Figure 4.

STAT3 directly regulates miR-125b-5p-1 expression. (A, B) LLC-PK1 cells were treated with PDCoV (100 TCID₅₀) and SeMet (150 μM) for 6, 24, and 48 h, and the level of STAT3 and phos-STAT3 (Tyr705) was determined by qRT-PCR or Western blot (n=3-6). (C) The base sequence and mutant sequence of miR-125b-5p-1 promoter. (D) The STAT3 protein expressions was determined by qRT-PCR and Western blot in LLC-PK1 cells when transfected with pcDNA3.1-vector or pcDNA3.1-STAT3 (n=3). (E) The pcDNA3.1-STAT3 or empty vector and WT-pGL3-miR-125b-1 or MUT- pGL3-miR-125b-1 were co-transfected into HEK293T cells. The luciferase reporter assay was used to detect whether STAT3 can target and bind on miR-125b-1 promoter (n=5). (F) The STAT3 protein expression was determined by qRT-PCR or Western blot in LLC-PK1 cells treated with siRNA-NC, siSTAT3, DMSO, and Stattic (STAT3 inhibitor, 0.75 μM) (n=3). (G) The miR-125b-5p-1 expression was measured by qRT-PCR when transfected with pcDNA3.1-STAT3, empty vector, siSTAT3, siRNA-NC, DMSO, Stattic (0.75 μM) (n=6). (H) The base sequence and mutant sequence of HK2 promoter. (I) The pcDNA3.1-STAT3 or empty vector and WT-pGL3-HK2 or MUT- pGL3-HK2 were co-transfected into HEK293T cells. The luciferase reporter assay was used to detect whether STAT3 can target and bind on HK2 promoter (n=5). (J, K) The HK2 mRNA and protein expressions were determined by qRT-PCR and Western blot in LLC-PK1 cells when transfected with pcDNA3.1-STAT3, empty vector, siSTAT3, siRNA-NC, DMSO, Stattic (0.75 μM) (n=3-6). (L, M) The siSTAT3 or siRNA-NC and miR-125b-5p-1 mimic or mimic-NC were co-transfected into LLC-PK1 cells. The HK2 mRNA and protein expressions were determined by qRT-PCR and Western blot (n=3-6). Means ± SD are shown. Statistical significiance was determined by Student t test. ***, P<0.001; **, P<0.01; *, P<0.05; ^###, P < 0.001; ^##, P<0.01; ^#, P<0.05; n.s: not significant. All experiments were repeated at least twice and representative results are shown.