The Long Non-Coding RNA Gene AC027288.3 Plays a Role in Human Endometrial Stromal Fibroblast Decidualization

During the secretory phase of the menstrual cycle, endometrial fibroblast cells begin to change into large epithelial-like cells called decidual cells in a process called decidualization. This differentiation continues more broadly in the endometrium and forms the decidual tissue during early pregnancy. The cells undergoing decidualization as well as the resulting decidual cells, support successful implantation and placentation during early pregnancy. This study was carried out to identify new potentially important long non-coding RNA (lncRNA) genes that may play a role in human endometrial stromal fibroblast cells (hESF) undergoing decidualization in vitro, and several were found. The expression of nine was further characterized. One of these, AC027288.3, showed a dramatic increase in the expression of hESF cells undergoing decidualization. When AC027288.3 expression was targeted, the ability of the cells to undergo decidualization as determined by the expression of decidualization marker protein-coding genes was significantly altered. The most affected markers of decidualization whose expression was significantly reduced were FOXO1, FZD4, and INHBA. Therefore, AC027288.3 may be a major upstream regulator of the WNT-FOXO1 pathway and activin-SMAD3 pathways previously shown as critical for hESF decidualization. Finally, we explored possible regulators of AC027288.3 expression during human ESF decidualization. Expression was regulated by cAMP and progesterone. Our results suggest that AC027288.3 plays a role in hESF decidualization and identifies several other lncRNA genes that may also play a role.


Introduction
The transdifferentiation or decidualization of endometrial fibroblasts into epitheloidlike decidual cells is critical in establishing pregnancy in many species.It begins during the secretory phase of the uterine cycle in humans and continues during early pregnancy, ultimately forming the decidual tissue [1].The decidual tissue supports the development of the conceptus during early pregnancy, including the development of the placenta, and thus is a primary driver of pregnancy health success [2].Initially, perivascular fibroblast cells begin to undergo decidualization as progesterone levels rise during the secretory phase of the menstrual cycle.This transdifferentiation then spreads more broadly in the endometrium during early pregnancy.Human endometrial stromal fibroblast (hESF) decidualization is often studied in vitro using cells isolated from the endometrium of non-pregnant or term placenta of pregnant patients [3].
Long non-coding RNAs (lncRNAs) commonly refer to RNA transcripts that are greater than 200 nucleotides that do not encode proteins that are produced by RNA pol I, II, or III [4,5].However, it has more recently been suggested that it would be more useful if lncRNAs were defined as at least 500 nt long and generated mainly by RNA pol II [4].Like a KAPA Stranded mRNA-Seq Kit as suggested by the manufacturer (KAPA Biosystems, Wilmington, MA, USA).Libraries were sequenced as single-end 75 base-pair reads on an Illumina NextSeq500 following the manufacturer's protocols (Illumina, San Diego, CA, USA).The sequencing files have been submitted to NCBI (Project PRJNA1068120).

RNA-Seq Analysis
The Poly A+ RNA-seq reads were mapped to the Genome Reference Consortium Human Build 38 (GRCh38.p14,Release 45) downloaded from Gencode [6], using Spliced Transcripts Alignment to a Reference (STAR) [28].Transcript read counts were then determined using the software package HTSeq (version 2.0.5)[29].Differential gene expression between vehicle-and EPC-treated cells isolated from 3 patients (N = 3) was determined using the Bioconductor package edgeR [30] as patient-matched samples as recommended in the user's guide [31].The false discovery rate (FDR) of differentially expressed genes (DEGs) was estimated using the Benjamini and Hochberg option, and those with an absolute fold-change (|FC|) ≥ 2 and an FDR < 0.01 were considered to be significantly differentially expressed between the vehicle and EPC-treated groups.To compare our Poly A + RNA-seq data from hESF cells isolated from pregnant patients to those isolated from non-pregnant patients, we also reanalyzed the raw data from previous studies [32,33] (PR-JNA368634) in a similar fashion.The data included hESF cells from non-pregnant patients with non-targeting, PGR-targeting, or FOXO1-targeting siRNA prior to treatment with vehicle or EPC for 72 h.Where possible, all RNA-seq data were annotated with HNGC gene symbols from an Ensembl GeneID using Ensembl BioMart [7].When this was impossible, the Human ENSEMBL Gene ID converter available at www.biotools.fr(accessed 10 January 2024) was used.Venn diagrams and Volcano plots of -Log 10 (FDR) versus Log 2 (FC) were prepared using GraphBio [34].Bigwig files were produced using the BamCoverage function of Deeptools2 [35] using bins per million normalization (bin size = 20) and displayed on the UCSC Genome Browser [36].

Reverse Transcription-Quantitative PCR (RT-qPCR)
Total RNA isolation, cDNA preparation, primer validation, and real-time qPCR were carried out as previously described [27].The qPCR data were analyzed using the ∆∆Ct method (31) using RPLP0 (36B4) as the housekeeping gene as previously used for human endometrial stromal cell decidualization experiments [27,37].The validated primer sequences are in Table 1.

Cell Fractionation
After trypsinization, the cells were centrifuged at 100× g for 5 min at 4 • C to obtain a cell pellet.The cells were washed and re-centrifuged thrice with Dulbecco's PBS (D-PBS).The final pellets of cells were gently resuspended in 0.1 mL hypotonic solution (10 mM Tris-HCl, 15 mM KCl, 1 mM EDTA, 0.5 mM dithiothreitol, pH 7.4) and incubated on ice for 3 min.Then, 100µL of lysis buffer (hypotonic solution containing 0.1% NP-40) was added to the cell suspension, and the mixture was gently mixed and placed on ice for 4 min.The entire lysate was overlaid on top of 0.5 mL of 24% sucrose solution (10 mM Tris-HCl, 15 mM KCl, 24% sucrose, 0.15 mM spermine, 0.5 M spermidine, 0.5 mM dithiothreitol, pH 7.5) and subjected to centrifugation at 6000× g for 10 min at 4 • C. The resulting upper 0.3 mL containing the cytoplasmic fraction was carefully transferred to a new tube, and the nuclear pellets were retained.The nuclear pellets were washed with D-PBS containing 0.1 mM EDTA, followed by centrifugation at 1000× g for 10 min at 4 • C. The nuclear pellet was resuspended in 0.3 mL of nuclear lysis buffer (20 mM HEPES, 0.5 M NaCl, 0.5 mM dithiothreitol and incubated on ice for 30 min.The nuclear and cytoplasmic fractions were used to prepare total RNA as above or subjected to Western blot analysis.

FOXO1 ChIP-PCR
Chromatin preparation, immunoprecipitation, RT-qPCR, and percent input calculations were carried out using methods previously described in detail [49] using control rabbit IgG (Cell Signaling Technology Cat# 2729, RRID:AB_1031062) and rabbit anti-human FOXO1 (Cell Signaling Technology Cat# 2880, RRID:AB_2106495).For ChIP qPCR primer validation, optimal annealing temperatures and PCR efficiencies were determined, and melt-curve analyses after qPCR verified the presence of single amplicons without primer dimers using diluted input samples.The sequence of the ChIP qPCR primers is in Table 2.

Statistical analysis
Graphs and statistical analysis of the RT-qPCR results for changes in RNA expression were prepared using Sigmaplot software version 15.X (SPSS Inc., Chicago, IL, USA) as previously described [27].For ChIP-PCR results, paired t-tests were used.Statistical significance in the results was noted at the p < 0.05 (a), p < 0.01 (b), p < 0.005 (c), or p < 0.001 (d) levels.

RNA-Seq Analysis
In order to find the identity of new lncRNA genes that may play a role in human endometrial decidualization, we carried out RNA-seq analysis of the transcriptome changes between EPC-(undergoing decidualization) and vehicle-treated (not undergoing decidualization) hESF cells isolated from pregnant patients.We then compared it with our reanalysis of previously published data obtained from hESF cells from non-pregnant patients.The complete listing of data obtained for protein-coding genes for hESF cells from pregnant and non-pregnant patients can be found in Supplemental Tables S1 and S2, respectively.A volcano plot of RNA-seq analysis of the differential mRNA expression between EPC-and vehicle-treated hESF cells from pregnant patients is shown in Figure 1A.The number of protein-coding genes that were significantly upregulated and downregulated during the decidualization of the pregnant hESF cells was 560 and 476, respectively.A comparison between RNA-seq analysis results between hESF cells from pregnant and non-pregnant patients is shown in the Venn diagram in Figure 1B.The number of protein-coding genes significantly upregulated and downregulated during decidualization in common between pregnant and non-pregnant hESF cells during decidualization was 551 and 466, respectively.
The complete listing of the protein-coding genes in the different categories of the Venn diagram is in Supplemental Table S3.The results show that many of the previously published protein-coding genes known to be differentially expressed during hESF decidualization were shared between the two RNA-seq datasets.For example, these genes include those whose expression increases (e.g., FOXO1, IGFBP1, PRL, TIMP3 [32], ATOH8, LEFTY2, and FZD4 [27]) and decreases (e.g., IGFBP5 [51]) during hESF decidualization.The complete listing of data obtained for LncRNA genes for hESF cells from pregnant and non-pregnant patients can be found in Supplemental Tables S4 and S5, respectively.A volcano plot of RNA-seq analysis of the differential lncRNA expression between EPC-(undergoing decidualization) and vehicle-treated (not undergoing decidualization) hESF cells from pregnant patients is shown in Figure 2A.The number of lncRNA genes that were significantly upregulated and downregulated during decidualization was 161 and 83, respectively.A comparison between RNA-seq analysis results between hESF cells from The complete listing of data obtained for LncRNA genes for hESF cells from pregnant and non-pregnant patients can be found in Supplemental Tables S4 and S5, respectively.A volcano plot of RNA-seq analysis of the differential lncRNA expression between EPC-(undergoing decidualization) and vehicle-treated (not undergoing decidualization) hESF Cells 2024, 13, 778 7 of 22 cells from pregnant patients is shown in Figure 2A.The number of lncRNA genes that were significantly upregulated and downregulated during decidualization was 161 and 83, respectively.A comparison between RNA-seq analysis results between hESF cells from pregnant and non-pregnant patients is shown in the Venn diagram in Figure 2B.The number of lncRNA genes significantly upregulated and downregulated in common between pregnant and non-pregnant hESF cells during decidualization was 157 and 80, respectively.The complete list of the LncRNA genes in the Venn diagram is in Supplemental Table S6.The results show that a couple of the previously published lncRNA genes known to be differentially expressed during hESF decidualization as common upregulated lncRNA genes, including HAND2-AS1 [18] and LNCSAMD11-1:1 (ENSG00000230699, ENST00000448179) [19].
Cells 2024, 13, x FOR PEER REVIEW 8 of 24 pregnant and non-pregnant patients is shown in the Venn diagram in Figure 2B.The number of lncRNA genes significantly upregulated and downregulated in common between pregnant and non-pregnant hESF cells during decidualization was 157 and 80, respectively.The complete list of the LncRNA genes in the Venn diagram is in Supplemental Table S6.The results show that a couple of the previously published lncRNA genes known to be differentially expressed during hESF decidualization as common upregulated lncRNA genes, including HAND2-AS1 [18] and LNCSAMD11-1:1 (ENSG00000230699, ENST00000448179) [19].

Verifying Differential Expression of 9 Novel LncRNA during hESF Decidualization
From the results, we focused further on nine different random lncRNAs that were similarly differentially expressed in both pregnant and non-pregnant hESF cells undergoing decidualization.For all 9, the Ensembl GeneID and all possible TranscriptIDs and the primary transcript expressed as determined by looking at the RNA-seq data on the UCSC genome browser (Supplemental Figures S1-S9) are summarized in Supplemental Table S7.To verify the differential expression of these lncRNAs, we carried out RT-qPCR on cells incubated with vehicle or EPC for 1, 2, or 3 days.For lncRNAs AC027288.3,AC023154.1,AC108861.1,BASP1-AS1, LINC02432, LINC02593, and LINC02600 expression was significantly greater in EPC-treated cells compared to vehicle-treated cells on each day examined (Figure 3A-G).The expression of LINC01605 was significantly greater in vehicle-treated cells compared to EPC-treated cells on each day examined (Figure 3H).Finally, AL121578.3RNA expression was only detected in vehicle-treated cells at 1-3 days (Figure 3I).

Verifying Differential Expression of 9 Novel LncRNA during hESF Decidualization
From the results, we focused further on nine different random lncRNAs that were similarly differentially expressed in both pregnant and non-pregnant hESF cells undergoing decidualization.For all 9, the Ensembl GeneID and all possible TranscriptIDs and the primary transcript expressed as determined by looking at the RNA-seq data on the UCSC genome browser (Supplemental Figures S1-S9) are summarized in Supplemental Table S7.To verify the differential expression of these lncRNAs, we carried out RT-qPCR on cells incubated with vehicle or EPC for 1, 2, or 3 days.For lncRNAs AC027288.3,AC023154.1,AC108861.1,BASP1-AS1, LINC02432, LINC02593, and LINC02600 expression was significantly greater in EPC-treated cells compared to vehicle-treated cells on each day examined (Figure 3A-G).The expression of LINC01605 was significantly greater in vehicle-treated cells compared to EPC-treated cells on each day examined (Figure 3H).Finally, AL121578.3RNA expression was only detected in vehicle-treated cells at 1-3 days (Figure 3I).

Localization of LncRNA Expression
We validated the fractionation method to isolate nuclear and cytoplasmic lncRNA in cells treated with EPC for 48 h.As shown in Figure 4A, the cytoplasmic GAPDH and nuclear SNRNP70 protein markers were only detected in the nuclear and cytoplasmic

Localization of LncRNA Expression
We validated the fractionation method to isolate nuclear and cytoplasmic lncRNA in cells treated with EPC for 48 h.As shown in Figure 4A, the cytoplasmic GAPDH and nuclear SNRNP70 protein markers were only detected in the nuclear and cytoplasmic fractions.As shown in Figure 4B, the known cytoplasmic DANCR and nuclear MALAT plus XIST lncRNA markers [52][53][54] were predominantly detected in the cytoplasmic and nuclear fractions, respectively.Additionally, shown in Figure 4B, AC027288.3and LINC02432 were predominantly cytoplasmic, while LINC02593 and BASP1-AS1 were mainly nuclear.Finally, significant amounts of AC023154.1,LINC02600, and LINC01605 were seen in both the nuclear and cytoplasmic fractions.

Steroid and cAMP Control of LncRNA Expression
Since both the steroids or cAMP analog components of the EPC treatment to induce decidualization in this study could be upstream regulators of lncRNA expression, we determined the effects of vehicle, steroids (estradiol plus progesterone), cAMP analog, and EPC on the expression of the nine lncRNA genes.For AC027288.3, both steroids (p < 0.01) and cAMP (p < 0.001) caused significant 3.4-and 9.5-fold increases in expression (Figure 5A) compared to vehicle-treated cells, respectively.Incubation of cells with cAMP analog but not steroids caused a significant increase in the expression of AC108861.3,LINC02432, AC023154.1,BASP1-AS1, and LINC02600 compared to vehicle-treated cells (Figure 5B-F).However, in the presence of cAMP analog, steroids caused a significant increase in the expression of all five lncRNAs., indicating a cAMP-dependent effect of the steroids.The expression of LINC02593 significantly (p < 0.001) increased in responses to cAMP analog compared to vehicle-treated cells (Figure 5G), while steroids had no effect.Both steroids and cAMP caused significant decreases in LINC01605 and AL121578.3expression compared to vehicle-treated cells, with the effect of the cAMP analog being much more pronounced for LINC01605 (Figure 5H-I).

Steroid and cAMP Control of LncRNA Expression
Since both the steroids or cAMP analog components of the EPC treatment to induce decidualization in this study could be upstream regulators of lncRNA expression, we determined the effects of vehicle, steroids (estradiol plus progesterone), cAMP analog, and EPC on the expression of the nine lncRNA genes.For AC027288.3, both steroids (p < 0.01) and cAMP (p < 0.001) caused significant 3.4-and 9.5-fold increases in expression (Figure 5A) compared to vehicle-treated cells, respectively.Incubation of cells with cAMP analog but not steroids caused a significant increase in the expression of AC108861.3,LINC02432, AC023154.1,BASP1-AS1, and LINC02600 compared to vehicle-treated cells (Figure 5B-F).However, in the presence of cAMP analog, steroids caused a significant increase in the expression of all five lncRNAs., indicating a cAMP-dependent effect of the steroids.The expression of LINC02593 significantly (p < 0.001) increased in responses to cAMP analog compared to vehicle-treated cells (Figure 5G), while steroids had no effect.Both steroids and cAMP caused significant decreases in LINC01605 and AL121578.3expression compared to vehicle-treated cells, with the effect of the cAMP analog being much more pronounced for LINC01605 (Figure 5H-I).

PGR and FOXO1 as Regulators of LncRNA Expression
PGR and FOXO1 are critical transcription factors that play vital roles in hESF decidualization [37,55].Since li le is known about what controls the differential expression of the lncRNA genes, we reanalyzed previously published RNA-seq data where cells were incubated with non-targeting (siNT) and transcription factor targeting (siPGR and siFOXO1) siRNAs for two days, followed by EPC for three days [32,33].The result of the analysis for targeting PGR expression (Supplemental Table S8) is summarized in the volcano plot in Figure 6A.Differential expression was compared to the consensus differentially expressed genes in pregnant relative and non-pregnant hESF cells undergoing decidualization (Supplemental Table S6).As shown in the Venn diagram in Figure 6B (see Supplemental Table S9 for gene lists), the expression of 22 genes that increased during decidualization was downregulated with siPGR treatment.On the other hand, the expression of 20 genes that decreased during decidualization was downregulated with siPGR treatment.Six of these 42 PGR-regulated genes include AC027288.3,AC108861.1,BASP1-AS1, LINC02593, AL121578.3and LINC01605.The result of the analysis for targe ing FOXO1 expression (Supplemental Table S10) is summarized in the volcano plot in Figure 7A.Differential expression in response to targe ing FOXO1 expression was compared to the consensus differentially expressed genes in pregnant and non-pregnant hESF cells undergoing decidualization (Supplemental Table S6).As shown in the Venn diagram in Figure 7B (see Supplemental Table S11 for gene lists), the expression of 3 genes that increased during decidualization was downregulated with siFOXO1 treatment.On the other hand, the expression of none of the genes that decreased during decidualization was upregulated with siFOXO1 treatment.Interestingly, one of these FOXO1-regulated genes included AC027288.3.Therefore, the AC027288.3expression appears to be regulated by both PGR and FOXO1 during EPC-induced decidualization of hESF cells in vitro.

PGR and FOXO1 as Regulators of LncRNA Expression
PGR and FOXO1 are critical transcription factors that play vital roles in hESF decidualization [37,55].Since little is known about what controls the differential expression of the lncRNA genes, we reanalyzed previously published RNA-seq data where cells were incubated with non-targeting (siNT) and transcription factor targeting (siPGR and siFOXO1) siRNAs for two days, followed by EPC for three days [32,33].The result of the analysis for targeting PGR expression (Supplemental Table S8) is summarized in the volcano plot in Figure 6A.Differential expression was compared to the consensus differentially expressed genes in pregnant relative and non-pregnant hESF cells undergoing decidualization (Supplemental Table S6).As shown in the Venn diagram in Figure 6B (see Supplemental Table S9 for gene lists), the expression of 22 genes that increased during decidualization was downregulated with siPGR treatment.On the other hand, the expression of 20 genes that decreased during decidualization was downregulated with siPGR treatment.Six of these 42 PGR-regulated genes include AC027288.3,AC108861.1,BASP1-AS1, LINC02593, AL121578.3and LINC01605.The result of the analysis for targetting FOXO1 expression (Supplemental Table S10) is summarized in the volcano plot in Figure 7A.Differential expression in response to targetting FOXO1 expression was compared to the consensus differentially expressed genes in pregnant and non-pregnant hESF cells undergoing decidualization (Supplemental Table S6).As shown in the Venn diagram in Figure 7B (see Supplemental Table S11 for gene lists), the expression of 3 genes that increased during decidualization was downregulated with siFOXO1 treatment.On the other hand, the expression of none of the genes that decreased during decidualization was upregulated with siFOXO1 treatment.Interestingly, one of these FOXO1-regulated genes included AC027288.3.Therefore, the AC027288.3expression appears to be regulated by both PGR and FOXO1 during EPC-induced decidualization of hESF cells in vitro.S6).Abbrev: P/NP-UP and -DO, common lncRNA genes between pregnant and non-pregnant hESF cells that increase and decrease during decidualization; NPsiPGR-UP and -DO, lncRNA's upregulated and downregulated in response to siPGR, respectively.S6).Abbrev: P/NP-UP and -DO, common lncRNA genes between pregnant and non-pregnant hESF cells that increase and decrease during decidualization; NPsiPGR-UP and -DO, lncRNA's upregulated and downregulated in response to siPGR, respectively.S6).Abbrev: P/NP-UP and -DO, common lncRNA genes between pregnant and non-pregnant hESF cells that increase and decrease during decidualization; NPsiFOXO1-UP and -DO, lncRNAs upregulated and downregulated in response to siFOXO1, respectively.

Potential Roles of Specific Transcription Factors on AC027288.3 Expression
Several transcription factors such as FOSL2, FOXO1, GATA2, NR2F2, NCOA2, PGR, and ZBTB16 are known to play critical roles in non-pregnant hESF cells undergoing decidualization based on several cistrome studies [32,33,[45][46][47][48].As shown in Figure 8A, there appears to be a cluster of binding sites for FOSL2, FOXO1, GATA2, NR2F2, and PGR in the first intron near the first exon of AC027288.3(ENST00000550268.2) during hESF decidualization.A further downstream cluster within the same intron includes binding regions for the same transcription factors (Supplemental Figure S10).Focusing on the first  S9 and S10.(B) Venn diagram comparing the RNA-seq results of the results in panel A to the common lncRNA's differentially expressed in NP plus pregnant (P) hESF cells undergoing decidualization (see Figure 2B and Supplemental Table S6).Abbrev: P/NP-UP and -DO, common lncRNA genes between pregnant and non-pregnant hESF cells that increase and decrease during decidualization; NPsiFOXO1-UP and -DO, lncRNAs upregulated and downregulated in response to siFOXO1, respectively.

Potential Roles of Specific Transcription Factors on AC027288.3 Expression
Several transcription factors such as FOSL2, FOXO1, GATA2, NR2F2, NCOA2, PGR, and ZBTB16 are known to play critical roles in non-pregnant hESF cells undergoing decidualization based on several cistrome studies [32,33,[45][46][47][48].As shown in Figure 8A, there appears to be a cluster of binding sites for FOSL2, FOXO1, GATA2, NR2F2, and PGR in the first intron near the first exon of AC027288.3(ENST00000550268.2) during hESF decidualization.A further downstream cluster within the same intron includes binding regions for the same transcription factors (Supplemental Figure S10).Focusing on the first cluster, we designed ChIP PCR primers for the two putative FOXO1 binding regions (FBR1 and FBR2) and a control (CON) region ~3 kb downstream from FBR2 (Figure 8B) to confirm FOXO1 binding in our hESF cells isolated from pregnant patients.As a positive control, we confirmed the enrichment of the binding of FOXO1 in our cells to a well-known region near the IGFBP1 gene (Figure 8C), as previously published [32].We were also able to confirm the enrichment of binding of FOXO1 in our cells to FBR1 and FBR2 of the first intronic region of AC027288.3,but not at a control region ~3 kb upstream of FBR2 (Figure 8D).Above, we showed that the combination of estradiol plus progesterone causes an increase in AC027288.3expression (Figure 5A).Given that there are PGR binding sites in the first intron of AC027288.3(Figure 9A), we hypothesized that the effect of steroidinduced expression was due to the actions of progesterone and not estradiol.To confirm this, we incubated the cells with the steroids individually and found that induction of AC027288.3expression is mediated by progesterone alone (Figure 8E).

Discussion
In the present study, we conducted a polyA+ RNA-Seq analysis of hESF cells from pregnant patients undergoing EP-induced decidualization in vitro.We compared this analysis to previously published data from hESF cells from non-pregnant patients under similar conditions.We found 158 and 82 common lncRNA genes whose expression is upregulated and downregulated during EPC-induced decidualization between the pregnant and non-pregnant hESF cell RNA-seq data.These genes included some, but not all, previously identified genes shown by other studies to be differentially expressed and play a function during hESF decidualization.Previously, we found that HAND2 plays a crucial role in hESF decidualization n [59], which was subsequently confirmed [60].The data of our current study confirms a previous finding that the expression of HAND2-AS1 is upregulated during hESF decidualization [18].This increased expression has previously been shown to play a role in regulating HAND2 expression [18].A second lncRNA identified in this study that increased during hESF decidualization was DANCR.To our knowledge, the function of DANCR in hESF decidualization has not been reported.However, the role of DANCR as a cytoplasmic lncRNA that "sponges" particular microRNAs or interacts with various proteins has been described in other cell types [61,62].Finally, TUNAR is known to decrease during hESF decidualization in vitro, and overexpressing it can negatively impact decidualization [21].Interestingly, TUNAR expression was not detected as common differentially expressed in this study.Notably, one caveat of our analysis is it would not include any non-polyadenylated lncRNAs since the RNA-seq libraries were constructed with poly A+ RNA.
AC027288.3 lncRNA appears to be an upstream regulator of FOXO1 expression during hESF decidualization.FOXO1 is a transcription factor whose expression is commonly used as a decidualization marker and is also known to play a vital role in decidualization [32,37,55,[63][64][65].FOXO1 controls the expression of several downstream target genes, such as IFBP1, PRL, and TIMP3.Targetting AC027288.3expression in hESF cells in our current study resulted in a marked downregulation of FOXO1 expression during decidualization.A BMP2-WNT/FZD-CTNBB1 pathway regulates FOXO1 expression during hESF decidualization, and BMP2 also controls FZD4 expression [56].In our current study, targeting AC027288.3expression during hESF decidualization did not influence BMP2 expression but significantly increased WNT4 and decreased FZD4 expression.Therefore, it may play a role in controlling FZD4 expression, which may be why FOXO1 expression decreased by targeting the expression of AC027288.3.Even though WNT4 expression is increased, the decreased expression of FZD4 with AC027288.3knockdown may lead to a resulting decrease in the BMP-WNT/FZD-CTNBB1 pathway signaling, which may have led to the decreased FOXO1 expression.Additional studies are needed to evaluate this.
Besides BMPs, activins are another set of TGFβ receptor ligands known to play a role in hESF decidualization.Human ESF cells show increased INHBA expression at the onset of decidualization in vivo and in vitro [66][67][68].Treatment of hESF cells with activins enhances the decidualization of hESF cells in vitro [57].Surprisingly, INHBA expression was dramatically reduced during decidualization when AC027288.3 expression was targeted in this study.FST is an inhibitor of activin signaling by binding to the ligand and preventing the binding and activation of their receptors [58].Our results indicated FST expression increases during hESF decidualization, and targetting AC027288.3expression prevented increases in FST expression.Strong evidence suggests that activin-SMAD2/3 signaling is crucial for normal decidualization in mice [69,70].This is also possible for human decidualization [71].The role of AC027288.3 in regulating the activin-SMAD2/3 signaling hESF decidualization needs to be investigated further.
Two major regulators of hESF decidualization are PGR and cAMP [71].Further, although progesterone alone can induce decidualization, part of the action of progesterone is to increase cAMP levels in the cells [72].Thus, PGR and increased levels of cAMP can individually or synergistically regulate decidualization and play a role in its maintenance.Of the nine lncRNA genes whose expression was examined more closely in this study, steroids alone only slightly influenced the expression of one lncRNA (AC027288.3),which was found to be dependent on the action of progesterone alone.On the other hand, the addition of cAMP influenced the expression of all nine.Of these, five showed a cAMP analog-dependent effect of steroids, the most pronounced being for the expression of AC027288.3.Further, reanalysis of RNA-seq data revealed that 42 lncRNA genes are differentially expressed during hESF decidualization and likely controlled directly or indirectly by PGR.These results show that, similarly to protein-coding genes, the expression of differentially expressed lncRNA genes during hESF decidualization can involve the actions of both cAMP and progesterone-PGR signaling.
FOXO1 is a significant regulator of hESF decidualization [32,33].In our current study, we found the expression of three lncRNA genes differentially expressed during hESF decidualization, which are likely controlled directly or indirectly by FOXO1.Indeed, FOXO1 ChIP-seq data from hESF cells undergoing decidualization revealed the binding of FOXO1 to regions of the first intron of AC027288.3.This FOXO1 binding was confirmed for some of these sites using ChIP-PC.These results seem confusing since the results of this study suggest that FOXO1 and AC027288.3may be regulating each other's expression in hESF cells undergoing decidualization.We speculate that this positive feedback might be involved in the commitment towards the decidual cell phenotype.Finally, previously published ChIPseq data show that other transcription factors may also be involved in directly regulating AC027288.3expression during decidualization, including PGR, FOSL2, GATA2, and NR2F2.Each of these is also a key regulator of hESF decidualization [32,33,[45][46][47][48].Therefore, the differential expression of at least some of the lncRNA genes during hESF decidualization is likely directly orchestrated by the same transcription factors that control protein-coding genes during hESF decidualization.
Besides AC027288.3, the precise function of the other eight lncRNAs of Table 1 in decidualization remains to be determined.Besides differential expression in other cells/tissues, the biological functions for AC023154.1,AC108861.1,AL121578.3,and LINC02593 lncRNAs do not currently appear in the literature.However, an interesting study reveals that during neuronal differentiation, BASP1-AS1 lncRNA regulates the expression of its adjacent protein-coding gene BASP1 [73].Together, BASP1-AS1 RNA and BASP1 protein form a molecular complex that also includes the ubiquitous bHLH protein TCF12 [73].The ability of BASP1-AS1 RNA to form a complex with TCF12 is interesting as several bHLH transcription factors play a key role in human endometrial decidualization [27,59,[74][75][76][77][78][79][80][81][82][83].Targetting LINC01605 expression inhibits autophagy and induces apoptosis in Tenon's capsule fibroblasts [84].In addition, LINC01605 RNA may promote aerobic glycolysis, proliferation, migration, and invasion of cancer cells [85,86].However, the functional role for decreasing expression of LINC01605 during hESF decidualization noted in our study remains to be determined.LINC02432 lncRNA may regulate HK2 and ferroptosis in cancer [87], which may contribute to the evidence that decidualization involves metabolic reprogramming and an increased reliance on aerobic glycolysis [83,88].Finally, it appears a bi-directional promotor possibly controls LINC02600 expression as its 5 ′ -end is very close to the 5 ′ -end of the protein-coding gene ADRA2C.The increased expression and function of ADRA2C in decidualization have been studied [89], but the role of LINC02600 lncRNA in regulating its expression during decidualization remains to be determined.
Collectively, our results provide the identity of several new lncRNA genes that are differentially expressed and may play a role in hESF decidualization.The results of this study further support the hypothesis that one lncRNA, AC027288.3,may play a role in hESF decidualization by regulating FZD4 and INHBA expression.In addition, our results suggest AC027288.3expression during hESF decidualization could be regulated using several transcription factors, including PGR, FOXO1, GATA2, NR2F2, and FOSL2.Additional experiments are warranted to determine the precise function and control of the expression of AC027288.3,and other lncRNAs demonstrated to be differentially expressed during hESF decidualization in this study.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/cells13090778/s1,Supplemental Table Legends.Table S1: RNA-seq results of protein-coding genes in hESF cells isolated from pregnant patients treated with VEH (not undergoing decidualization) or EPC (undergoing decidualization).FDR, false-discovery rate; Log2FC, log (base 2) Fold-Change (EPC vs. VEH).Cells were used from three independent patients (N = 3).Table S2: RNA-seq results of protein-coding genes in hESF cells isolated from non-pregnant patients treated with VEH (not undergoing decidualization) or EPC (undergoing decidualization).FDR, false-discovery rate; Log2FC, log base 2 Fold-Change (EPC vs. VEH).Cells were used from three independent patients (N = 3).Table S3: Combined RNA-seq results of protein-coding genes from hESF cells isolated from both pregnant and non-pregnant patients treated with VEH (not undergoing decidualization) or EPC (undergoing decidualization).See Figure 1B.Table S4: RNA-seq results of lncRNA genes in hESF cells isolated from pregnant patients treated with VEH (not undergoing decidualization) or EPC (undergoing decidualization).FDR, false-discovery rate; Log2FC, log base 2 Fold-Change (EPC vs. VEH).Cells were used from three independent patients (N = 3).Table S5: RNAseq results of lncRNA genes in hESF cells isolated from non-pregnant patients treated with VEH (not undergoing decidualization) or EPC (undergoing decidualization).FDR, false-discovery rate; Log2FC, log base 2 Fold-Change (EPC vs. VEH).Cells were used from three independent patients (N = 3).Table S6: Combined RNA-seq results of lncRNA genes from hESF cells isolated from both pregnant and non-pregnant patients treated with VEH (not undergoing decidualization) or EPC (undergoing decidualization).See Figure 2B.Table S7: Information on the nine lncRNA genes of interest, including their Symbol, ENSEMBL GeneID, TranscriptIDs, and primary transcripts (or exons, as determined in Supplemental Figures S1-S9).Table S8: lncRNA genes regulated by PGR during decidualization.RNA-seq results of lncRNA genes in hESF cells isolated from non-pregnant patients treated with siNT or siPGR for two days, followed by EPC for three days.FDR, false-discovery rate; Log2FC, log base 2 Fold-Change (EPC vs. VEH).Cells were used from three independent patients (N = 3).Table S9: Combined RNA-seq results of lncRNA genes differentially expressed during decidualization (common between hESF cells isolated from both pregnant and non-pregnant patients) plus regulated by PGR.See Figure 6B.Table S10: lncRNA genes regulated by FOXO1 during decidualization.RNAseq results of lncRNA genes in hESF cells isolated from non-pregnant patients treated with siNT or siFOXO1 for two days, followed by EPC for three days.FDR, false-discovery rate; Log2FC, log base 2 Fold-Change (EPC vs. VEH).Cells were used from three independent patients (N = 3).Table S11: Combined RNA-seq results of lncRNA genes differentially expressed during decidualization (common between hESF cells isolated from both pregnant and non-pregnant patients) plus regulated by FOXO1.See Figure 7B.Supplemental Figures. Figure S1.RNA-Seq results at the AC027288.3(ENSG00000257894) gene region on chromosome 12.Three transcripts (in green) are associated with this gene, and only one (ENST00000550268.2) expression is upregulated in EPC-treated cells compared to VEH-treated cells.The other transcripts do not seem to be expressed in the samples.This transcript is antisense to the SYT1 gene with no overlapping exonic regions.The picture was taken from the UCSC Genome Browser (http://genome.ucsc.edu).Figure S2.RNA-Seq results at the AC023154.1 (ENSG00000248115) gene region on chromosome 4. Three transcripts (in green) are associated with this gene, and only one (ENST00000510351.1)expression is upregulated in EPC-treated cells compared to VEH-treated cells.The gene is antisense to an intron region of the protein-coding gene SCFD2.The picture was taken from the UCSC Genome Browser (http://genome.ucsc.edu).Figure S3.RNA-Seq results at the AC108861.1 (ENSG00000278408) gene region on chromosome 15.One transcript (in green) is associated with this gene (ENST00000611634.1),and its expression is upregulated in EPC-treated cells compared to VEH-treated cells.The gene is antisense to the protein-coding THSD4 and has four different protein-coding transcripts (not all shown), and one (ENST00000357769.4)has its first exon overlapping with the third exon of AC108861.1.The picture was taken from the UCSC Genome Browser (http://genome.ucsc.edu).Figure S4.RNA-Seq results at the AL121578.3(ENSG00000259977) gene region on chromosome X.One transcript (in green) is associated with this gene (ENST00000567273.1)and its expression is downregulated in EPC-treated compared to VEHtreated cells.The gene is antisense and overlaps with an intron of another gene, ENSG00000250349, which encodes a tetraspanin family member.The picture was taken from the UCSC Genome Browser (http://genome.ucsc.edu).Figure S5.RNA-Seq results at the BASP1-AS1 gene region on chromosome 5. A. Five transcripts (in green) are associated with this gene on chromosome 5.It is antisense to BASP1 (which has two alternate transcripts) and a close inspection of the first exons of BASP1-AS1 and BASP1.B. The first exon of the main BASP1 transcript does not overlap with the first exon of the BASP1-AS1 transcripts.However, the first exon of a minor BASP1 transcript overlaps with two BASP1-AS1 transcripts (ENST00000399760.2,ENST00000667677.1).Although the expression of this gene is upregulated in EPC-treated cells compared to VEH-treated cells, it is unclear which of the five transcripts is involved.The picture was taken from the UCSC Genome Browser (http://genome.ucsc.edu).Figure S6.RNA-Seq results at the LINC01605 gene region on chromosome 8.Four transcripts (in green) are associated with this gene, and two (ENST00000522718.5,ENST00000520422.5)appear to be more highly expressed in VEH-treated cells than EPC-treated cells undergoing decidualization.The picture was taken from the UCSC Genome Browser (http://genome.ucsc.edu).Figure S7.Table of information of the lncRNA genes of interest, including their Symbol, ENSEMBL GeneID, TranscriptIDs, and primary transcripts (or exons, as determined in Supplemental Figures S1-S9).RNA-Seq results at the LINC02432 gene region on chromosome 4. Three transcripts (in green) are associated with this gene, and one (ENST00000701566.1)appears to be more highly expressed in EPC-treated cells than in VEH-treated cells undergoing decidualization.The picture was taken from the UCSC Genome Browser (http://genome.ucsc.edu).Figure S8.RNA-Seq results at the LINC02593 gene region on chromosome 1.One transcript (in green) is associated with this gene and appears more highly expressed in EPC-treated cells than VEH-treated cells undergoing decidualization.The closest gene is approximately 3 kb away and is SAMD11, with their 5" ends facing each other.The 5 ′ -end of LINC02593 may be much closer to that of SAMD11 than is currently annotated on the UCSC Genome Browser.The picture was taken from the UCSC Genome Browser (http://genome.ucsc.edu).Figure S9.RNA-Seq results at the LINC02600 gene region on chromosome 4.One transcript (in green) is associated with this gene and appears more highly expressed in EPC-treated cells than VEH-treated cells undergoing decidualization.The closest gene is approximately 2 kb away and is ADRA2C, with their 5" ends facing each other.The 5 ′ -ends of LINC02600 and ADRA2C may be much closer than is currently annotated on the UCSC Genome Browser.The picture was taken from the UCSC Genome Browser (http://genome.ucsc.edu).Figure S10.Transcription factor binding site cluster 2 in the first intron of ENST00000550268.2 (AC027288.3)from multiple ChIP-seq publications.A Bed file containing the data were downloaded from Chip Atlas and displayed on the IGV genome browser.A Color gradient represents −10*Log 10 (MACS2 Q-value) with blue (50), cyan (250), green (500), yellow (750), and red (>1000).

Cells 2024 , 24 Figure 1 .
Figure 1.RNA−seq results reveal a large number of protein-coding genes are differentially expressed during hESF cell decidualization.(A) Volcano plot of RNA-seq results of hESF cells isolated from pregnant patients treated with vehicle (not undergoing decidualization) or EPC (undergoing decidualization).Note that fold-change represents EPC-compared to VEH-treated cells.(B) Venn diagram comparing the RNA-seq results of hESF cells isolated from pregnant (P) patients to those isolated from non-pregnant (NP) patients.Abbrev: FDR, false-discovery rate; FC, fold change EPCcompared to VEH-treated cells; UP increases during decidualization; DO, decreases during decidualization.Data are provided in Supplemental Tables S1-S3.

Figure 1 .
Figure 1.RNA-seq results reveal a large number of protein-coding genes are differentially expressed during hESF cell decidualization.(A) Volcano plot of RNA-seq results of hESF cells isolated from pregnant patients treated with vehicle (not undergoing decidualization) or EPC (undergoing decidualization).Note that fold-change represents EPC-compared to VEH-treated cells.(B) Venn diagram comparing the RNA-seq results of hESF cells isolated from pregnant (P) patients to those isolated from non-pregnant (NP) patients.Abbrev: FDR, false-discovery rate; FC, fold change EPC-compared to VEH-treated cells; UP increases during decidualization; DO, decreases during decidualization.Data are provided in Supplemental Tables S1-S3.

Figure 2 .
Figure 2. RNA−seq results reveal a moderate number of long non-coding genes are differentially expressed during hESF cell decidualization.(A) Volcano plot of RNA-seq results of hESF cells isolated from pregnant patients treated with vehicle (not undergoing decidualization) or EPC (undergoing decidualization).(B).Venn diagram comparing the RNA-seq results of hESF cells isolated from pregnant patients (P) to those isolated from non-pregnant (NP) patients.Abbrev: FDR, falsediscovery rate; FC, fold change EPC-compared to VEH-treated cells; NC, no change; UP increases

Figure 2 .
Figure 2. RNA-seq results reveal a moderate number of long non-coding genes are differentially expressed during hESF cell decidualization.(A) Volcano plot of RNA-seq results of hESF cells isolated from pregnant patients treated with vehicle (not undergoing decidualization) or EPC (undergoing decidualization).(B).Venn diagram comparing the RNA-seq results of hESF cells isolated from pregnant patients (P) to those isolated from non-pregnant (NP) patients.Abbrev: FDR, false-discovery rate; FC, fold change EPC-compared to VEH-treated cells; NC, no change; UP increases during decidualization; DO, decreases during decidualization.Data are provided in Supplemental Tables S4-S6.

Cells 2024 , 24 Figure 6 .
Figure 6.RNA−seq results of lncRNA genes regulated by PGR in non-pregnant (NP) hESF cells undergoing EPC-induced decidualization.(A) Volcano plot of reanalyzed RNA-seq results of NP hESF incubated with non-targeting (siNT) and PGR-targeting (siPGR) siRNA for two days, then EPC for three days.Abbrev: FDR, false-discovery rate; FC, fold change siPGR-compared to siNTtreated cells; NC, no change; UP increases in response to siPGR; DO, decreases in response to siPGR.Data are provided in Supplemental TablesS7 and S8.(B) Venn diagram comparing the RNA-seq results of the results in panel A to the common lncRNA's differentially expressed in NP plus pregnant (P) hESF cells undergoing decidualization (see Figure2Band Supplemental TableS6).Abbrev: P/NP-UP and -DO, common lncRNA genes between pregnant and non-pregnant hESF cells that increase and decrease during decidualization; NPsiPGR-UP and -DO, lncRNA's upregulated and downregulated in response to siPGR, respectively.

Figure 6 .
Figure 6.RNA-seq results of lncRNA genes regulated by PGR in non-pregnant (NP) hESF cells undergoing EPC-induced decidualization.(A) Volcano plot of reanalyzed RNA-seq results of NP hESF incubated with non-targeting (siNT) and PGR-targeting (siPGR) siRNA for two days, then EPC for three days.Abbrev: FDR, false-discovery rate; FC, fold change siPGR-compared to siNT-treated cells; NC, no change; UP increases in response to siPGR; DO, decreases in response to siPGR.Data are provided in Supplemental TablesS7 and S8.(B) Venn diagram comparing the RNA-seq results of the results in panel A to the common lncRNA's differentially expressed in NP plus pregnant (P) hESF cells undergoing decidualization (see Figure2Band Supplemental TableS6).Abbrev: P/NP-UP and -DO, common lncRNA genes between pregnant and non-pregnant hESF cells that increase and decrease during decidualization; NPsiPGR-UP and -DO, lncRNA's upregulated and downregulated in response to siPGR, respectively.

Figure 7 .
Figure 7. RNA−seq results of lncRNA genes regulated by FOXO1 in hESF cells undergoing EPCinduced decidualization.(A) Volcano plot of reanalyzed RNA-seq results of non-pregnant (NP) hESF incubated with non-targeting (siNT) and PGR-targeting (siPGR) siRNA for two days, then EPC for three days.Abbrev: FDR, false-discovery rate; FC, fold change sFOXO1-compared to siNTtreated cells; NC, no change; UP increases in response to siFOXO1; DO, decreases in response to siFOXO1.Data are provided in Supplemental TablesS9 and S10.(B) Venn diagram comparing the RNA-seq results of the results in panel A to the common lncRNA's differentially expressed in NP plus pregnant (P) hESF cells undergoing decidualization (see Figure2Band Supplemental TableS6).Abbrev: P/NP-UP and -DO, common lncRNA genes between pregnant and non-pregnant hESF cells that increase and decrease during decidualization; NPsiFOXO1-UP and -DO, lncRNAs upregulated and downregulated in response to siFOXO1, respectively.

Figure 7 .
Figure 7. RNA-seq results of lncRNA genes regulated by FOXO1 in hESF cells undergoing EPCinduced decidualization.(A) Volcano plot of reanalyzed RNA-seq results of non-pregnant (NP) hESF incubated with non-targeting (siNT) and PGR-targeting (siPGR) siRNA for two days, then EPC for three days.Abbrev: FDR, false-discovery rate; FC, fold change sFOXO1-compared to siNT-treated cells; NC, no change; UP increases in response to siFOXO1; DO, decreases in response to siFOXO1.Data are provided in Supplemental TablesS9 and S10.(B) Venn diagram comparing the RNA-seq results of the results in panel A to the common lncRNA's differentially expressed in NP plus pregnant (P) hESF cells undergoing decidualization (see Figure2Band Supplemental TableS6).Abbrev: P/NP-UP and -DO, common lncRNA genes between pregnant and non-pregnant hESF cells that increase and decrease during decidualization; NPsiFOXO1-UP and -DO, lncRNAs upregulated and downregulated in response to siFOXO1, respectively.

Cells 2024 , 24 Figure 8 .
Figure 8. FOXO1 and progesterone/PGR may directly regulate AC027288.3expression during hESF decidualization.(A) Map of select transcription factor binding sites near the ENST00000550268.2transcript of the AC027288.3gene during hESF decidualization from previous ChIP-seq studies downloaded from ChIP-Atlas.A Color gradient represents −10*Log10(MACS2 Q-value) with blue (50), cyan (250), green (500), yellow (750), and red (>1000).(B) Map of FOXO1-binding sites near the ENST00000550268.2transcript of the AC027288.3gene during hESF decidualization from reanalysis of a previous ChIP-seq study.FBR1 and FBR2 represent the FOXO1-binding region primer amplicon region used in ChIP-PCR, while CON represents a control region ~3 kb downstream of FBR2.The picture was taken from the UCSC Genome Browser (h p://genome.ucsc.edu).(C) ChIP-PCR of a well-known FOXO binding region near the IGFBP1 gene region.(D) ChIP-PCR results for FOXO1 binding to the FBR1 and FBR2 in the first intron of ENST00000550268.2 near the transcriptional start site.Bars represent mean ± SEM using cells from four independent patients (N = 4).(E) Effects of vehicle (V), estradiol (E) versus progesterone (P), or both estradiol plus progesterone (B) on AC027288.3expression.Bars represent mean ± SEM using cells from six independent patients (N = 6).Statistical significance denoted as a, c, and d represents P less than 0.05, 0.005, and 0.001, respectively.

Figure 8 .
Figure 8. FOXO1 and progesterone/PGR may directly regulate AC027288.3expression during hESF decidualization.(A) Map of select transcription factor binding sites near the ENST00000550268.2transcript of the AC027288.3gene during hESF decidualization from previous ChIP-seq studies downloaded from ChIP-Atlas.A Color gradient represents −10*Log 10 (MACS2 Q-value) with blue (50), cyan (250), green (500), yellow (750), and red (>1000).(B) Map of FOXO1-binding sites near the ENST00000550268.2transcript of the AC027288.3gene during hESF decidualization from reanalysis of a previous ChIP-seq study.FBR1 and FBR2 represent the FOXO1-binding region primer amplicon region used in ChIP-PCR, while CON represents a control region ~3 kb downstream of FBR2.The picture was taken from the UCSC Genome Browser (http://genome.ucsc.edu).(C) ChIP-PCR of a well-known FOXO binding region near the IGFBP1 gene region.(D) ChIP-PCR results for FOXO1 binding to the FBR1 and FBR2 in the first intron of ENST00000550268.2 near the transcriptional start site.Bars represent mean ± SEM using cells from four independent patients (N = 4).(E) Effects of vehicle (V), estradiol (E) versus progesterone (P), or both estradiol plus progesterone (B) on AC027288.3expression.Bars represent mean ± SEM using cells from six independent patients (N = 6).Statistical significance denoted as a, c, and d represents P less than 0.05, 0.005, and 0.001, respectively.

Table 2 .
DNA oligonucleotide sequences (5 ′ -to 3 ′ -prime) that were used for ChIP-qPCR in this study.