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

Abstract

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 down-regulated 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 STATEMENT: We 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.

Fig. 1.

Deregulated lncRNAs in HepaRG cells exposed to APAP. (A and B) show the volcano plots in HepaRG cells treated with 5 and 10 mM APAP for 24 hours, respectively. The x-axis represents the log₂-fold change, and the y-axis represents the P value (−log₁₀ based). A P value <0.05 and absolute log₂-fold change >1 were used as thresholds to differentiate significantly up- and down- expressed lncRNAs. Significantly up- and down- expressed LincRNAs are labeled in red and green colors, respectively. (C) displays the specific fold changes of LINC00574 in cells treated with 5 or 10 mM APAP. (D) shows the changes of the expression of UGT2B15 upon exposure to 5 or 10 mM APAP. **P < 0.01. FPKM, fragments per kilobase of transcript per million mapped reads.

Fig. 2.

LINC00574 is a liver-specific lncRNA with a stable secondary structure. (A) Schematic representation of LINC00574 on chromosome 6 based on University of California Santa Cruz Genome Browser tracks showing CpG island information, peak signal of H3K27Ac, mammalian conservation. (B) Secondary structure (color indicates positional entropy) and mountain plot of LINC00574. A secondary structure of LINC00574 was predicted based on minimal free energy (–1060.10 kcal/mol, left panel). Right panel shows a mountain plot of minimum free energy structure, centroid structures, and partition function structure. The closely related status for these curves indicated the stable secondary structure of RNA molecule. The height (y-axis) of the mountain plot indicates the number of base pairs enclosing a sequence position indicated in the x-axis. pe indicates partition function structure; Centroid indicates centroid structure. (C) RNA levels (log₂ fragments per kilobase of transcript per million mapped reads) of LINC00574 in 15 different types of tissues (tumors and adjacent normal tissues) from TCGA data base. BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; COAD, colon adenocarcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; THCA, thyroid carcinoma; UCEC, uterine corpus endometrial carcinoma.

Fig. 3.

LINC00574 level correlated with the expression UGT2B15. (A) Significantly positive correlation between the expression of LINC00574 and the expression of UGT2B15 in liver tissues (tumors and adjacent normal tissues) in TCGA data base. (B) and (C) Significant down-regulation of relative LINC00574 and UGT2B15 in HepaRG cells obtained at multiple time points after 10 mM APAP exposure. Each assay was carried out in triplicate. **P < 0.01. FPKM, fragments per kilobase of transcript per million mapped reads.

Fig. 4.

Knockdown of LINC00574 decreases UGT2B15 expression. (A) Enriched LINC00574 RNAs were observed in the nuclear fraction, compared with the cytoplasmic fraction. (B–D) Differentiated HepaRG cells were transfected with 20 nM siRNA, ASO, or their cognate controls. Both siRNA and ASO, designed to target LINC00574, decreased LINC00574 (B) and the expression of UGT2B15 mRNA (C) and protein (D) in HepaRG cells. Each assay was carried out in triplicate. *P < 0.05; **P < 0.01. ASO-LINC00574, antisense oligonucleotide (ASO) against LINC00574; ASO-NC, negative control ASO; si-LINC00574, small interfering RNA (siRNA) against LINC00574; siRNA-NC, negative control siRNA.

Fig. 5.

hsa-miR-129-5p modulates the expression of UGT2B15 by interacting with LINC00574. (A) Free energy analysis shows that hsa-miR-129-5p is able to target LINC00574 with an MFE of –25.2 kcal/mol. ΔG indicates Gibbs energy. (B) hsa-miR-129-5p oligonucleotides interact with LINC00574 in vitro. Lanes 1 and 2 indicate the mobility of hsa-miR-129-5p and LINC00574 oligonucleotides, respectively; lane 3 indicates the mobility shift of the miRNA-lncRNA; lanes 4 and 5 indicate the competition assays to diminish the complex formed by hsa-miR-129-5p and LINC00574 oligonucleotides using excess unlabeled nonspecific competitors (cold negative control, cold-NC) or hsa-miR-129-5p (cold-miRNA). Lane 6 indicates the RNA-protein complexes formed by cytoplasmic extracts from HepaRG cells together with hsa-miR-129-5p or LINC00574 oligonucleotides. Lanes 7 and 8 show the competition assays to diminish the RNA-protein complex. Lanes 9–12 indicate the mobility status of RNA-protein complex together with antibodies against Ago1, Ago2, Ago3, and Ago4, respectively. The arrow indicates the oligonucleotide complexes in lane 3. The hollow triangle indicates the RNA-protein complexes in lanes 6–12. (C) Differentially expressed miR129-5p upon treating cells with 5 or 10 mM APAP. (D) The luciferase reporter gene activity in HepG2 cells was suppressed by transfection with hsa-miR-129-5p mimics. The luciferase reporter plasmid (100 ng) containing response elements of hsa-miR-129-5p in LINC00574 was cotransfected with 50 nM of miRNA negative control (miR-NC), hsa-miR-129-5p mimics, miRNA inhibitor negative control (IH-NC), or miR-129-5p-inhibitor (miR-129-5p-IH). (E) Exogeneous hsa-miR-129-5p inhibited the endogenous expression of LINC00574 in HepaRG cells. Differentiated HepaRG cells were transiently transfected with 50 nM of miRNA negative control (miR-NC), hsa-miR-129-5p mimics, miRNA inhibitor negative control (IH-NC), or miR-129-5p-inhibitor (miR-129-5p-IH). A decreased LINC00574 level was observed in cells transfected with hsa-miR-129-5p mimics. (F) Exogeneous hsa-miR-129-5p inhibited the protein expression of UGT2B15 in HepaRG cells detected by Western blot. Each assay was conducted in triplicate. Data are presented as means ± S.D. *P < 0.05; **P < 0.01.