1 Introduction
Successful implantation depends on normal development of trophoblast cells and endometrial stroma cell decidualization. Zygotes cleavage and differentiate into inner cell mass (ICM) and trophectodermal cells (TE). Then, TE cells proliferate and differentiate into placenta, which is vital for exchange between mother and fetus. Defects in trophoblast cells could lead to adverse pregnancy outcomes. Insufficient placental trophoblast invasion impaired spiral artery remodeling, contributing to the occurrence of pre-eclampsia. Meanwhile, dysgenesis of trophoblast cells may cause intrauterine growth restriction and early miscarriage. On the other hand, excessive proliferation and invasion can lead to hydatidiform mole, choriocarcinoma, or other trophoblastic tumors.
Trophoblast proliferation, migration, and invasion are controlled by many factors [
1]. It is difficult to elucidate the function of human trophoblast cells
in vivo, therefore study of proliferation, migration, and invasion by trophoblast cell lines
in vitro can help to gain a deeper understanding of the developmental process of trophoblast cells and placenta. The HTR-8/SVneo cell line is an immortalized trophoblast cell line, which was established from first trimester human trophoblasts, sharing similar characteristics with parental trophoblast cells [
2]. Therefore, HTR-8/SVneo cells are suitable for investigating the function of genes in trophoblast cells.
RRS1, located on chromosome 8q, is a conserved essential gene encoding novel regulatory protein for ribosome biogenesis [
3]. It is reported that
RRS1 was involved in promoting malignant tumor cell activities. Some studies have reported that
RRS1 might serve as a tumor biomarker in hepatocellular carcinoma (HCC) [
4], and be overexpressed in breast cancer [
5–
7]. Meanwhile, in human HCC cells, downregulation of
RRS1 expression by RNAi inhibited the proliferation while promoting cell apoptosis [
8]. Given that trophoblast cells and malignant tumor cells share some similar characteristics in proliferation, migration, and invasion [
9,
10], we hypothesize that
RRS1 is also functional in trophoblast cells. However, there is no relevant research on
RRS1 in trophoblast cells currently. In our study, we found that TE highly expressed
RRS1. We then explored the potential function of
RRS1 in human trophoblast cell line HTR-8/SVneo and found that reduction of
RRS1 led to impaired proliferation, migration, and invasion in HTR-8/SVneo cells, and was related to early spontaneous abortion.
2 Materials and methods
2.1 Cell culture
Human trophoblast cell line HTR-8/SVneo was cultured in RPMI 1640 medium (Gibco, C11875500BT, USA) containing 10% fetal bovine serum (Gibco, A5670701, USA), 1% penicillin and streptomycin (Gibco, A5873601, USA). HTR-8/SVneo cells were incubated at 37 °C and 5% CO2 and routinely passaged every 2 or 3 days.
2.2 Human subjects
Participants were recruited from Peking University Third Hospital. Approval was granted by the Ethics Committee (Project number: IRB00006761-M2022688). All participants signed the research informed consent before the pregnancy termination. There were 8 normal chorionic villus samples collected from individuals getting an elective abortion, used as control, and 7 chorionic villus samples from patients undergoing spontaneous abortion. All participants were at < 60 days gestational age.
2.3 siRNA transfection
si-RRS1-1, siRRS1-2, siRRS1-3, and si-NC (empty vector) were purchased from Ribobio (Guangzhou, China). Cells were transfected using Lipofectamine RNAiMAX transfection reagent (ThermoFisher, 13778030, USA). After 48 h and 72 h of transfection, the cells were used for subsequent experiments.
2.4 RNA extraction and quantitative real-time PCR
Total RNA was extracted from 105–107 HTR-8/SVneo cells and chorionic villus samples using TRIzol (Invitrogen, 15596018CN, USA). RNA was reversed using PrimeScript RT reagent Kit (Takara, RR037Q, Japan). qPCR was performed using SYBR Green Universal Master Mix (Applied Biosystem, 4309155, USA) on ABI 7500 system. The PCR primers were listed in Table S1. There were at least three technical replicates and three biological replicates.
2.5 Western blot
The protein was extracted from 106–107 HTR-8/SVneo cells using RIPA lysis buffer (Beyotime, P0013B, China) and the concentration was detected by BCA Protein Assay Kit (Beyotime, P0009, China). 100 μg proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to polyvinylidene difluoride (PVDF) membranes, then blocked with 5% dry milk in Tris-buffer for 1 h. The membranes were incubated with primary antibodies at 4 °C overnight, then incubated with second antibodies at 25 °C for 2 h. Protein bands were detected using ECL (Beyotime, P0018S, China). Relative protein levels were analyzed using ImageJ. There were at least three biological replicates. Antibodies include RRS1 antibody (1:2000, ab188161, abcam, USA), β-tubulin antibody (1:2000, ab188161, abcam, USA), FOS (1:2000, ab208942, abcam, USA), VEGFA (1:2000, ab46154, abcam, USA), TP53 (1:2000, ab131442, abcam, USA), and CTSH (1:2000, ab133641, abcam, USA).
2.6 Immunofluorescence staining
Cells were fixed in 5% PFA for 1 h at 25 °C, treated with permeabilization buffer with Triton X-100 (Beyotime, P0096, China), blocked with blocking buffer (Beyotime, P0102, China), then incubated with primary antibodies at 4 °C overnight, washed by PBS, and incubated with second antibodies at 25 °C for 2 h and 0.5–10 μg/mL DAPI solution. Images were taken from confocal microscope platform (Zeiss LSM 880). There were at least three biological replicates. Antibodies include RRS1 antibody (1:200, ab188161, abcam, USA), ki-67 antibody (1:200, ab15580, abcam, USA), and γH2AX antibody (1:200, ab81299, abcam, USA). EdU cell proliferation assay used BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime, C0078S, China).
2.7 IncuCyte® proliferation assay for live-cell analysis
Transfected cells were used for proliferation assay. Following 48 h of transfection with siRNAs targeting RRS1, cells were seeded into a 96-well plate at a density of 5000 cells per well and incubated for 12–24 h until cells adhered and reached 10%–20% confluency. Then we placed the cell plate into the IncuCyte® live-cell analysis system, monitored cell growth by capturing phase contrast images every three hours, and analyzed the results using the integrated confluence algorithm.
2.8 Incucyte® scratch wound invasion assay
Migration and invasion capacities were assessed using the Incucyte® scratch wound invasion assay. For migration, transfected cells were seeded into a 96-well plate, then placed the plate into a 37 °C incubator with 5% CO2 overnight. Wounds were simultaneously created in all wells using wound maker. After wounding, the cells were washed twice with culture media to remove the free cells. Then the cell plate was placed into the IncuCyte® live-cell analysis system, the images were scanned every three hours. For invasion, after wounds were created, 30 µL matrigel matrix (Corning, 356231, USA) was added to the wells. Subsequent steps were the same as migration protocol.
2.9 RNA-seq data processing
TrimGalore [
11] was used to trim the raw reads and remove low-quality reads (–quality 20). Then the clean reads were mapped to hg19 genome reference using STAR [
12] and then counted using featureCounts [
13]. Transcript integrity number (TIN) was calculated by RSeQC [
14]. Quality control of samples was performed as follows: clean reads mapping rate > 80%, mitochondrial gene mapping rate < 5%, TIN > 70; > 18 000 000 counts and 16 000 genes per sample. Then 8 control group samples and 7 knockdown samples were kept for later analysis.
2.10 Identification of differentially expressed genes
Genes with an expression count of 3 or more in at least the number of samples corresponding to the smallest group size were retained for subsequent analysis using DESeq2 (v1.40.2) [
15]. Differentially expressed genes (DEGs) were defined by the threshold of absolute log
2FC value greater than 1,
P value < 0.05, and mean TPM of higher-expression group < 1.
2.11 Enrichment analysis
GO, KEGG enrichment analysis, and GSEA were performed by clusterProfile R package [
16]. All enriched terms were filtered out by
P value < 0.05. Disease enrichment analysis was performed using online tools metascape [
17] with DisGeNET database.
2.12 Statistical analysis
Wilcox test was used to compare differences among different groups in our figures. Statistically significant values were defined as follows: *, **, and *** indicate P < 0.05, P < 0.01, and P < 0.001, respectively.
3 Results
3.1 Trophoblast cells highly expressed RRS1
We investigated the expression pattern and DNA methylation pattern of
RRS1 in TE and ICM from published data using single-cell multi-omics sequencing technologies [
18]. We found that TE cells expressed significantly higher levels of
RRS1 in human blastocyst (Fig. 1A), indicating that
RRS1 may play an important role in TE rather than primitive endoderm (PE) and epiblast (EPI) cells. No significant differences in DNA methylation patterns were observed among TE, PE, and EPI lineages, indicating that the differential expression of
RRS1 is not regulated by DNA methylation (Fig. 1B). We examined the RRS1 protein in HUVEC, HeLa, and HTR-8/SVneo cell lines using Western blot, respectively. All three cell lines expressed RRS1 protein (Fig. 1C), HTR-8/SVneo cells expressed the highest levels of RRS1 among them (Fig. 1D). Next, we detected the subcellular expression of RRS1 by immunofluorescence. It is shown that RRS1 was mainly expressed in the nucleus of HTR-8/SVneo cells (Fig. 1G). To determine the role of
RRS1 in trophoblast cells, we performed knockdown of
RRS1 in HTR-8/SVneo cells. To select the optimal knockdown strategy, siRNAs were evaluated for their ability to knock down gene expression. Three different siRNAs targeting
RRS1 (si
RRS1-1, si
RRS1-2, and si
RRS1-3) were transfected into HTR-8/SVneo cells at the concentration of 10 nmol/L and 50 nmol/L. The sequences were shown in Table S1. After 48 h and 72 h, cells from each group were collected for RNA extraction and RT-qPCR. The results showed that si
RRS1-1 and si
RRS1-2 effectively knocked down the mRNA level of
RRS1 in HTR-8/SVneo cells (Fig. 1E). Then, we performed Western blot to detect the protein level of RRS1. After 72 h of transfection, RRS1 expression was significantly reduced in si
RRS1-1 and si
RRS1-2 transfected cells compared to the control group (Fig. 1F). Immunofluorescence analysis further showed that the cellular localization of RRS1 remained unchanged in the nucleus but relative fluorescent intensity reduced significantly in knockdown cells (Fig. 1G). Based on these findings, we selected si
RRS1-1 and si
RRS1-2 at 50 nmol/L with a 72 h transfection duration for follow-up experiments.
3.2 RRS1 regulated proliferation of HTR-8/SVneo cells
We first investigated the effects of RRS1 on the proliferation of HTR-8/SVneo cells by IncuCyte® S3 live-cell analysis system. The process of cell growth was analyzed based on cell confluence and cell count metrics with visual verification via live-cell images. After 48 h of transfection and 12–24 h of incubation, until the cells reached 10%–20% confluence, cell growth was monitored continuously for a further 96 h. Continuous live-cell assays and real-time data analysis revealed that knockdown of RRS1 impaired the proliferation of HTR-8/SVneo cells. The speed of cell confluence decreased significantly after 18 h and 21 h onward in siRRS1-1 and siRRS1-2, respectively (Fig. 2A, 2B, and 2C). Detailed data at each time point were shown in Table S2.
Ki-67 is a cellular marker for proliferation that is present during active phases of cell cycle but absent in quiescent cells [
19]. RT-qPCR and immunofluorescence analysis further showed that Ki-67 mRNA (Fig. S2A) and protein expression (Fig. 2D) decreased in
RRS1-knockdown cells. The fluorescence intensity of 5-ethynyl-2´-deoxyuridine (EdU), which can label dividing cells to detect status of cell proliferation [
20], also decreased in
RRS1-knockdown cells (Fig. 2E). These results additionally demonstrated that
RRS1 knockdown significantly inhibited cell proliferation in HTR-8/SVneo cells. Besides, we found that depletion of
RRS1 resulted in DNA double-strand break and cell apoptosis, as evidenced by the fluorescence intensity of γ-H2AX, a sensitive molecular marker of DNA damage and repair [
21], was enhanced after
RRS1 knockdown (Figs. 2F and 5B).
3.3 RRS1 regulated migration and invasion of HTR-8/SVneo cells
In addition to proliferation, migration and invasion are also important functions of trophoblast cells, thus we further investigated the impact of RRS1 knockdown on the migration and invasion of HTR-8/SVneo cells. After transfection of siRNAs targeting RRS1 for 48 h, cells were seeded at a density of 2.5×104 cells per well into 96-well plate, and incubated for 12–24 h to ensure that the cell confluence reached 85%–95%. Then, we used Incucyte® 96-well wound-making tool to create precise and reproducible wounds in all wells. Evaluation of migration ability of HTR-8/SVneo cells was conducted by Incucyte® 96-well scratch wound cell migration assays based on live-cell time-lapse imaging for 3 days. The results of the cell scratch assay showed a significant reduction in migration distance of RRS1-knockdown group. On day 3, cells of control group have covered the entire surface of the wells, but there was still a certain distance between the wounds in siRRS1-1 and siRRS1-2 cells (Fig. 3A). Relative wound density and wound confluence (Fig. 3B) were analyzed by Incucyte ZOOM™, indicating that the wound healing speed was slower in RRS1-knockdown cells compared to control group. The specific data were shown in Table S3.
Besides, we observed the invasion ability of two groups using Incucyte® 96-well scratch wound invasion assay. We seeded the cells in the 96-well plate and created the wounds in all wells using the same way as described in the migration assay, and covered the cells with matrigel matrix, then observed the cells for 3 days. On day 3, there was a shallower degree of invasion in the matrix in siRRS1-1 and siRRS1-2 cells (Fig. 3C). The relative wound density and wound confluence of siRRS1-1 and siRRS1-2 cells was lower than that of the control (Fig. 3D). Detailed data at each time point were shown in Table S4. All these results confirmed that RRS1 knockdown impaired migration and invasion abilities of HTR-8/SVneo cells.
3.4 Knockdown of RRS1 affected transcription in HTR-8/SVneo cells
After transfection of siRNA for 72 h, cells were collected for RNA-seq. Sequencing quality were assessed by multiple indicators (TIN > 70; mapping rate > 80%, mitochondrial gene mapping rate < 5%) (Fig. S1A) and samples with more than 18 000 000 counts and 16 000 genes detected were retained for subsequent analytic pipelines (Fig. S1B). Principal-component analysis (PCA) showed a strong heterogeneity in expression patterns between knockdown and control groups and consistency within each group (Fig. 4A). A total of 767 differentially expressed genes (DEGs) were identified between the two groups. 341 genes were upregulated and 426 genes were downregulated in RRS1-knockdown cells, as shown in heatmap (Fig. 4B) and Table S5. We then performed enrichment analysis on DEGs by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. KEGG analysis revealed that downregulated genes were enriched in pathways involved in cytokine–cytokine receptor interaction, TNF signaling, JAK–STAT signaling, IL-17 signaling (Fig. 4C). GO analysis showed that upregulated genes were highly involved in extracellular matrix organization, hormone metabolic process, DNA damage response, and intrinsic apoptotic signaling pathway (Figs. 4D and S3A), indicating that RRS1 depletion resulted in cell dysfunction and apoptosis. Downregulated genes were enriched in female pregnancy, embryo implantation, trophoblast cell migration, and wound healing, indicating that RRS1 depletion in trophoblast cells may affect placental function (Figs. 4E and S3B). Gene set enrichment analysis (GSEA) revealed that genes associated with DNA damage response by P53, intrinsic apoptotic signaling pathway were upregulated in RRS1-knockdown cells, whereas genes related with trophoblast cell migration, maternal placenta development were downregulated in RRS1-knockdown cells (Fig. 4F).
3.5 RRS1 deficiency was associated with human early spontaneous abortion
Classic genes regulating apoptosis and ferroptosis were significantly upregulated in RRS1-knockdown group, such as TP53, CTSD, FTH1. DNA damage response genes were also upregulated in RRS1-knockdown group, such as ZNF385A, TFAP4, indicating that aggravated cellular DNA damage led to cell proliferation impairment due to RRS1 depletion, which is consistent with immunofluorescence staining results. Migration and wound healing related genes were significantly downregulated in RRS1-knockdown group, such as PLAU, PPBP, PLAT, ABAT. Genes related to pregnancy were also downregulated, such as LIF, CYP28B1, PRDM1 (Fig. 5A).
We then performed RT-qPCR analysis of DEGs related to key cellular function, such as cell proliferation and apoptosis related genes TP53, CTSH, migration related gene VEGFA, and pregnancy related gene FOS to verify the expression changes in RRS1-knockdown cells. The results showed that TP53, CTSH were upregulated in RRS1-knockdown cells, while FOS and VEGFA were downregulated in RRS1-knockdown cells, consistent with our transcriptomic data (Fig. 5B). Western blot analysis further confirmed the protein level alterations for TP53, CTSH, FOS and VEGFA (Figs. 5C and S2B), demonstrating concordance across transcriptomic, qPCR, and protein level analyses.
To predict which diseases are associated with RRS1 deficiency, we performed DisGeNET analysis using DEGs. The results showed that pregnancy related diseases like miscarriage, eclampsia, pre-eclampsia, chorioamnionitis were significantly enriched, suggesting that RRS1 defects may lead to damage in placental development (Fig. 5D). DEGs were enriched and shared in specific diseases and their relationships were shown in a network plot (Figs. 5E and S3). Notably, genes such as HMOX1, PPARG, THBD may play a pivotal role in placental development, as they have been linked to multiple diseases. To further investigate the correlation between RRS1 deficiency and early spontaneous abortion, we collected chorionic villus samples from patients undergoing early spontaneous abortions and normal individuals with a gestational age less than 60 days. RRS1 expression significantly reduced in the spontaneous abortion group compared to the control by RT-qPCR (Fig. 5F), which suggested that RRS1 deficiency in chorionic villus was associated with early spontaneous abortion.
4 Discussion
The placenta is a critical part of embryo
in utero, derived from the differentiation of trophoblast cells, and supports fetal development. A study analyzing mutations and phenotypes in mice revealed that abnormalities in placental morphology accounted for a large proportion of embryonic lethality, suggesting the importance of further research in this area [
22]. However, studies focused on gene mutations and mechanisms underlying trophoblast function and placental development are still largely needed [
23]. In this study, we found that
RRS1 regulated the proliferation, migration, and invasion of trophoblast cells, and that
RRS1 deficiency in chorionic villus was associated with early spontaneous abortion. Proliferation, migration, and invasion are fundamental features of trophoblast cells, similar to the characteristics of tumor cells to some extent. Unlike tumor cells, these processes in trophoblast cells are tightly controlled [
24]. During early pregnancy, insufficient cell proliferation can lead to miscarriage, while excessive proliferation may result in trophoblastic diseases such as hydatidiform mole or choriocarcinoma [
25].
Previous studies showed that Ki-67 expression decreased in cytotrophoblast cells and decidua in miscarriage patients [
26]. In our study, after knockdown of
RRS1, Ki-67 expression significantly decreased, while γ-H2AX and other DNA damage-related genes were significantly upregulated, confirming that
RRS1 depletion led to impaired proliferation associated with DNA replication damage. In chorionic villus from patients undergoing early spontaneous abortion,
RRS1 levels decreased compared with those in normal chorionic villus, verifying that
RRS1 deficiency impairs the function of trophoblast cells and is associated with early spontaneous abortion. Recent studies have also shown that
RRS1 is overexpressed in some human cancers and plays a role in regulating tumor proliferation.
RRS1 knockdown suppresses tumor cell growth and activates p53 and p21 signaling [
4,
8,
27,
28]. Additionally,
RRS1 is overexpressed in tumor tissues and correlates with poor survival [
5,
7,
29]. In this study, we also found that p53 signaling was upregulated after
RRS1 knockdown, indicating that
RRS1 participates in the regulation of cell proliferation, which is consistent with previous studies.
The migration and invasion of trophoblasts are key processes in implantation and the establishment of the maternal-fetal interface, with significant implications for pregnancy outcomes [
30,
31]. In pre-eclampsia and eclampsia, a characteristic feature of trophoblasts is shallow invasion, which can cause hypertension and edema, ultimately leading to fetal hypoxia, threatening the lives of both the mother and fetus [
32,
33]. In our study,
RRS1 knockdown significantly suppressed the migratory and invasive abilities of HTR-8/SVneo cells. Additionally, wound healing-related genes were significantly decreased, confirming that the
RRS1 depletion impaired migration and invasion. The regulation of extracellular matrix (ECM) plays a crucial role in trophoblast proliferation, differentiation, and invasion. Extensive ECM remodeling is involved in pre-eclampsia and eclampsia [
34,
35]. A previous study analyzed gene expression patterns in early-onset pre-eclampsia compared to the control group and found that DEGs were significantly enriched in ECM structural constituents [
36]. In our study, genes involved in extracellular matrix organization were upregulated in
RRS1-knockdown cells, and genes associated with pre-eclampsia and eclampsia, such as
FGFR4,
INHBA,
NOTCH2,
PTG5, and
PTGS2, were significantly altered. These findings are consistent with previous studies and indicate that
RRS1 defects led to impaired placental development due to trophoblast dysfunction.
Additionally,
RRS1 regulates ribosome biogenesis in eukaryotes as a ribosome assembly factor, and it is required for recruiting RPL5, RPL11, and 5S rRNA into pre-ribosomes. The absence of
RRS1 blocks nascent ribosome assembly, potentially affecting protein synthesis and cellular activities [
37]. In our study, ribosome related genes, such as
NSA2,
MRPL42,
RPL21P28, and
RPS6KA5, were downregulated in the
RRS1-knockdown cells.
The HTR-8/SVneo cell line has been widely used as a stable model for trophoblast and placental research. However, a gap remains between cell line studies and actual placental development in vivo. Further studies, such as whole-exome sequencing (WES) and RNA-seq of patients with early spontaneous abortion, could help verify the impact of RRS1 on trophoblast function.
In conclusion, TE exhibit high RRS1 expression, which plays a crucial role in regulating the proliferation, migration, and invasion of trophoblast cells. RRS1 depletion impairs these functions, and RRS1 deficiency in human chorionic villus is associated with early spontaneous abortion. These findings improve the mechanistic understanding of trophoblast function and may potentially benefit patients suffering from early spontaneous abortion.
4.0.0.0.1 Acknowledgements
This project is funded by National Key Research and Development Program (Nos. 2022YFC2702200, 2023YFA1800300, and 2022YFC2702401), and National Natural Science Foundation of China (Nos. 82288102 and 82201838).
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