**1. Introduction**

Endometriosis is defined by the presence of endometrial tissue in ectopic locations, typically in or around the peritoneal cavity [1,2]. While the exact prevalence of endometriosis is likely underrepresented, most sources cite that a minimum of 10% of women in their reproductive years have this disease [3–5]. Primarily described as a hormonal disorder, the pathogenesis of endometriosis has also been linked to immunological/inflammatory, genetic, and environmental factors. More recently, the role of epigenetics in the development and progression of this disorder has been investigated [6–12]. Epigenetic mechanisms are heritable changes to one's phenotype that are not associated with a change in nucleotide sequence and include DNA methylation, post-translational modifications to histone proteins, and often microRNAs [13,14].

In addition to heterochromatin-like protein 1 (HP-1), polycomb (PcG) and trithorax (TrxG) complexes are at the heart of epigenetics. Responsible for maintaining gene repression and activity, respectively [15], the latter two complexes function antagonistically to establish epigenetic regulation [15]. Polycomb repressive complex 1 (PRC1), polycomb repressive complex 2 (PRC2), and Pho repressive complex (PhoRC) all form the PcG complexes, with the former two typically being the subject of extensive epigenetic research. The Polycomb Repressive Complex 2 (PRC2) consists of four core proteins, RbAp46/48, Embryonic Ectoderm Development (EED), Suppressor of Zeste 12 (SUZ12),

**Citation:** Brunty, S.; Ray Wright, K.; Mitchell, B.; Santanam, N. Peritoneal Modulators of EZH2-miR-155 Cross-Talk in Endometriosis. *Int. J. Mol. Sci.* **2021**, *22*, 3492. https://doi.org/10.3390/ijms22073492

Academic Editor: Antonio Simone Laganà

Received: 19 February 2021 Accepted: 26 March 2021 Published: 28 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and Enhancer of Zeste Homolog 2 (EZH2), the catalytic subunit of the PRC2 complex. These components work together to regulate chromatin structure via tri-methylation of lysine 27 on histone 3 (H3K27me3) [16,17], which is also known to interact with PRC1. EED binds the histone site while EZH2 methylates it, with the help of SUZ12 [18]. This modification leads to the formation of closed chromatin structure (heterochromatin) and thus marks transcriptional repression, as further demonstrated by the presence of other co-factors [19–21].

There is very little known about the mechanistic role of PRC2 complex and how it is regulated in the endometriosis disease process. While an in vivo study showed heightened expression of EZH2 and trimethylation of H3K27 in secretory endometrium and endometriotic lesions [22,23], another cell culture study showed that inhibition of PGE2 receptors EP3 and EP4 occur concurrently with decreased EZH2 expression [24], supporting a role for PRC2 in endometriosis-associated pain.

It has been shown that the PRC2 complex (specifically EZH2) is, at least partly, regulated by Jumonji and AT-Rich Interaction Domain Containing 2 (JARID2) [25], a member of the largest family of histone demethylases, the jumonji family, where all but JARID2 contain the catalytic JmjC domain responsible for histone demethylation [26,27]. Research has found that JARID2 is a cofactor for PRC2 [28]. Additionally, its methylation by the PRC2 complex at K116 is part of a regulatory mechanism that controls the PRC2 enzymatic activity where the methylated JARID2 binds to the EED component of the PRC2 complex. This is required for efficient deposition of H3K27me3 during cell differentiation and fine-tunes the PRC2 activity [29]. JARDI2 is thought to be crucial in the development and progression of cancer. This is due to its cross-talk with EZH2 and PRC2 activity in embryonic stem cells (ESC), as JARID2 is necessary for proper ESC differentiation [25,30,31].

JARID2 is suggested to be modulated by few mechanisms. For example, iron oxidation, which occurs due to increased reactive oxygen species generation and known to be present in excess in women with endometriosis, blocks the catalytic activity of JARID2 [32]. JARID2 is also a common target of microRNAs some of which have been identified by our laboratory to be differentially expressed (miR-30b, miR-30c, miR-10a, miR-29a, miR-26a, miR-148a, miR-181a, miR-30e) in endometriotic lesions compared to control tissues [33]. Palma et al. showed in acute lymphoid leukemia that miR-155-5p induced cell death via a network of mechanisms, including regulation of cyclinD1 by JARID2 [34]. Other such studies support the possibility that miR-155-5p could have been evolved to regulate PRC2 by tweaking JARID2 expression [35]. Interestingly, miR-155-5p is an established promoter of inflammation via regulation of macrophages and cytokines [35–37]. Thus, targeting this demethylase (JARID2) via modulators such as microRNAs, could be a novel method of treatment for endometriosis.

miR-155 is highly expressed in regulatory T-cells (Tregs), where it is targeted by transcription factor forkhead box P3 (FOXP3) [38]. Though limited in evidence, FOXP3 also plays a role in the inflammatory aspect of endometriosis, which correlates with miR-155-5p being a promoter of inflammation. The prevalence of FOXP3<sup>+</sup> Tregs in an endometriotic environment during secretory phase prevent leukocyte recruitment to the sites of endometriosis [39]. Additionally, peritoneal fluid (PF) from women with endometriosis has a higher concentration of FOXP3-expressing TCD4<sup>+</sup> CD25high cells than the PF of control patients [40,41]. Other studies have also shown that FoxP3 is an inducer of miR-155 [42].

It is important to note that FOXP3 also has an indirect relationship with the EZH2 component of PRC2. Overexpression of the FOXP3 protein not only lessened the proliferative effects of EZH2, but also enhanced degradation of the EZH2 protein in breast cancer models [43]. Conversely, there is evidence that trimethylation of H3K27 by EZH2 is capable of silencing FOXP3 promoter regions, therefore leading to aberrant Treg cell differentiation and function [44]. These studies suggest a complex interplay between epigenetic mediators, PRC2 complex, miR-155-5p, JARID2 and the inflammatory mediator FOXP3. In this study, it is hypothesized that the imbalance in this crosstalk triggers inflammatory responses and possibly nociception in endometriosis. This current study investigated the

sis cell model.

**2. Results** 

crosstalk between these mediators in endometriotic patient tissues and in an endometriosis cell model. or women with endometriosis (EuE, *n* = 10) and ectopic tissue from women with endometriosis (EcE, *n* = 6) (Figure 1A). When compared to the EuN tissues, expression of all three

### **2. Results** PRC2 protein complex (*SUZ12*, *EED* and *EZH2*) and *JARID2*, was higher in both the eu-

*Int. J. Mol. Sci.* **2021**, *22*, 3492 3 of 19

#### *2.1. PRC2 Complex and JARID2 mRNA and Protein Expression in Endometriotic Tissues* topic (EuE) and ectopic (EcE) tissue from endometriosis patients. Compared to EuN tis-

crosstalk between these mediators in endometriotic patient tissues and in an endometrio-

first determined. qPCR was used to determine the mRNA expression of PRC2 components SUZ12, EED, and EZH2 in eutopic tissue from women with no endometriosis (EuN, *n* = 5)

*2.1. PRC2 Complex and JARID2 mRNA and Protein Expression in Endometriotic Tissues* 

The endogenous expression of PRC2 complex proteins in endometriotic tissues were first determined. qPCR was used to determine the mRNA expression of PRC2 components SUZ12, EED, and EZH2 in eutopic tissue from women with no endometriosis (EuN, *n* = 5) or women with endometriosis (EuE, *n* = 10) and ectopic tissue from women with endometriosis (EcE, *n* = 6) (Figure 1A). When compared to the EuN tissues, expression of all three PRC2 protein complex (*SUZ12*, *EED* and *EZH2*) and *JARID2*, was higher in both the eutopic (EuE) and ectopic (EcE) tissue from endometriosis patients. Compared to EuN tissues, *SUZ12* levels increased close to 2-fold for EcE but was not significant, however there was a significant increase in *EED* expression by 5.07-fold in EuE (*p* = 0.0153) and 7.13-fold (*p* = 0.0067) in EcE. *EZH2* expression was also increased 2.35-fold in the EuE and 3.10-fold in the EcE but did not reach significance. Expression for *JARID2* increased over 2-fold in EcE tissues, but this was not significant. sues, *SUZ12* levels increased close to 2-fold for EcE but was not significant, however there was a significant increase in *EED* expression by 5.07-fold in EuE (*p* = 0.0153) and 7.13-fold (*p* = 0.0067) in EcE. *EZH2* expression was also increased 2.35-fold in the EuE and 3.10-fold in the EcE but did not reach significance. Expression for *JARID2* increased over 2-fold in EcE tissues, but this was not significant. Protein expression was also determined using the automated Western blotting system, WES. While EZH2 showed a significant increase of >7 fold (*p* = 0.0219) in EcE tissues compared to EuN, no significant difference was seen in expression of H3K27me3 or JARID2 (Figure S1). This lack of change in JARID2 expression might be attributed to its altered regulation

**Figure 1.** mRNA expression of PRC2 complex and JARID2 and miRNAs that target JARID2 in endometriotic tissues. (**A**) Relative mRNA expression of polycomb repressor complex 2 (PRC2) elements and *JARID2* in eutopic tissues from control women, EuN (*n* = 5), or eutopic and ectopic tissues from women with endometriosis, EuE (*n* = 10) and EcE tissues (*n* = 6). In general, these elements were upregulated in both eutopic and ectopic endo tissues compared to control tissue with *EED* showing significant upregulation in both the eutopic (*p* = 0.0153) and ectopic (*p* = 0.0067). *JARID2* expression was higher in EcE. \* *p* < 0.05, \*\* *p* < 0.01 when compared to EuN tissues. (**B**) Compared **Figure 1.** mRNA expression of PRC2 complex and JARID2 and miRNAs that target JARID2 in endometriotic tissues. (**A**) Relative mRNA expression of polycomb repressor complex 2 (PRC2) elements and *JARID2* in eutopic tissues from control women, EuN (*n* = 5), or eutopic and ectopic tissues from women with endometriosis, EuE (*n* = 10) and EcE tissues (*n* = 6). In general, these elements were upregulated in both eutopic and ectopic endo tissues compared to control tissue with *EED* showing significant upregulation in both the eutopic (*p* = 0.0153) and ectopic (*p* = 0.0067). *JARID2* expression was higher in EcE. \* *p* < 0.05, \*\* *p* < 0.01 when compared to EuN tissues. (**B**) Compared to control tissues (*n* = 7), expression of miR-148a, miR-29a, and miR-155 (miRNAs that target JARID2) were all higher in endo tissues (both eutopic and ectopic, *n* = 8).

JARID2) were all higher in endo tissues (both eutopic and ectopic, *n* = 8).

to control tissues (*n* = 7), expression of miR-148a, miR-29a, and miR-155 (miRNAs that target

Protein expression was also determined using the automated Western blotting system, WES. While EZH2 showed a significant increase of >7 fold (*p* = 0.0219) in EcE tissues compared to EuN, no significant difference was seen in expression of H3K27me3 or JARID2 (Figure S1). This lack of change in JARID2 expression might be attributed to its altered regulation. *Int. J. Mol. Sci.* **2021**, *22*, 3492 4 of 19 *2.2. miRNAs Targeting JARID2 in Endometriotic Tissues* 

#### *2.2. miRNAs Targeting JARID2 in Endometriotic Tissues* The expression levels of miRNAs that regulate JARID2 was next determined in the

The expression levels of miRNAs that regulate JARID2 was next determined in the patient tissues. miRNA qPCR assays were used to measure expression of miR-148a, miR-29a, and miR-155, which, among others, target JARID2 (Targetscan 7.1 and Ingenuity Pathway Analysis Qiagen, Germantown, MD, USA). Interestingly, all three miRNAs were overexpressed in both EuE and EcE tissues compared to EuN tissues (Figure 1B). Both miR-148a and miR-155 showed an over 5-fold increase in expression for the EuE tissues and were also shown to be induced more than 2.5–14-fold, respectively on EcE, while miR-29a expression increased 2–4-fold with levels higher in EuE and EcE tissues. patient tissues. miRNA qPCR assays were used to measure expression of miR-148a, miR-29a, and miR-155, which, among others, target JARID2 (Targetscan 7.1 and Ingenuity Pathway Analysis Qiagen, Germantown, MD, USA). Interestingly, all three miRNAs were overexpressed in both EuE and EcE tissues compared to EuN tissues (Figure 1B). Both miR-148a and miR-155 showed an over 5-fold increase in expression for the EuE tissues and were also shown to be induced more than 2.5–14-fold, respectively on EcE, while miR-29a expression increased 2–4-fold with levels higher in EuE and EcE tissues.

#### *2.3. PRC2 Complex mRNA and Protein Expression in PF Treated Endometrial Cells* Peritoneal cavity is one of the major sites for endometriotic lesions in women with

*2.3. PRC2 Complex mRNA and Protein Expression in PF Treated Endometrial Cells* 

Peritoneal cavity is one of the major sites for endometriotic lesions in women with endometriosis [45,46]. These patients also exhibit larger volumes of PF rich in inflammatory and nociceptive molecules [47,48]. Current theories propose a dynamic role for PF in modulating the growth of endometriotic lesions, which might be epigenetically regulated by the altered expression of certain miRNAs previously shown in endometriosis [49,50]. Whether PF from patients with and without endometriosis differentially regulated the PRC2 complex proteins in endometrial cells was determined. For this, human endometrial cells were exposed to 1% PF from women with (*n* = 13) or without endometriosis (*n* = 12) for 48 h followed by the measurement of both mRNA and protein expression of PRC2 complex proteins using similar techniques as described for the endometriotic tissues. Cells treated with both 1% control or endo PF had increased *SUZ12*, *EED*, and *EZH2* mRNA expression but none were shown to be statistically significant (Figure 2A). When protein expression was determined using the automated Western Blotting system, WES, EZH2 showed no significant difference in expression levels when compared to the media control. While H3K27me3 did show an upregulation of over 2-fold for endo PF treated cells, this was not significant. (Figure 2B,C). endometriosis [45,46]. These patients also exhibit larger volumes of PF rich in inflammatory and nociceptive molecules [47,48]. Current theories propose a dynamic role for PF in modulating the growth of endometriotic lesions, which might be epigenetically regulated by the altered expression of certain miRNAs previously shown in endometriosis [49,50]. Whether PF from patients with and without endometriosis differentially regulated the PRC2 complex proteins in endometrial cells was determined. For this, human endometrial cells were exposed to 1% PF from women with (*n* = 13) or without endometriosis (*n* = 12) for 48 h followed by the measurement of both mRNA and protein expression of PRC2 complex proteins using similar techniques as described for the endometriotic tissues. Cells treated with both 1% control or endo PF had increased *SUZ12*, *EED*, and *EZH2* mRNA expression but none were shown to be statistically significant (Figure 2A). When protein expression was determined using the automated Western Blotting system, WES, EZH2 showed no significant difference in expression levels when compared to the media control. While H3K27me3 did show an upregulation of over 2-fold for endo PF treated cells, this was not significant. (Figure 2B,C).

**Figure 2.** mRNA and protein expression of PRC2 complex proteins in PF treated endometrial cells. (**A**) mRNA expression of *SUZ12*, *EED*, and *EZH2* in cells treated with control PF (*n* = 12) and endo PF (*n* = 13), relative to expression in a media **Figure 2.** mRNA and protein expression of PRC2 complex proteins in PF treated endometrial cells. (**A**) mRNA expression of *SUZ12*, *EED*, and *EZH2* in cells treated with control PF (*n* = 12) and endo PF (*n* = 13), relative to expression in a media control

control (*p* > 0.05). (**B**) Representative WES images and densitometric analysis for EZH2, and H3K27me3 in PF-treated cells.

It is to be noted that molecular weights of protein bands in automated WES system differs from the traditional Western

blotting, due to differences in technology.

(*p* > 0.05). (**B**) Representative WES images and densitometric analysis for EZH2, and H3K27me3 in PF-treated cells. **(C**) Relative protein expression of EZH2, and H3K27me3 in PF-treated cells was calculated in relation to a media control and presented as a ratio in which media alone is 1.Densities of protein bands obtained were normalized to β-actin or H3. It is to be noted that molecular weights of protein bands in automated WES system differs from the traditional Western blotting, due to differences in technology. *Int. J. Mol. Sci.* **2021**, *22*, 3492 5 of 19

#### *2.4. JARID2 and miRNAs Targeting It in PF Treated Endometrial Cells* The expression of JARID2 in the peritoneal fluid treated endometrial cells was also

*2.4. JARID2 and miRNAs Targeting It in PF Treated Endometrial Cells* 

The expression of JARID2 in the peritoneal fluid treated endometrial cells was also examined. While both the control and endo PF treated cells showed an increase in mRNA expression of *JARID2* when compared to media alone, neither was shown to be significant (Figure 3A). Analysis of protein expression of JARID2 showed a significant upregulation of expression when cells were treated with endo PF of 3.61-fold (*p* = 0.0027) and by about 2-fold compared to control PF (*p* = 0.0096) treated cells (Figure 3B,C). examined. While both the control and endo PF treated cells showed an increase in mRNA expression of *JARID2* when compared to media alone, neither was shown to be significant (Figure 3A). Analysis of protein expression of JARID2 showed a significant upregulation of expression when cells were treated with endo PF of 3.61-fold (*p* = 0.0027) and by about 2-fold compared to control PF (*p* = 0.0096) treated cells (Figure 3B,C).

**Figure 3.** mRNA and protein expression of JARID2 in PF treated endometrial cells. (**A**) mRNA expression of JARID2 in cells treated with control PF (*n* = 12) and endo PF (*n* = 13), relative to expression in a media control (*n* = 6) (*p* > 0.05). (**B**) Representative WES images and densitometric analysis for JARID2 in PF-treated cells. (**C**) Relative protein expression of JARID2 in PF-treated cells was calculated in relation to a media control and presented as a ratio in which media alone is 1. Significant upregulation of JARID2 of 3.61-fold was seen in the endo PF treated cells when compared to media alone cells (*p* = 0.0027) and by about 2-fold compared to control PF (*p* = 0.0096) treated cells. \*\* significant difference (*p* < 0.01) when compared to media alone. \$\$ Significant difference (*p* < 0.05) in mean compared to control PF. Densities of the protein bands obtained were normalized to β-actin or H3. It is to be noted that molecular weights of protein bands in automated WES system differs from the traditional Western blotting, due to differences in technology. **Figure 3.** mRNA and protein expression of JARID2 in PF treated endometrial cells. (**A**) mRNA expression of JARID2 in cells treated with control PF (*n* = 12) and endo PF (*n* = 13), relative to expression in a media control (*n* = 6) (*p* > 0.05). (**B**) Representative WES images and densitometric analysis for JARID2 in PF-treated cells. (**C**) Relative protein expression of JARID2 in PF-treated cells was calculated in relation to a media control and presented as a ratio in which media alone is 1. Significant upregulation of JARID2 of 3.61-fold was seen in the endo PF treated cells when compared to media alone cells (*p* = 0.0027) and by about 2-fold compared to control PF (*p* = 0.0096) treated cells. \*\* significant difference (*p* < 0.01) when compared to media alone. \$\$ Significant difference (*p* < 0.05) in mean compared to control PF. Densities of the protein bands obtained were normalized to β-actin or H3. It is to be noted that molecular weights of protein bands in automated WES system differs from the traditional Western blotting, due to differences in technology.

> Next, it was assessed if the addition of the PF to the endometrial cells changed the expression levels of the miRNAs that regulates JARID2 levels. miR-148a and miR-29a showed a decrease in expression in both control and endo PF treated endometrial cells, while miR-29a showed a decrease in expression for the control PF treated cells but a slight increase in the endo PF treated cells. Surprisingly, but consistent with what was observed earlier in the EcE tissues, miR-155 showed an increase in expression in both control and endo PF treated cells, but no results were shown to be statistically significant. (Figure 4). Next, it was assessed if the addition of the PF to the endometrial cells changed the expression levels of the miRNAs that regulates JARID2 levels. miR-148a and miR-29a showed a decrease in expression in both control and endo PF treated endometrial cells, while miR-29a showed a decrease in expression for the control PF treated cells but a slight increase in the endo PF treated cells. Surprisingly, but consistent with what was observed earlier in the EcE tissues, miR-155 showed an increase in expression in both control and endo PF treated cells, but no results were shown to be statistically significant. (Figure 4). This increase in miR-155 might have lowered the JARID2 expression.

This increase in miR-155 might have lowered the JARID2 expression.

*Int. J. Mol. Sci.* **2021**, *22*, 3492 6 of 19

**Figure 4.** Expression of miRNAs that target JARID2 in PF treated endometrial cells. Compared to media control cells (*n* = 4), expression of miR-148a was shown to be lower in the endo PF treated cells while miR-155 and miR-29a was increased in expression but none were significant. control PF (*n* = 12), endo PF (*n* = 13). **Figure 4.** Expression of miRNAs that target JARID2 in PF treated endometrial cells. Compared to media control cells (*n* = 4), expression of miR-148a was shown to be lower in the endo PF treated cells while miR-155 and miR-29a was increased in expression but none were significant. control PF (*n* = 12), endo PF (*n* = 13). With the knowledge that FOXP3 is a regulator of miR-155, the expression levels of FOXP3 in the endometrial tissues and in the PF treated endometrial cells were determined. qPCR showed that while the tissues from patients with endometriosis (EuE and EcE) were slightly upregulated compared to EuN, there was no significance between the expressions

#### *2.5. FOXP3 mRNA and Protein Expression in Endometrial Tissues and PF Treated Endometrial 2.5. FOXP3 mRNA and Protein Expression in Endometrial Tissues and PF Treated Endometrial Cells* (Figure 5A). Relative protein expressions of FOXP3 are shown in Figure 5B. No significant difference was seen between the mean density of endo tissue and control tissue bands.

*Cells*  With the knowledge that FOXP3 is a regulator of miR-155, the expression levels of FOXP3 in the endometrial tissues and in the PF treated endometrial cells were determined. qPCR showed that while the tissues from patients with endometriosis (EuE and EcE) were slightly upregulated compared to EuN, there was no significance between the expressions (Figure 5A). Relative protein expressions of FOXP3 are shown in Figure 5B. No significant With the knowledge that FOXP3 is a regulator of miR-155, the expression levels of FOXP3 in the endometrial tissues and in the PF treated endometrial cells were determined. qPCR showed that while the tissues from patients with endometriosis (EuE and EcE) were slightly upregulated compared to EuN, there was no significance between the expressions (Figure 5A). Relative protein expressions of FOXP3 are shown in Figure 5B. No significant difference was seen between the mean density of endo tissue and control tissue bands. *FOXP3* mRNA expression in the endo PF treated cells were shown to be increased in expression but was not significant (Figure 5C). For protein expression using the automated Western blotting system, WES, FOXP3 was increased in cells treated with both control and endo PF, but a statistically significant change in expression was only shown with endo PF treatment (2.32-fold, *p* = 0.0493) (Figure 5D).

**Figure 5.** mRNA and protein expression of FOXP3 in patient tissues and PF treated cells. (**A**) Relative mRNA expression of *FOXP3* in EuN (*n* = 5), EuE (*n* = 10), and EcE (*n* = 6). Upregulation of the *FOXP3* mRNA expression in the endometriotic patient tissues was observed but was not significant; (**B**) Relative protein expression of FOXP3 in EuE and EcE was calculated in relation to EuN. Down-**Figure 5.** mRNA and protein expression of FOXP3 in patient tissues and PF treated cells. (**A**) Relative mRNA expression of *FOXP3* in EuN (*n* = 5), EuE (*n* = 10), and EcE (*n* = 6). Upregulation of the *FOXP3* mRNA expression in the endometriotic patient tissues was observed but was not significant; (**B**) Relative protein expression of FOXP3 in EuE and EcE was calculated in relation to EuN. Down-regulation of EuE and EcE were seen when compared to EuN. For all comparisons made,

**Figure 5.** mRNA and protein expression of FOXP3 in patient tissues and PF treated cells. (**A**) Relative mRNA expression of *FOXP3* in EuN (*n* = 5), EuE (*n* = 10), and EcE (*n* = 6). Upregulation of the *FOXP3* mRNA expression in the endometriotic patient tissues was observed but was not significant; (**B**) Relative protein expression of FOXP3 in EuE and EcE was calculated in relation to EuN. Down-

(Figure 6B,C).

*p* > 0.05. (**C**) mRNA expression of FOXP3 in cells treated with control PF (*n* = 12) and endo PF (*n* = 13), relative to expression in a media control (*p* > 0.05). (**D**) Relative protein expression of FOXP3 in PF-treated cells was calculated in relation to a media control and presented as a ratio, in which media alone or EuN is 1. FOXP3 expression was 2.32-fold higher in endo PF (*p* = 0.0493) than in control media alone treated cells. \* *p* < 0.05. Densities of the protein bands obtained were normalized to β-actin or H3. regulation of EuE and EcE were seen when compared to EuN. For all comparisons made, *p* > 0.05. (**C**) mRNA expression of FOXP3 in cells treated with control PF (*n* = 12) and endo PF (*n* = 13), relative to expression in a media control (*p* > 0.05). (**D**) Relative protein expression of FOXP3 in PF-treated cells was calculated in relation to a media control and presented as a ratio, in which media alone or

*FOXP3* mRNA expression in the endo PF treated cells were shown to be increased in expression but was not significant (Figure 5C). For protein expression using the automated Western blotting system, WES, FOXP3 was increased in cells treated with both control and endo PF, but a statistically significant change in expression was only shown with endo PF treatment (2.32-fold, *p* = 0.0493) (Figure 5D). EuN is 1. FOXP3 expression was 2.32-fold higher in endo PF (*p* = 0.0493) than in control media alone treated cells. \* *p* < 0.05. Densities of the protein bands obtained were normalized to β-actin or H3. *2.6. miR-155 Regulates PRC2 Complex and FOXP3*  Since JARID2 and FOXP3 are targets of miR-155 and miR-155 was upregulated in

#### *2.6. miR-155 Regulates PRC2 Complex and FOXP3* endometriotic tissues and PF treated cells, it was investigated if modulating miR-155 lev-

*Int. J. Mol. Sci.* **2021**, *22*, 3492 7 of 19

Since JARID2 and FOXP3 are targets of miR-155 and miR-155 was upregulated in endometriotic tissues and PF treated cells, it was investigated if modulating miR-155 levels using a mimic or inhibitor will alter these target genes. To test this, the expression of JARID2, PRC2 complex and FOXP3 were determined in endometrial cells transfected with a miR-155 mimic or inhibitor (antagonist). Transfection efficiency of miR-155 is shown in Figure 6A. In cells transfected with the mimic, treatment with control PF increased the expression of miR-155 by over 3-fold, compared to when treated with the inhibitor, where the expression decreased below 0.50-fold. In contrast, in cells transfected with the mimic, treatment with endo PF increased miR-155 expression by 1.5-fold but decreased below 0.50-fold after treatment in cells transfected with the inhibitor. Upon PF treatment of the cells transfected with the miR-155 mimic, there was minimal effect on *JARID2* mRNA expression (*p* > 0.05), but seemed to increase *FOXP3* expression in cells treated with control PF and even more so in cells treated with endo PF. In contrast, PF treatment of miR-155 inhibitor transfected cells had no major effect on *JARID2* or *FOXP3* mRNA expression (Figure 6B,C). els using a mimic or inhibitor will alter these target genes. To test this, the expression of JARID2, PRC2 complex and FOXP3 were determined in endometrial cells transfected with a miR-155 mimic or inhibitor (antagonist). Transfection efficiency of miR-155 is shown in Figure 6A. In cells transfected with the mimic, treatment with control PF increased the expression of miR-155 by over 3-fold, compared to when treated with the inhibitor, where the expression decreased below 0.50-fold. In contrast, in cells transfected with the mimic, treatment with endo PF increased miR-155 expression by 1.5-fold but decreased below 0.50-fold after treatment in cells transfected with the inhibitor. Upon PF treatment of the cells transfected with the miR-155 mimic, there was minimal effect on *JARID2* mRNA expression (*p* > 0.05), but seemed to increase *FOXP3* expression in cells treated with control PF and even more so in cells treated with endo PF. In contrast, PF treatment of miR-155 inhibitor transfected cells had no major effect on *JARID2* or *FOXP3* mRNA expression

**Figure 6.** Key mRNA levels in cells transfected with a miR-155 mimic and inhibitor. (**A**) Levels of miR-155 showing transfection efficiency. (**B**) Transfection with a miR-155 mimic had little effect on *JARID2* expression in PF-treated cells (*p* > 0.05), (**C**) but seemed to increase *FOXP3* expression in cells treated with control PF. Compared to control media, the miR-155 inhibitor had no major effect **Figure 6.** Key mRNA levels in cells transfected with a miR-155 mimic and inhibitor. (**A**) Levels of miR-155 showing transfection efficiency. (**B**) Transfection with a miR-155 mimic had little effect on *JARID2* expression in PF-treated cells (*p* > 0.05), (**C**) but seemed to increase *FOXP3* expression in cells treated with control PF. Compared to control media, the miR-155 inhibitor had no major effect on *JARID2* or *FOXP3* expression in PF treated cells.

Western blotting analysis showed that overexpression of miR-155 resulted in significantly lower JARID2 protein expression in control PF-treated cells compared to endo PFtreated cells (*p* = 0.0106). Neither EZH2 nor H2K27me3 protein expression showed any significant up or downregulation in cells overexpressing miR-155 (Figure 7A). In contrast, while both JARID2 and EZH2 showed an upregulation in protein expression in the endo PF treatment groups, when miR-155 was inhibited, no significance was achieved. The protein expression of H3K27me3 in the control PF-treated cells in miR-155 inhibited cells, was

Western blotting analysis showed that overexpression of miR-155 resulted in significantly lower JARID2 protein expression in control PF-treated cells compared to endo PF-treated cells (*p* = 0.0106). Neither EZH2 nor H2K27me3 protein expression showed any significant up or downregulation in cells overexpressing miR-155 (Figure 7A). In contrast, while both JARID2 and EZH2 showed an upregulation in protein expression in the endo PF treatment groups, when miR-155 was inhibited, no significance was achieved. The protein expression of H3K27me3 in the control PF-treated cells in miR-155 inhibited cells, was significantly upregulated when compared to both the transfected media alone cells and the endo PF-treated cells (*p* = 0.0105 and 0.0138, respectively) (Figure 7B). In miR-155 overexpressing cells, both control and endo PF significantly increased FOXP3 protein expression when compared to transfected media alone (*p* = 0.0005 and 0.0079, respectively). In contrast no significant difference in FOXP3 protein expression was seen in cells transfected with an miR-155 inhibitor (Figure 7C). *Int. J. Mol. Sci.* **2021**, *22*, 3492 8 of 19 significantly upregulated when compared to both the transfected media alone cells and the endo PF-treated cells (*p* = 0.0105 and 0.0138, respectively) (Figure 7B). In miR-155 overexpressing cells, both control and endo PF significantly increased FOXP3 protein expression when compared to transfected media alone (*p* = 0.0005 and 0.0079, respectively). In contrast no significant difference in FOXP3 protein expression was seen in cells transfected with an miR-155 inhibitor (Figure 7C).

**Figure 7.** Expression of key targets in cells transfected with miR-155 mimic and inhibitor. (**A**) Transfection with a miR-155 mimic resulted in significantly lower JARID2 expression in control PF-treated cells (*n* = 6) compared to endo PF-treated cells (*n* = 6) (*p* = 0.0106). No significant difference in expression was seen in EZH2 or H3K27me3. (**B**) Transfection with a miR-155 inhibitor resulted in higher H3K27me3 for the control PF-treated cells when compared to transfected media and endo PF-treated cells (*p* = 0.0105, 0.0138, respectively). Relative expression of EZH2 and JARID2 showed no significant increase or decrease in any of the treated groups. (**C**) Transfection with an miR-155 mimic showed significant upregulation of FOXP3 in both control and endo PF-treated cells (*p* = 0.0005, 0.0079) when compared to transfected media alone. Though FOXP3 was induced by 3 or 6-fold in control or endo PF treated cells transfected with miR-155 inhibitor, no significance was observed. \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.005 compared to transfected media alone, \$ Significant difference (*p* < 0.05) **Figure 7.** Expression of key targets in cells transfected with miR-155 mimic and inhibitor. (**A**) Transfection with a miR-155 mimic resulted in significantly lower JARID2 expression in control PF-treated cells (*n* = 6) compared to endo PF-treated cells (*n* = 6) (*p* = 0.0106). No significant difference in expression was seen in EZH2 or H3K27me3. (**B**) Transfection with a miR-155 inhibitor resulted in higher H3K27me3 for the control PF-treated cells when compared to transfected media and endo PF-treated cells (*p* = 0.0105, 0.0138, respectively). Relative expression of EZH2 and JARID2 showed no significant increase or decrease in any of the treated groups. (**C**) Transfection with an miR-155 mimic showed significant upregulation of FOXP3 in both control and endo PF-treated cells (*p* = 0.0005, 0.0079) when compared to transfected media alone. Though FOXP3 was induced by 3 or 6-fold in control or endo PF treated cells transfected with miR-155 inhibitor, no significance was observed. \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.005 compared to transfected media alone, \$ Significant difference (*p* < 0.05) in mean compared to endo PF. Density of protein bands obtained was normalized to β-actin or H3.
