Placental microRNAs as potential regulators in tumour processes

Data obtention Raw miRNAs counts from five Placental Tissues were obtained from the GEO database (ncbi.nlm.nih.gov/geo). Sample accession IDs taken were GSM1901233, GSM1901234, GSM1901235, GSM1901236, and GSM1901237 from the series GSE73713. The tissue compartment from which these samples were taken was villous trophoblast from individuals with a mean gestational age of 39.1 weeks. For Uterus, Breast, Cervical, and Prostate, Normal and Cancerous Tissue samples (Table 1), raw miRNAs counts were obtained from the GDC database (portal.gdc.cancer.gov). Also, the complete miRNA quantification Transcriptome Profiles available for these types of cancer were obtained from The Cancer Genome Atlas Project (TCGA): TCGA-BRCA (1207 files from 1079 cases), TCGA-PRAD (551 files from 494 cases), TCGA-CESC (312 files from 307 cases), and for TCGA-UCEC (579 files from 550 cases).

Making use of R version 4.0.2 and Bioconductor project version 3.11, RNA-seq analysis was performed using limma, Glimma and EdgeR packages (Law et al. 2016). Considering miRNAs counts as an input, and putting them through pre-processing and exploratory data analysis before obtaining lists of differentially expressed (DE) miRNAs. DE microRNAs were divided into Up- or Down-regulated miRNAs, a P-value < 0.05 and a fold-change ≥ 5 were set as the cut-off values of DE miRNAs. For TCGA data analysis DESeq2, GenomicDataCommons and TCGAbiolinks packages were used for Normalization and Differential Expression Analysis (Colaprico et al. 2015). Functional annotation of DE miRNAs For obtaining the functional annotation based on enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways of DE microRNAs, the tool DIANA-miRPath v3.0 was used (Vlachos et al. 2015), which employs TargetScan (www.targetscan.org), microT-CDS (www.microrna.gr/microT-CDS) and TarBase v7.0 (www.microrna.gr/tarbase) databases for experimentally supported interactions for DE miRNAs.

To validate the robustness of miRNA–target interactions, we performed an independent enrichment analysis using miRNet 2.0, miRTarBase (experimentally validated interactions), and TargetScan consensus predictions. Overlap analysis showed that 82% of MAPK-pathway genes identified via DIANA-miRPath were also retrieved using miRTarBase, and 75% were supported by at least two independent tools. GSEA of MAPK-, TGF-β-, and Wnt-associated gene sets confirmed that these pathways are significantly enriched (FDR < 0.05) among targets of C19MC miRNAs. This multi-tool agreement strengthens the conclusion that C19MC miRNAs impose broad regulatory control over developmental signalling pathways.

After performing the differential expression analysis of Placenta vs Tumour samples, 78 Up-regulated and 25 Down-regulated miRNAs were found considering a fold-change above 5 and under -5 respectively, these results showed data homogeneity for both comparisons (Figure 1). As part of the 78 differentially expressed (DE) miRNAs, those from the C19MC have shown to be the most Up-regulated placental miRNAs compared to cancerous tissues (Figure 2). Figure 1. (Left) Differentially Expressed miRNAs between Placenta and Cancerous Tissues, (Right) and between Placenta and Normal Tissues.

Figure 2. Volcano plot for DE miRNAs between Placenta and Cancerous Tissues. The vertical line at zero divides upand down-regulated miRNAs, horizontal line sets significantly DE miRNAs above the fold change value. Placental and TCGA data for breast (BRCA), prostate (PRAD), cervical (CERC) and uterine (UCEC) cancer, DE analysis showed 78 shared up-regulated miRNAs and 15 down-regulated miRNAs in placental samples when compared with the four types of cancer considered (Figure 3). Functional annotation of C19MC miRNAs exhibited a high gene regulation in the placenta for both embryonic and tumour progression key KEGG metabolic pathways (Table 2). Suggesting a meticulous epigenetic regulation by miRNAs of these pathways in Placenta but not in the evaluated cancerous tissues. Figure 3. Venn Diagram of (Left) Up-regulated and (Right) Down-regulated DE miRNAs between Placenta and TCGA samples. In red: TCGA-BRCA; green: TCGA-PRAD; purple: TCGA-CESC; yellow: TCGA-UCEC.

Several miRNAs have been identified as predominantly or almost exclusively expressed in placental tissue, particularly those belonging to the C19MC gene cluster. This supports a central role for placental miRNAs in fetal development, trophoblast behaviour, and the coordination of implantation and placentation (Donker et al. 2012; Thamotharan et al. 2017). More recent work has strengthened this view: Guo et al. (2025) highlighted that placental miRNAs regulate proliferation, differentiation, invasion, angiogenesis and immune modulation, reinforcing their importance in trophoblast physiology. Decreased expression or dysregulation of these ncRNAs has also been associated with processes related to the Hallmarks of Cancer, including angiogenesis, tumour invasion, cell proliferation, migration, immune evasion and apoptosis resistance, mechanistic overlaps long proposed by the Trophoblastic Thesis of Cancer. The Trophoblastic Thesis sustains that similarities between tumour cells and trophoblasts are not coincidental but reflect shared developmental programs that are aberrantly reactivated in cancer (Ross 2015; Vidal et al. 2018). Although trophoblasts and cancer cells share biological pathways, the placenta preserves strict molecular regulation, while cancer displays loss of control and persistent activation of these programs. Recent evidence supports this distinction: placenta-derived miRNAs, particularly C19MC miRNAs, can exert tumour-suppressive functions under physiological conditions. Zhang et al. (2024) demonstrated that placental extracellular vesicles enriched in C19MC miRNAs can inhibit proliferation and modulate signalling in ovarian and endometrial cancer cells,

suggesting that trophoblastic regulatory systems, when correctly controlled ,act to maintain normal developmental boundaries rather than to promote uncontrolled proliferation. In this research, 78 miRNAs were found to be highly expressed in placental tissue compared with the cancer tissues evaluated. Of these, 45 belong to the C19MC cluster (out of 52 reported miRNAs). Upregulation of these miRNAs is associated with pathways involved in apoptosis, pluripotency regulation, adherens junctions, and multiple signalling pathways such as MAPK, FoxO, Wnt, AMPK, ErbB, TGF-beta, Ras, mTOR, TNF and p53. These findings remain consistent with the updated literature, which increasingly describes placental miRNAs as master regulators of developmental signalling networks (Xu et al. 2021; Guo et al. 2025). The mitogen-activated protein kinase (MAPK) pathway emerged as the pathway regulated by the largest number of placental miRNAs. MAPK regulation has been associated with apoptosis avoidance and cellular proliferation via kinase cascades (Pérez-Pérez et al. 2008). In humans, MAPKs are grouped into the ERK, JNK, and p38/SAPK subfamilies, each associated with developmental and pathological processes (Morrison 2012; Sun et al. 2015). MAPK pathways are essential for trophoblast invasion, placental morphogenesis, and vascular development (Anton et al. 2012; Nadeau & Charron 2014). Recent placental research confirms that MAPK signalling integrates miRNA-mediated regulation of trophoblast proliferation and immune tolerance (Guo et al. 2025), strengthening the relevance of the MAPK pathway in the placenta–cancer parallel. The MAPK pathway is frequently activated in human cancers, promoting malignant traits such as autonomous proliferation. DNA-damaging agents modulate MEK/ERK activity independently of p53, leading to apoptosis in cervical cancer cells (Sun et al. 2015). Aberrant ERK/MAPK activation is widely recognised as a hallmark of cancer, often driven by hyperactivation of upstream regulators such as RAS and BRAF (Cotto-Rios et al. 2020). A total of 135 MAPK-related genes were identified as regulated by the 45 upregulated C19MC miRNAs. These genes include key regulators of embryogenesis and cancer progression: CACN, SOS, RAS, BRAF, JNK, IL1R, TRAF, TAB, NFKB, ELK, ATF2, MAPK, DUSP and FGF family members. These molecular nodes serve as points of convergence between trophoblast and cancer biology (Figure 4).

Figure 4. Regulation of placental C19MC miRNAs over genes associated with the MAPK signaling pathway, resulting in inhibition of cell proliferation, angiogenesis and migration/invasion. SOS proteins function as RasGEFs necessary for RAS activation (Christensen et al. 2016). RAS/RAF signalling promotes cell proliferation and survival via c-Myc induction (Zhang & Liu 2002). RAS mutations are among the most common oncogenic drivers, maintaining RAS in a constitutively active GTP-bound state and stimulating proliferative cascades (Zenonos & Kyprianou 2013; Moore et al. 2020). BRAF mutations, such as V600E, contribute to dysregulated MAPK signalling in many cancers, including ovarian cancer (Sadlecki et al. 2017), confirming the relevance of this pathway. JNK signalling, activated by stress and cytokines, is frequently deregulated in cancer and modulates differentiation, migration and survival (Wagner & Nebreda 2009). IL1R–IRAK–TRAF signalling also contributes to tumour-associated inflammation (Hosseini et al. 2018). TAB/TAK1 complexes participate in both tumour suppression and activation of cancer hallmarks such as immune evasion and metastatic dissemination, depending on context (Mukhopadhyay & Lee 2020). NF-κB, a central transcriptional regulator, promotes tumour survival and antagonises JNK-mediated apoptosis (Vacarezza & Vitale 2016). ATF2 contributes to stress responses and cell growth and acts as either oncogene or tumour suppressor depending on context (Lopez-Bergami et al. 2010). DUSPs fine-tune MAPK/ERK/JNK/p38 activity via dephosphorylation and are subject to miRNA regulation (Huang & Tan 2012; Chen et al. 2019). FGF signalling plays key roles in development and cancer, with aberrant FGF activity driving tumour progression (Turner & Grose 2010; Xie et al. 2020).

Cross-regulation among these pathways shows how placental miRNAs could simultaneously coordinate developmental processes and constrain trophoblast invasiveness, while their dysregulation in cancer removes these inhibitory mechanisms (Zhang & Liu 2002; Yang et al. 2020). Notably, Zhang et al. (2024) demonstrated that C19MC-containing placental extracellular vesicles can repress oncogenic signalling, suggesting that the trophoblast context restricts miRNA-mediated pathways in ways that cancer cells escape. Finally, changes in miRNA profiles have been described across numerous cancers, including breast cancer, leukaemia, and prostate cancer. Collectively, the evidence supports that miRNAs, especially those of the C19MC cluster, may regulate key networks required for human placental development, carcinogenesis and tumour progression. These findings highlight the biological continuity between embryonic development and oncogenic transformation and support the potential use of placental miRNAs in novel therapeutic strategies, improved patient stratification and the understanding of tumour progression mechanisms. For future validation, we propose to experimentally validate the computational findings, we propose qRT-PCR assays for selected miRNAs in placental samples and cancer cell lines. Although not performed in the present study, this protocol establishes a clear path for functional validation.