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. 2019 May;9(3):639-647.
doi: 10.1016/j.apsb.2018.12.002. Epub 2018 Dec 11.

Bioengineered miR-27b-3p and miR-328-3p modulate drug metabolism and disposition via the regulation of _target ADME gene expression

Xin Li  1   2 , Ye Tian  3   2 , Mei-Juan Tu  2 , Pui Yan Ho  2 , Neelu Batra  2 , Ai-Ming Yu  2
Affiliations

Bioengineered miR-27b-3p and miR-328-3p modulate drug metabolism and disposition via the regulation of _target ADME gene expression

Xin Li et al. Acta Pharm Sin B. 2019 May.

Abstract

Drug-metabolizing enzymes, transporters, and nuclear receptors are essential for the absorption, distribution, metabolism, and excretion (ADME) of drugs and xenobiotics. MicroRNAs participate in the regulation of ADME gene expression via imperfect complementary Watson-Crick base pairings with _target transcripts. We have previously reported that Cytochrome P450 3A4 (CYP3A4) and ATP-binding cassette sub-family G member 2 (ABCG2) are regulated by miR-27b-3p and miR-328-3p, respectively. Here we employed our newly established RNA bioengineering technology to produce bioengineered RNA agents (BERA), namely BERA/miR-27b-3p and BERA/miR-328-3p, via fermentation. When introduced into human cells, BERA/miR-27b-3p and BERA/miR-328-3p were selectively processed to _target miRNAs and thus knock down CYP3A4 and ABCG2 mRNA and their protein levels, respectively, as compared to cells treated with vehicle or control RNA. Consequently, BERA/miR-27b-3p led to a lower midazolam 1'-hydroxylase activity, indicating the reduction of CYP3A4 activity. Likewise, BERA/miR-328-3p treatment elevated the intracellular accumulation of anticancer drug mitoxantrone, a classic substrate of ABCG2, hence sensitized the cells to chemotherapy. The results indicate that biologic miRNA agents made by RNA biotechnology may be applied to research on miRNA functions in the regulation of drug metabolism and disposition that could provide insights into the development of more effective therapies.

Keywords: 3′-UTR, 3′-untranslated region;, VDR, vitamin D receptor; ABCG2; ABCG2, ATP-binding cassette sub-family G member 2;, ADME, absorption, distribution, metabolism, and excretion; BERA, bioengineered RNA agent;, CYP, cytochrome P450; Bioengineered RNA; CYP3A4; Drug disposition; E. coli, Escherichia coli;, FPLC, fast protein liquid chromatography; LC--MS/MS, liquid chromatographytandem mass spectroscopy;, microRNA, miR or miRNA; RNAi, RNA interference;, RT-qPCR, reverse transcription quantitative real-time polymerase chain reaction; RXRα, retinoid X receptor α;, tRNA, transfer RNA; miR-27b; miR-328; ncRNA, noncoding RNA;, PAGE, polyacrylamide gel electrophoresis.

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Figures

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Graphical abstract
Fig. 1
Figure 1
Bioengineering of new BERA/miR-27b-3p molecule. (A) Schematic illustration of the tRNA/pre-miR-34a carrier used for the production of BERA/miR-27b-3p. (B) Urea-PAGE analysis of total bacterial RNAs showed that chimeric miR-27b-3p was heterogeneously expressed in E. coli using the tRNA/pre-miR-34a scaffold. Total RNAs isolated from untransformed HST08 (WT) E. coli were used as control, and our previously constructed tRNA-hsa-miR-27b-123nt plasmid transformed HST08 E. coli was utilized for comparison. MiR-27b-3p was expressed at much higher levels when using the tRNA/pre-miR-34a carrier. (C) Representative FPLC traces during the purification of BERA/miR-27b-3p. Inserts were corresponding urea-PAGE analyses of collected fractions (1, 2, 3 and 4) eluted at 8.7 min, which confirmed the purity of isolated recombinant ncRNAs.
Fig. 2
Figure 2
BERA/miR-27b-3p is processed to miR-27b-3p in human LS-180 cells, effectively reduces VDR and CYP3A4 protein expression levels, and thus alters cellular CYP3A4 drug metabolism capacity. LS-180 cells was transfected with 50 nmol/L BERA/miR-27b-3p or control RNA for 24, 48, 72 and 96 h. Levels of miR-27b-3p in BERA/miR-27b-3p treated cells were significantly higher than control RNA and the high level of miR-27b-3p persisted 96 h (A). Treatment of 1α-VD3 greatly induced CYP3A4 mRNA (B) and protein (C) expression, whereas had no effect on VDR mRNA (B) or protein (C) levels. Following the induction by 1α-VD3, CYP3A4 (B) and VDR (C) mRNA levels, as well as their protein levels (D) were significantly reduced by BERA/miR-27b-3p, compared to control RNA (C). CYP3A4 enzymatic activity, as measured by [1′-OH-MDZ]/[MDZ] metabolic ratio after exposure to MDZ for 1.5 h (at 48 h post-transfection), was dramatically increased by 1α-VD3 treatment. 1′-OH-MDZ formation was significantly lower in LS-180 cells treated with BERA/miR-27b-3p than control RNA (E). Values are mean ± SD of triplicate treatments. *P<0.05; **P<0.01; ***P<0.001, compared to vehicle or control RNA group (Student׳s t-test).
Fig. 3
Figure 3
BERA/miR-328-3p is processed to miR-328-3p in human cancer cells and consequently knocks down ABCG2 levels, leading to higher intracellular accumulation of mitoxantrone and greater sensitivity to mitoxantrone. Human MCF7/MX100 and BeWo cells were transfected with 10 nmol/L BERA/miR-328-3p, control RNA or vehicle for 48 or 72 h. Level of miR-328-3p in BERA/miR-328-3p treated cells was significantly higher than tRNA control (A). (n = 3/group; ***P<0.001, **P<0.01, compared to the vehicle or tRNA control; one-way ANOVA). As a result, ABCG2 mRNA (B) and protein (C) levels were significantly reduced in BERA/miR-328-3p treated MCF/MX100 cells but not in BeWo cells. (***P<0.001, **P<0.01, compared to the vehicle or control RNA; n=3 per group; one-way ANOVA). Flow cytometry analyses revealed that compared to the vehicle control, MCF-7/MX100 cells transfected with BERA/miR-328-3p had significantly higher mitoxantrone fluorescence intensity (D). (*P<0.05, compared to the vehicle control; n=3/group; one-way ANOVA). BERA/miR-328-3p significantly sensitized MCF7/MX100 cells but not BeWo cells to mitoxantrone. The estimated EC50 and Hill slope for mitoxantrone (MX) cytotoxicity against BERA/miR-328-3p- and control tRNA-treated MCF7/MX100 and BeWo cells (***P<0.001, compared to vehicle and control RNA; n=4/group; one-way ANOVA).
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