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. 2016 Apr 27;6:24854. doi: 10.1038/srep24854

Figure 4. Direct targeting of TLR4 mRNA by ssc-miR-30d_R-1.

Figure 4

The predicted conserved ssc-miR-30d_R-1-binding site (underlined) in the 3′-UTR of pig TLR4 mRNA (A). Luciferase activity in MARC-145 cell lysates transfected with constructs encoding wild-type (WT) or mutated (Mut) target gene 3′-UTRs plus mimics (B) or inhibitors (C) of ssc-miR-30d_R-1 (or the appropriate control). Validation of ssc-miR-30d_R-1 targeting of the TLR4 3′-UTR by treatment with different doses of inhibitor at 48 h after transfection (D). *P < 0.05 and **P < 0.01 (Student’s t test). Data are from three independent experiments (mean ± SD). (E) MARC-145 cells transfected with ssc-miR-30d_R-1 (miRNA) mimic or inhibitor, control siRNA, pc-DNA-TLR4 or TLR4 siRNA plasmids were stimulated by LPS. Expression of β-actin (internal control), TLR4, MyD88, and NF-κB were detected by Western blot. Data shown are representative of three independent experiments.