*4.3. LncRNAs Modulating Cellular Signaling Pathways in Endometriosis*

Cell signaling pathways have a pivotal role in the regulation of a variety of cellular processes in response to intracellular or extracellular stimuli. The regulation of components of a signaling pathway by lncRNAs can be direct or indirect and result in functional changes in the signaling cascades. Direct regulation can be achieved by direct binding of the lncRNA to signaling proteins leading to changes in either their free cellular levels or their activity. We define indirect regulation as cases where no direct lncRNA binding to signaling molecules has been shown and where the lncRNA is thought to alter the transcription of genes associated with the signaling pathway resulting in an altered cellular response. In Table 3, we summarize currently known cases where a lncRNA directly or indirectly affects a signaling pathway in endometriosis.


**Table 3.** Mechanisms of cell signaling regulation via lncRNAs in endometriosis.

In an example of direct regulation of a signaling pathway in endometriosis, the lncRNA *MEG3-210* has been shown to regulate endometriosis stromal cell migration, invasion, and apoptosis through the p38 MAPK and PKA/SERCA2 signaling pathways. In this case, *MEG3-210* directly interacts with Galectin-1 in vitro and affects the growth of endometriotic lesions in vivo in a murine model of the disease [64]. Mechanistically, *MEG3- 210* titrates away the cellular levels of Galectin-1, preventing its action on the p38 MAPK and PKA/SERCA2 signaling cascades in endometrial stromal cells. In endometriosis, the levels of *MEG3-210* are downregulated and the levels of free Galectin-1 are upregulated, which is associated with the subsequent activation of p38 MAPK signaling–mediated phosphorylation of ATF2. Activated ATF2 increases the expression of BCL-2 and MMP contributing to the anti-apoptotic, pro-migratory, and invasive phenotype of endometriosis cells. Simultaneously, *MEG3-210* downregulation leads to suppression of the PKA/SERCA2 signaling cascade [64].

In an example of indirect regulation of signaling pathway, knockdown of *MALAT1* lncRNA in endometriosis cells leads to enhanced cell death, reduced migration and invasion associated with activation of Caspase-3, and downregulation of MMP-9 and the NFkB/iNOS signaling pathway (Figure 3a) [65]. In contrast to endometriosis tissue where *MALAT1* is overexpressed, in the granulosa cells of women with endometriosis *MALAT1* expression is reduced [66]. This is associated with a reduced follicle count, due to impaired cell proliferation resulting from ERK/MAPK-dependent p21/p53 cell cycle arrest. This implicates altered expression of *MALAT1* in endometriosis-related infertility. In cultured primary endometrial stromal cells, depletion of *MALAT1* by siRNA knockdown results in the suppression of hypoxia-induced autophagy, as indicated by a reduction in the expression of autophagy markers Beclin-1 and LC3-II [67]. In this signaling cascade, expression of *MALAT1* is regulated by the HIF1α transcription factor, known to be overexpressed

in endometriosis lesions and to regulate multiple gene targets in response to hypoxia (Figure 3a) (reviewed in [76]).

**Figure 3.** (**a**) *MALAT1* was identified as a sponge of *miR-200c*. This regulation is not restricted to *miR-200c* and might include the entire *miR-200* family (*miR-200s*), consisting of *miR-200a*, *miR-200b*, *miR-200c*, *miR-141*, and *miR-429*. Upregulation of *MALAT1* in women with endometriosis leads to enhanced sponging of *miR200s* and promotes zinc finger E-box binding homeobox transcription factor 1 (ZEB1) and ZEB2 expression leading to higher EMT. In HESCs, the lncRNA *MALAT1* directly interacts with *miR-126-5p*, which regulates cAMP responsive element-binding protein (CREB1) expression. Upregulation of *MALAT1* inhibits apoptosis probably via activation of the PI3K–AKT pathway through the *miR-126-5p*–CREB1 axis. *MALAT1* lncRNA can also lead to reduced apoptosis in HESCs through the upregulation of the NFkB/iNOS signaling pathway activity, which also enhances migration and invasion of cells. In cultured primary endometrial stromal cells, *MALAT1* leads to upregulation of hypoxia-induced autophagy. In this signaling cascade, regulation of *MALAT1* expression is under the control of the HIF1α transcription factor. (**b**) In granulosa cells (GCs) of women with endometriosis, significant downregulation of *MALAT1* expression was reported. *MALAT1* knockdown induced an increase in phosphorylated ERK1/2 (p-ERK1/2) that was associated with altered follicle count, due to impaired cell proliferation resulting from ERK/MAPKdependent activation of p21/p53 cell cycle arrest. In an autograft transplantation rat model of endometriosis, inhibition of the lncRNA *BANCR* led to a decrease in ectopic tissue volume associated with a significant reduction in serum levels of *VEGF*, *MMP-2*, and *MMP-9, ERK*, and *MAPK* mRNA and in phosphorylated ERK and MAPK protein levels in tissues. HESCs: human endometrial stromal cells.

In another example, the most downregulated lncRNA in ectopic tissue of women with ovarian endometriosis was *LINC01541* [33], which has been shown respond to levels of estradiol [68]. A gene-targeting in vitro study in human endometrial stromal cells showed that the cellular levels of *LINC01541* affect the activity of WNT/β-catenin, proand anti-apoptotic signaling regulators Caspase-3 and BCL2 and the levels of VEGFA production [68].

The presence of extracellular lncRNA in exosomes or microvesicles raises the possibility that these lncRNAs may be able to serve as extracellular signals for cell signaling regulation in endometriosis. This was supported by reports that exosomal lncRNAs promote angiogenesis in endometriosis [77]. Based on an in vitro co-culture model and patient serum analysis, the authors developed a novel mechanistic model explaining how endometriosis stromal cells of the lesion induce angiogenesis. They suggested that these cells in an ectopic environment can produce exosomes that are enriched in *aHIF* lncRNA, a pro-angiogenic lncRNA, highly expressed in ectopic endometrial stromal tissue. These *aHIF*-rich exosomes are then taken up by recipient macrovascular cells, where they cause upregulation of the angiogenesis-related genes VEGF-A, VEGF-D, and bFGF.

Endometriosis is an estrogen-dependent disease, and therefore, lncRNAs involved in the estrogen pathway or those targeted by estrogen signaling could play a role in the disease. The lncRNA *H19* is positively regulated by estrogen, and its expression in the endometrium increases during the proliferative stage of the menstrual cycle [78,79]. *H19* has been shown regulate several pathways that are relevant in endometriosis including IGF1R, ITGB3, IER3, and ACTA2 [53–55,80]. Another lncRNA, *steroid receptor RNA activator 1* (*SRA1*) lncRNA, has been reported to act in concert with SRA1 to regulate the expression of estrogen receptors by affecting alternative splicing, and thereby the growth of stromal cells in ovarian endometriosis [81]. These examples illustrate how lncRNAs could play a role in endometriosis via the estrogen pathway or as targets of estrogen regulation.

Conceptually, the mechanism of lncRNAs' action in endometriosis may involve influencing cellular pathways, where one lncRNA may regulate several targets using different molecular strategies (Figure 3a) or one targeted signaling pathway may be affected by multiple lncRNAs (Figure 3b). To better understand this complexity, integrative in silico and experimental analyses interrogating the molecular networks in endometriosis cells need to be applied. This approach should help to dissect the relationship between lncRNA, mRNA, miRNA expression, genetic- and epigenetic-driven chromatin remodeling, and signal transduction activity and to enable lncRNA target identification. The use of such in silico bioinformatics algorithms for cellular network construction in endometriosis has already been attempted by several studies [41,43,46]. For example, Wang et al. [41] constructed an lncRNA–miRNA–mRNA network, revealing lncRNAs that act as competing endogenous RNAs (ceRNA), the miRNAs they sponge, and the target genes that were involved in regulating endometrial receptivity in endometriosis. Moreover, Jiang et al. [43] identified the lncRNAs (*SMIM25*, *LINC01018*), miRNA (*miR-182-5p*), and mRNA (*CHLI*) as part of a ceRNA network implicated in the regulation of immune responses in endometriosis. These in silico studies may be valuable to predict how lncRNAs may function in endometriosis, but experimental validation is required in order for these predictions to be confirmed.
