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. 2021 Sep 8;12:729062. doi: 10.3389/fphar.2021.729062

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Copyright © 2021 Zhang, Guan, Zhang, Li, Xu, Gong, Chen and Lu.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Nqo1-AS1 increases Nqo1 mRNA stability and expression. (A–C) RNase protection assay was performed to examine the RNA duplexes formation between Nqo1-AS1 and Nqo1 mRNA. PcDNA3.1-Nqo1-AS1 overlapping region (pc-Nqo1-AS1-OL), PcDNA3.1-Nqo1-AS1 non-overlapping region (pc-Nqo1-AS1-NOL) or pcDNA3.1 (control) vector was cotransfected with pcDNA3.1- Nqo1 (pc-Nqo1) into mle-12 cells. After transfection for 48 h, the total RNA was extracted from the cells. RNA sample was digested with increasing amounts of RNAse A + T cocktail (represented as the black wedge and multiple “+++”) in various samples.Then the remaining double-stranded RNA was reversely transcribed to cDNA and amplified the overlapping part of Nqo1 (Nqo1-OL) mRNA (A) and the non-overlapping part of Nqo1 (Nqo1-NOL) mRNA (B) by PCR. Gapdh PCR product (C) was used as a control. (D) Schematic diagram displayed the RNA duplexes formation between the overlapping part of Nqo1-AS1 (Nqo1-AS1-OL) and the overlapping part of Nqo1 (Nqo1-OL) mRNA, which protected both of them from RNase digestion. “E” followed by number represented exon. The sites of primers used in RNase protection assay were indicated as follows:1 Nqo1-OL PCR primer; 2Nqo1-NOL PCR primer. (E–F) Line chart shown the stability of Nqo1 mRNA over time relative to time 0 after blocking new RNA synthesis with a-amanitin treatment (10 μg/ml). 18S rRNA was used as an internal control, which was a product of RNA polymerase I and was unchanged after a-amanitin treatment. (E) Nqo1-AS1 siRNA or siRNA CTL was transfected into the mle-12 cells for 24 h, and followed by a-amanitin treatment for 0 h, 6 h, 12 h, 18 and 24 h. Then the Nqo1 mRNA expression level was measured by qRT-PCR. (F) The pc-Nqo1-AS1 or pcDNA3.1 vector was transfected into the mle-12 cells for 24 h and then treated with a-amanitin for 0 h, 6 h, 12 h, 18 and 24 h. Subsequently, the Nqo1 mRNA expression level was detected by qRT-PCR. (G) The luciferase activity of pmirGLO-Nqo1 3′UTR was markedly decreased in the mle-12 cells transfected with Nqo1-AS1 siRNA compared to cells transfected with siRNA CTL. (H) The luciferase activity of pmirGLO-Nqo1 3′UTR was increased significantly in the mle-12 cells that overexpressing the Nqo1-OL of Nqo1-AS1, but not the Nqo1-NOL of Nqo1-AS1. *p < 0.05. Data represented the mean ± SEM from three independent experiments.