RNA-ATB

As with many other lncRNAs, lncRNA-activated by TGFβ (RNA-ATB) was first discovered in cancer, upregulated in hepatocellular carcinoma, it promotes cell proliferation, migration and invasion [247]. It has been reported that in hepatocells RNA-ATB is expressed in response to TGFβ and in fibroblasts; it can create positive feedback regulation by promoting TGFβ paracrine release. Lnc RNA-ATB was found to be significantly downregulated in placental samples from women with preeclampsia. Moreover patients with EOPE showed an even stronger deregulation [248]. Given the proliferative, invasive and migratory features of trophoblasts and in particular extravillous trophoblast, Liu and coworkers (2017) investigated lncRNA-ATB function in trophoblast cell line HTR-8/SVneo, which is a standard in vitro model of extravillous trophoblast. While overexpression of lncRNA-ATB increased the proliferative, migratory and invasive potential of HTR-8/SVneo cells, the downregulation

caused a steep decrease in proliferation, migration and invasion, proving that this gene has an important role on the physiology of the extravillous trophoblast and that its deregulation could explain an aberrant implantation and endometrium invasion in preeclampsia, potentially being linked to incomplete spiral artery remodeling. Whether RNA-ATB regulates trophoblast function through the interaction with members of the miR200 family is ye<sup>t</sup> to be determined. However, increased miR200 has been found to affect the development of endometrium receptivity, negatively impacting implantation [249]. In labor, miR200 is upregulated in the human uterus and has been associated with pre-term labor in murine studies [250]. It seems likely that an interaction between RNA-ATB and miR200 is required for correct placental development, gestation and delivery.

PVT1, TUG1 and DIAPH2-AS1: Regulating Gene Expression through Recruitment of Chromatin Remodeling Complexes

lncRNAs work through different mechanisms, depending on the specific lncRNA, the cell type, the downstream targets [231]. In the past few years a few lncRNAs have been identified in preeclampsia as potential modulators, among which PVT1, TUG1 and DIAPH2-AS1 adopt the same mechanism of action. lncRNA TUG1 is downregulated in preeclamptic placentas. Interference of TUG1 in trophoblast cell lines (JEG3 and HTR-8/SVneo) negatively affected cell proliferation and growth, migration and invasion, network formation, while it increased apoptosis [31]. Transcriptome analysis by RNA-sequencing of HTR-/SVneo cells in which TUG1 was downregulated showed a prevalence of affected genes involved in cell growth, migration and apoptosis. Xu and coworkers (2017) identified RND3 as main downstream factor involved in the phenotypic effects of TUG1 downregulation. RND3 mRNA and protein levels were strongly upregulated in response to TUG1 interference in vitro and RND3 mRNA was upregulated in preeclamptic placenta. RND3 is also known as RhoE, a GTPase that acts as a tumor suppressor, negatively regulating proliferation, migration and invasion [251]. In vitro experiments beautifully elucidated the mechanism by which TUG1 modulates RND3 expression—TUG1 directly interacts with the histone modification factor Enhancer of Zeste Homolog 2 (EZH2) and recruits it to the RND3 promoter, where EZH2 drives the silencing of RND3 by tri-methylating H3K27, resulting in strong RND3 downregulation [31]. A year later, Xu and coworkers [252] identified another lncRNA PVT1, strongly downregulated in preeclamptic placenta, whose downregulation negatively affects proliferation and increases apoptosis of trophoblast cell lines. PVT1 was found to recruit EZH2 to the promoter of the transcription factor ANGPTL4, driving its repression by increase in repressive chromatin markers: this could partially explain the phenotypic effects of PVT1 deregulation. Feng and coworkers [253] uncovered a complicated regulatory network behind PAX3 deregulation in preeclampsia which is linked with decreased proliferation, invasion and migration of trophoblast cells [254]. PAX3 is a transcription factor downregulated in preeclamptic placentas and this correlates with DNA hypermethylation of the promoter region [171,254]. In this study, Feng and coworkers (2019) found that in preeclamptic placentas lncRNA DIAPH2-AS1 is upregulated along with the transcription factor HOXD8. In vitro experiments in HTR-8/SVneo cells clarified the regulatory network: under hypoxia the transcription factor HOXD8 is upregulated and induces expression of the lncRNA DIAPH2-AS1. DIAPH2-AS1 recruits lysine-specific demethylase 1 (LSD1) to the promoter of PAX3 where it alters the chromatin modification state, decreasing methylation of Histone H3. LSD1 can also modify DNA methyl-transferase 1 (DNMT1), stabilizing it. ChIP experiments showed enrichment of LSD1 and DNMT1 at the PAX3 promoter, which correlated with increased DNA methylation and mRNA repression. Interference of DIAPH2-AS1 was enough to reverse the phenotype and increase PAX3 levels [253]. These studies underscore that different epigenetic mechanisms regulate gene expression. It is possible that certain mechanisms are favored in different cell types and future studies will help identify the conserved regulatory networks that plays a role in the etiology of preeclampsia.

### 3.2.2. micro RNA and Preeclampsia
