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. Author manuscript; available in PMC: 2020 Dec 30.
Published in final edited form as: Arch Toxicol. 2020 Mar 28;94(5):1637–1653. doi: 10.1007/s00204-020-02706-5

Fig. 6.

Fig. 6

LINC00844 serves as an hsa-miR-486-5p sponge to protect SULT2A1 from hsa-miR-486-5p-mediated suppression in HepaRG cells. a A miRNA targeting network for SULT2A1 and LINC00844. LncBase Predicted v.2 predicted that LINC00844 (octagon) was potentially targeted by 23 miRNAs (oval), including hsa-miR-486-5p, which was among the 12 miRNA candidates for SULT2A1 (rectangle) predicted by miRanda. hsa-miR-486-5p and hsa-miR-495-3p are two experimentally-verified miRNAs (blue oval) that repress SULT2A1 in HepG2 cells. b LINC00844 directly interacts with hsa-miR-486-5p. Top, RNAhybrid analysis predicted that hsa-miR-486-5p may hybridize with LINC00844 beginning at position 38 (transcriptional start site as position 1) with an MFE of −22.4 kcal/mol. Bottom, FREMSAs verified the direct binding between miR-486-MRE on LINC00844 and hsa-miR-486-5p. Individually incubated probes for hsa-miR-486-5p (Lane 1) and miR-486-MRE (Lane 2) exhibited single bands. Co-incubation of hsa-miR-486-5p and miR-486-MRE probes resulted in a gel shift (Lane 3, arrow) and reduction of the band for either hsa-miR-486-5p and miR-486-MRE. The intensity of the shifted band was reduced by the addition of cold-miR-486–5p (Lane 5) at a 50 × concentration, but not by cold-NC (Lane 4). c LINC00844 is a specific target of hsa-miR-486-5p. LINC00844 was significantly upregulated in HepaRG cells transfected with hsa-miR-486-5p mimics, but not hsa-miR-495-3p, compared to control. d Confirmation of LINC00844 knockdown in HepaRG cells overexpressing hsa-miR-486-5p. Transfection of si-LINC00844 (pink bar), but not si-NC (blue bar), significantly reduced the level of LINC00844 in HepaRG cells overexpressing hsa-miR-486-5p. e LINC00844 protects SULT2A1 from hsa-miR-486-5p-mediated gene suppression. The level of SULT2A1 mRNA significantly increased in HepaRG cells overexpressing hsa-miR-486-5p (blue bar), compared to mimic-NC (white bar). However, when LINC00844 was knocked down, SULT2A1 mRNA expression was suppressed by hsa-miR-486-5p overexpression (pink bar). f Differential expression of hsa-miR-486-5p and LINC00844 between HepaRG and HepG2 cells. LINC00844 expression was > 500-fold higher in HepaRG cells than HepG2 cells, whereas hsa-miR-486-5p expression only exhibited a 5.6-fold elevation in HepaRG cells compared to HepG2 cells. Results shown are mean ± SD from three independent experiments. *p < 0.05 (color figure online)