Nicotine Suppresses Phagocytic Ability of Macrophages by Regulating the miR-296-3p–SIRPα Axis

Figure 3

MiR-296-3p directly targets the 3′-UTR of SIRPα and inhibits SIRPα expression in macrophages. (a) In the presence of actinomycin D (Act D; 2.5 mg/mL) or both Act D (2.5 mg/mL) and nicotine (1 μM) for 0, 2, 4, 6, 8, and 10 hours, respectively. The level of SIRPα mRNA was analyzed by qRT-PCR and GAPDH mRNA served as an internal control. (b) The sequences of binding sites of miR-296-3p to SIRPα mRNA 3′-UTR and their mutant sequences. (c and d) 48 hours after U937-derived macrophages were transfected with negative control (NC), miR-296-3p mimics, or miR-296-3p inhibitors, the levels of SIRPα mRNA and protein were analyzed by qRT-PCR and western blotting, respectively. GAPDH mRNA served as an internal control. (e) 48 hours after 293T cells were transfected, the firefly luciferase activity was analyzed and renal luciferase activity served as a transfection control. (f) Bars show the miR-296-3p or Actin beta (ACTB) levels in the complex pulled down by the sense or antisense of the SIRPα mRNA 3′-UTR. ACTB served as a negative control. *, **, ***, and ****.

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