RRS1 regulates proliferation, migration, and invasion of HTR-8/SVneo human trophoblasts

RRS1, a ribosome-biogenesis factor previously linked to tumor growth, is now shown to be indispensable for normal human trophoblast behaviour: its depletion cripples proliferation, migration and invasion of HTR-8/SVneo cells and is markedly down-regulated in chorionic villi from early spontaneous abortions, positioning RRS1 insufficiency as a candidate driver of placental insufficiency. Single-cell multi-omics revealed robust RRS1 mRNA in trophectoderm but not in primitive endoderm or epiblast, and nuclear-localised protein was highest in the immortalised first-trimester trophoblast line HTR-8/SVneo. Three siRNAs reduced RRS1 transcript by 75% and protein by 70%; confluence curves generated by IncuCyte live-cell imaging showed an 18-h lag in proliferation that translated into a 35% drop in Ki-67 and a 42% reduction in EdU incorporation. Simultaneously, γ-H2AX foci increased 2.3-fold, indicating DNA double-strand breaks, while qPCR and Western blot confirmed up-regulation of TP53, CTSH and other apoptosis-related genes.

Wound-healing and Matrigel invasion assays demonstrated that RRS1 silencing decreased relative wound density by 30% and 40% respectively after 72 h, mirroring clinical shallow invasion phenotypes. RNA-seq identified 767 differentially expressed genes; down-regulated transcripts were enriched for cytokine–cytokine receptor interaction, TNF and IL-17 signalling, while up-regulated sets clustered around extracellular-matrix organisation, intrinsic apoptotic signalling and DNA-damage response. GSEA showed negative enrichment of "trophoblast migration" and "maternal placenta development" gene sets, whereas p53-mediated DNA-damage pathways were positively enriched. Key placental genes—LIF, VEGFA, FOS, PLAU—were suppressed, and DisGeNET analysis linked the signature to miscarriage, pre-eclampsia and chorioamnionitis.

Clinical relevance was tested with chorionic villi collected < 60 days gestation: RRS1 mRNA was 2.4-fold lower in seven spontaneous-abortion samples than in eight elective-termination controls matched for maternal age, suggesting that physiological down-regulation compromises early placentation. No methylation differences were detected, implying transcriptional rather than epigenetic control. Collectively, the data outline a model in which RRS1 sustains ribosome assembly, maintains genomic integrity and supports the migratory/invasive programme that transforms trophoblasts into endovascular plugs; when levels fall, replication stress activates p53, ECM remodelling falters and implantation fails. Future work will explore whether RRS1 supplementation or p53 modulation can rescue trophoblast function and whether germ-line or somatic RRS1 variants predispose to recurrent pregnancy loss.