FREMSA: A Method That Provides Direct Evidence of the Interaction between microRNA and mRNA

Yu, Dianke; Chen, Si; Li, Dongying; Knox, Bridgett; Guo, Lei

doi:10.1007/978-1-0716-0223-2_30

Cited by 16 publications

(7 citation statements)

References 15 publications

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“…Fluorescent-based RNA electrophoretic mobility shift assay (FREMSA) was carried out according to the protocol described in our previous report (Yu et al, 2020). Briefly, 200 fmol dye-miR-1301-3p and cognate dye-ADH6, dye-ALDH5A1, and dye-ALDH8A1 oligonucleotides were mixed in basic buffer containing 10 mM HEPES buffer (pH 7.3), 0.5% glycerol, 20 mM KCl, and 10 mM MgCl 2 , respectively.…”

Section: Methodsmentioning

confidence: 99%

MicroRNA hsa-miR-1301-3p Regulates Human ADH6, ALDH5A1 and ALDH8A1 in the Ethanol-Acetaldehyde-Acetate Metabolic Pathway

et al. 2020

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Alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs) are vital enzymes involved in the metabolism of a variety of alcohols. Differences in the expression and enzymatic activity of human ADHs and ALDHs correlate with individual variability in metabolizing alcohols and drugs and in the susceptibility to alcoholic liver disease. MicroRNAs (miRNAs) function as epigenetic modulators to regulate the expression of drug-metabolizing enzymes. To characterize miRNAs that target ADHs and ALDHs in human liver cells, we carried out a systematic bioinformatics analysis to analyze free energies of the interaction between miRNAs and their cognate sequences in ADH and ALDH transcripts and then calculated expression correlations between miRNAs and their targeting ADH and ALDH genes using a public data base. Candidate miRNAs were selected to evaluate bioinformatic predictions using a series of biochemical assays. Our results showed that 11 miRNAs have the potential to modulate the expression of two ADH and seven ALDH genes in the human liver. We found that hsa-miR-1301-3p suppressed the expression of ADH6, ALDH5A1, and ALDH8A1 in liver cells and blocked their induction by ethanol. In summary, our results revealed that hsa-miR-1301-3p plays an important role in ethanol metabolism by regulating ADH and ALDH gene expression. SIGNIFICANCE STATEMENTSystematic bioinformatics analysis showed that 11 microRNAs might play regulatory roles in the expression of two alcohol dehydrogenase (ADH) and seven aldehyde dehydrogenase (ALDH) genes in the human liver. Experimental evidences proved that hsa-miR-1301-3p suppressed the expression of ADH6, ALDH5A1, and ALDH8A1 in liver cells and decreased their inducibility by ethanol.Disclaimer: The information in this paper is not an official guidance or policy statement of the US Food and Drug Administration (FDA). No official support or endorsement by the US FDA is intended or should be inferred.

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Section: Methodsmentioning

confidence: 99%

MicroRNA hsa-miR-1301-3p Regulates Human ADH6, ALDH5A1 and ALDH8A1 in the Ethanol-Acetaldehyde-Acetate Metabolic Pathway

et al. 2020

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“…The detailed protocol for fluorescence-based RNA electrophoretic mobility shift assay (FREMSA) has been introduced in Molecular Toxicology Protocols (Yu et al, 2020). Briefly, 200 fmol hsa-miR-129-5p and cognate LINC00574 oligonucleotides were added to the reaction buffer containing 10 mM HEPES buffer (pH 7.3), 0.5% glycerol, 20 mM KCl, and 10 mM MgCl 2 .…”

Section: Methodsmentioning

confidence: 99%

Coordinated Regulation of UGT2B15 Expression by Long Noncoding RNA LINC00574 and hsa-miR-129-5p in HepaRG Cells

Yu¹,

Chen

et al. 2020

Drug Metab Dispos

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Recent studies have shown that microRNAs and long noncoding RNAs (lncRNAs) regulate the expression of drug metabolizing enzymes (DMEs) in human hepatic cells and that a set of DMEs, including UDP glucuronosyltransferase (UGT) 2B15, is downregulated dramatically in liver cells by toxic acetaminophen (APAP) concentrations. In this study we analyzed mRNA, microRNA, and lncRNA expression profiles in APAP-treated HepaRG cells to explore noncoding RNA-dependent regulation of DME expression. The expression of UGT2B15 and lncRNA LINC00574 was decreased in APAP-treated HepaRG cells. UGT2B15 levels were diminished by LINC00574 suppression using antisense oligonucleotides or small interfering RNA. Furthermore, we found that hsa-miR-129-5p suppressed LINC00574 and decreased UGT2B15 expression via LINC00574 in HepaRG cells. In conclusion, our results indicate that LINC00574 acts as an important regulator of UGT2B15 expression in human hepatic cells, providing experimental evidence and new clues to understand the role of cross-talk between noncoding RNAs. SIGNIFICANCE STATEMENTWe showed a molecular network that displays the cross-talk and consequences among mRNA, micro RNA, long noncoding RNA, and proteins in acetaminophen (APAP)-treated HepaRG cells. APAP treatment increased the level of hsa-miR-129-5p and decreased that of LINC00574, ultimately decreasing the production of UDP glucuronosyltransferase (UGT) 2B15. The proposed regulatory network suppresses UGT2B15 expression through interaction of hsa-miR-129-5p and LINC00574, which may be achieved potentially by recruiting RNA binding proteins.

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“…FREMSA is an in vitro assay that could visually and accurately observe miRNA binding to its target RNA ( Yu et al, 2020 ). The oligonucleotides for hsa-miR-15a-3p were synthesized and 5′-labeled with cy5.5TM dye .…”

Section: Methodsmentioning

confidence: 99%

miR-15a-3p Protects Against Isoniazid-Induced Liver Injury via Suppressing N-Acetyltransferase 2 Expression

Zhang

et al. 2021

Front. Mol. Biosci.

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Isoniazid (INH), an effective first-line drug for tuberculosis treatment, has been reported to be associated with hepatotoxicity for decades, but the underlying mechanisms are poorly understood. N-acetyltransferase 2 (NAT2) is a Phase II enzyme that specifically catalyzes the acetylation of INH, and NAT2 expression/activity play pivotal roles in INH metabolism, drug efficacy, and toxicity. In this study, we systematically investigated the regulatory roles of microRNA (miRNA) in NAT2 expression and INH-induced liver injury via a series of in silico, in vitro, and in vivo analyses. Four mature miRNAs, including hsa-miR-15a-3p, hsa-miR-628-5p, hsa-miR-1262, and hsa-miR-3132, were predicted to target the NAT2 transcript, and a negative correlation was observed between hsa-miR-15a-3p and NAT2 transcripts in liver samples. Further experiments serially revealed that hsa-miR-15a-3p was able to interact with the 3′-untranslated region (UTR) of NAT2 directly, suppressed the endogenous NAT2 expression, and then inhibited INH-induced NAT2 overexpression as well as INH-induced liver injury, both in liver cells and mouse model. In summary, our results identified hsa-miR-15a-3p as a novel epigenetic factor modulating NAT2 expression and as a protective module against INH-induced liver injury, and provided new clues to elucidate the epigenetic regulatory mechanisms concerning drug-induced liver injury (DILI).

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FREMSA: A Method That Provides Direct Evidence of the Interaction between microRNA and mRNA

Cited by 16 publications

References 15 publications

MicroRNA hsa-miR-1301-3p Regulates Human ADH6, ALDH5A1 and ALDH8A1 in the Ethanol-Acetaldehyde-Acetate Metabolic Pathway

MicroRNA hsa-miR-1301-3p Regulates Human ADH6, ALDH5A1 and ALDH8A1 in the Ethanol-Acetaldehyde-Acetate Metabolic Pathway

Coordinated Regulation of UGT2B15 Expression by Long Noncoding RNA LINC00574 and hsa-miR-129-5p in HepaRG Cells

miR-15a-3p Protects Against Isoniazid-Induced Liver Injury via Suppressing N-Acetyltransferase 2 Expression

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