**3. Discussion**

LncRNAs are epigenetic regulators that have been implicated in development and disease, but whose role in the pathogenesis of endometriosis remains relatively unknown. Endometriosis shares some features with cancer, including EMT, therefore we chose to investigate the role of *LINC01133* in endometriosis, a well-characterized lncRNA that has been associated with EMT in cervical [21], breast [22], colorectal [23] and gastric [24] cancer. We found that *LINC01133* is significantly upregulated in ectopic endometriosis lesions, and that knockdown in an epithelial endometriosis cell line indicates that it promotes cell proliferation and suppresses cell migration and invasion in endometriosis, but that it does not regulate EMT in this disease. Our results indicate that *LINC01133* affects cell proliferation by affecting the cell cycle via the p21/cyclin pathway, and cellular invasion and cytoskeleton remodeling due to Cofilin phosphorylation and inactivation by the TESK1 kinase. A caveat of our study is that we used the immortalized endometriosis epithelial cell line 12Z for our functional experiments, although this cell line is widely accepted in the field as a cell model of endometriosis [30].

The effects on cell proliferation were associated with cell cycle arrest in G1 and impaired S-phase entry due to significant up-regulation of cell cycle checkpoint protein p21 and concomitant downregulation of Cyclin A. The mechanism by which *LINC01133* may regulate these genes in endometriosis remains unclear. In non-small cell lung carcinoma *LINC01133* suppresses the transcription of *CDKN1A* (*p21*) via a direct EZH2-mediated chromatin remodeling mechanism [31]. In another context, *LINC01133* promotes the progression of cervical cancer by sponging miR-4784 to cause the up-regulation of AT-hook DNA-binding motif-containing protein 1 (AHDC1) promoting EMT [21]. The high basal level of p21 expression and moderate transcriptional activation upon *LINC01133* knockdown (~2.5-fold) indicates that sponging rather than an EZH2 mediated p21 activation may be a more likely mechanism of regulation, although this remains to be tested.

Impaired expression of *LINC01133* has been associated with the regulation of EMT in cancer [23,31,32]. EMT is a multi-stage process leading to the gradual remodeling of the epithelial into a mesenchymal phenotype. This includes the loss of epithelial markers and concomitant acquisition of mesenchymal markers, an increase in cell migration and invasion, disruption of cell-cell contacts, impaired adhesion and the remodeling of the cytoskeleton [19]. This molecular remodeling also takes place during the establishment of endometriosis lesions [17]. However, although we saw an enrichment of some mesenchymal gene sets among differentially expressed genes in 12Z endometriosis epithelial cells following *LINC01133* knockdown, we found little phenotypic evidence for EMT. Our data showed a significant upregulation of *TGFβ2* following *LINC01133* knockdown is associated with an increase in the levels of expression of the master regulator of EMT, *SOX4* [33,34] and subsequent down-regulation of *CDH1* and *CDH2*. However, expression of the epithelial markers *KDR7* and *KDR19* [35], along with the EMT regulators *TWIST*, *SNAIL*, *ZEB1* and *ZEB2* [36] were not significantly affected by *LINC01133* knockdown. CDH1 is a tumor suppressor and cell polarity regulator [37] and the loss of CDH1 promotes motility and invasion. There is also some evidence that in endometriosis lesions the loss of CDH2 expression may be associated with increased invasive capacity of endometrial epithelial cells. Matsuzaki et al. [38] have shown that deep infiltrating endometriosis lesions express less CDH2, compared to early peritoneal lesions. In normal endometrial tissue high levels of CDH2 were associated with the proliferative phase of the cycle [39]. However, whether activation of CDH2 by *LINC01133* is responsible for the loss of proliferation capacity and increased invasiveness of endometriosis epithelial cells needs to be tested.

Recently, we have shown that the levels of expression of *VCAM-1* are increased in tissue samples of women with endometriosis, compared to women without the disease [40]. The loss of *VCAM-1* was shown to attenuate the TGF-β1 induced proliferation, migration and invasion of endometriosis stroma cells derived from ovarian endometriomas [41]. Our data showed that the function of the protein as a regulator of cell invasion of epithelial endometriosis cells might differ from those in stroma, while downregulation of *VCAM-1*

following *LINC01133* knockdown was associated with an increase of cellular invasion. As up-regulation of *VCAM-1* in malignant cells is associated with recruitment of tumorassociated monocytes and macrophages and immune escape of the tumors [42,43], we postulate that *LINC01133* dependent *VCAM-1* regulation in endometriosis epithelial cells may be related to immune surveillance of the lesions.

A central event in cellular invasion is the dynamic cytoskeleton remodeling leading to changes in cellular morphology. We [44] and others [45,46] have shown that the dysregulation of cytoskeleton dynamics and related signaling pathways are linked to pathogenesis of endometriosis and disease-associated infertility. Actin filaments, microtubules, and intermediate filaments involved in the formation of cytoskeletal structures, such as stress fibers and pseudopodia promote the invasion of normal cells and invasion and metastasis of tumor cells. Here we showed that the increase in the invasion capacity of 12Z cell under *LINC01133* knockdown is associated with changes in cellular morphology to more flattened and larger phenotype with an increased number of stress fibers, provoked by the activation of TESK1 expression and inactivation of Cofilin. TESK1 is a serine/threonine kinase with kinase domain similar to those of LIM-kinases and a unique C-terminal proline-rich domain [47] known to phosphorylate Cofilin at Ser-3 [29]. Cofilin plays an essential role in actin filament dynamics by enhancing depolymerization and severance of actin filaments [48]. These activities of Cofilin are abolished by phosphorylation at Ser-3. Therefore, the changes in Cofilin phosphorylation at Ser-3 are regarded as one of the most important mechanisms for regulating Cofilin activity and actin filament dynamics. It has been shown that induction of stress fibers require an active Rho-ROCK signaling pathway independent of TESK1 [29]. Several studies also indicate that the balance between Rho and Rac activity in cells determines the patterns of actin organization, cell morphology and motility [49]. Thus, the effects of *LINC01133* on the cellular filament and cytoskeleton dynamics may not be restricted only to the TESK1/Cofilin pathway.

Overall, in this study, we found that *LINC01133* is overexpressed in ectopic endometriosis lesions compared to the eutopic endometrium of women both with and without the disease. By knocking down *LINC01133* in endometriosis epithelial cells we were able to show that the lncRNA promotes cellular proliferation and inhibits cell invasion in these cells, and to identify components of these pathways that were affected, including p21, Cyclin A and TESK. These results indicate that *LINC01133* may be a clinically relevant player in endometriosis, although this remains to be tested in vivo.
