PM_2.5 Upregulates MicroRNA-146a-3p and Induces M1 Polarization in RAW264.7 Cells by Targeting Sirtuin1

Background: Fine particulate matter (PM_2.5) exposure is proved to be associated with illnesses, but the mechanism is not clear. Potential effects of PM_2.5 on innate immunity have become a hotspot recently. Confronting PM_2.5, macrophages are able to be activated and induce inflammatory responses. Whether PM_2.5 exposure affects macrophage polarization and associated mechanisms remains to be further explored. Afterwards, whether Sirtuin1 (SIRT1) an important intermediate regulator in various physiological processes takes part in the macrophage polarization induced by PM_2.5 is unknown. MiRNAs are acknowledged as key regulator in posttranscriptional modification and our previous study found that miR-146a is a novel biomarker of PM_2.5 exposure. Thus, we propose a hypothesis, PM_2.5 exposure induces M1 polarization and miR-146a-3p is a potential upstream regulator by targeting SIRT1. Methods: RAW264.7 cells were treated with different concentrations of PM_2.5 for 24h. The expressions of cytokines and key molecular markers were detected by qRT-PCR, Western blotting and ELISA. The activation degree of TLRs and NF-κB was assessed by Western blotting. The specific agonist and antagonist of SIRT1 were used to explore the potential role of SIRT1 in M1 polarization induced by PM_2.5. MiR-146a-3p mimic and inhibitor were pre-transfected into RAW264.7 cells and the effects on M1 polarization induced by PM_2.5 were evaluated. Luciferase analysis was used to identify the binding site of miR-146a-3p and SIRT1. Results: PM_2.5 increased the mRNA and protein expression of M1 markers including interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase (iNOS) in RAW264.7 cells. The protein level of TLR4 was significantly increased and the ratio of phosphorylated NF-κB p65 versus p65 subunit was also elevated in PM_2.5 group. PM_2.5 decreased the protein level of SIRT1 but not the mRNA expression in vitro and in vivo experiments. Pre-treatment with SIRT1 agonist SRT1720 rescued the PM_2.5 induced M1 response. Whereas, SIRT1 antagonist EX527 augment the effect. MiR-146a-3p was upregulated in PM_2.5 treated RAW264.7 cells. Luciferase experiments reported that SIRT1 was directly targeted by miR-146a-3p. Overexpression of miR-146a-3p downregulated the expression of SIRT1 protein in untreated RAW264.7 cells. Importantly, inhibition of miR-146a-3p upregulated SIRT1 protein and suppressed M1 polarization in PM_2.5 treated RAW264.7 cells. Conclusions: These results suggested that PM_2.5 induces the inflammatory M1 polarization and TLR4/NF-κB signal transduction pathway might be involved in the process. MiR-146a-3p is a novel regulator of PM_2.5 exerted M1 polarization by targeting SIRT1.