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Inhibition of Aberrant MicroRNA-133a Expression in Endothelial Cells by Statin Prevents Endothelial Dysfunction by Targeting GTP Cyclohydrolase 1 in Vivo

View Article: PubMed Central - PubMed

ABSTRACT

Background:: GTP cyclohydrolase 1 (GCH1) deficiency is critical for endothelial nitric oxide synthase uncoupling in endothelial dysfunction. MicroRNAs (miRs) are a class of regulatory RNAs that negatively regulate gene expression. We investigated whether statins prevent endothelial dysfunction via miR-dependent GCH1 upregulation.

Methods:: Endothelial function was assessed by measuring acetylcholine-induced vasorelaxation in the organ chamber. MiR-133a expression was assessed by quantitative reverse transcription polymerase chain reaction and fluorescence in situ hybridization.

Results:: We first demonstrated that GCH1 mRNA is a target of miR-133a. In endothelial cells, miR-133a was robustly induced by cytokines/oxidants and inhibited by lovastatin. Furthermore, lovastatin upregulated GCH1 and tetrahydrobiopterin, and recoupled endothelial nitric oxide synthase in stressed endothelial cells. These actions of lovastatin were abolished by enforced miR-133a expression and were mirrored by a miR-133a antagomir. In mice, hyperlipidemia- or hyperglycemia-induced ectopic miR-133a expression in the vascular endothelium, reduced GCH1 protein and tetrahydrobiopterin levels, and impaired endothelial function, which were reversed by lovastatin or miR-133a antagomir. These beneficial effects of lovastatin in mice were abrogated by in vivo miR-133a overexpression or GCH1 knockdown. In rats, multiple cardiovascular risk factors including hyperglycemia, dyslipidemia, and hyperhomocysteinemia resulted in increased miR-133a vascular expression, reduced GCH1 expression, uncoupled endothelial nitric oxide synthase function, and induced endothelial dysfunction, which were prevented by lovastatin.

Conclusions:: Statin inhibits aberrant miR-133a expression in the vascular endothelium to prevent endothelial dysfunction by targeting GCH1. Therefore, miR-133a represents an important therapeutic target for preventing cardiovascular diseases.

No MeSH data available.


GTP cyclohydrolase 1 (GCH1) mRNA is a target of miR-133.(A) Prediction of miRs targeting the 3′-UTR of the GCH1 mRNA by Target Scan. The GCH1 3′-UTR has 2 isoforms, the longer one (1–2011) and the shorter one (1–1033), where the shorter isoform is equal to 978 to 2011 in the longer isoform. (B) The luciferase reporter plasmid containing the 3′-UTR of GCH1 mRNA (GCH1-UTR) or the mutant of the GCH1-UTR (MT-GCH1-UTR) was cotransfected with scr-miR, premiR-1, premiR-206, premiR-613, premiR-133a, premiR-133b into HEK293 cells. The luciferase activity in the total cell lysates was assayed. n=5 per group. *P<0.05 versus scr-miR alone. NS indicates no significance. (C) HEK293 cells transfected with the plasmid harboring GCH1 cDNA were cotransfected with scr-miR or premiR-133a for 48 hours. Then, actinomycin D (ActD, 10 µg/mL) was added to the culture medium for the indicated time. The half-life of GCH1 mRNA was analyzed by RT-PCR. n=5 per group. *P<0.05 versus Scr-miR alone. miR indicates microRNA; premiR, preliminary miR-133a; RT-PCR, reverse transcription polymerase chain reaction; scr-miR, scrambled miR; and UTR, untranslated region.
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Figure 1: GTP cyclohydrolase 1 (GCH1) mRNA is a target of miR-133.(A) Prediction of miRs targeting the 3′-UTR of the GCH1 mRNA by Target Scan. The GCH1 3′-UTR has 2 isoforms, the longer one (1–2011) and the shorter one (1–1033), where the shorter isoform is equal to 978 to 2011 in the longer isoform. (B) The luciferase reporter plasmid containing the 3′-UTR of GCH1 mRNA (GCH1-UTR) or the mutant of the GCH1-UTR (MT-GCH1-UTR) was cotransfected with scr-miR, premiR-1, premiR-206, premiR-613, premiR-133a, premiR-133b into HEK293 cells. The luciferase activity in the total cell lysates was assayed. n=5 per group. *P<0.05 versus scr-miR alone. NS indicates no significance. (C) HEK293 cells transfected with the plasmid harboring GCH1 cDNA were cotransfected with scr-miR or premiR-133a for 48 hours. Then, actinomycin D (ActD, 10 µg/mL) was added to the culture medium for the indicated time. The half-life of GCH1 mRNA was analyzed by RT-PCR. n=5 per group. *P<0.05 versus Scr-miR alone. miR indicates microRNA; premiR, preliminary miR-133a; RT-PCR, reverse transcription polymerase chain reaction; scr-miR, scrambled miR; and UTR, untranslated region.

Mentions: Computational target-scan analysis (Figure 1A) showed that miR-1, miR-206, and miR-613 could potentially bind the highly conserved target site (76–83, 5′-ACAUUCC-3′) in the longer isoform (1–2011) of the 3′-UTR of GCH1 mRNA (online-only Data Supplement Figure IA). miR-133a/b can target the site (5′-GGACCAAA-3′) at 1685 to 1692 in the longer isoform or at 707 to 714 in the shorter isoform (1–1033) of the 3′-UTR of GCH1 mRNA, suggesting that GCH1 gene expression may be posttranscriptionally regulated by miRs.


Inhibition of Aberrant MicroRNA-133a Expression in Endothelial Cells by Statin Prevents Endothelial Dysfunction by Targeting GTP Cyclohydrolase 1 in Vivo
GTP cyclohydrolase 1 (GCH1) mRNA is a target of miR-133.(A) Prediction of miRs targeting the 3′-UTR of the GCH1 mRNA by Target Scan. The GCH1 3′-UTR has 2 isoforms, the longer one (1–2011) and the shorter one (1–1033), where the shorter isoform is equal to 978 to 2011 in the longer isoform. (B) The luciferase reporter plasmid containing the 3′-UTR of GCH1 mRNA (GCH1-UTR) or the mutant of the GCH1-UTR (MT-GCH1-UTR) was cotransfected with scr-miR, premiR-1, premiR-206, premiR-613, premiR-133a, premiR-133b into HEK293 cells. The luciferase activity in the total cell lysates was assayed. n=5 per group. *P<0.05 versus scr-miR alone. NS indicates no significance. (C) HEK293 cells transfected with the plasmid harboring GCH1 cDNA were cotransfected with scr-miR or premiR-133a for 48 hours. Then, actinomycin D (ActD, 10 µg/mL) was added to the culture medium for the indicated time. The half-life of GCH1 mRNA was analyzed by RT-PCR. n=5 per group. *P<0.05 versus Scr-miR alone. miR indicates microRNA; premiR, preliminary miR-133a; RT-PCR, reverse transcription polymerase chain reaction; scr-miR, scrambled miR; and UTR, untranslated region.
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Figure 1: GTP cyclohydrolase 1 (GCH1) mRNA is a target of miR-133.(A) Prediction of miRs targeting the 3′-UTR of the GCH1 mRNA by Target Scan. The GCH1 3′-UTR has 2 isoforms, the longer one (1–2011) and the shorter one (1–1033), where the shorter isoform is equal to 978 to 2011 in the longer isoform. (B) The luciferase reporter plasmid containing the 3′-UTR of GCH1 mRNA (GCH1-UTR) or the mutant of the GCH1-UTR (MT-GCH1-UTR) was cotransfected with scr-miR, premiR-1, premiR-206, premiR-613, premiR-133a, premiR-133b into HEK293 cells. The luciferase activity in the total cell lysates was assayed. n=5 per group. *P<0.05 versus scr-miR alone. NS indicates no significance. (C) HEK293 cells transfected with the plasmid harboring GCH1 cDNA were cotransfected with scr-miR or premiR-133a for 48 hours. Then, actinomycin D (ActD, 10 µg/mL) was added to the culture medium for the indicated time. The half-life of GCH1 mRNA was analyzed by RT-PCR. n=5 per group. *P<0.05 versus Scr-miR alone. miR indicates microRNA; premiR, preliminary miR-133a; RT-PCR, reverse transcription polymerase chain reaction; scr-miR, scrambled miR; and UTR, untranslated region.
Mentions: Computational target-scan analysis (Figure 1A) showed that miR-1, miR-206, and miR-613 could potentially bind the highly conserved target site (76–83, 5′-ACAUUCC-3′) in the longer isoform (1–2011) of the 3′-UTR of GCH1 mRNA (online-only Data Supplement Figure IA). miR-133a/b can target the site (5′-GGACCAAA-3′) at 1685 to 1692 in the longer isoform or at 707 to 714 in the shorter isoform (1–1033) of the 3′-UTR of GCH1 mRNA, suggesting that GCH1 gene expression may be posttranscriptionally regulated by miRs.

View Article: PubMed Central - PubMed

ABSTRACT

Background:: GTP cyclohydrolase 1 (GCH1) deficiency is critical for endothelial nitric oxide synthase uncoupling in endothelial dysfunction. MicroRNAs (miRs) are a class of regulatory RNAs that negatively regulate gene expression. We investigated whether statins prevent endothelial dysfunction via miR-dependent GCH1 upregulation.

Methods:: Endothelial function was assessed by measuring acetylcholine-induced vasorelaxation in the organ chamber. MiR-133a expression was assessed by quantitative reverse transcription polymerase chain reaction and fluorescence in situ hybridization.

Results:: We first demonstrated that GCH1 mRNA is a target of miR-133a. In endothelial cells, miR-133a was robustly induced by cytokines/oxidants and inhibited by lovastatin. Furthermore, lovastatin upregulated GCH1 and tetrahydrobiopterin, and recoupled endothelial nitric oxide synthase in stressed endothelial cells. These actions of lovastatin were abolished by enforced miR-133a expression and were mirrored by a miR-133a antagomir. In mice, hyperlipidemia- or hyperglycemia-induced ectopic miR-133a expression in the vascular endothelium, reduced GCH1 protein and tetrahydrobiopterin levels, and impaired endothelial function, which were reversed by lovastatin or miR-133a antagomir. These beneficial effects of lovastatin in mice were abrogated by in vivo miR-133a overexpression or GCH1 knockdown. In rats, multiple cardiovascular risk factors including hyperglycemia, dyslipidemia, and hyperhomocysteinemia resulted in increased miR-133a vascular expression, reduced GCH1 expression, uncoupled endothelial nitric oxide synthase function, and induced endothelial dysfunction, which were prevented by lovastatin.

Conclusions:: Statin inhibits aberrant miR-133a expression in the vascular endothelium to prevent endothelial dysfunction by targeting GCH1. Therefore, miR-133a represents an important therapeutic target for preventing cardiovascular diseases.

No MeSH data available.