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1.
Fig. 1

Fig. 1. The potential binding sites of miRNAs in the 3’-UTR of human HNF-4A mRNA. From: The role of microRNAs in hepatocyte nuclear factor-4alpha expression and transactivation.

Alignments and locations of the predicted target sequences of miRNAs (miR-34a, miR-34c-5p, miR-449a, miR-143 and miR-216b) within the 3’-UTR of HNF-4A mRNA are shown. The numbering refers to the 5′-end of the 3’-UTR as 1.

Zhongyan Wang, et al. Biochim Biophys Acta. 2013 May;1829(5):436-442.
2.
Fig. 8

Fig. 8. Down-regulation of HNF-4α target genes by miR-34a, miR-34c-5p and miR-449a. From: The role of microRNAs in hepatocyte nuclear factor-4alpha expression and transactivation.

The mRNA expression levels of HNF-4α target genes, TTR, ApoB and α1-AT in HepG2 cells, transfected with indicated miRNA mimics as described in Fig. 4 legend, were examined by RTqPCR. Each column represents the mean ± SD of three independent experiments. The data are relative to that transfected with siControl (miR-Ctr), ** p<0.01.

Zhongyan Wang, et al. Biochim Biophys Acta. 2013 May;1829(5):436-442.
3.
Fig. 5

Fig. 5. Over-expression of miR-34a, miR-34c-5p and miR-449a reduces HNF-4α binding to its target genes, ApoB, TTR, and α1-AT. From: The role of microRNAs in hepatocyte nuclear factor-4alpha expression and transactivation.

EMSAs were performed to detect the effect of miRNAs on HNF-4α DNA binding activity. HepG2 cells were transfected with miRNA mimics as indicated. Nuclear extracts were prepared. The 32P-labelled oligonucleotide probes based on the HNF-4α-specific binding site in the promoter of ApoB, TTR or α1-AT genes were used [11]. Representative EMSAs of HNF-4α binding are shown. Histograms (right-hand panels) show densitometric analyses of HNF-4α-DNA complexes. Values represent mean ± SD of three separate experiments. ** p<0.01 versus untreated control (−).

Zhongyan Wang, et al. Biochim Biophys Acta. 2013 May;1829(5):436-442.
4.
Fig. 7

Fig. 7. The transactivation potential of a HNF-4α target gene, TTR, is inhibited by miR-34a, miR-34c-5p and miR-449a. From: The role of microRNAs in hepatocyte nuclear factor-4alpha expression and transactivation.

The miRNA mimics were cotransfected into HepG2 cells with a luciferase construct containing the promoter region of TTR spanning nucleotides (191 to +5) or empty pGL4.11[luc2P] vector (pGL4). The data shown are the relative luciferase activity, the ratio of the Firefly luciferase activity to Renilla luciferase activity, and represent the mean ± SD of three independent experiments. **p<0.01 indicates a significant difference compared to controls without mimic treatment (−).

Zhongyan Wang, et al. Biochim Biophys Acta. 2013 May;1829(5):436-442.
5.
Fig. 4

Fig. 4. The effect of miRNAs on HNF-4α expression. From: The role of microRNAs in hepatocyte nuclear factor-4alpha expression and transactivation.

HepG2 cells, seeded into 6-well plates, were transfected with 100 pmol of miRIDIAN miRNA mimics, negative control miRNA mimic (miR-Ctr) or were untreated (−). At 48 h or 72 h after transfection, mRNA and nuclear extracts were prepared, the expression levels of HNF-4α mRNA (A) and protein (B) were determined by RT-qPCR and Western blot, respectively. The bar graph in panel B summarizes the densitometric analyses of the effects of miRNAs on HNF-4α protein expression normalized by β-actin levels. The experiments were performed in triplicate, and presented as mean ± SD. *p<0.05 and **p<0.01 versus untreated control (−) or miR-Ctr.

Zhongyan Wang, et al. Biochim Biophys Acta. 2013 May;1829(5):436-442.
6.
Fig. 6

Fig. 6. The inhibitory effect of miR-34a, miR-34c-5p and miR-449a on HNF-4α binding activity in intact cells. From: The role of microRNAs in hepatocyte nuclear factor-4alpha expression and transactivation.

HepG2 cells were transfected with miRNA mimics as indicated. The abilities of HNF-4α to bind to the HNF-4α-specific binding site in the promoter of ApoB, TTR or α1-AT gene were determined by ChIP assay with either antibody against HNF-4α or mouse IgG (IgG, negative control). Chromatin-immunoprecipitated DNA was analyzed by qPCR with primers and probes specific for the HNF-4α-binding sites in ApoB, TTR or α1-AT gene promoter [20]. The control samples were set at 1. The results are mean ± SD (n =3) assayed in triplicate by qPCR. * p<0.05 and ** p<0.01 indicate that the value is significantly different from control.

Zhongyan Wang, et al. Biochim Biophys Acta. 2013 May;1829(5):436-442.
7.
Fig. 2

Fig. 2. Analyzing the role of miRNAs in the HNF-4A 3’-UTR with luciferase reporters. From: The role of microRNAs in hepatocyte nuclear factor-4alpha expression and transactivation.

A. HepG2 cells were transfected with pmirGLO-HNF-4A 3’UTR (pmirGLO-HNF-4) or luciferase reporter vector alone (pmirGLO). B. The reporter (pmirGLO-HNF4) was cotransfected into the cells with siDicer I, siControl (siCtr) or alone (−). C. Cotransfections were carried out with reporter (pmirGLO-HNF4) and various miRNAs mimics as indicated. At 48 h after transfection, the cells were lysed and analyzed for Firefly and Renilla luciferase activity using a dual reporter assay system. The data shown are the ratio of the Firefly luciferase activity to the Renilla luciferase activity, and represent the mean ± SD obtained from three individual wells. The experiments were performed in triplicate. **p<0.01 versus pmirGLO-HNF4 (A), and versus the cells transfected with pmirGLO-HNF4 alone (−) (B and C).

Zhongyan Wang, et al. Biochim Biophys Acta. 2013 May;1829(5):436-442.
8.
Fig. 3

Fig. 3. Deletion of the target seed region(s) for miR-34a/miR-34c-5p/miR-449a in HNF-4A 3’-UTR eliminates the repressive effect induced by the corresponding miRNAs. From: The role of microRNAs in hepatocyte nuclear factor-4alpha expression and transactivation.

Three different luciferase constructs are shown: (1) HNF4 3’UTR (black color), a full length of HNF-4A 3’-UTR with the seed sequences of all miRNAs tested, Site A and B for miR-34a/miR-34c-5p/miR-449a, Site C for miR-143, Site D and E for miR-216b. (2) HNF4 Del I 3’UTR (grey color), HNF-4A 3’-UTR carrying only one binding site (Site A), for miR-34a/miR-34c-5p/miR-449a and Site C/D/E. (3) HNF4 Del II 3’UTR (white color), HNF-4A 3’-UTR with two binding sites (sites A and B) deleted for miR-34a/miR-34c-5p/miR-449a. The luciferase constructs with various miRNAs mimics as indicated were introduced into HepG2 cells. The relative luciferase activities were analyzed as described in Fig. 2 legend. *p<0.05 and **p<0.01 versus the cells transfected with luciferase reporter alone (−).

Zhongyan Wang, et al. Biochim Biophys Acta. 2013 May;1829(5):436-442.

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