## 2 Results
### Lnc-_Nr6a1_ Depletion Reduces Cell Adhesion and Alters Gene Expression
Since one of the Inc-_Nr6a1_ isoforms is transcribed as a precursor containing miR-181a2 and miR-181b2 genes, we investigated the effect of precursor over-expression using a CRISPR/Cas9 synergistic activation mediator (SAM) system (Figure 6A) [17]. To this end, we designed 10 single-guide RNA (sgRNAs) targeting regions close to the transcription start site (Table S8) and found that sgRNA 2 and sgRNA 8 notably increased both Inc-_Nr6a1_ isoforms and miR-181 genes expression, as shown by qPCR analysis (Figure 6B), supporting that regulation of _Luc-Nr6a1_ and miR-181a2/b2 is driven by the same promoter. Cells overexpressing the _Luc-Nr6a1_ gene showed the epithelial phenotype. However, both immunofluorescent and immunoblotting analysis revealed an increased expression of  
Figure 5: _Luc-Nr6a1_ depletion affects gene expression changes by TGF-\(\beta\). (**A**) Venn diagrams showing the intersection of upregulated (left panel) and downregulated genes (right panel) in control and _Luc-Nr6a1_-depleted cells treated with TGF-\(\beta\) for 2 h. Probability of overlapping based on hypergeometric distribution is provided. (**B**) Significant Gene Ontology annotation enrichments of genes affected by TGF-\(\beta\) treatment independently of the presence or absence of _Luc-Nr6a1_. (**C**) Migration of control, _Luc-Nr6a1_-depleted, Inc-_Nr6a1_-_1-_upperguated and Inc-_Nr6a1_-2-upperguated NMuMG cells were tested in wound-healing assays. Cells were imaged at 10 min intervals for 26 h. Frames of the movie at 12 and 18 h are shown. (**D**) Percentage of surface covered using frames at 9 and 12 h. Experiments were performed in triplicate samples. Values represent the percentage of wound closures. _p_-values are indicated for comparison. Error bars represent S.D. ***_p_ < 0.001. (**E**) A representative wound-density quantification of the time-lapse video frames from different cells using Wimasis image analysis. the mesenchymal marker N-cadherin and a decrease in the epithelial marker E-cadherin expression (Figure 6C,D). As _Lnc-Nr6a1_ upregulation induced expression changes associated with mesenchymal cells, we wondered whether _Lnc-Nr6a1_ upregulation could affect features of the EMT program. Neither proliferation nor adhesion capacity were affected in precursor-overexpressing cells (Figure S9); however, wound healing and Matrigel invasion assays revealed that these cells migrated significantly faster and were more invasive than _lnc-Nr6a1-1_ overexpressing cells (Figure 6E-G, Video S2). A feature of EMT programs is they contribute to the resistance of cells to anoikis. We determined the percentage of apoptotic cells in _lnc-Nr6a1-1_- and _Lnc-Nr6a1_-upregulated cells growing in the presence of methylcellulose and on Poly-HEMA-coated plates to prevent both adherence among them and their adherence to plastic, thereby eliciting anoikis. Cells overexpressing _Lnc-Nr6a1_ presented higher anoikis resistance than cells overexpressing the _lnc-Nr6a1-1_ isoform and control cells (Figure 6H). These results indicate, on the one hand, that miR-181a2/b2 expression increases the migratory and invasive ability of the cells and confers resistance to anoikis; and on the other hand, that the mature transcripts and embedded miRNAs from _Lnc-Nr6a1_ genomic loci perform independent functions. Figure 6: _Cont._  
Figure 6: Over-expression of _Lnc-Nr6a1_. (**A**) Diagram of CRISPR SAM-CRISPRR/Cas9 activation system. Nuclease-dead Cas9 (dCas9), fused to the transcriptional activator VP64, complexes with the CRISPR guide RNA. MS2 bacteriophage coat protein is fused to the p65 subunit of NF-kappaB and the activation domain of human heat-shock factor 1 (HSF1). MS2 coactivator proteins bind to MS2 RNA aptamer in the gRNA and with VP64 synergistically activate gene expression. (**B**) Expression levels of lnc-_Nr6a1_ isoforms and miR-181a2-3p in cells lines with different sgRNAs (within the region from \(-\)285 to +105 bp, distance to TSS). _Hprt_ and miR-425-5p genes were used to normalize expression of lnc-_Nr6a1_ isoforms and miR-181a2/b2, respectively.