Long noncoding RNA MARL regulates antiviral responses through suppression miR-122-dependent MAVS downregulation in lower vertebrates
Fig 6
(A) MARL sequence contains two sites complementary to miR-122. miR-122 binding sites in MARL wild-type form (Luc-MARL-wt) and the mutated form (Luc-MARL-mut) were shown. (B) EPC cells were transfected with NC or miR-122, together with Luc-MARL-wt or Luc-MARL-mut. At 48 h post-transaction, the luciferase activity was analyzed and normalized to renilla luciferase activity. (C and D) MARL could downregulate GFP expression. EPC cells were cotransfected with the wild type of mVenus-MARL or the mutated type, together with NC or miR-122. At 48 h post-transfection, the fluorescence intensity (C) and the GFP expression (D) were evaluated by enzyme-labeled instrument and western blotting, respectively. Scale bar, 20 μm; original magnification × 10. (E) MIC cells were transfected with NC-i or miR-122-i for 48 h. The expression of MARL were measured by qPCR. (F) The wild and mutated forms of biotinylated miR-122 sequence were shown (left panel). MIC cells were transfected with the biotinylated wild type of miR-122 (Bio-miR-122-wt) or the biotinylated mutated type of miR-122 (Bio-miR-122-mut) for 48 h. Cells were harvested for biotin-based pulldown assay. MARL expression were analyzed by qPCR (right panel). (G) The schematic diagram of the RNA pull down method to identify the binding between MARL and miR-122 (left panel). MIC lysates were incubated with biotin-labeled MARL and MARL-mut. miRNA real-time PCR was performed after pull down process (right panel). (H) The schematic diagram of RIP method (left panel). The qPCR results of the MS2-RIP method used to identify the binding between MARL and miR-122 in MIC cells. miRNA real-time qPCR was performed after RNA immunoprecipitation process (right panel). All data represented the mean ± SE from three independent triplicated experiments. *, p < 0.05.