MicroRNA-143 modulates the expression of Natriuretic Peptide Receptor 3 in cardiac cells

Figures

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  Figure 1. Expression of miR-143 in human HF plasma, animal model and in vitro experimental platforms. (a) Upregulation of miR-143 in HF plasma compared with non-HF controls. In the Box and Whisker plot, center lines represent the median; lower and upper box limits indicate the 25th and 75th percentiles respectively, crosses represent sample means; data points are plotted as open circles. n = 42, 56, 44, 12 for control, HF, HFREF and HFPEF respectively. Student’s t test, p values as indicated. Relative expression levels of miR-143 in (b) rat plasma and (c) rat left ventricular tissue at 7 days post-surgery as compared to sham controls (n = 6). (d) Expression of miR-143 in HCMa cells after 48 h hypoxic treatment. Data in (b,c and d) are presented as mean ± SD, Student’s t test, *p < 0.05.

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  Figure 2. Regulatory effects of miR-143 on the expression of NPR3. (a) Luciferase reporter assay to demonstrate the interaction between miR-143 and NPR3 3′UTR. (b) Overexpression of miR-143 (miR-143 mimic) suppressed the expression of NPR3 and knockdown of miR-143 (antagomiR-143) increased NPR3 transcripts level in HCMa. (c) NPR3 was upregulated in antagomiR-143 treated normoxic cells. Downregulation of NPR3 level in hypoxia-treated HCMa was reversed when co-treated with antagomiR-143. Data are presented as mean ± SD, n = 3, Student’s t test, *p < 0.05.

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  Figure 3. MIR-143HG promoter activity is modulated by hypoxia. (a) Hypoxic treatment increases the promoter activity of HIF1a. (b) Western blot analysis of HIF-1a protein level in HCMa after hypoxic treatments (c) Mutagenesis analyses of MIR143HG promoter demonstrate the essential roles of hypoxia response elements in hypoxia induced transcriptional activation for MIR143HG. The mutated HRE sites are indicated by underlined gray fonts. No change in the NPR3 promoter activity was observed after hypoxia treatment. Data are presented as mean ± SD, n = 3, Student’s t test, *p < 0.05.

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  Figure 4. Differential promoter activities of MIR143HG and MIR100HG in the presence of exogenous miR-100 or miR-143 in HCMa cells. (a) Promoter activities of MIR143HG were enhanced and inhibited in the presence of exogenous miR-143 and miR-100 respectively. (b) Promoter activity of MIR100HG was attenuated in the presence of miR-143 mimics. (c) Mutagenesis analyses of MIR143HG promoter showing that miR-143 and miR-100 failed to interact with promoter constructs containing the mutated transcriptional response elements (MTRE) representing the respective microRNA binding sites. Inset in (c) depicts the relative locations of the TREs tested in this study. Data are presented as mean ± SD, n = 3, Student’s t test, *p < 0.05. n.s, non-significance, p > 0.05.

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  Figure 5. Regulatory effects of miR-143 on the expression of NPPA, NPPC, NR3C2 and CRHR2. (a) Luciferase reporter assay to demonstrate the interactions between miR-143 and the 3′UTRs of NPPA, NPPC, NR3C2 and CRHR2. (b) Overexpression of miR-143 (miR-143 mimic) suppressed expression of NPPA, NPPC, and NR3C2 in HCMa cells. However, knockdown of NPR3 level with antagomiR-143 did not lead to enhancement of NPPA, NPPC, and NR3C2 levels in HCMa cells. Data are presented as mean ± SD, n = 3, Student’s t test, *p < 0.05.

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  Figure 6. Schematic diagram of hypoxia-induced transcriptional activation of MIR143HG and the proposed microRNA directed transcriptional modification by miR-143 and miR100 in cardiac cells.