## 2 Results
### Upregulation of Inc-Nr6a1 and Embedded miR-181a2 and miR-181b2 during EMT
Likewise, _Nr6a1_ transcript levels were low in most tissues and organs analyzed except in testis, while expression profiles of Inc-_Nr6a1_ isoforms in multiple adult tissues showed that they were expressed notably in skeletal muscle, and differed markedly in other tissues or organs; therefore, Inc-_Nr6a1-1_ was expressed in the eyes while Inc-_Nr6a1-2_ was expressed in bone marrow and the thymus, suggesting that they could regulate different biological processes (Figure 1H,I). Together these data demonstrate that the expression of _Luc-Nr6a1_ and _Nr6a1_ genes are not matched. Sequence analysis using ORF Finder (National Centre for Biotechnology Information) predicted open reading frames smaller than 43 amino acids without a valid Kozack sequence. In addition, a low-coding potential score (\(-\)0.83 and \(-\)0.86) and a low-coding probability (0.028 and 0.028) returned by the Coding Potential Calculator software [13] and Coding-Potential Assessment Tool [14], respectively, confirmed a negligible protein coding potential for Inc-_Nr6a1-1_ and Inc-_Nr6a1-2_, respectively (Figure S3). A human _Luc-Nr6a1_ orthologue transcript produced by the syntenic locus in chromosome 9 displays an overall 60% and 68% sequence identity with Inc-_Nr6a1-1_ and Inc-_Nr6a1-2_, respectively. It is composed of 2 exons of 330 and 387 bp and an intron of 40,220 bp that harbors mir-181a2 and mir-181b2 genes. In a similar way to the murine counterpart, expression of the human transcript was induced in MCF-10A human breast epithelial cells and primary human omental mesothelial cells after TGF-\(\beta\) treatment (Figure S4A,B). Figure 1: _Cont._  
Figure 1: Genomic context and expression profiles of _Inc-Nr6a1_ and _Nr6a1_ genes. (**A**) Schematic illustration of the genomic location and transcript structure of the _Inc-Nr6a1_ gene embedded within the third intron of the _Nr6a1_ gene. The annotated nascent and processed transcripts of _Inc-Nr6a1_ (Inc-_Nr6a1_-_1_ and _Inc-Nr6a1_-_2_) and _Inc-_Nr6a1_-_2_) are shown. The red arrows indicate the transcription orientation. (**B**) Schematic representation of spliced Inc-pri-miR-181a2/b2 transcript and (**C**) electrophoretic gel showing PCR products generated with a forward primer located in exon2 and reverse primers located in miR-181a2 and mir181b2, respectively. (**D**) Rate of poly(A\({}^{-}\)) RNA relative to poly(A\({}^{+}\)) RNA for the indicated transcripts, quantified by RT-qPCR. (**E**) Relative levels of _Nr6a1_ mRNA in cells treated with TGF-\(\beta\) at the indicated times. Experiments were performed in triplicate samples and error bars represent S.D. (**F**) Relative levels of Inc-_Nr6a1_ isoforms in cells treated with TGF-\(\beta\) at the indicated times. (**G**) Expression of miR-181a2/b2 in cells treated with TGF-\(\beta\) for 24 h. Experiments were repeated twice in triplicate samples; error bars represent S.D. (**H,I**) relative expression of _Nr6a1_ mRNA and _Inc-Nr6a1_ isoforms, respectively, in a broad range of adult mouse tissues. Results are presented relative to a fixed reference sample (thymus and liver in (**H,J**), respectively). Experiments were performed in triplicate samples; error bars represent S.D. ***\(p<0.001\), **\(p<0.01\)** by two-tailed Student’s _t_-test. Figure 2: Signaling pathways involved in Inc-_Nr6a1_ isoforms upregulation by TGF-\(\beta\). **(A)** NMuMG cells were transfected with DisRNAs against Smad4 (DisSmad4) or scramble DisRNA (SRC) and treated with TGF-\(\beta\) for 2 h. Levels of Inc-_Nr6a1-1_ (top) and Inc-_Nr6a1-2_ (bottom) were measured by RT-qPCR analysis. Experiments were performed in triplicate samples; error bars represent S.D. **(B)** Effect of p38 kinase inhibitor, SB203580, on Inc-_Nr6a1_ isoforms upregulation. Cells were pretreated with 10 \(\upmu\)M inhibitor for 1 h and next treated with TGF-\(\beta\) for 2 h where indicated. Levels of Inc-_Nr6a1-1_ (top) and Inc-_Nr6a1-2_ (bottom) were measured by RT-qPCR analysis. Experiments were performed in triplicate samples; error bars represent S.D.