## 2 Results
### Lnc-_Nr6a1_ Depletion Reduces Cell Adhesion and Alters Gene Expression  
To examine the physiological relevance of _Lnc-Nr6a1_ expression in EMT, we used CRISPR-Cas9 technology to remove _Lnc-Nr6a1_ transcription. NMuMG cells were edited by CRISPR using two pairs of single guide RNAs (sgRNAs) located in the promoter and first exon of _Lnc-Nr6a1_, respectively, obtaining a deletion of 600 bp (Figure 3A). Complete inhibition of Inc-_Nr6a1_ isoforms expression was achieved in homozygous clones (Figure 3B,C). Furthermore, disruption of the _Lnc-Nr6a1_ host gene expression affected the miR181a2/b2 expression (Figure 3D). It should be noted that expression of identical mature miR-181a and miR-181b, encoded by mir-181a1 and mir-181b1 genes on chromosome 1, was not affected by TGF-\(\beta\) treatment. Previous chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) assays in the same cell line [16] showed a remarkable H3K4 me3 and H3K27 ac-enriched region with high chromatin accessibility (via ATAC-seq) around the transcription start site of the _Lnc-Nr6a1_ gene. Neither H3K4 me3 and H3K27 ac enrichment nor chromatin accessibility were found in the miR181a2/b2 region (Figure S5A). In addition, querying the PanCancer Atlas expression data reveals a strong co-expression relationship between _Lnc-Nr6a1_ and embedded miR-181a2/b2 in different tumors (Figure S5B). Altogether, these results support that up-regulation of miR-181a2/b2 and _Lnc-Nr6a1_ after TGF-\(\beta\) treatment are driven by activation of the same promoter. To reveal the functional importance of the _Lnc-Nr6a1_ transcripts, we performed transcription profiling of the _Lnc-Nr6a1_-depleted cells. _Lnc-Nr6a1_ knockout resulted in statistically significant changes in the expression levels of 218 genes (adjusted \(p\)-value \(<0.05\) and \(|\log_{2}\)FC\(|>0.5\)) compared with the control cells; among them 95 and 123 were downregulated and upregulated, re spectively (Table S1 and Figure 3E). Some of these changes were confirmed by RT-qPCR, as downregulation (\(>\)100-fold) of _Dsg2_, _Fam111a_, _Mal2_, _FoxF1_ and upregulation of _Prune_, _Pkp1_, _Adgr13_, (Figure S6). Gene ontology analysis of the downregulated genes in _Lnc-Nr6a1_\({}^{-/-}\) cells showed an enrichment in genes involved in cell adhesion and the glycolytic process (Figure 3F and Table S2). Next, we investigated the effect of _Lnc-Nr6a1_ knock-down on the adhesion capacity of NMuMG cells to substrate and cells. _Lnc-Nr6a1_-depleted cells showed a lower capacity to adhere to the substrate; this effect was observed over a long and short time (Figure 4A,B). Furthermore, neither cell cycle changes nor alterations in proliferation were observed in _Lnc-Nr6a1_-depleted cells (Figure S7A,B). In order to determine the effect of _Lnc-Nr6a1_ absence on cell-cell adhesion, monolayers of NMuMG cells were incubated with calcine-labelled _Lnc-Nr6a1_-depleted cells or control cells for 2 h. Flow cytometry analysis showed a lower ability of _Lnc-Nr6a1_-depleted cells to adhere to cells (Figure 4D,E). The loss of adhesion capacity to substrate and cells were partially recovered after expressing Inc-Nr6a1-1 isoform (Figure 4C-E). Together, the combination of IncRNA profiling and functional assays identified _Lnc-Nr6a1_ as a IncRNA involved in cell adhesion to substrate and cells. Since _Lnc-Nr6a1_ is induced early by TGF-\(\beta\), we studied whether expression changes produced by TGF-\(\beta\) were dependent on the presence of _Lnc-Nr6a1_. We analyzed the transcriptomic profiles of the control cells versus _Lnc-Nr6a1_-depleted cells after 2 h of TGF-\(\beta\) treatment (Tables S3 and S4). In total, 895 genes were upregulated and 747 genes downregulated in the control cells and 641 and 459 genes, respectively, in the _Lnc-Nr6a1_-depleted cells (adjusted \(p\)-value \(<\) 0.05 and 1 log2 FC1 \(>\) 0.5).