Figure 3.

The mutual regulation between miRNAs and IFN-β protein in PAMs. (A) 10 or 40 nM four miRNA mimics and negative control miRNA mimic (CmiR), and (B) 20 or 100 nM four miRNA inhibitors and negative control miRNA inhibitor (CmiRi), were transfected transiently into primary PAMs, which were then stimulated by 50 µg/ml poly I:C for 24 h. The protein expression levels of porcine IFN-β in the supernatant of PAMs at 24 hpi were measured by ELISA. The IFN-β protein expression could be suppressed by miRNA mimics (A) and the native endogenous miRNAs inhibition effect on IFN-β protein expression could be relieved by miRNA inhibitors (B) in primary PAMs. (C) Primary PAMs were treated by 50 µg/ml poly I:C to stimulate the production of porcine IFN-β, whose protein levels were monitored by a commercial porcine IFN-β ELISA kit at 1, 3 8 and 24 h post-treatment. Mock-treated PAMs were used as control. (D) qRT-PCR analysis of miRNAs expression including let-7b, miR-26a, miR-34a and miR-145, in primary PAMs stimulated by 50 µg/ml poly I:C at 1, 3, 8 and 24 h after treatment. The native IFN-β protein induction stimulated by poly I:C could upregulate the expression of endogenous miRNAs in primary PAMs. The results were normalized to U6 expression and mock-treated controls according to ∆∆Ct method. The data represent means ± standard error of the mean of three independent experiments.