Figure 7.

The introduction of gga-miR-6631-5p inhibitor hindered LaSota NDV replication by up-regulating INSIG1 in DF-1 cells. (A) The effect of gga-miR-6631-5p overexpression on E2F1, INSIG1, HSPBP1, SERPING1 and PSMD11 mRNA expression was tested by RT-qPCR assay in DF-1 cells at 48 h after transfection. (B) Putative complementary sites between gga-miR-6631-5p and INSIG1 3’UTR, and mutant sites in INSIG1-mut reporter. (C) DF-1 cells were co-transfected with NC mimic or gga-miR-6631-5p mimic and INSIG1-wt reporter or INSIG1-mut reporter, followed by the detection of luciferase activities at 48 h after transfection. (D) The level of gga-miR-6631-5p was measured by RT-qPCR assay at 48 h post transfection in DF-1 cells transfected with in-miR-6631-5p or in-NC. (E) DF-1 cells were transfected with si-con or si-INSIG1. Forty-eight hours later, INSIG1 mRNA level was measured through RT-qPCR assay. (F) DF-1 cells were transfected with in-NC (100 nM), in-miR-6631-5p (100 nM), in-miR-6631-5p (100 nM) + si-con (100 nM), or in-miR-6631-5p (100 nM) + si-INSIG1 (100 nM). Non-transfected cells acted as the control group. At 24 h after transfection, cells were infected with LaSota NDV at the MOI of 1. Next, TCID₅₀ assay was performed at the indicated time points (12, 24, 36, 48 h) after LaSota infection.