Abstract
Epigenetic changes alter the expression of genes at both pre- and post-transcriptional levels without changing their DNA sequence. Accumulating evidence suggests that such changes can modify cellular behavior and characteristics required during development and in response to various extracellular stimuli. Trophoblast cells develop from the outermost trophectoderm layer of the blastocyst and undergo many phenotypic changes as the placenta develops. One such phenotypic change is differentiation of the epithelial natured cytotrophoblasts into the mesenchymal natured extravillous trophoblasts. The extravillous trophoblasts are primarily responsible for invading into the maternal decidua and thus establishing connection with the maternal spiral arteries. Any dysregulation of this process can have adverse effects on the pregnancy outcome. Hence, tight regulation of this epithelial–mesenchymal transition (EMT) is critical for successful pregnancy. This review summarizes the recent research on the epigenetic regulation of the EMT occurring in the trophoblast cells during placental development. The functional significance of chemical modifications of DNA and histone, which regulate transcription, as well as non-coding RNAs, which control gene expression post-transcriptionally, is discussed in relation to trophoblast biology.
Introduction
The transient extra-embryonic organ, placenta, is pivotal for appropriate in utero development of complex organisms like mammals. Abnormalities in placental development can lead to several feto-maternal pathological complications including preeclampsia (PE), intrauterine growth restriction (IUGR), recurrent abortion etc (Pollheimer et al., 2018; Turco and Moffett, 2019). An integrative insight into trophoblast biology is therefore a prerequisite to develop therapeutic measures for these multi-faceted morbidities.
The epithelial cytotrophoblast cells (CTBs) trans-differentiate into invasive extravillous trophoblast cells (EVTs) (mesenchymal) and demonstrate an altered gene expression pattern (DaSilva-Arnold et al., 2015). Modulation of the gene expression signature is regulated largely by the epigenome of a cell, thereby directing the cellular fate (Lanzuolo and Orlando, 2007). Regulation of the gene expression pattern by various epigenetic modifications during epithelial–mesenchymal transition (EMT) has been described in several pathophysiological conditions including cancer (O'Leary et al., 2018; Kim et al., 2020; Nowak and Bednarek, 2021). Few attempts have been made to summarize the role of epigenetic regulation on placental development and dissecting its relevance in PE (Kohan-Ghadr et al., 2016; Apicella et al., 2019; Ashraf et al., 2021). However, the intricate epigenetic regulation of the EMT process in trophoblast differentiation has not been comprehensively reviewed yet.
Here, we have reviewed, analyzed and summarized recent findings that depict epigenetic regulation of EMT in human trophoblast cells. A search of published literature was performed using the PubMed database from the beginning to October 2021 using variations of search terms like ‘EMT or epithelial to mesenchymal transition’, ‘invasion’, ‘non-coding RNA’, ‘DNA methylation’, ‘histone modification’, etc in combination with ‘trophoblast’. Only English language articles were included. The identified publications were screened and analyzed manually to extract information which has been summarized in this review. Further, its functional consequences in physiological and pathological trophoblast phenotype have also been analyzed.
Trophoblast differentiation in human placenta
Successful placentation and its maintenance are critical for pregnancy preservation. Apart from its role in prenatal development, placental function influences adult health as well (Kratimenos and Penn, 2019; Lane-Cordova et al., 2019). The outer cell layer at the blastocyst stage, the trophectoderm (TE), is the origin of the diverse trophoblast lineages of the placenta. These trophoblast cells provide the primary architectural and functional support to the placenta. During placental development in humans, the TE layer generates CTBs of the chorionic villi (Fig. 1). To some extent, these CTBs act as committed progenitor cells in the placenta (James et al., 2015; Gamage et al., 2016). These CTBs further differentiate to produce morphologically and functionally diverse trophoblast cell types. The multinucleated syncytiotrophoblast cells (STBs) are formed through the fusion of trophoblast cells and are aligned with the placental villi at the fetal–maternal interface. On the other hand, some of the epithelial CTBs also differentiate into the migratory EVTs. EVTs originate from the proliferating CTBs at the tip of the anchoring villi. As the anchoring villi tips are exposed to a plethora of cytokines and growth factors secreted from the decidual stromal cells and uterine NK cells, the differentiation and functions of EVTs may be regulated by decidual paracrine signaling (Pollheimer et al., 2018). The EVTs detach from the villi and start invading the maternal endometrium. These invaded trophoblast cells are called interstitial EVTs. A moderate number of the invading EVTs replace some of the endothelial cells of the maternal spiral artery. These cells partially mimic endothelial function and are called endovascular EVTs. This arterial remodeling ensures higher blood flow through the fetal–maternal interface, which in turn aids in higher nutrition and gaseous exchange to accommodate the needs of the growing fetus (Douglas et al., 2009; Gamage et al., 2016; Boss et al., 2018; Knofler et al., 2019). It is a well-known fact that improper trophoblast invasion is associated with diverse pregnancy-related pathological complications including PE and IUGR. These pathologies are one of the major causes of feto-maternal morbidity and mortality (Burton and Jauniaux, 2018; Huppertz, 2018).
Figure 1.
Different lineages of human trophoblast cells. The trophectoderm cells in the blastocyst develop into progenitor trophoblasts which further form the cytotrophoblasts. The cytotrophoblast (CTB) differentiate into multi-nucleated syncytiotrophoblast (STB) or the invasive extravillous trophoblast (EVT). Further differentiation of EVTs is also indicated.
The differentiation of cytotrophoblast to extravillous trophoblast therefore, holds enormous importance in maintaining a properly functional placenta. During this differentiation process, the EVTs lose firm attachment with the villi tip and acquire the ability to migrate (Fig. 2). This ability to migrate is a prominent mesenchymal characteristic. Upon receiving the appropriate extracellular signals, which induce CTB to EVT differentiation, it is expected that there is an alteration in the gene expression profile of these cells (Knofler et al., 2019; Meinhardt et al., 2020). This alteration is required to achieve the invasion ability of EVTs. Therefore, the differentiation of epithelial villous CTBs to invasive EVTs suggests the crucial involvement of EMT (Davies et al., 2016). This hypothesis has been supported by a recent study, which demonstrated changes in the expression of multiple EMT-associated genes using paired CTBs and EVTs isolated from first-trimester placenta (DaSilva-Arnold et al., 2015).
Figure 2.
Cell populations at maternal–fetal interface. Schematic diagram showing different types of trophoblast cells at the maternal–fetal interface during the first trimester. The cytotrophoblast cells are present in the villous columns, the syncytiotrophoblasts are at the villous lining, and the extravillous trophoblast (EVT) cells are at the tip of the villous column. The invaded interstitial trophoblasts are located in the endometrium layer of the uterus. The endovascular trophoblasts are at the uterine spiral artery lining and the circulating trophoblast cells are present in the blood flow. The uterine NK cells are also indicated.
Epithelial to mesenchymal transition
EMT is a developmentally conserved trans-differentiation process. The epithelial cells lose their apicobasal polarity and obtain the ability to migrate and invade, a mesenchymal trait. Along with invasiveness, these trans-differentiated cells demonstrate enhanced resistance to apoptotic signals (Zavadil et al., 2008; Kalluri and Weinberg, 2009; Chaffer et al., 2016). EMT is generally encountered in three well-studied and biologically distinctive functional setups—type I in embryonic development; type II in wound healing and tissue fibrosis; and type III in cancer metastasis. Whereas the first two subtypes are tightly regulated and transient, the third type is robust and stable (Baum et al., 2008; Acloque et al., 2009; Thiery et al., 2009; Chen et al., 2017b).
The manifestation of EMT in a cell is marked by a precise alteration in the expression profile of a particular subset of genes. This subset of genes is defined as EMT-associated genes and hence, can be utilized as a marker of EMT. Upon induction of EMT, expression of the epithelial marker gene E-cadherin (CDH1) diminishes while expression of the mesenchymal marker genes, N-cadherin (CDH2), Vimentin (VIM) and Fibronectin 1 (FN1) increases remarkably. These four genes are often referred to as the core EMT signature. However, EMT-associated genes are not limited to these four genes only and include various transcription factors, cell signaling molecules, cell–cell junction proteins, etc (Teng et al., 2007; Zeisberg and Neilson, 2009). Upregulation of some transcription factors such as Twist, Zeb and Snail is necessary and sufficient to bring about EMT and therefore they are defined as EMT master regulators. These transcription factors directly modulate the expression profile of EMT core genes and alter the cellular phenotype (Kang et al., 2021). The main genes involved in EMT which can act as markers are depicted in Fig. 3.
Figure 3.
Epithelial to mesenchymal transition (EMT). Schematic representation showing genes which change their expression during the EMT process. The green box shows the cell membrane proteins; the red box shows the transcription factors involved; the blue box shows the extracellular matrix proteins; and the purple box shows the matrix modulating enzymes.
A cohort of extracellular signals is found to induce this transition, making notable functional and morphological changes in the cells (Moustakas and Heldin, 2007). Growth factors, cytokines, Notch, Wnt and transforming growth factor β (TGFβ) signaling are a few that can favor these alterations (Grego-Bessa et al., 2004; Howard et al., 2011; Misra et al., 2012; Ghahhari and Babashah, 2015; Tan et al., 2015; Xu et al., 2015; Brittain et al., 2017).
Epigenetic regulation of gene expression
The functional identity of a cell is governed by its expression profile in terms of RNAs and proteins. Therefore, the induction and maintenance of cellular identity are controlled by a broad range of regulatory mechanisms of gene expression. These regulations do not involve any alteration of genomic sequence; hence, are known as epigenetic regulations. Functionally, four different regulatory mechanisms are studied, which include (i) three-dimensional chromatin remodeling and architecture-based regulation; (ii) non-coding RNA-mediated gene regulation; (iii) post-translational modifications in histone proteins; and (iv) chemical modifications in DNA nucleotides. Together, these modifications can be defined as the epigenome of a particular cell. It is highly dynamic and responds to any variation in the cellular niche. Thus, the gene expression profile is influenced in an epigenetically regulated manner, as a response to different extrinsic factors or environmental cues. Occasionally, the cellular response is memorized through the incorporation of diverse stable epigenetic marks (Kim et al., 2010; Kim and Costello, 2017). We summarize these different modes of epigenetic regulation, their role in gene expression, and biological relevance in Table I.
Table IEpigenetic regulators and their function in gene expression.
| Epigenetic regulation
. | Modifiers
. | Target
. | Effect
. | Reference
. |
|---|
| 3D-chromatin architecture | Position | | Both* | Fedorova and Zink (2008), Hubner et al. (2013) |
| Histone modifications | Acetylation | H2BK5 | Increase | Galasinski et al. (2002) |
| | H3K9 | Increase | Vaquero et al. (2004) |
| | H3K36 | Increase | Edmunds et al. (2008) |
| | H4K5 | Increase | Kawasaki et al. (2000), Kruhlak et al. (2001) |
| Methylation | H3K4 | Increase | Bernstein et al. (2002), Santos-Rosa et al. (2002), Lee and Skalnik (2005), Greer and Shi (2012) |
| | H3K9 | Reduce | Nielsen et al. (2001), Vandel et al. (2001), Schultz et al. (2002), Dodge et al. (2004) |
| | H3K27 | Reduce | Kuzmichev et al. (2002), Su et al. (2003) |
| | H4K20 | Reduce | Fang et al. (2002), Rice et al. (2002) |
| Ubiquitination | H2AK119 | Reduce | Wang et al. (2004), Kapetanaki et al. (2006) |
| | H2BK120 | Increase | Kao et al. (2004), Zhu et al. (2005) |
| Biotinylation | H2AK126 | Reduce | Stanley et al. (2001), Kothapalli et al. (2005a), Chew et al. (2006) |
| | H4K12 | DNA damage response | Stanley et al. (2001), Kothapalli et al. (2005a,b) |
| Phosphorylation | H2A.XY142 | DNA damage response | Xiao et al. (2009) |
| | H2A.XS139 | DNA repair | Rogakou et al. (1998, 1999), Burma et al. (2001), Ward and Chen (2001), Stiff et al. (2004), Ichijima et al. (2005), Mukherjee et al. (2006) |
| | H1S27 | HP1 binding blocks | Garcia et al. (2004), Daujat et al. (2005) |
| | H3T45 | DNA replication | Hurd et al. (2009) |
| Sumoylation | H4K59 | Reduce | Shiio and Eisenman (2003) |
| DNA modifications | Methylation | Cytosine | Reduce | Ehrlich and Lacey (2013) |
| | Adenine | Reduce | Heyn and Esteller (2015) |
| Hydroxymethylation | Cytosine | Both | Guibert and Weber (2013) |
| Non-coding RNAs | lncRNA | | Both | Dykes and Emanueli (2017), Sun et al. (2017), Sun et al. (2018) |
| micro RNA | | Both | Ma et al. (2010), Moreno-Moya et al. (2014), Xiao et al. (2017) |
| pi RNA | | Reduce | Watanabe and Lin (2014), Weick and Miska (2014) |
| Circular RNA | | Increase | Ebbesen et al. (2017), Hsiao et al. (2017), Huang et al. (2017) |
| Enhancer RNA | | Increase | Chen et al. (2017a), Liu (2017) |
| Fragmented RNA derivatives | | Both | Kumar et al. (2016), Chen et al. (2017c) |
| Epigenetic regulation
. | Modifiers
. | Target
. | Effect
. | Reference
. |
|---|
| 3D-chromatin architecture | Position | | Both* | Fedorova and Zink (2008), Hubner et al. (2013) |
| Histone modifications | Acetylation | H2BK5 | Increase | Galasinski et al. (2002) |
| | H3K9 | Increase | Vaquero et al. (2004) |
| | H3K36 | Increase | Edmunds et al. (2008) |
| | H4K5 | Increase | Kawasaki et al. (2000), Kruhlak et al. (2001) |
| Methylation | H3K4 | Increase | Bernstein et al. (2002), Santos-Rosa et al. (2002), Lee and Skalnik (2005), Greer and Shi (2012) |
| | H3K9 | Reduce | Nielsen et al. (2001), Vandel et al. (2001), Schultz et al. (2002), Dodge et al. (2004) |
| | H3K27 | Reduce | Kuzmichev et al. (2002), Su et al. (2003) |
| | H4K20 | Reduce | Fang et al. (2002), Rice et al. (2002) |
| Ubiquitination | H2AK119 | Reduce | Wang et al. (2004), Kapetanaki et al. (2006) |
| | H2BK120 | Increase | Kao et al. (2004), Zhu et al. (2005) |
| Biotinylation | H2AK126 | Reduce | Stanley et al. (2001), Kothapalli et al. (2005a), Chew et al. (2006) |
| | H4K12 | DNA damage response | Stanley et al. (2001), Kothapalli et al. (2005a,b) |
| Phosphorylation | H2A.XY142 | DNA damage response | Xiao et al. (2009) |
| | H2A.XS139 | DNA repair | Rogakou et al. (1998, 1999), Burma et al. (2001), Ward and Chen (2001), Stiff et al. (2004), Ichijima et al. (2005), Mukherjee et al. (2006) |
| | H1S27 | HP1 binding blocks | Garcia et al. (2004), Daujat et al. (2005) |
| | H3T45 | DNA replication | Hurd et al. (2009) |
| Sumoylation | H4K59 | Reduce | Shiio and Eisenman (2003) |
| DNA modifications | Methylation | Cytosine | Reduce | Ehrlich and Lacey (2013) |
| | Adenine | Reduce | Heyn and Esteller (2015) |
| Hydroxymethylation | Cytosine | Both | Guibert and Weber (2013) |
| Non-coding RNAs | lncRNA | | Both | Dykes and Emanueli (2017), Sun et al. (2017), Sun et al. (2018) |
| micro RNA | | Both | Ma et al. (2010), Moreno-Moya et al. (2014), Xiao et al. (2017) |
| pi RNA | | Reduce | Watanabe and Lin (2014), Weick and Miska (2014) |
| Circular RNA | | Increase | Ebbesen et al. (2017), Hsiao et al. (2017), Huang et al. (2017) |
| Enhancer RNA | | Increase | Chen et al. (2017a), Liu (2017) |
| Fragmented RNA derivatives | | Both | Kumar et al. (2016), Chen et al. (2017c) |
Table IEpigenetic regulators and their function in gene expression.
| Epigenetic regulation
. | Modifiers
. | Target
. | Effect
. | Reference
. |
|---|
| 3D-chromatin architecture | Position | | Both* | Fedorova and Zink (2008), Hubner et al. (2013) |
| Histone modifications | Acetylation | H2BK5 | Increase | Galasinski et al. (2002) |
| | H3K9 | Increase | Vaquero et al. (2004) |
| | H3K36 | Increase | Edmunds et al. (2008) |
| | H4K5 | Increase | Kawasaki et al. (2000), Kruhlak et al. (2001) |
| Methylation | H3K4 | Increase | Bernstein et al. (2002), Santos-Rosa et al. (2002), Lee and Skalnik (2005), Greer and Shi (2012) |
| | H3K9 | Reduce | Nielsen et al. (2001), Vandel et al. (2001), Schultz et al. (2002), Dodge et al. (2004) |
| | H3K27 | Reduce | Kuzmichev et al. (2002), Su et al. (2003) |
| | H4K20 | Reduce | Fang et al. (2002), Rice et al. (2002) |
| Ubiquitination | H2AK119 | Reduce | Wang et al. (2004), Kapetanaki et al. (2006) |
| | H2BK120 | Increase | Kao et al. (2004), Zhu et al. (2005) |
| Biotinylation | H2AK126 | Reduce | Stanley et al. (2001), Kothapalli et al. (2005a), Chew et al. (2006) |
| | H4K12 | DNA damage response | Stanley et al. (2001), Kothapalli et al. (2005a,b) |
| Phosphorylation | H2A.XY142 | DNA damage response | Xiao et al. (2009) |
| | H2A.XS139 | DNA repair | Rogakou et al. (1998, 1999), Burma et al. (2001), Ward and Chen (2001), Stiff et al. (2004), Ichijima et al. (2005), Mukherjee et al. (2006) |
| | H1S27 | HP1 binding blocks | Garcia et al. (2004), Daujat et al. (2005) |
| | H3T45 | DNA replication | Hurd et al. (2009) |
| Sumoylation | H4K59 | Reduce | Shiio and Eisenman (2003) |
| DNA modifications | Methylation | Cytosine | Reduce | Ehrlich and Lacey (2013) |
| | Adenine | Reduce | Heyn and Esteller (2015) |
| Hydroxymethylation | Cytosine | Both | Guibert and Weber (2013) |
| Non-coding RNAs | lncRNA | | Both | Dykes and Emanueli (2017), Sun et al. (2017), Sun et al. (2018) |
| micro RNA | | Both | Ma et al. (2010), Moreno-Moya et al. (2014), Xiao et al. (2017) |
| pi RNA | | Reduce | Watanabe and Lin (2014), Weick and Miska (2014) |
| Circular RNA | | Increase | Ebbesen et al. (2017), Hsiao et al. (2017), Huang et al. (2017) |
| Enhancer RNA | | Increase | Chen et al. (2017a), Liu (2017) |
| Fragmented RNA derivatives | | Both | Kumar et al. (2016), Chen et al. (2017c) |
| Epigenetic regulation
. | Modifiers
. | Target
. | Effect
. | Reference
. |
|---|
| 3D-chromatin architecture | Position | | Both* | Fedorova and Zink (2008), Hubner et al. (2013) |
| Histone modifications | Acetylation | H2BK5 | Increase | Galasinski et al. (2002) |
| | H3K9 | Increase | Vaquero et al. (2004) |
| | H3K36 | Increase | Edmunds et al. (2008) |
| | H4K5 | Increase | Kawasaki et al. (2000), Kruhlak et al. (2001) |
| Methylation | H3K4 | Increase | Bernstein et al. (2002), Santos-Rosa et al. (2002), Lee and Skalnik (2005), Greer and Shi (2012) |
| | H3K9 | Reduce | Nielsen et al. (2001), Vandel et al. (2001), Schultz et al. (2002), Dodge et al. (2004) |
| | H3K27 | Reduce | Kuzmichev et al. (2002), Su et al. (2003) |
| | H4K20 | Reduce | Fang et al. (2002), Rice et al. (2002) |
| Ubiquitination | H2AK119 | Reduce | Wang et al. (2004), Kapetanaki et al. (2006) |
| | H2BK120 | Increase | Kao et al. (2004), Zhu et al. (2005) |
| Biotinylation | H2AK126 | Reduce | Stanley et al. (2001), Kothapalli et al. (2005a), Chew et al. (2006) |
| | H4K12 | DNA damage response | Stanley et al. (2001), Kothapalli et al. (2005a,b) |
| Phosphorylation | H2A.XY142 | DNA damage response | Xiao et al. (2009) |
| | H2A.XS139 | DNA repair | Rogakou et al. (1998, 1999), Burma et al. (2001), Ward and Chen (2001), Stiff et al. (2004), Ichijima et al. (2005), Mukherjee et al. (2006) |
| | H1S27 | HP1 binding blocks | Garcia et al. (2004), Daujat et al. (2005) |
| | H3T45 | DNA replication | Hurd et al. (2009) |
| Sumoylation | H4K59 | Reduce | Shiio and Eisenman (2003) |
| DNA modifications | Methylation | Cytosine | Reduce | Ehrlich and Lacey (2013) |
| | Adenine | Reduce | Heyn and Esteller (2015) |
| Hydroxymethylation | Cytosine | Both | Guibert and Weber (2013) |
| Non-coding RNAs | lncRNA | | Both | Dykes and Emanueli (2017), Sun et al. (2017), Sun et al. (2018) |
| micro RNA | | Both | Ma et al. (2010), Moreno-Moya et al. (2014), Xiao et al. (2017) |
| pi RNA | | Reduce | Watanabe and Lin (2014), Weick and Miska (2014) |
| Circular RNA | | Increase | Ebbesen et al. (2017), Hsiao et al. (2017), Huang et al. (2017) |
| Enhancer RNA | | Increase | Chen et al. (2017a), Liu (2017) |
| Fragmented RNA derivatives | | Both | Kumar et al. (2016), Chen et al. (2017c) |
Since the functional character of a cell is defined by its epigenome, consequently, alteration of its gene expression profile for executing the mesenchymal transition would require modification of its epigenetic status. Several reviews discuss the epigenetic regulation of EMT in various cancer models (O'Leary et al., 2018; Kim et al., 2020; Nowak and Bednarek, 2021). In tumorigenesis and cancer metastasis, the EMT process is controlled at both pre- and post-transcriptional level by employing various non-coding RNAs as well as histone and DNA modifications etc (Huangyang and Shang, 2013; Lin et al., 2016; Sekhon et al., 2016; Serrano-Gomez et al., 2016; Sun and Fang, 2016; Xu et al., 2016; Ambrosio et al., 2017; Skrypek et al., 2017; Träger and Dhayat, 2017; Drak Alsibai and Meseure, 2018).
EMT in trophoblast cells in developing placenta and related pathologies
Similar to other physiological and pathological events, the trophoblast lineage differentiation in the developing placenta involves EMT (DaSilva-Arnold et al., 2015, 2018). Using PCR array, DaSilva-Arnold et al. (2015) clearly demonstrated that CTB differentiation into EVTs is an EMT phenomenon. Other research also demonstrated an EMT-specific expression pattern of several genes during placentation and trophoblast differentiation. During normal placentation, epithelial E-cadherin is highly expressed in anchoring villous cytotrophoblasts whereas mesenchymal N-cadherin is found in EVT cells (Kokkinos et al., 2010). Furthermore, trophoblast motility in EVTs is controlled by snail family transcription factors (Kokkinos et al., 2010). Another study showed that EMT master regulator ZEB2 is highly expressed in EVTs as compared to CTBs (DaSilva-Arnold et al., 2019). Interestingly, studies demonstrated that these changes in expression are transient (DaSilva-Arnold et al., 2018; Yang and Meng, 2019) as the ability of migration is again reduced in interstitial and endovascular trophoblast population, which may be considered as further differentiated forms of EVTs.
The EMT required for normal placental development could be driven spontaneously by trophoblasts themselves or influenced by other cell types present in the uterine microenvironment. The trophoblast–macrophage interaction is one of those which has been investigated to uncover the role of this interaction for sustaining healthy pregnancy. Ding et al. (2021) showed that trophoblasts co-cultured with activated macrophages (M2) have decreased E-cadherin and increased vimentin expression. Further analysis indicated that M2 macrophage-derived granulocyte colony-stimulating factor (G-CSF) induced EMT in trophoblast by activating Akt and MAPK (ERK1/2) pathways (Ding et al., 2021).
A dysregulated expression pattern of EMT-associated genes has been observed in different placenta-related pathologies. Higher expression of E-cadherin in preeclamptic placenta is associated with the loss of trophoblast invasion (Kokkinos et al., 2010). On the contrary, the highly invasive trophoblasts in placenta accreta show a significant loss in E-cadherin expression (Duzyj et al., 2015). Overall reduction of EMT in PE is also supported by various studies showing reduced expression of the mesenchymal markers, vimentin and N-cadherin (Shen et al., 2019; Li et al., 2020; Wu et al., 2020a). Additionally, the EMT inducing transcription factors, ZEB1, SNAI1, NOTCH1 and cFOS are reduced in PE (Wang et al., 2019; Yang and Meng, 2019; Li et al., 2020; Wu et al., 2020a) while expression of the epithelial transcription factor grainyhead like 2 (GRHL2) is increased (Shen et al., 2019). Furthermore, in vitro studies confirmed that modulating the expression of these transcription factors resulted in the altered levels of the EMT core marker genes. The reduced expression of SNAI1 and higher expression of GRHL2 in PE correlated well with higher expression of E-cadherin while silencing of cFOS expression resulted in reduced expression of the mesenchymal markers, vimentin and N-cadherin (Shen et al., 2019; Li et al., 2020; Wu et al., 2020a).
Protein ubiquitination and degradation dynamics may also play critical roles in controlling the expression levels of the EMT core genes in preeclamptic placenta. Wu et al. (2020a) showed that the ubiquitination regulator, beta-transducin repeat containing E3 ubiquitin protein (β-TrCP) is upregulated in the preeclamptic placenta both at mRNA and protein levels. This also reduces the trophoblast cell migration. Overexpression of TrCP downregulated the EMT transcription factor, Snail (SNAI1) and the mesenchymal markers, vimentin and N-cadherin. Together, this indicates the involvement of protein level dynamics in modulating EMT in trophoblast cells (Wu et al., 2020a).
Similar to PE, reduced EMT has also been implicated in spontaneous abortion. Villous samples from spontaneous abortion showed higher expression of the extracellular matrix protein, secreted protein acidic and rich in cysteine like-1 (SPARCL1). In parallel, in vitro over-expression of SPARCL1 reduced the mesenchymal marker genes, vimentin and N-cadherin while upregulating the epithelial marker, E-cadherin (Liu et al., 2020). Another study showed reduced levels of G-CSF in villous samples of recurrent spontaneous abortion patients. G-CSF produced by activated M2 macrophages is responsible for decreased E-cadherin and increased vimentin expression in trophoblasts, thus linking improper EMT in trophoblasts with spontaneous abortion (Ding et al., 2021).
Hence, it is clear that the expression pattern alteration that favors EMT is necessary for appropriate placenta development while dysregulation of this process may result in placenta-related pregnancy disorders. Since EMT requires a change in gene expression pattern, it can be hypothesized that this is controlled by various epigenetic modifiers. Some recent studies have investigated the involvement of epigenetic regulators of EMT in trophoblast cells and placental development. However, the field remains to be explored further.
Epigenetic regulation of EMT in trophoblast cells and placenta development
The trophoblast cells undergoing EMT process demonstrate altered gene expression patterns. Modulation of cell invasion is a mesenchymal aspect; therefore, precise alteration of invasion supporting gene expression pattern is required. This indicates the inevitable involvement of epigenetic regulation for shaping this particular expression profile. Several studies support the active involvement of epigenetic regulation in controlling EMT and thus in regulating trophoblast biology. Here, we analyzed the available literature on the regulation of EMT in trophoblast cells through several epigenetic modifications (Table II).
Table IIEpigenetic regulation of EMT-associated genes in trophoblast.
| Molecule
. | Epigenetic regulators
. | Reference
. |
|---|
| AQPEP | DNA methylation | Gamage et al. (2018) |
| CAPN2 | DNA methylation | Gamage et al. (2018) |
| CDH1 | MEG-3, SNHG12, SNHG14, miR-431, DNA methylation | Chen et al. (2013a), Yu et al. (2018), Yang and Meng (2019), Yang et al. (2019), Zhou et al. (2020), Zhang and Zhang (2021) |
| CDH11 | DNA methylation | Anton et al. (2014) |
| CDH18 | DNA methylation | Chen et al. (2013a) |
| CDH2 | MEG-3, SNHG5/miR-26a-5p | Yu et al. (2018), Yang et al. (2019) |
| COL5A1 | DNA methylation | Anton et al. (2014) |
| CTNNB1 | SNHG14, DNA methylation | Chen et al. (2013a), Zhang and Zhang (2021) |
| DLX4 | DNA methylation | Gamage et al. (2018) |
| FERMT3 | DNA methylation | Gamage et al. (2018) |
| FN1 | DNA methylation | Zhao et al. (2017) |
| GRHL2 | circTNRC18/miR-762 | Shen et al. (2019) |
| IGF1 | MALAT-1/miR-206 | Wu et al. (2020b) |
| IGF2BP1 | miR-423-5p | Zirkel et al. (2013), Guo et al. (2018) |
| IGF2BP2 | miR-181a-5p | Wu et al. (2018) |
| IGFBP5 | DNA methylation | Jia et al. (2017) |
| ITGA5 | DNA methylation | Zhao et al. (2017) |
| JGD4 | DNA methylation | Chen et al. (2013a) |
| LHX2 | DNA methylation | Gamage et al. (2018) |
| MASPIN | H3K9ac, H3K4me3, DNA methylation | Dokras et al. (2006), Shi et al. (2015) |
| MEST | DNA methylation | Peng et al. (2016) |
| MMP2 | TUG-1/miR-29b | Li et al. (2019) |
| MMP2/MMP9 | lncRNA uc 187, SNHG12, SNHG14/miR-330-5p | Cao et al. (2017), Zhou et al. (2020), Zhang and Zhang (2021) |
| MMP9 | DNA methylation | Li et al. (2018) |
| NCAM1 | DNA methylation | Anton et al. (2014) |
| NOTCH1 | miR 210 | Wang et al. (2019) |
| SMAD4 | miR-34a-5p | Xue et al. (2019) |
| SMAD7 | MEG-3 | Yu et al. (2018) |
| SNAI1 | DNA methylation | Chen et al. (2013b) |
| SNAI2 | DNA methylation | Chen et al. (2013b) |
| STAT3 | circPAPPA/miR-384 | Zhou et al. (2019) |
| TGFB1 | miR-142-3p, DNA methylation | Gamage et al. (2018) |
| TIMP1 | lncRNA uc 187 | Cao et al. (2017) |
| TIMP3 | HDAC9 | Xie et al. (2019) |
| VEGFA | TUG-1/miR-29b | Li et al. (2019) |
| VIMENTIN | MEG-3, SNHG12, SNHG14, DNA methylation | Chen et al. (2013a), Yu et al. (2018), Yang and Meng (2019), Zhou et al. (2020), Zhang and Zhang (2021) |
| WNT1 | DNA methylation | Gamage et al. (2018) |
| ZEB1 | miRNA-431 | Yang and Meng (2019) |
| Molecule
. | Epigenetic regulators
. | Reference
. |
|---|
| AQPEP | DNA methylation | Gamage et al. (2018) |
| CAPN2 | DNA methylation | Gamage et al. (2018) |
| CDH1 | MEG-3, SNHG12, SNHG14, miR-431, DNA methylation | Chen et al. (2013a), Yu et al. (2018), Yang and Meng (2019), Yang et al. (2019), Zhou et al. (2020), Zhang and Zhang (2021) |
| CDH11 | DNA methylation | Anton et al. (2014) |
| CDH18 | DNA methylation | Chen et al. (2013a) |
| CDH2 | MEG-3, SNHG5/miR-26a-5p | Yu et al. (2018), Yang et al. (2019) |
| COL5A1 | DNA methylation | Anton et al. (2014) |
| CTNNB1 | SNHG14, DNA methylation | Chen et al. (2013a), Zhang and Zhang (2021) |
| DLX4 | DNA methylation | Gamage et al. (2018) |
| FERMT3 | DNA methylation | Gamage et al. (2018) |
| FN1 | DNA methylation | Zhao et al. (2017) |
| GRHL2 | circTNRC18/miR-762 | Shen et al. (2019) |
| IGF1 | MALAT-1/miR-206 | Wu et al. (2020b) |
| IGF2BP1 | miR-423-5p | Zirkel et al. (2013), Guo et al. (2018) |
| IGF2BP2 | miR-181a-5p | Wu et al. (2018) |
| IGFBP5 | DNA methylation | Jia et al. (2017) |
| ITGA5 | DNA methylation | Zhao et al. (2017) |
| JGD4 | DNA methylation | Chen et al. (2013a) |
| LHX2 | DNA methylation | Gamage et al. (2018) |
| MASPIN | H3K9ac, H3K4me3, DNA methylation | Dokras et al. (2006), Shi et al. (2015) |
| MEST | DNA methylation | Peng et al. (2016) |
| MMP2 | TUG-1/miR-29b | Li et al. (2019) |
| MMP2/MMP9 | lncRNA uc 187, SNHG12, SNHG14/miR-330-5p | Cao et al. (2017), Zhou et al. (2020), Zhang and Zhang (2021) |
| MMP9 | DNA methylation | Li et al. (2018) |
| NCAM1 | DNA methylation | Anton et al. (2014) |
| NOTCH1 | miR 210 | Wang et al. (2019) |
| SMAD4 | miR-34a-5p | Xue et al. (2019) |
| SMAD7 | MEG-3 | Yu et al. (2018) |
| SNAI1 | DNA methylation | Chen et al. (2013b) |
| SNAI2 | DNA methylation | Chen et al. (2013b) |
| STAT3 | circPAPPA/miR-384 | Zhou et al. (2019) |
| TGFB1 | miR-142-3p, DNA methylation | Gamage et al. (2018) |
| TIMP1 | lncRNA uc 187 | Cao et al. (2017) |
| TIMP3 | HDAC9 | Xie et al. (2019) |
| VEGFA | TUG-1/miR-29b | Li et al. (2019) |
| VIMENTIN | MEG-3, SNHG12, SNHG14, DNA methylation | Chen et al. (2013a), Yu et al. (2018), Yang and Meng (2019), Zhou et al. (2020), Zhang and Zhang (2021) |
| WNT1 | DNA methylation | Gamage et al. (2018) |
| ZEB1 | miRNA-431 | Yang and Meng (2019) |
Table IIEpigenetic regulation of EMT-associated genes in trophoblast.
| Molecule
. | Epigenetic regulators
. | Reference
. |
|---|
| AQPEP | DNA methylation | Gamage et al. (2018) |
| CAPN2 | DNA methylation | Gamage et al. (2018) |
| CDH1 | MEG-3, SNHG12, SNHG14, miR-431, DNA methylation | Chen et al. (2013a), Yu et al. (2018), Yang and Meng (2019), Yang et al. (2019), Zhou et al. (2020), Zhang and Zhang (2021) |
| CDH11 | DNA methylation | Anton et al. (2014) |
| CDH18 | DNA methylation | Chen et al. (2013a) |
| CDH2 | MEG-3, SNHG5/miR-26a-5p | Yu et al. (2018), Yang et al. (2019) |
| COL5A1 | DNA methylation | Anton et al. (2014) |
| CTNNB1 | SNHG14, DNA methylation | Chen et al. (2013a), Zhang and Zhang (2021) |
| DLX4 | DNA methylation | Gamage et al. (2018) |
| FERMT3 | DNA methylation | Gamage et al. (2018) |
| FN1 | DNA methylation | Zhao et al. (2017) |
| GRHL2 | circTNRC18/miR-762 | Shen et al. (2019) |
| IGF1 | MALAT-1/miR-206 | Wu et al. (2020b) |
| IGF2BP1 | miR-423-5p | Zirkel et al. (2013), Guo et al. (2018) |
| IGF2BP2 | miR-181a-5p | Wu et al. (2018) |
| IGFBP5 | DNA methylation | Jia et al. (2017) |
| ITGA5 | DNA methylation | Zhao et al. (2017) |
| JGD4 | DNA methylation | Chen et al. (2013a) |
| LHX2 | DNA methylation | Gamage et al. (2018) |
| MASPIN | H3K9ac, H3K4me3, DNA methylation | Dokras et al. (2006), Shi et al. (2015) |
| MEST | DNA methylation | Peng et al. (2016) |
| MMP2 | TUG-1/miR-29b | Li et al. (2019) |
| MMP2/MMP9 | lncRNA uc 187, SNHG12, SNHG14/miR-330-5p | Cao et al. (2017), Zhou et al. (2020), Zhang and Zhang (2021) |
| MMP9 | DNA methylation | Li et al. (2018) |
| NCAM1 | DNA methylation | Anton et al. (2014) |
| NOTCH1 | miR 210 | Wang et al. (2019) |
| SMAD4 | miR-34a-5p | Xue et al. (2019) |
| SMAD7 | MEG-3 | Yu et al. (2018) |
| SNAI1 | DNA methylation | Chen et al. (2013b) |
| SNAI2 | DNA methylation | Chen et al. (2013b) |
| STAT3 | circPAPPA/miR-384 | Zhou et al. (2019) |
| TGFB1 | miR-142-3p, DNA methylation | Gamage et al. (2018) |
| TIMP1 | lncRNA uc 187 | Cao et al. (2017) |
| TIMP3 | HDAC9 | Xie et al. (2019) |
| VEGFA | TUG-1/miR-29b | Li et al. (2019) |
| VIMENTIN | MEG-3, SNHG12, SNHG14, DNA methylation | Chen et al. (2013a), Yu et al. (2018), Yang and Meng (2019), Zhou et al. (2020), Zhang and Zhang (2021) |
| WNT1 | DNA methylation | Gamage et al. (2018) |
| ZEB1 | miRNA-431 | Yang and Meng (2019) |
| Molecule
. | Epigenetic regulators
. | Reference
. |
|---|
| AQPEP | DNA methylation | Gamage et al. (2018) |
| CAPN2 | DNA methylation | Gamage et al. (2018) |
| CDH1 | MEG-3, SNHG12, SNHG14, miR-431, DNA methylation | Chen et al. (2013a), Yu et al. (2018), Yang and Meng (2019), Yang et al. (2019), Zhou et al. (2020), Zhang and Zhang (2021) |
| CDH11 | DNA methylation | Anton et al. (2014) |
| CDH18 | DNA methylation | Chen et al. (2013a) |
| CDH2 | MEG-3, SNHG5/miR-26a-5p | Yu et al. (2018), Yang et al. (2019) |
| COL5A1 | DNA methylation | Anton et al. (2014) |
| CTNNB1 | SNHG14, DNA methylation | Chen et al. (2013a), Zhang and Zhang (2021) |
| DLX4 | DNA methylation | Gamage et al. (2018) |
| FERMT3 | DNA methylation | Gamage et al. (2018) |
| FN1 | DNA methylation | Zhao et al. (2017) |
| GRHL2 | circTNRC18/miR-762 | Shen et al. (2019) |
| IGF1 | MALAT-1/miR-206 | Wu et al. (2020b) |
| IGF2BP1 | miR-423-5p | Zirkel et al. (2013), Guo et al. (2018) |
| IGF2BP2 | miR-181a-5p | Wu et al. (2018) |
| IGFBP5 | DNA methylation | Jia et al. (2017) |
| ITGA5 | DNA methylation | Zhao et al. (2017) |
| JGD4 | DNA methylation | Chen et al. (2013a) |
| LHX2 | DNA methylation | Gamage et al. (2018) |
| MASPIN | H3K9ac, H3K4me3, DNA methylation | Dokras et al. (2006), Shi et al. (2015) |
| MEST | DNA methylation | Peng et al. (2016) |
| MMP2 | TUG-1/miR-29b | Li et al. (2019) |
| MMP2/MMP9 | lncRNA uc 187, SNHG12, SNHG14/miR-330-5p | Cao et al. (2017), Zhou et al. (2020), Zhang and Zhang (2021) |
| MMP9 | DNA methylation | Li et al. (2018) |
| NCAM1 | DNA methylation | Anton et al. (2014) |
| NOTCH1 | miR 210 | Wang et al. (2019) |
| SMAD4 | miR-34a-5p | Xue et al. (2019) |
| SMAD7 | MEG-3 | Yu et al. (2018) |
| SNAI1 | DNA methylation | Chen et al. (2013b) |
| SNAI2 | DNA methylation | Chen et al. (2013b) |
| STAT3 | circPAPPA/miR-384 | Zhou et al. (2019) |
| TGFB1 | miR-142-3p, DNA methylation | Gamage et al. (2018) |
| TIMP1 | lncRNA uc 187 | Cao et al. (2017) |
| TIMP3 | HDAC9 | Xie et al. (2019) |
| VEGFA | TUG-1/miR-29b | Li et al. (2019) |
| VIMENTIN | MEG-3, SNHG12, SNHG14, DNA methylation | Chen et al. (2013a), Yu et al. (2018), Yang and Meng (2019), Zhou et al. (2020), Zhang and Zhang (2021) |
| WNT1 | DNA methylation | Gamage et al. (2018) |
| ZEB1 | miRNA-431 | Yang and Meng (2019) |
DNA methylation-mediated regulation
Many studies have investigated the role of DNA methylation in controlling several physiological aspects and functions of the trophoblast cells, including invasion and differentiation (Hemberger, 2007; Logan et al., 2013; Tanaka et al., 2014). In peri-implantation mice embryos, the installation of trophoblast-specific DNA methylation marks is essential for the establishment of TE identity (Nakanishi et al., 2012; Senner et al., 2012; Oda et al., 2013). Further differentiation of CTBs into STBs happens in developing human placenta and the two cell types demonstrate differential DNA methylation patterns. STBs have reduced DNA methylation but higher levels of DNA hydroxy-methylation compared to CTBs (Fogarty et al., 2015; Wilson et al., 2019). In vitro studies on trophoblast cell syncytialization using BeWo cells corroborates the same (Shankar et al., 2015). Interestingly, the amount of both 5-methyl cytosine and 5-hydroxymethyl cytosine increase in CTBs with gestational age (Wilson et al., 2019).
Comparison of mature trophoblast lineages, CTB and EVT, isolated from first-trimester placenta revealed the presence of distinct differentially methylated regions (Gamage et al., 2018). Clustal analysis indicated that the CTB methylome was similar to that of the candidate trophoblast stem cell population while EVT methylome was most distinct. Moreover, the differential pattern of methylation in EVT associated with 41 genes involved in EMT and cancer metastasis. This confirms the crucial involvement of DNA methylation in trophoblast differentiation from CTB to EVT (Gamage et al., 2018). Furthermore, a study on the immortalized trophoblast cell line HTR8/SVneo demonstrates that DNA methylation is a critical regulator for the EMT in trophoblast. Inhibition of DNA methyl transferase (DNMT) activity using 5-aza-cytidine diminishes migration ability and induces the expression of the epithelial genes, E-cadherin and β-catenin. Deregulated DNMT activity is thus an activator of EMT, which provides strong evidence for the involvement of DNA methylation in the EMT process in trophoblast cells (Chen et al., 2013a). Additionally, TET2-mediated demethylation and subsequent expression of the EMT effector MMP9 are crucial for maintaining the normal physiology of human trophoblasts (Li et al., 2018).
Several studies revealed that an altered methylation profile of specific genes like MEST, FN1, ITGA5, IGFBP5, MASPIN has a direct effect on the trophoblast differentiation and invasion (Shi et al., 2015; Peng et al., 2016; Jia et al., 2017; Zhao et al., 2017). Among these, FN1 is one of the members of the EMT core signature gene set. Also, DNA methylation-dependent regulation of expression of EMT master regulator Snail and Slug transcription factors were demonstrated during trophoblast differentiation (Chen et al., 2013b).
A known modulator of DNA methylation in primary cytotrophoblasts is hypoxia (Yuen et al., 2013; Smith et al., 2017). Further, in humans, hypoxia induces CTB differentiation to EVT, which have invading ability (Wakeland et al., 2017). Combined together, it can be hypothesized that hypoxia induces differentiation to EVTs by modulating DNA methylation.
The DNA methylation pattern of cell adhesion molecules in the placenta differs between preeclamptic and healthy pregnancy (Anton et al., 2014). Also, higher amount of 5-methyl cytosine was found in preeclamptic term placenta compared to healthy term placenta (Wilson et al., 2019). These findings indicate that deregulated DNA methylation may lead to placenta-related pathologies like PE.
Histone modification-based regulation
Modified histones are one of the critical components in epigenetic regulation of gene expression and cellular phenotype. The role of histone acetylation, methylation and ribosylation in modulating trophoblast differentiation is known, however, the majority of the studies have been conducted in the murine model (Kohan-Ghadr et al., 2016). It is reported that the kinase enzyme, MAP3K4 may regulate EMT in mouse trophoblast stem cells. Knockout of MAP3K4 results in enhanced activity of histone deacetylase HDAC6 and reduction in H2BK5 acetylation level leading to decreased E-cadherin expression. Also, with knock down HDAC6 background, mouse TS cells demonstrate restoration of claudin and zona occludin level. Together, these point to the involvement of histone acetylation in the EMT process in trophoblast cells (Mobley et al., 2017; Mobley and Abell, 2017).
In humans, cytotrophoblast fusion during the formation of syncytiotrophoblast is regulated by histone acetylation on specific genes viz, OVOL1, TEAD4, TP63, which are associated with trophoblast differentiation. It was found that the acetylated histone H3 level is reduced during in vitro differentiation of cytotrophoblasts (Jaju Bhattad et al., 2020). Kwak et al. (2019) compared ChIP-seq data of cytotrophoblast and syncytiotrophoblast to analyze alteration of histone acetylation and methylation profile in trophoblast differentiation. H3K9 methylation increased while H3K9 acetylation decreased for ERG1, cFOS, cJUN transcription factors during syncytialization of human trophoblast (Kwak et al., 2019). This was accompanied by increased activity of histone deacetylase 1 enzyme (Kwak et al., 2019). Though modulation of histone modifications has been demonstrated to have a role in differentiation of cytotrophoblast to syncytiotrophoblast, its involvement in differentiation along the CTB to EVT path has not been investigated. A few studies have investigated the involvement of histone acetylation in modulating trophoblast invasion but the area is largely unexplored.
As gestational age advances, the expression of the EMT-related gene, Maspin increases in the CTBs thereby reducing their invasion ability (Dokras et al., 2002). In the second- and third-trimester placenta, the transcription activating histone marks, acetylated H3K9 and methylated H3K4, were higher in the Maspin promoter region. Furthermore, treatment of placental explants from all the trimesters with the HDAC inhibitor trichostatin A could significantly increase the expression of Maspin (Dokras et al., 2006).
In preeclamptic placenta, the histone deacetylase enzyme HDAC9 was found to be reduced. Further, modulating HDAC9 in vitro inhibited trophoblast invasion by upregulating TIMP3 (Xie et al., 2019). This suggests that HDAC9 influences EMT but its direct role has not been established. Interestingly, despite being a well-known epigenetic regulator, the role of histone modifications in differentiation of human trophoblast is poorly explored.
Non-coding RNA-mediated regulation
The role of micro RNAs (miRNAs) and long non-coding RNAs (lncRNAs) in regulating trophoblast function and placental development has received a lot of attention. In PE, the trophoblast invasion is compromised, and several studies have identified the association of different ncRNAs with trophoblast invasion and proliferation.
Long non-coding RNAs
lncRNA uc.187 is upregulated in PE as compared to age-matched normal term placenta. Downregulation of this lncRNA in vitro increases invasiveness of trophoblasts by increasing the expression of MMP2 and MMP9 while reducing TIMP1. Conversely, higher expression of lncRNA uc.187 induces trophoblast apoptosis (Cao et al., 2017). Similarly, another lncRNA, SPRY4-IT1 is upregulated in PE and negatively regulates trophoblast migration in vitro (Zou et al., 2013).
On the contrary, expression of many lncRNAs is decreased in PE namely, lncRNA-ATB, MEG3, SNHG5, SNHG12, SNHG14, TUG1 and MALAT1. Strong staining of lncRNA-ATB was demonstrated in trophoblasts of normal healthy placenta but was decreased in PE. This lncRNA is involved in trophoblast invasion and endovascular mimicry as evidenced by impaired tube formation in RNAi-mediated downregulation of endogenous lncRNA-ATB (Liu et al., 2017).
MEG-3 expression is positively correlated with EMT induction as evidenced by lower expression of E-cadherin and higher expression of N-cadherin and vimnetin in MEG-3 overexpressed trophoblast cells. Ectopic overexpression of MEG-3 also enhanced the ability of invasion and migration (Yu et al., 2018).
Phenotype analysis of HTR8/SVneo cells overexpressing lncRNA SNHG5 demonstrated an increase in cell proliferation, invasion, and migration, while inhibiting cell apoptosis. Bioinformatic analysis and reporter assay identified an interaction between SNHG5 and miR-26a-5p, which in turn controls expression of the EMT marker N-cadherin. The same authors also showed that in PE placentas, the expression pattern of N-cadherin and lncRNA SNHG5 are positively correlated. Taken together, this suggests that SNHG5 regulates trophoblast EMT through miR-26a-5p/N-cadherin axis (Yang et al., 2019).
Overexpression of the lncRNAs SNHG12 and SNHG14 in HTR8/SVneo cells enhanced cell proliferation and invasion. A reduction in the epithelial markers E-cadherin and β-catenin along with an increase in the mesenchymal protein vimentin was observed after overexpression of SNHG14 (Zhang and Zhang, 2021). In contrast, silencing of SNHG12 resulted in lower cell migration; reduced MMP2 and MMP9 expression; and reversal of EMT by increasing E-cadherin expression with concomitant reduction in vimentin expression (Zhou et al., 2020). Ectopic expression of TUG1 also promotes trophoblast invasion and tube formation by regulating MMP2 and VEGFA (Li et al., 2019). Inhibition of MALAT1 in trophoblast decreases cell migration (Wu et al., 2020b).
Many of the lncRNAs exert their biological effect by acting as molecular sponges of specific miRNAs, e.g. SNHG5-miR-26a-5p; SNHG14-miR-330-5p; TUG1-miR-29b and MALAT1-miR-206. Computational analysis suggested potential binding of SNHG14 with miR-330-5p which was confirmed by a Luciferase assay. This interaction explains the inverse relationship in expression of these two non-coding RNAs. In turn, miR-330-5p regulates the MMP2 and MMP9 levels in trophoblast cells, thereby modulating cell migration. Similarly, miR-206 showed negative correlation with MALAT1 expression in PE as well as in a trophoblast cell line overexpressing MALAT1. Luciferase reporter assay affirms the interaction between miR-206 and MALAT1. miR-206 controls IGF1 expression in trophoblast and hence, negatively regulates trophoblast migration and invasion (Wu et al., 2020b). These data indicate that altered lncRNA expression may be involved in the etiology of PE.
Micro RNAs
miRNAs are another class of well-studied non-coding RNA molecules that are implicated in several cellular functions including proliferation, apoptosis and invasion. In fact, miRNA profiling in primary trophoblast subpopulations reveals specific miRNA expression set in different trophoblast cell types (Gamage et al., 2018). Here, we have limited our review to include studies that show the role of miRNAs in regulating EMT- and EMT-associated gene regulation in trophoblasts.
miR-431 is overexpressed in PE and its expression is inversely related to its target ZEB1, which is one of the key transcription factors of EMT induction. Exposure of HTR8/SVneo cells to miR-431 mimic downregulates cell invasion and affects the ZEB1 expression level. Furthermore, it has been shown to induce E-cadherin expression and reduce vimentin expression. This suggests that miR-431 is capable of negatively regulating EMT in trophoblast (Yang and Meng, 2019).
Various miRNAs affect the signaling pathways which are known to induce EMT. miR-34a-5p and miR-142-3p are upregulated in PE and influence the TGF/Smad pathway. Their overexpression inhibited invasion and migration of trophoblasts by directly targeting TGFΒ1 and SMAD4 expression, respectively (Liu et al., 2019; Xue et al., 2019). IGF2BP1 and IGF2BP2 induce EMT by enhancing the expression or stability of transcription factors such as LEF1, SNAI2 or ZEB1 (Zirkel et al., 2013; Shen et al., 2021). Two miRNAs, namely miR-423-5p and miR-181a-5p, which target these IGF2BPs, are highly expressed in PE placenta (Guo et al., 2018; Wu et al., 2018). Also, overexpression of these miRNAs negatively regulates trophoblast invasion in vitro (Guo et al., 2018; Wu et al., 2018; Huang et al., 2019). Notch signaling is another pathway which controls cell invasion ability. miR-210, which is also increased in PE, reduces the expression of NOTCH1 and thus trophoblast invasion, migration, and tube formation ability are impaired (Wang et al., 2019).
Interestingly, miR-210 inhibits EMT in HTR8/SVneo cells by decreasing expression of the lncRNA MEG3 (Wang et al., 2021). This indicates that interaction of lncRNA and miRNA is bi-directional as not only lncRNAs can act as molecular sponges for miRNAs but upregulation of miRNAs can also influence levels of lncRNAs. It has also been found that the miRNome of a particular cell type is partially dependent on the DNA methylation status. The miRNAs under the influence of differentially methylated regions in trophoblast DNA are involved in pluripotency regulation (Gamage et al., 2018). Hence, the expression pattern of these miRNAs may act as a potential regulator of trophoblast functions in vivo.
Circular RNAs
Much attention is also focused on the functional consequences of the circular RNAs in diverse biological systems. Two discrete pieces of research have studied the role of different circRNAs in the regulation of trophoblast biology. Upregulation of circTNRC18 was found in the blood and placental tissue of PE patients. In the trophoblast cell line, circTNRC18 reduces cell migration and expression levels of mesenchymal-associated gene vimentin. It could act as a sponge of miR-762, which in turn promotes EMT by directly targeting the inhibitory transcription factor grainy head like 2 (GRHL2). Therefore, circTNRC18 may regulate trophoblast EMT through the miR-762/GRHL2 pathway (Shen et al., 2019).
On the other hand, circPAPPA is reduced in PE patients, both in tissue and plasma. Knock-down of circPAPPA inhibits trophoblast invasion and proliferation possibly since it acts as a sponge for miR-384, and thereby modulates the STAT3 activity (Zhou et al., 2019). The STAT pathway is known to be involved in EMT induction (Liu et al., 2014).
There is no evidence available to date on the functional relevance of eRNAs, and fragmented RNAs on the trophoblast biology and placenta-related disorders. Also, evidence is scarce on the chromatin architecture alteration during trophoblast differentiation and EMT. Therefore, it is a very promising yet unploughed field of trophoblast research.
Conclusion
Differentiation of cytotrophoblast into invasive extravillous trophoblast during placental development is associated with changes in the expression pattern of a specific set of genes. This change in expression follows the basic expression pattern of EMT markers. However, it is very likely that the pattern differs from other well-known biological contexts of EMT (Kalluri and Weinberg, 2009; Zeisberg and Neilson, 2009). Although only a limited number of EMT-associated gene expression studies have been conducted during early placental development, the unique pattern of gene expression corroborates this difference in EMT pattern (DaSilva-Arnold et al., 2015).
Since the epithelial-nature cytotrophoblasts transiently trans-differentiate to the mesenchymal-nature extravillous trophoblasts, it reinforces the hypothesis of epigenetic regulation of EMT in trophoblast cells. The expression profile alteration of EMT-related genes during trophoblast differentiation is found to be regulated by several epigenetic modifications at pre- and post-transcriptional level. This review identifies potential research questions that need to be addressed for adding a valuable understanding to the field of trophoblast biology. Regulation and biological functions of 5-hydroxymethyl cytosine and 6-methyl adenine in trophoblast are among them. Apart from DNA modification-based regulation, it would also be intriguing to study the effects of diverse histone modifications in controlling trophoblast biology. Since the epigenetic modifiers and readers have become lucrative targets of biologically active molecules in recent times, deeper research in this area may yield potential therapeutic agents for trophoblast-related pathologies.
Data availability
No new data were generated or analyzed in support of this research.
Acknowledgements
The authors would like to thank Mr Ramchandra B. Pokale, Chief Artist, Center for Community Medicine, AIIMS, New Delhi and Ms Lavi Bhati, MSc student, Department of Reproductive Biology, AIIMS, New Delhi for help in preparing the figures.
Authors’ roles
J.C. and S.G. conceived the idea and did literature search. All authors contributed to analysis of literature, critical discussions and the writing of the manuscript. All authors have approved the final version.
Funding
This research was funded by the Science and Engineering Research Board, Department of Science and Technology, Government of India (EMR/2017/005430/BHS) and Indian Council of Medical Research, Government of India (5/10/FR/65/2020-RBMCH). J.C. received fellowship from Indian Council of Medical Research.
Conflict of interest
The authors have no conflicts of interest to disclose.
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