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Regulation of Cytochrome b 5 Expression by miR-223 in Human Liver: Effects on Cytochrome P450 Activities

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ABSTRACT

Purpose

Cytochrome b 5 (b 5) is a hemoprotein that transfers electrons to several enzymes to fulfill functions in fatty acid desaturation, methemoglobin reduction, steroidogenesis, and drug metabolism. Despite the importance of b 5, the regulation of b 5 expression in human liver remains largely unknown. We investigated whether microRNA (miRNA) might be involved in the regulation of human b 5.

Methods

Twenty-four human liver specimens were used for correlation analysis. In silico analysis and luciferase assay were performed to determine whether the predicted miRNAs functionally target to b 5. The miR-223 was overexpressed into HepG2 cells infected with adenovirus expressing human cytochrome P450.

Results

In human livers, the b 5 protein levels were not positively correlated with the b 5 mRNA levels, and miR-223 levels were inversely correlated with the b 5 mRNA levels or the translational efficiencies. The luciferase assay showed that miR-223 functionally binds to the element in the 3′-untranslated region of b 5 mRNA. The overexpression of miR-223 significantly reduced the endogenous b 5 protein level and the mRNA stability in HepG2 cells. Moreover, the overexpression of miR-223 significantly reduced CYP3A4-catalyzed testosterone 6β-hydroxylation activity and CYP2E1-catalyzed chlorzoxazone 6-hydroxylase activity but not CYP1A2-catalyzed 7-ethoxyresorufin O-deethylase activity.

Conclusions

miR-223 down-regulates b 5 expression in the human liver, modulating P450 activities.

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Abbreviations

Ad:

Adenovirus

b 5 :

Cytochrome b 5

CYP:

Cytochrome P450

DMEM:

Dulbecco’s modified Eagle’s medium

FBS:

Fetal bovine serum

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

HPLC:

High-performance liquid chromatography

miRNA:

MicroRNA

MOI:

Multiplicity of infection

MRE:

miRNA recognition element

NPR:

NADPH-cytochrome P450 reductase

P450:

Cytochrome P450

PAGE:

Polyacrylamide gel electrophoresis

pre-miRNA:

Precursor miRNA

RT-PCR:

Reverse transcription-polymerase chain reaction

siRNA:

Small interfering RNA

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ACKNOWLEDGMENTS AND DISCLOSURES

The authors would like to thank Dr. Satoru Asahi (Takeda Pharmaceutical, Osaka, Japan) for providing Hepc/3A4.2-30, Hepc/2E1.3-8, and Hepc/1A2.9 cells. The authors would also like to thank Dr. Masataka Takamiya and Yasuhiro Aoki (Department of Legal Medicine, Iwate Medical University School of Medicine, Japan) for providing human liver samples. This work was support by Grant-in-Aid for Challenging Exploratory Research from Japan Society for the Promotion of Science [Grant 24659074].

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Author notes

  1. Tsuyoshi Yokoi

    Present address: Department of Drug Safety Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan

Authors and Affiliations

  1. Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japan

    Kei Takahashi, Yuki Oda, Yasuyuki Toyoda, Tatsuki Fukami, Tsuyoshi Yokoi & Miki Nakajima

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  1. Kei Takahashi
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  3. Yasuyuki Toyoda
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Correspondence to Miki Nakajima.

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Takahashi, K., Oda, Y., Toyoda, Y. et al. Regulation of Cytochrome b 5 Expression by miR-223 in Human Liver: Effects on Cytochrome P450 Activities. Pharm Res 31, 780–794 (2014). https://doi.org/10.1007/s11095-013-1200-7

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  • DOI: https://doi.org/10.1007/s11095-013-1200-7

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