*2.1. LINC01133 Is Upregulated in Ectopic Endometriosis Lesions*

In order to identify changes that may occur in *LINC01133* expression levels during endometriosis pathogenesis, we compared control eutopic endometrium from women without endometriosis with eutopic endometrium from endometriosis patients, and ectopic endometriosis lesions using quantitative reverse transcription PCR (qRT-PCR). This showed that *LINC01133* expression is significantly upregulated in endometriosis lesions compared to eutopic tissue of both patients and controls (Figure 1A). These changes were independent of disease stage and menstrual cycle phase (Figure 1B,C). We next used RNA Scope in situ hybridization to determine *LINC01133* spatial localization within endometrium tissue. We found that *LINC01133* is expressed in both stromal and epithelial cells of the eutopic endometrium of women with endometriosis, but appeared to show higher levels in glandular epithelial cells (Figure S1A). Quantification confirmed this observation, with positive glandular epithelial cells around five times more frequent than positive stroma cells (*p* = 0.0012) (Figure S1B).

**Figure 1.** *LINC01133* expression is upregulated in endometriosis lesions. (**A**) Relative expression of *LINC01133* is significantly increased in ectopic endometriotic lesions compared to the eutopic endometrium of women with and without endometriosis. Expression in the eutopic endometrium does not differ between women with and without the disease. (**B**) In endometriosis patients, *LINC01133* expression does not significantly differ between mild (rAF I+II) and more severe (rAF III+IV) disease stages, in either the eutopic endometrium or ectopic lesions. (**C**) *LINC01133* expression does not significantly differ between the proliferative and secretory stages of the menstrual cycle in control eutopic endometrium, eutopic endometrium from patients, or ectopic lesions. Data in (**A**–**C**) are presented as dot plots including the mean relative expression levels and standard deviation in each group As the sample sizes were not equal, data were analyzed by fitting a mixed model, rather than repeated measures ANOVA (which requires equal sample size). Adjusted *p*-values values (adjp) < 0.05 were considered significant with non-significant differences indicated by ns. Control: endometrial tissue of women without endometriosis, Eutop: endometrial tissue of women with endometriosis, Ectop: endometriosis lesions. **Figure 1.** *LINC01133* expression is upregulated in endometriosis lesions. (**A**) Relative expression of *LINC01133* is significantly increased in ectopic endometriotic lesions compared to the eutopic endometrium of women with and without endometriosis. Expression in the eutopic endometrium does not differ between women with and without the disease. (**B**) In endometriosis patients, *LINC01133* expression does not significantly differ between mild (rAF I + II) and more severe (rAF III + IV) disease stages, in either the eutopic endometrium or ectopic lesions. (**C**) *LINC01133* expression does not significantly differ between the proliferative and secretory stages of the menstrual cycle in control eutopic endometrium, eutopic endometrium from patients, or ectopic lesions. Data in (**A**–**C**) are presented as dot plots including the mean relative expression levels and standard deviation in each group As the sample sizes were not equal, data were analyzed by fitting a mixed model, rather than repeated measures ANOVA (which requires equal sample size). Adjusted *p*-values values (adjp) < 0.05 were considered significant with non-significant differences indicated by ns. Control: endometrial tissue of women without endometriosis, Eutop: endometrial tissue of women with endometriosis, Ectop: endometriosis lesions.

#### *Transcriptional Deregulation of 1210 Genes* To evaluate the role of *LINC01133* within the epithelial cell compartment of endome-*2.2. LINC01133 siRNA Knockdown in 12Z Endometriosis Epithelial Cells Leads to Transcriptional Deregulation of 1210 Genes*

*2.2. LINC01133 siRNA Knockdown in 12Z Endometriosis Epithelial Cells Leads to* 

triosis lesions, we performed transient siRNAs-based knockdown of *LINC01133* in the 12Z endometriosis epithelial cell line, followed by RNA- sequencing. First, using qRT-PCR we confirmed the efficiency of *LINC01133* knockdown using three independent siRNA oligos. To evaluate the role of *LINC01133* within the epithelial cell compartment of endometriosis lesions, we performed transient siRNAs-based knockdown of *LINC01133* in the 12Z endometriosis epithelial cell line, followed by RNA- sequencing. First, using qRT-PCR we confirmed the efficiency of *LINC01133* knockdown using three independent siRNA oligos. Significant knockdown was achieved for all 3 oligos (*p* ≤ 0.0001), with

the most efficient siRNA *LINC01133a* reducing *LINC01133* expression by more than 90% 72 h after transfection (Figure 2A). In order to identify any genes and pathways affected by *LINC01133* knockdown, we then conducted RNA-sequencing comparing *LINC01133a* knockdown cells with non-targeting siRNA control cells 72 h after transfection (3 biological replicates each). We identified four *LINC01133* transcript isoforms that are expressed in 12Z cells and confirmed that all were efficiently targeted by the *LINC01133a* siRNA oligo (Figure 2B). Further, analysis revealed 1210 differentially expressed (DE) transcripts in 12Z knockdown cells, compared to controls using a fold change cutoff of >1.5 and adjp < 0.05. From those DE genes, 703 were down-regulated and 507 up-regulated in knockdown cells (Table S4). These transcriptional changes enabled a clear separation of *LINC01133* knockdown and control samples by unsupervised hierarchical clustering (Figure 2C). *Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 5 of 19

(**A**) qRT-PCR shows relative expression of *LINC01133* is significantly reduced in 12Z cells for all 3 siRNA oligos that were used. (**B**) Tissue-specific *LINC01133* isoforms are efficiently knocked down in 12Z cells. Top: A UCSC genome browser screenshot shows RNA sequencing reads mapping to *LINC01133* are dramatically reduced in a knockdown with siRNA oligo *LINC01133a*. Bottom: Genome assembly using Cufflinks reveals multiple *LINC01133* isoforms in 12Z cells that differ Right: Hundreds of genes are up- or down-regulated in the *LINC01133a* knockdown in 12Z cells. **Figure 2.** *LINC01133* knockdown leads to the deregulation of hundreds of genes in an endometriosis epithelial cell line.

**Figure 2.** *LINC01133* knockdown leads to the deregulation of hundreds of genes in an endometriosis epithelial cell line.

(**A**) qRT-PCR shows relative expression of *LINC01133* is significantly reduced in 12Z cells for all 3 siRNA oligos that were used. (**B**) Tissue-specific *LINC01133* isoforms are efficiently knocked down in 12Z cells. Top: A UCSC genome browser screenshot shows RNA sequencing reads mapping to *LINC01133* are dramatically reduced in a knockdown with siRNA oligo *LINC01133a*. Bottom: Genome assembly using Cufflinks reveals multiple *LINC01133* isoforms in 12Z cells that differ from the annotated GENCODE and Refseq transcripts. (**C**) Left: Hierarchical clustering of differentially expressed genes from RNA sequencing biological replicates shows that the control and *LINC01133a* siRNA samples cluster separately. Right: Hundreds of genes are up- or down-regulated in the *LINC01133a* knockdown in 12Z cells. *Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 6 of 19

#### *2.3. The Knockdown of LINC01133 in 12Z Cells Effects Genes and Pathways with a Known Function in Endometriosis Lesion Formation Function in Endometriosis Lesion Formation*  To gain insight into the function of the genes being regulated by *LINC01133*, we car-

*2.3. The Knockdown of LINC01133 in 12Z Cells Effects Genes and Pathways with a Known* 

To gain insight into the function of the genes being regulated by *LINC01133*, we carried out gene ontology enrichment analysis (GOEA) (http://bioinformatics.sdstate.edu/go/) (accessed on 2 December 2020) [26] and gene set enrichment analysis (GSEA) (http:// www.gsea-msigdb.org/gsea) (accessed on 2 December 2020) [27,28]. The GOEA showed enrichment in genes that control cell proliferation, migration and angiogenesis (Figure S2A and Table S5), all processes to be involved in the pathogenesis of the disease. GSEA enrichment in targets of the mammalian histone methyltransferase EZH2, adult tissue stem cells and mesenchymal cells (Figure S2B and Table S6), Together these results suggest that *LINC01133* may be involved in the regulation of epithelial cell proliferation, invasion and cell fate conversion (EMT), processes that are known to support ectopic lesion growth. ried out gene ontology enrichment analysis (GOEA) (http://bioinformatics. sdstate.edu/go/) (accessed on 2 December 2020) [26] and gene set enrichment analysis (GSEA) (http://www.gsea- msigdb.org/gsea) (accessed on 2 December 2020) [27,28]. The GOEA showed enrichment in genes that control cell proliferation, migration and angiogenesis (Figure S2A and Table S5), all processes to be involved in the pathogenesis of the disease. GSEA enrichment in targets of the mammalian histone methyltransferase EZH2, adult tissue stem cells and mesenchymal cells (Figure S2B and Table S6), Together these results suggest that *LINC01133* may be involved in the regulation of epithelial cell proliferation, invasion and cell fate conversion (EMT), processes that are known to support ectopic lesion growth.

#### *2.4. LINC01133 Regulates Proliferation and Invasion of Endometriosis Epithelial Cells In Vitro 2.4. LINC01133 Regulates Proliferation and Invasion of Endometriosis Epithelial Cells In Vitro*

To determine if the phenotypic changes predicted by GOEA and GSEA analysis following *LINC01133* knockdown in 12Z cells occur, we evaluated changes in cell proliferation and invasion in 12Z cells 72 h post knockdown. The results showed that *LINC01133* knockdown leads to a slight, but significant downregulation of 12Z proliferation (Figure 3A) and significantly enhances the invasion phenotype of knockdown cells (Figure 3B). The relative proliferation rate of *LINC01133* knockdown cells was 30% lower (adjp < 0.0001), and the invasion rate was 1.5 times higher (adjp < 0.05) when compared to cells transfected with the siRNA control oligo. To determine if the phenotypic changes predicted by GOEA and GSEA analysis following *LINC01133* knockdown in 12Z cells occur, we evaluated changes in cell proliferation and invasion in 12Z cells 72 h post knockdown. The results showed that *LINC01133* knockdown leads to a slight, but significant downregulation of 12Z proliferation (Figure 3A) and significantly enhances the invasion phenotype of knockdown cells (Figure 3B). The relative proliferation rate of *LINC01133* knockdown cells was 30% lower (adjp < 0.0001), and the invasion rate was 1.5 times higher (adjp < 0.05) when compared to cells transfected with the siRNA control oligo.

**Figure 3.** *LINC01133* knockdown reduces proliferation and increases invasion of 12Z endometriosis epithelial cells. (**A**)

median and box boundaries at the 25th and 75th percentiles from three biological replicates. Six independent fields were counted and the mean values taken for analysis. Statistical analysis of the data between the groups in A and B was done using Kruskal–Wallis ANOVA test with Dunn's test for multiple comparison. Adjp < 0.05 were considered significant.

(**A**) Relative number of proliferating cells is significantly reduced in *LINC01133* knockdowns using 3 different siRNA oligos. Data are presented as bar plots of mean values from three biological replicates +SD. (**B**) Invading cells are significantly increased following *LINC01133* knockdown using 2 different siRNA oligos. Left: Quantification of the number of invading cells after *LINC01133* knockdown analyzed by the trans-well method. Right: Representative images of control and knockdown cells at 10× magnification stained with CyQuant fluorescence dye (green), which shows fluorescence enrichment when bound to cellular nucleic acids. Data are presented as a box plot ranging from minimum to maximum, including the median and box boundaries at the 25th and 75th percentiles from three biological replicates. Six independent fields were counted and the mean values taken for analysis. Statistical analysis of the data between the groups in A and B was done using Kruskal–Wallis ANOVA test with Dunn's test for multiple comparison. Adjp < 0.05 were considered significant.
