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. 2013 Apr 22;2(2):e000121.
doi: 10.1161/JAHA.113.000121.

MicroRNA-210 decreases heme levels by targeting ferrochelatase in cardiomyocytes

Aijun Qiao  1 , Arineh Khechaduri, R Kannan Mutharasan, Rongxue Wu, Varun Nagpal, Hossein Ardehali
Affiliations

MicroRNA-210 decreases heme levels by targeting ferrochelatase in cardiomyocytes

Aijun Qiao et al. J Am Heart Assoc. .

Abstract

Background: MicroRNA-210 (miR-210) increases in hypoxia and regulates mitochondrial respiration through modulation of iron-sulfur cluster assembly proteins (ISCU1/2), a protein that is involved in Fe/S cluster synthesis VSports手机版. However, it is not known how miR-210 affects cellular iron levels or production of heme, another iron containing molecule that is also needed for cellular and mitochondrial function. .

Methods and results: To screen for micro-ribonucleic acids (miRNAs) regulated by iron, we performed a miRNA gene array in neonatal rat cardiomyocytes treated with iron chelators. Levels of miR-210 are significantly increased with iron chelation, however, this response was mediated entirely through the hypoxia-inducible factor (HIF) pathway V体育安卓版. Furthermore, miR-210 reduced cellular heme levels and the activity of mitochondrial and cytosolic heme-containing proteins by modulating ferrochelatase (FECH), the last enzyme in heme biosynthesis. Mutation of the 2 miR-210 binding sites in the 3' untranslated region (UTR) of FECH reversed the miR-210 response, while mutation of either binding site in isolation did not exert any effects. Changes mediated by miR-210 in heme and FECH were independent of ISCU, as overexpression of an ISCU construct lacking the 3' UTR does not alter miR-210 regulation of heme and FECH. Finally, FECH levels increased in hypoxia, and this effect was not reversed by miR-210 knockdown, suggesting that the effects of miR-210 on heme are restricted to normoxic conditions, and that the pathway is overriden in hypoxia. .

Conclusions: Our results identify a role for miR-210 in the regulation of heme production by targeting and inhibiting FECH under normoxic conditions V体育ios版. .

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Figures

Figure 1.
Figure 1.
DFO increases miR‐210 levels through a HIF‐dependent pathway. A, Heatmap plot of microRNA expression in response to FAC and DFO in neonatal rat cardiomyocytes. B, Difference in log median ratios (DLMR) for miR‐210 in response to DFO and FAC; n=3 in each group. C, Changes in miR‐210 levels in NRCM treated with DFO in the presence and absence of ARNT or HIF‐1α siRNA. D, Changes in miR‐210 in wild type and ARNT knockout MEFs in response to DFO. There is no increase in the levels of miR‐210 after DFO treatment in ARNT knockout MEFs. Data are presented as mean±standard error of the mean (SEM). *P<0.05, n=6 in each group for (C) and (D). DFO indicates desferoxamine; HIF, hypoxia‐inducible factor; FAC, ferric ammonium citrate; NS, nonsignificant; siRNA, small interfering RNA; NRCM, neonatal rat cardiomyocytes; ARNT, aryl hydrocarbon receptor nuclear translocator; MEF, mouse embryonic fibroblast; KO, knockout.
Figure 2.
Figure 2.
Modulation of miR‐210 in H9c2 cells. A, miR‐210 levels after transfection of H9c2 cells with pre‐miR‐210. B, miR‐210 levels after transfection with anti‐miR‐210. Data are presented as mean±standard error of the mean (SEM). *P<0.05, n=6 in each group.
Figure 3.
Figure 3.
miR‐210 influences the levels of heme in H9c2 cells. Heme levels are decreased with miR‐210 overexpression (A), while its levels are increased in response to miR‐210 downregulation (B). Cellular levels of iron are not altered in response to miR‐210 overexpression (C) or downregulation (D). Data are presented as mean±standard error of the mean (SEM). *P<0.05, n=5 in each group.
Figure 4.
Figure 4.
miR‐210 alters the activity of heme‐containing proteins. Mitochondrial complex IV activity is decreased or increased in response to miR‐210 overexpression (A) or downregulation (B). The activity of peroxidase is decreased with miR‐210 overexpression (C), while its levels are increased in response to miR‐210 downregulation (D). Data are presented as mean±standard error of the mean (SEM). *P<0.05, n=6 in each group.
Figure 5.
Figure 5.
Among genes involved in heme synthesis or degradation, only FECH mRNA and protein levels are altered in response to miR‐210 modulation. mRNA levels of genes involved in heme synthesis or degradation, as assessed by RT‐PCR, in response to miR‐210 overexpression (A) or downregulation (B). miR‐210 inhibits FECH protein by targeting the 3′ UTR of its respective mRNA. FECH protein levels are reduced in response to miR‐210 overexpression (C), while they are increased with miR‐210 downregulation (D). Summary bar graphs are shown below the Western blots. Data are presented as mean±standard error of the mean (SEM). *P<0.05, n=3 in each group. FECH indicates ferrochelatase; RT‐PCR indicates reverse transcription polymerase chain reaction; UTR, untranslated region; ISCU, iron‐sulfur cluster assembly protein.
Figure 6.
Figure 6.
miR‐210 does not alter the levels of heme or FECH in HEK293 cells. A, Cellular heme levels after transfection of HEK293 cells with pre‐miR‐210. B and C, FECH levels after transfection of HEK293 cells with pre‐miR‐210; n=6 in each group. FECH indicates ferrochelatase; HEK, human embryonic kidney cells.
Figure 7.
Figure 7.
FECH mRNA is a direct target of miR‐210. A, miR‐210 overexpression in H9c2 cells inhibits luciferase activity of a construct containing the 3′ UTR of FECH. Positive control include 2 perfect matches to miR‐210 and negative control use pMIR‐report empty plasmid. B, miR‐210 downregulation results in an increase in luciferase activity. C, Schematic representation of the 2 putative miR‐210 binding sites in the FECH mRNA 3′ UTR. The site that was mutated is “GCAC.” D, While mutation of each site individually did not later the FECH response to miR‐210, mutation of both sites completely ameliorated the effect. Data are presented as mean±standard error of the mean (SEM). *P<0.05, n=6 in each group. FECH indicates ferrochelatase; UTR, untranslated region.
Figure 8.
Figure 8.
miR‐210 targeting of FECH is responsible for the changes in cellular heme levels. A, While the endogenous FECH protein levels are reduced with miR‐210 overexpression, the myc‐tagged protein lacking the 3′ UTR is unresponsive. B, Overexpression of FECH (lacking the 3′ UTR) does not lead to a reduction in cellular heme levels. Data are presented as mean±standard error of the mean (SEM). *P<0.05, n=4 to 6 in each group. FECH indicates ferrochelatase; UTR, untranslated region; GFP, green fluorescent protein.
Figure 9.
Figure 9.
Overexpression of ISCU lacking the 3′ UTR does not alter the effects of miR‐210 on FECH or heme. ISCU mRNA (A) and protein (B) levels in response to miR‐210 overexpression in H9c2 cells. Human ISCU (C) levels are significantly increased after cotransfection of miR‐210 and human ISCU. Data are presented as mean±standard error of the mean (SEM). *P<0.05, n=3 to 6 in each group. Two‐way ANOVA with Tukey post hoc analysis was used for (C). ISCU indicates iron‐sulfur cluster assembly protein; FECH, ferrochelatase; UTR, untranslated region; ANOVA, analysis of variance; GFP, green fluorescent protein.
Figure 10.
Figure 10.
Effects of ISCU overexpression on FECH and heme levels in response to miR‐210. A, FECH mRNA levels are reduced with miR‐210 in the presence and absence of overexpression of ISCU construct lacking the 3′ UTR. B, The response of cellular heme levels to miR‐210 is not altered with overexpression of ISCU construct lacking the 3′ UTR. C, The reduction in complex IV activity with miR‐210 is not reversed by overexpression of the truncated ISCU1/2. Data are presented as mean±standard error of the mean (SEM). *P<0.05, n=3 to 5 in each group. ISCU indicates iron‐sulfur cluster assembly protein; FECH, ferrochelatase; UTR, untranslated region; GFP, green fluorescent protein.
Figure 11.
Figure 11.
The increase in FECH levels in response to hypoxia is not reversed by miR‐210. A, miR‐210 levels are increased in response to 1.0% oxygen for 12 hours in H9c2 cells and the effect is reversed with miR‐210 downregulation. B, FECH levels are increased in response to hypoxia, however, the increase is not reversed with miR‐210 knockdown. C and D, Proposed model for the effects of HIF and miR‐210 on FECH levels in response to hypoxia. In normoxia the miR‐210 regulation of FECH predominates (C) whereas in hypoxia the direct effects of HIF‐1 on FECH induction predominate (D). Data are presented as mean±standard error of the mean (SEM). *P<0.05, n=5 to 6 in each group. Two‐way ANOVA with Tukey post hoc analysis was used for (A) and (B). FECH indicates ferrochelatase; HIF, hypoxia‐inducible factor; ANOVA, analysis of variance.
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