Figure 2.

Mir214-3p downregulated Ulk1 expression through direct binding to the Ulk1 gene. (A) The regulatory network of microRNAs and TFs in the regulation of target mRNA. (B) miRNA levels were analyzed using a TLDA kit. The RNA samples from the livers were pooled by group, and PCR arrays were analyzed in triplicate. (C) The miRNA levels in the liver of a human NAFLD patient were compared to those of a person without fatty liver (Con, control) (n = 5). (D) miRNA–autophagy-related gene interactions were analyzed using miRWalk and TargetScan. (E) miRNA–autophagy-related gene interactions were analyzed using a target-prediction website. Red indicates higher expression, and green indicates lower expression, relative to the LFD group. The intensity of the color, and the size, indicate the degree of change in expression. (F) The expression of miRNAs that were upregulated according to TLDA analysis was validated using qRT-PCR. Values represent mean ± SEM (n = 10). * p < 0.05; ** p < 0.01; *** p < 0.001, compared to the LFD group. (G–H) The expression of autophagy-related genes in (G) miRNA inhibitor-transfected or (H) mimic-transfected cells. Increase is indicated in red, and decrease is indicated in blue, relative to the negative control (n = 3). (I) Hepatocytes were transfected with the Mir214-3p inhibitor or mimic for 48 h; the protein levels of the target autophagy-related genes were then measured using immunoblotting. (J) Dual reporter assay was performed using the GLuc-SEAP vector containing 3′ UTR of Ulk1 mRNA. Hepatocytes were co-transfected with the GLuc-SEAP vector and Mir214-3p mimic for 48 h. After incubation, luciferase activity in the medium was measured, and Mir214-3p levels were quantified in cell lysates. (K) Luciferase activity was measured in hepatocytes transfected with the GLuc-SEAP vector of Ulk1 3′ UTR containing the wild-type or mutated Mir214-3p binding sites. Values represent mean ± SD (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001, compared to the NC. GLuc: Gaussia luciferase; HFD: high-fat diet; LFD: low-fat diet; miRNA: microRNA; mRNA: messenger RNA; NAFLD: nonalcoholic fatty liver disease; NC: negative control; PCR: polymerase chain reaction; qRT-PCR: quantitative reverse transcription PCR; SD: standard deviation; SEAP: secreted alkaline phosphatase; SEM: standard error of mean; TF: transcription factor; TLDA: TaqMan low-density array; UTR: untranslated region