**5. Therapeutic Opportunities for lncRNAs**

As lncRNAs function to regulate gene expression, lncRNAs represent novel therapeutic molecules. Therapeutic noncoding RNAs as targeting molecules, including small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), miRNA mimics, miRNA sponges, and CRISPR–Cas9-based gene-editing technologies, have been experimentally developed to regulate gene expression and potentially treat disease, but therapeutic targeting using noncoding RNAs is in its infancy [103]. To date, 11 RNA-based therapeutics are approved by the United States Food and Drug Administration (US FDA) and/or the European Medicines Agency (EMA) [104]. While no RNA-based therapeutics are indicated for endometriosis, therapeutic linear lncRNAs and circRNAs may act to inhibit downstream genes and subsequent cellular function and offer significant promise for non-hormonal therapy. Understanding the precise mechanisms of lncRNAs and their antagonists is the first step towards translational applications, as indicated by several preclinical studies highlighted below.

First, the lncRNA H19 imprinted maternally expressed transcript (H19) regulates insulin grown factor receptor (IGF1R) expression by acting as a molecular sponge to let-7 [105]. An in vitro knockdown of H19 with siRNA led to the higher expression of let-7 by real-time quantitative polymerase chain reaction (qPCR) and subsequent inhibition of IGF1R transcript and protein. Functionally, the H19 knockdown resulted in the reduced proliferation of primary endometrial stromal cells isolated from the eutopic endometrium of subjects with endometriosis [105]. Secondly, the molecular sponge mechanism in a preclinical in vitro model can also be found for long intergenic non-protein coding RNA 261 (LINC00261), which binds miR-132-3p and subsequently acts as a regulator of BCL-2-like 11 (BCL2L11) expression. The overexpression of LINC00261 inhibited the proliferation and invasion of the endometriosis cell line CRL-7566 through the BCL2L11 network. Further, the overexpression of LINC00261 revealed a decrease in miR-132-3p expression and increased BCL2L11 expression [76]. The role of BCL2L11 in endometriosis was investigated by siRNA knockdown. BCL2L11 knockdown reduced epithelial-mesenchymal transition (EMT) markers and reduced invasion [76]. While clinically promising, the scientific reproducibility of this effect has not been tested due to the original study being performed in a single cell line, CRL-7566. The CRL-7566 cell line is derived from an ovarian endometrioma. While it was commercially available from American Type Tissue Culture Collection (ATCC), it

is no longer available due to its slow growth rate. Further, while the CRL-7566 line was authenticated with short tandem repeat (STR) profiling, it was not well characterized in terms of molecular markers for endometrial epithelium and endometrial stroma [106,107]. Thus, these promising effects need to be replicated.

The in vitro studies on lncRNAs in endometriosis above led to preclinical mouse model studies. First, the lncRNA AFAP1 antisense RNA1 (AFAP1-AS1) mediates the signal transducer and activator of the transcription-transforming growth factor beta-SMAD (STAT3/TGF-β/SMAD) signaling pathway through miR-424-5p to influence endometriosis progression. Huan et al. reported that AFAP1-AS1 knockdown inhibited proliferation and migration and promoted apoptosis in an SV40-transformed, endometriosis eutopic endometrium stromal cell line, hEM15a [108]. Additionally, AFAP1-AS1 regulates EMT. Specifically, AFAP1-AS1 is thought to act in concert with steroid hormones, such as estradiol, to induce the expression of the transcription factor zinc finger E-box binding homeobox 1 (ZEB1). Interestingly, the shRNA knockdown of AFAP1-AS1 reduced the expression of ZEB1 in the spontaneously transformed endometrial cancer cell line Ishikawa. Further, Ishikawa cells with a knockdown of AFAP1-AS2 showed reduced tumor dimensions in a nude mouse model compared to non-targeted Ishikawa cells [109]. These studies highlight the impact of AFAP1-AS1 on proliferation and growth. While promising, the studies in an endometrial cancer cell line highlight the potential lack of clinical applicability to endometriosis. Finally, endometriosis is a disease of significant immunologic features. The use of nude mice, which are immunocompromised, may not be biologically applicable to endometriosis. Improved in vivo and in vitro models are needed to improve the translatability of studies.

Second, Liu and colleagues studied the lncRNA small nucleolar host gene 4 (SNHG4) in a heterologous mouse model of endometriosis. In this model, nude mice were injected subcutaneously with primary endometrial stromal cells (ESCs) isolated from ectopic endometrium and transfected with either NC-si, SNHG4-si1, or SNHG4-si1 combined with anti-miR-148-3p. After silencing SNHG4, the volume of endometriotic lesions was considerably reduced compared to the non-targeting control. Further, the expression of MET proto-oncogene receptor tyrosine kinase (MET) was inhibited, while miR-148a-3p was upregulated. The inhibitor of miR-148a-3p combined with SNHG4 knockdown rescued endometriotic lesions growth and upregulated the MET expression. The authors postulated that SNHG4 might upregulate proto-oncogene expression, in particular MET, via the suppression of miR-148a-3p, to promote the increased growth of endometrial tissue outside the uterine cavity and endometriosis lesions [110]. The impact of oncogenes and the manipulation of oncogenes in therapy for endometriosis deserves future study, particularly as non-hormonal therapies.

Third, studies showed that lncRNA maternally expressed 3 (MEG3-210) has a regulatory mechanism in endometriosis. MEG3-210 was downregulated in the eutopic endometrium of endometriosis patients and the primary cultures of endometrial stromal cells from women with endometriosis. The overexpression of MEG3-210 in the primary cultures of endometrial stromal cells from women with endometriosis revealed reduced invasion and migration. Further, flow cytometry detected a reduction in apoptosis. They examined two molecular pathways, including p38 signaling for its role in the endometriosis inflammatory response and PKA/SERCA2 signaling for its effects on cell motility and apoptosis. Western blotting showed that the protein levels of phosphorylated mitogen-activated protein kinase 14 (better known as p38) and phosphorylated activating transcription factor 2 (ATF2) were significantly increased after the downregulation of MEG3-210. Furthermore, the protein levels of protein kinase cAMP-activated catalytic subunit alpha (PRKACA, better known as PKA) and ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 (SERCA2) were decreased after MEG3-210 downregulation [111]. Previously, p38 activity was found to be higher in the eutopic and ectopic endometria in endometriosis patients. Increased p38 MAPK activity in endometriotic cells correlated with the activation of inflammatory cytokines, such as interleukin one beta (IL1b) and tumor necrosis factor-alpha

(TNFα) [112]. Finally, they found that p38/MAPK and PKA/SERCA2 signaling pathways act through Galectin1. Galectin-1 is a member of the sub-family of galectins that play a role in intracellular signal processing, molecular modification, cell motility, and malignant biological behavior [111,113]. Recently, MEG3 has regulated transforming growth factor-beta (TGFβ) signaling [114]. Previous work has shown the role of TGFβ signaling in ovarian endometriomas through small RNA signaling [115]. Further studies should examine the connection of lncRNA MEG3 in TGFβ signaling in endometriomas.

Like lncRNA, circular RNAs share the sponge mechanism of action. For example, the circular RNA circ\_0007331 targets miR-200c-3p and, consequently, targets hypoxiainducible factor 1 subunit alpha (HIF1A), a key transcription factor for angiogenesis and hypoxia mechanisms. Through this mechanistic axis, circ\_0007331 knockdown, with the cooperation of HIF1A downstream, reduced the proliferation and invasion of primary endometrial cell cultures from women with endometriosis. With the overexpression of miR-200c-3p, proliferation and invasion increased, as did HIF1A. The inhibition of miR-200c-3p, conversely, reduced the proliferation and invasion caused by circ\_0007331 knockdown, confirming that the circ\_0007331/miR-200c-3p/HIF-1α axis has an important role in cell proliferation and invasion in endometriosis [116]. Using a homologous endometriosis mouse model, treatment with circ\_0007331 shRNA, shRNA NC, or anti-miR-200c-3p showed that circ\_0007331 knockdown reduced the lesion sizes. Further, treatment with anti-miR-200c-3p did not. Using immunohistochemistry, endometriosis lesions from mice treated with circ\_0007331 shRNA were negative for HIF1A, but mice treated with anti-miR-200c-3p treatment maintained HIF1A expression [116]. These results show the importance of the circ\_0007331/miR-200c-3p/HIF-1α axis in the endometrium of endometriosis patients.

Finally, the sponge mechanism of action has been proposed for the circular RNA circ\_0004712, and miR-148a-3p. Notably, this axis plays an important role in estradiol (E2)-induced EMT processes in the development of endometriosis, potentially through the β-catenin pathway. The E2 treatment of either the endometrial cancer cell line Ishikawa or the human papillomavirus (HPV)-16 E6/E7 transformed endometriosis endocervical cell line End1/E6E7 showed the overexpression of circ\_0004712. Further, E2 treatment increased migration in transwell assays and the induction of EMT through the b-catenin pathway. The E2- treatment effect was suppressed with the knockdown of circ\_0004712 [117]. Interest in circRNA application in endometriosis is a relatively new area of research. However, the exciting data to date support additional preclinical studies.

While lncRNAs offer opportunities for targeting cellular function, lncRNAs themselves offer options as therapeutic targets. The dysregulation of lncRNA expression has been linked to diseases and complex biological processes [118]. Recently, lncRNA HOX transcript antisense RNA (HOTAIR) has been associated with a genetic susceptibility to endometriosis. Functional single nucleotide polymorphisms, including rs1838169 and rs17720428, were frequently found in endometriosis patients [119]. Moreover, endometriosis pathogenesis may revolve around a functional axis of HOTAIR/homeobox D10 and HO-TAIR/homeobox A5. Homeobox proteins (HOXs) are critical in maintaining endometrium homeostasis during embryo implantation and menstrual cycles, highlighting their importance in endometriosis [120]. HOTAIR knockdown reduced cell proliferation and migration and increased HOXD10 and HOXA5 expression in two ovarian clear cell cancer cell lines, ES-2 and TOV-21G [119]. The overexpression of HOTAIR in epithelial ovarian cancer cells increases cancer invasiveness and metastasis. Moreover, the involvement of HOTAIR in cancer progression and response to standard chemotherapy, possibly promoting mesenchymal stem cell formation, has been highlighted [121,122]. Since endometriosis shares features with cancer, these results make HOTAIR a possible target for future endometriosis or ovarian cancer therapies. Secondly, Zhang et al. discovered that another potential target, CCDC144NL antisense RNA1 (CCDC144NL-AS1), was found to be upregulated in ectopic endometriosis and eutopic endometrium from women with endometriosis. The in vitro knockdown of CCDC144NL-AS1 in the SV40-transformed, endometriosis eutopic endometrium stromal cell line hEM15a was associated with decreased migration and invasion. Assuming that alterations in motility and invasion were related to cytoskeleton alteration, the authors found an altered distribution of cytoskeletal F-actin stress fibers compared to lower protein levels of vimentin filaments and matrix metallopeptidase 9 (MMP9) after CCDC144NL-AS1 knockdown [123]. Although there are yet no clinical studies, preclinical studies reveal a potential application of lncRNAs.

In the last few years, a growing interest in diet and nutrition as complementary therapeutic support for endometriosis was established even if randomized clinical trials do not show benefits [124]. Moreover, a connection between nutrition and ncRNA epigenetics has been found with sulforaphane, epigallocatechin gallate (EGCG), genistein, resveratrol, and curcumin in female reproductive tract cancers. A possible therapeutic role of these compounds combined with traditional therapies has been highlighted. As we know, endometriosis shares some pathways with neoplastic disease. Phytochemicals and nutraceuticals have been shown to influence pathways involving the miR-200 family, let-7 family, or miR-34a that can interact with inflammatory and oxidation mechanisms that play an important role in endometriosis [125].
