclerotic plaque. Among these members, TLR4 has drawn more attention during the development progress of atherosclerosis. TLR4 is known as the receptor of LPS, and its deficiency significantly attenuated aortic atherosclerosis in ApoE-/- mice. Moreover, lipid accumulation in circulating monocytes was significantly reduced in TLR4-deficient mice. Growing evidence indicates that TLR4 plays a very important role in macrophage foam cells formation, indicating a critical roles of TLR4 in atherosclerosis via regulating lipid deposition[32, 33]. To elucidate the underlying mechanism involved in miR-21-regulated lipid-laden macrophage foam cell formation, TLR4 pathway was discussed. As expected, miR-21 overexpression remarkably dampened the activation of TLR4 and its downstream NF-κB, while miR-21 expression inhibition reversely augmented the activation of TLR4-NF-κB. When blocking TLR4 expression with its specific antibody, LPS-induced lipid accumulation was strikingly decreased in macrophages transfected with anti-miR-21 inhibitor. Simultaneously, a similar reduction in lipid deposition was also confirmed when pretreatment with PDTC. Hence, these results suggested that miR-21 could negatively regulated lipid accumulation via TLR4-NF-κB pathway in LPS-stimulated macrophages, implying an important role in the development of atherosclerosis.
During the past decade, a prominent role for inflammation in atherosclerosis and its implications have been appreciated. Inflammation ranks as a major characterization for atherosclerosis, and the release of abundant inflammatory molecules will give rise to abnormal foam cell formation and initiate the development of atherosclerotic lesions. Blocking macrophage inflammation by TGR5 activation attenuates atherosclerosis lesions, indicating a potential therapeutic aspect in anti-atherosclerosis. LPS is known as a potent inducer of the inflammatory response. Therefore, we further analyzed the effect of miR-21 in LPS-triggered inflammation in macrophages. Following transfection with miR-21, the levels of pro-inflammatory cytokine IL-6 was dramatically attenuated, accompany with an increase of anti-inflammatory cytokine IL-10. The corresponding changes of IL-6 and IL-10 were also confirmed when silencing miR-21 levels, indicating an important function of miR-21 on LPS-induced macrophage inflammation. As a key component of innate immune response, TLR4 possesses a pivotal role in the initiation and progression of atherosclerosis, and can regulate the inflammatory response in macrophages via its downstream NF-κB signaling[29, 35]. To further elucidate the underlying mechanism involved in miR-21-mediated inflammation cytokines secretion, we blocked the activation of TLR4 and NF-κB. After blocking TLR4 expression, the increase in IL-6 and decrease in IL-10 was significantly mitigated in miR-21-silencing cells. The similar changes in IL-6 and IL-10 were also corroborated when preconditioning with PDTC in anti-miR-21 inhibitor-transfected macrophages. Together, these results told that miR-21 could regulate macrophage inflammation and lipid accumulation via the TLR4- NF-κB signaling pathway. However, the mechanism involved in miR-21-induced inhibitory effect on TLR4-NF-κB is still unclear, which needs to be explored in our next plan.
In conclusion, our research investigated for a potential role of miR-21 in atherosclerosis. In this study, LPS induced the expression of miR-21 in a time- and dose-dependent manner. Further analysis manifested that miR-21 negatively regulated lipid-laden foam cell formation and inflammatory responses in LPS-stimulated macrophages through the TLR4-NF-κB pathway, indicating a critical roles of miR-21 in the progression of atherosclerosis. Hence, the beneficial clinical effects of miR-21 overexpression in the prevention and treatment of atherosclerosis deserve further investigations.