Figure 7
Figure 7. Functional significance of putative miR-223 complementary DNA binding sites and miR-223 levels on NFI-A transcription. (A) Top panel: Schematic representation of the NFI-A promoter region (nt −1400 to 24) containing the complementary putative miR-223 DNA binding sites (nt −1239 to −1231; nt −1055 to −1046). Numbers are relative to the NFI-A transcriptional start site (+1). Bottom panel: HL60 cells stably transfected with pGL4.20 luciferase reporter vectors containing the wild-type NFI-A promoter sequence (NF-wt), the mutagenized forms of the putative miR-223 DNA binding sites (NF-mut-1, NF-mut-2, and NF-mut 1-2), or the “empty” vector were treated (+) or not (−) with 1μM RA for the indicated time points. Data are expressed as the luciferase activity normalized by the total protein amount in each sample. Bars represent the mean of 3 independent experiments performed in duplicate ± SEM. Statistical significance was calculated between RA-treated and untreated samples. *P < .05. (B) HL60 wild-type cells (wt), stably infected with a lentiviral vector expressing miR-223 (Lenti-223) or an empty viral vector (pgk) were tested by quantitative RT-PCR and Northern blot assays to measure mature miR-223 level. U6 detection is shown as RNA loading control. NFI-A mRNA and protein levels were tested by quantitative RT-PCR and immunoblot analysis. β-tubulin is a loading control. Quantitative RT-PCR results represent the average of 3 independent evaluations ± SD. (C) WT HL60 cells (wt) ectopically carrying the miRZip-223 anti–miR-223 (miRZip-223), or the scramble hairpin control (miRZip-scr) constructs, were treated with the indicated concentrations of RA for 72 hours. NFI-A mRNA and protein levels were tested by quantitative RT-PCR and immunoblotting (β-tubulin is a loading control). Quantitative RT-PCR results represent the average of 3 independent evaluations ± SD. (D) ChIP assays performed in HL60 cells ectopically expressing (Lenti-223) or not (pgk) miR-223, or in HL60 cells expressing the miRZip-223 or the miRZip-scr, using the indicated antibodies. Quantitative RT-PCR was performed to amplify NFI-A region 1. Data are plotted as the Lenti-223/pgk and miRZip-223/miRZip-scr infected cell ratio. Error bars represent SD of 3 independent evaluations. *P < .05. **P < .01.

Functional significance of putative miR-223 complementary DNA binding sites and miR-223 levels on NFI-A transcription. (A) Top panel: Schematic representation of the NFI-A promoter region (nt −1400 to 24) containing the complementary putative miR-223 DNA binding sites (nt −1239 to −1231; nt −1055 to −1046). Numbers are relative to the NFI-A transcriptional start site (+1). Bottom panel: HL60 cells stably transfected with pGL4.20 luciferase reporter vectors containing the wild-type NFI-A promoter sequence (NF-wt), the mutagenized forms of the putative miR-223 DNA binding sites (NF-mut-1, NF-mut-2, and NF-mut 1-2), or the “empty” vector were treated (+) or not (−) with 1μM RA for the indicated time points. Data are expressed as the luciferase activity normalized by the total protein amount in each sample. Bars represent the mean of 3 independent experiments performed in duplicate ± SEM. Statistical significance was calculated between RA-treated and untreated samples. *P < .05. (B) HL60 wild-type cells (wt), stably infected with a lentiviral vector expressing miR-223 (Lenti-223) or an empty viral vector (pgk) were tested by quantitative RT-PCR and Northern blot assays to measure mature miR-223 level. U6 detection is shown as RNA loading control. NFI-A mRNA and protein levels were tested by quantitative RT-PCR and immunoblot analysis. β-tubulin is a loading control. Quantitative RT-PCR results represent the average of 3 independent evaluations ± SD. (C) WT HL60 cells (wt) ectopically carrying the miRZip-223 anti–miR-223 (miRZip-223), or the scramble hairpin control (miRZip-scr) constructs, were treated with the indicated concentrations of RA for 72 hours. NFI-A mRNA and protein levels were tested by quantitative RT-PCR and immunoblotting (β-tubulin is a loading control). Quantitative RT-PCR results represent the average of 3 independent evaluations ± SD. (D) ChIP assays performed in HL60 cells ectopically expressing (Lenti-223) or not (pgk) miR-223, or in HL60 cells expressing the miRZip-223 or the miRZip-scr, using the indicated antibodies. Quantitative RT-PCR was performed to amplify NFI-A region 1. Data are plotted as the Lenti-223/pgk and miRZip-223/miRZip-scr infected cell ratio. Error bars represent SD of 3 independent evaluations. *P < .05. **P < .01.

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