Liquid biopsy recently became a very promising diagnostic method that has several advantages over conventional invasive methods. Liquid biopsy may serve as a source of several important biomarkers including cell-free nucleic acids (cf-NAs). Cf-DNA is widely used in prenatal testing in order to characterize fetal genetic disorders. Analysis of cf-DNA may provide information about the mutation profile of tumor cells, while cell-free non-coding RNAs are promising biomarker candidates in the diagnosis and prognosis of cancer. Many of these markers have the potential to help clinicians in therapy selection and in the follow-up of patients. Thus, cf-NA-based diagnostics represent a new path in personalized medicine. Although several reviews are available in the field, most of them focus on a limited number of cf-NA types. In this review, we give an overview about all known cf-NAs including cf-DNA, cf-mtDNA and cell-free non-coding RNA (miRNA, lncRNA, circRNA, piRNA, YRNA, and vtRNA) by discussing their biogenesis, biological function and potential as biomarker candidates in liquid biopsy. We also outline possible future directions in the field.

We are on the brink of a paradigm shift in both theoretical and clinical oncology. Genomic and transcriptomic profiling, alongside personalized approaches that account for individual patient variability, are increasingly shaping discourse. Discussions on the future of personalized cancer medicine are mainly dominated by the potential of non-coding RNAs (ncRNAs), which play a prominent role in cancer progression and metastasis formation by regulating the expression of oncogenic or tumor suppressor proteins at transcriptional and post-transcriptional levels; furthermore, their cell-free counterparts might be involved in intercellular communication. Non-coding RNAs are considered to be promising biomarker candidates for early diagnosis of cancer as well as potential therapeutic agents. This review aims to provide clarity amidst the vast body of literature by focusing on diverse species of ncRNAs, exploring the structure, origin, function, and potential clinical applications of miRNAs, siRNAs, lncRNAs, circRNAs, snRNAs, snoRNAs, eRNAs, paRNAs, YRNAs, vtRNAs, and piRNAs. We discuss molecular methods used for their detection or functional studies both in vitro and in vivo. We also address the challenges that must be overcome to enter a new era of cancer diagnosis and therapy that will reshape the future of oncology.

Exposure to physiological estrogens or xenoestrogens (e.g., zearalenone or bisphenol A) increases the risk for cancer. However, little information is available on their significance in ovarian cancer. We present a comprehensive study on the effect of estradiol, zearalenone and bisphenol A on the phenotype, mRNA, intracellular and cell-free miRNA expression of human epithelial ovarian cell lines. Estrogens induced a comparable effect on the rate of cell proliferation and migration as well as on the expression of estrogen-responsive genes (GREB1, CA12, DEPTOR, RBBP8) in the estrogen receptor α (ERα)-expressing PEO1 cell line, which was not observable in the absence of this receptor (in A2780 cells). The basal intracellular and cell-free expression of miR200s and miR203a was higher in PEO1, which was accompanied with low ZEB1 and high E-cadherin expression. These miRNAs showed a rapid but intermittent upregulation in response to estrogens that was diminished by an ERα-specific antagonist. The role of ERα in the regulation of the MIR200B-MIR200A-MIR429 locus was further supported by publicly available ChIP-seq data. MiRNA expression of cell lysates correlated well with cell-free miRNA expression. We conclude that cell-free miR200s might be promising biomarkers to assess estrogen sensitivity of ovarian cells.

MicroRNAs are short non-coding RNA molecules that are involved in tumor development and are considered to be promising candidates in cancer therapy. Here, we studied the role of miR-30s in the pathophysiology of ovarian cancer. According to our results miR-30a-5p, miR-30d-5p, and miR-30e-5p were overexpressed in the estrogen receptor α (ERα)-expressing PEO1 cell line compared to A2780 that lacks this receptor. Furthermore, the expression of miR-30a-5p, miR-30d-5p, and miR-30e-5p were induced in response to high-dose estrogen treatment in PEO1 where intensive cell death was observed according to the induction of apoptosis and autophagy. Lacking or blocking ERα function reduced tolerance to high-dose estrogen that suggests the importance of ERα-mediated estrogen response in the maintenance of proliferation. MiR-30d-5p mimic reduced cell proliferation in both A2780 and PEO1. Furthermore, it decreased the tolerance of PEO1 cells to high-dose estrogen by blocking the ERα-mediated estrogen response. This was accompanied by decreased SOX4 expression that is thought to be involved in the regulation of the PI3K/AKT pathway. Blocking this pathway by AZD8835 led to the same results. MiR-30d-5p or AZD8835 sensitized PEO1 cells to tamoxifen. We suggest that miR-30d-5p might be a promising candidate in the therapy of ovarian cancer.

Xenoestrogens are natural or synthetic compounds that mimic the effect of endogenous estrogens and might cause cancer. We aimed to compare the global transcriptomic response to zearalenone (ZEA; mycotoxin) and bisphenol A (BPA; plastic additive) with the effect of physiological estradiol (E2) in the PEO1 human ovarian cell line by mRNA and microRNA sequencing. Estrogen exposure induced remarkable transcriptomic changes: 308, 288 and 63 genes were upregulated (log₂FC > 1); 292, 260 and 45 genes were downregulated (log₂FC < −1) in response to E2 (10 nM), ZEA (10 nM) and BPA (100 nM), respectively. Furthermore, the expression of 13, 11 and 10 miRNAs changed significantly (log₂FC > 1, or log₂FC < −1) after exposure to E2, ZEA and BPA, respectively. Functional enrichment analysis of the significantly differentially expressed genes and miRNAs revealed several pathways related to the regulation of cell proliferation and migration. The effect of E2 and ZEA was highly comparable: 407 genes were coregulated by these molecules. We could identify 83 genes that were regulated by all three treatments that might have a significant role in the estrogen response of ovarian cells. Furthermore, the downregulation of several miRNAs (miR-501-5p, let-7a-2-3p, miR-26a-2-3p, miR-197-5p and miR-582-3p) was confirmed by qPCR, which might support the proliferative effect of estrogens in ovarian cells.

The term liquid biopsy reveals a non-invasive diagnostic method that might be based on the quantification of cell-free microRNAs in body fluids. However, the identification of candidates for liquid biopsy is challenging. Our aim was to compare the cell-free expression of miR-483-5p, miR-205-5p, and let-7f-5p in ovarian cell cultures and plasma samples of patients with ovarian cancer. Both the intracellular and cell-free expression of miR-205-5p and let-7f-5p proved to be higher in the Estrogen Receptor α (ERα) expressing PEO1 cell-line than in the estrogen non-sensitive A2780. Moreover, the expression of let-7f-5p was up-regulated in response to estradiol exposure that was diminished after the addition of an ERα selective antagonist. MiR-483-5p had lower intracellular and cell-free expression in PEO1. All these miRNAs had detectable expression level in plasma samples, among which miR-205-5p proved to be overexpressed in the plasma samples of patients with ovarian tumors compared to healthy controls and possessed an acceptable diagnostic potential with ROC-AUC 0.683 (95% CI 0.57–0.795). Functional annotation clustering of the target genes of miR-205-5p revealed several clusters involved in cancer development. We suggest that miR-205-5p might be a promising biomarker candidate in ovarian cancer that should be further analyzed in larger sample size.