Potential function of miRNA as a diagnostic biomarker for human ovarian cancer.

In 1993, Lee et al. found that the gene lin-4 controlling the development of Caenorhabditis elegans resulted in the downregulation of lin-14 protein expression [ 23 ]. Subsequently, Reinhart et al. identified another miRNA let-7 in the same nematode species [ 24 ]. Owing to the work in the past 30 years, researchers have found miRNAs to be widely expressed in multiple human cells. The first evidence of miRNA involvement in human cancer progression was obtained in chronic lymphocytic leukemia, with the detected downregulation of miR-15a/miR-16-1 [ 25 ]. miRNA was first identified as a biomarker for cancer in 2008, when it was used by Lawrie et al.. to identify diffuse large B-cell lymphoma in patients’ serum [ 26 ]. Currently, the knowledge of miRNAs as biomarkers remain new, and additional research is needed in future studies. miRNAs measure 20–25 nt and downregulate the expression of target genes at the post-transcriptional level [ 27 – 29 ]. Its production begins in the nucleus, where its precursor and primary miRNA are transcribed into precursor molecules of primary miRNA (pri-miRNA) under the influence of ribonucleic acid polymerases II and III [ 30 ]. The Drosha and DGCR8 enzyme complexes cleave pri-miRNAs into pre-miRNAs with a hairpin structure of 60–70 nt, which are then exported from the nucleus to the cytoplasm through the enzyme complex exportin-5/Ran GTP [ 31 ]. After entering the cytoplasm, pre-miRNAs are cleaved and further processed into short dsmiRNA (20–22 nt) by a second RNase endonuclease and its dsRNA binding partner, forming an RNA induced silencing complex (RISC), which refers to mature miRNAs, while the other strand is degraded [ 32 – 34 ] (Fig. 1 ). miRNAs regulate the RNA sequences of target genes based on their complementarity, typically through RISC-mediated RNA degradation and/or post-translational inhibition. A single miRNA can target multiple gene transcripts, and multiple miRNAs can simultaneously target a single mRNA transcript, regulating over 60% of human genes [ 35 , 36 ]. Recently, miRNAs have been considered to be closely related to the occurrence and development of cancer through the dysregulation of growth signals, dysregulation of cellular energetics, immune evasion, evasion of apoptosis, sustained angiogenesis, tissue invasion, and metastasis [ 37 – 39 ] (Fig. 1 ). Fig. 1 miRNA biogenesis and functioning mechanism miRNA biogenesis and functioning mechanism Another important role of miRNAs is in intercellular signal transduction. Most miRNAs exist within the cells; however, a large proportion migrate to the extracellular space [ 40 , 41 ]. Approximately 10% of these circulating miRNAs are secreted in exosomes, while the other 90% form complexes with proteins such as argonaute 2, nuclear phosphoprotein 1, and high-density lipoprotein, stabilizing their presence [ 42 ]. They are eliminated from the body through tissue damage, apoptosis, and necrosis, or through active channels, microbubbles, or binding to proteins in the blood, saliva, breast milk, semen, urine, and other fluids [ 43 ].

It is well known that miRNA can regulate gene expression, and epigenetic modifications such as high/low methylation and histone modifications have a direct impact on miRNA regulation [ 44 , 45 ]. Therefore, dysregulated miRNAs may affect one or more cellular pathways, such as cell cycle regulation and transposon silencing, which can lead to various pathological conditions. Chromosomal rearrangements, changes in genome copy number, epigenetic modifications, and defects in miRNA biogenesis pathways give rise to sudden expression of miRNAs [ 46 , 47 ]. miRNA dysregulation in cancer occurs through two possible epigenetic mechanisms, and miRNAs functioning in OC progression can be divided into two categories: (1) tumor suppressor miRNAs, which have tumor suppressor gene-like functions with upregulated expression, and (2) oncogenic miRNAs, which have oncogene-like functions with downregulated expression. Compared to protein markers, miRNAs undergo changes before protein expression and with tumor progression. In pre-operative patients with OC, the test results showed overexpression of certain miRNAs, while CA-125 test results remained unchanged [ 48 ], indicating that miRNAs may detect OC earlier than proteins, highlighting their advantage in the early detection and monitoring of different types of cancer. The differential expression profiles of miRNAs in the tissues, serum, plasma, and urine of patients with OC and controls further suggest their potential for early diagnosis. The expression of miRNAs in early OC tissues shows significant changes and has high specificity in the diagnosis of OC, particularly at an early stage. Differential expression of miRNAs may correspond to the pathogenesis of OC in different tissue types. For example, Lorio et al.. discovered a miRNA that could distinguish between normal ovarian and EOC tissues [ 49 ]. By comparing miRNAs in 29 tissues between patients with EOC and healthy controls, their results showed that the expression of miR-141, miR-200a, miR-200b, and miR-200c was significantly up-regulated, whereas the expression of 25 miRNAs was down-regulated, among which, miR-125b-1, miR-140, miR-145, and miR-199a were the most significantly downregulated. Recently, an increasing number of researchers have focused on the relationship between miRNAs and OC, with the aim of distinguishing the tissue types of OC (serous, endometrioid, clear cell, and mucinous) by studying the differential expression of different miRNAs. Previous studies have shown that, in a sample compared with benign ovarian tissue, the expression of miR-200b-3p, miR-135b-5p, and miR-182-5p is consistently overexpressed in tumor tissue and ascites, suggesting that they may be oncogenes, and the expression of miR-200 family members is significantly increased in the differentially expressed group [ 50 ]. Considering the wide range of miRNAs detected in tissues, Wang and colleagues established an EOC model using 41 ovarian tissue samples, 7 borderline serous ovarian tumors, and 15 normal ovarian controls [ 51 ]. Ten candidate miRNAs were identified in one miRNA microarray group, and it was found that, the expression of miR-1271-5p and miR-5743p was significantly down-regulated while that of miR-182-5p, miR-183-5p, miR-96-5p, miR-15b-5p, miR-182-3p, miR-141-5p, miR-130b-5p, and miR-135b-3p was up-regulated. Using these 10 miRNAs, this study distinguished OC tissues from normal tissue with 97% sensitivity and 92% specificity. A multi-cohort, retrospective, prospective study was conducted to detect stage I serous OC. Kandimalla et al. used a systematic biomarker validation method to identify miRNA biomarkers for the early detection of stage I high-grade serous OC [ 52 ]. A group consisting of eight miRNAs, including miR-182, miR-183, miR-202, miR-205, miR-508, miR-509-3, miR-513b, and miR-513 C, was subjected to miRNA sequencing in stage I high-grade serous OC tissues, and it was found that the area under the curve (AUC) of these miRNAs for detecting stage I OC was as high as 0.99. However, these promising data require validation in large, prospective, multi-center trials. To evaluate the diagnostic accuracy of these eight miRNAs for stage I OC, a logistic regression model (using TCGA and GSE65819 datasets) was applied for fitting analysis. The results showed that, the AUC of the eight miRNAs for detecting stage I OC in the TCGA cohort was 0.96, and the AUC for detecting all stages of OC in the TCGA and GSE65819 cohorts were 0.89 and 0.83, respectively. These results confirm the ability of these eight miRNAs to detect OC at the tissue level. More importantly, compared with CA-125 levels, these eight miRNAs showed significantly superior diagnostic performance, with an AUC of 0.73 for detecting CA-125 in patients with stage I-OC. This indicates that miRNA markers are highly accurate in detecting stage I OC in the serum, particularly superior to CA-125, and have a high specificity for OC detection, highlighting their potential role in the screening and early detection of OC. The AUC of the 8 miRNAs in OC is 0.85, while the detection accuracy in other diseases ranges from 0.19 to 0.71, reinforcing the notion that the 8 miRNA signals are superior to the conventional marker CA-125 and have high specificity for detecting OC. Thus, their potential for future clinical translation in non-invasive detection and population screening of OC at early stages is emphasized. Recent studies have also identified novel miRNAs, whose dysregulation is correlated with clinical stages, prognosis, and response to therapy in patients with OC (Table 1 ), shedding light on their potential as both diagnostic biomarkers and therapeutic targets. For example, miRNA-3652 is intricately involved in OC cell proliferation, migration, and invasion, and the dysregulation of miRNA-3652 is closely linked to the altered expression of critical genes involved in OC growth and metastasis [ 53 ]. Yang et al. reported that, miRNA-584 expression is significantly decreased in OC cells, and its level is correlated with lymphatic metastasis, distant metastasis, and poor prognosis in patients with OC [ 54 ], while miRNA‑199b‑3p declined OC progression by directly targeting ZEB1, providing a promising therapeutic target for OC [ 55 ]. Kumar and colleagues further found that the expression of targets of miRNA-205 ( BCL2 , ZEB1 , E2F1 , and TP53 ) and miRNA-34a ( MDM4 , MAPK3 , BRCA1 , and AREG ) was significantly downregulated and upregulated in EOC, respectively, highlighting the therapeutic potential of miRNAs in EOC management [ 56 ]. Similarly, miRNA-34a presents inhibitory effects on OC cells via directly binding and suppressing HDAC1 expression, subsequently decreasing resistance to cisplatin and proliferation in OC cells [ 57 ], miRNA-142-3p acts as an OC suppressor by directly targeting FAM83D in OC progression [ 58 ], miRNA-193a-5p hinders proliferation, migration and invasion of OC cells by targeting RAB11A [ 59 ], and miRNA-1271 suppresses OC cell growth by targeting Cyclin G1 [ 60 ], miRNA-613 suppresses OC cell proliferation and invasion by directly regulating KRAS [ 61 ], miRNA-125b-5p disrupts OC cell proliferation and metastasis by targeting CD147 [ 62 ], miRNA-424-5p positively regulates ferroptosis by targeting ACSL4 in OC cells [ 63 ], miRNA-1224-5p down-regulates the proliferation and invasion of OC cells by targeting SND 1 [ 64 ], miRNA-383 suppresses OC cell proliferation, invasion and aerobic glycolysis by targeting LDHA [ 65 ], miRNA-142-5p enhances cisplatin-induced apoptosis in OC cells by targeting multiple anti-apoptotic genes including baculoviral IAP repeat-containing 3 ( BIRC3 ), B-cell lymphoma-2 ( BCL-2 ), BCL2-like 2 ( BCL2L2 ), and myeloid cell leukemia sequence 1 ( MCL1 ) [ 66 ], miRNA-324-3p suppresses the aggressive OC through targeting the WNK2 /RAS pathway [ 67 ], miRNA-505-3p inhibits EOC cell motility by targeting PEAK1 [ 68 ], miRNA-204-5p suppresses OC cell proliferation by decreasing USP47 [ 69 ], and miRNA‑145 suppresses OC development via targeting cyclin D2 ( CCND2 ) and E2F transcription factor 3 ( E2F3 ) [ 70 ]. Table 1. A table summarizing the negative role of miRNAs in OC progression A table summarizing the negative role of miRNAs in OC progression In contrast, some miRNAs promote OC development (Table 2 ). For example, miRNA-96 accelerates the malignant progression of OC cells by directly targeting FOXO3a [ 71 ], miRNA-27a promotes progression of OC by mediating FOXO1 [ 72 ], miRNA-600 induces OC cells stemness, proliferation and metastasis by targeting Kruppel-like factor 9 (KLF9) [ 73 ], and intriguingly, exosomal miRNA-4516 derived from OC stem cells (OCSCs) enhances cisplatin tolerance in OC by suppressing GAS7 [ 74 ], miRNA-301b-3p accelerates migration and invasion of high-grade ovarian serous tumor by targeting the cytoplasmic polyadenylation element binding protein 3 (CPEB3)/EGFR axis [ 75 ], miRNA-135b improves proliferation and regulates chemotherapy resistance in OC cells [ 76 ], and miRNA-663 facilitates the growth, migration and invasion of OC cells via targeting tumor suppressor candidate 2 ( TUSC2 ) [ 77 ]. Therefore, targeting these miRNAs may be a promising strategy for the treatment of OC. Table 2. A table summarizing the positive role of miRNAs in OC progression A table summarizing the positive role of miRNAs in OC progression

Specific metabolic processes in kidney and urinary tract epithelial tissues can alter the expression patterns of miRNAs, thereby widening these differences. High levels of RNase in the urinary tract can lead to the complete degradation of free RNA. Compared with RNA, miRNAs are more resistant to nuclease degradation, mainly because of their smaller volume, and thus detected in urine [ 91 ]. The relatively small number of detectable miRNAs in the urine indicates that most circulating miRNAs are either metabolized by the kidneys through unknown mechanisms or disrupted in the urine. Yun et al. reported that the stability of miRNAs in urine supernatants remained unchanged even after seven cycles of freezing and thawing or 72 h of storage at room temperature [ 92 ]. In the long term, considering the potential convenience of urine testing in screening populations, further exploration of urine biomarkers may be desirable. Owing to their stability and non-invasive nature, this will be beneficial for the diagnosis of OC at an early stage (Fig. 2 ). Relatively few studies have used urine as a research object to detect OC; among these, important studies have discovered the role of urine miRNAs (Table 3 ) . Zhou et al.. studied miRNAs in the urine of patients with ovarian serous adenocarcinoma, analyzing urine samples from 39 patients with ovarian serous adenocarcinoma, 26 patients with benign gynecological diseases, and 30 healthy controls to determine the clinical value of urine miRNAs in detecting ovarian serous adenocarcinoma [ 93 ]. Compared to healthy samples, only miR-30a-5p was up-regulated (>2-fold) in the urine samples of patients with serous adenocarcinoma, whereas 37 miRNAs were down-regulated. Upregulation of miR-30a-5p in urine is closely related to early ovarian serous adenocarcinoma and lymph node metastasis, reflecting its association with early serous epithelial cancer screening. To verify this result, an miR-30a-5p -knockout model was established in OC cell lines, which was found to inhibit growth, indicating that this study is verifiable and may provide a more accurate screening test for serous epithelial cancer. In addition, urinary miR-30a-5p was significantly reduced in patients with OC after surgical tissue resection, whereas no reduction was observed in other solid tumors, such as gastric and colorectal cancer, after resection of cancer tissue. This indicates that urinary miR-30a-5p originates from ovarian serous adenocarcinoma tissue, and the same pattern was not observed in other solid tumors such as colorectal cancer. Therefore, these results suggest that the upregulation of miR-30a-5p in urine is specific for screening ovarian serous adenocarcinomas. Table 3. Summary of recent studies on miRNA detection in urine Meng et al. used a combination of miR-200a, miR-200b, and miR-200c to detect patients with EOC, resulting in 100% specificity and 83% sensitivity, with AUC values of 0.853, 0.953, and 0.903, respectively [ 94 ], indicating increased cancer specificity. Zavesky et al. detected the expression of miRNAs in the urine of patients with OC and endometrial cancer and recruited patients with EOC, fallopian tube cancer, endometrial cancer, and benign diagnosis of OC and endometrial cancer who underwent gynecological surgery [ 95 ]. The experiment compared the expression in preoperative and postoperative OC samples to determine whether cell-free miRNAs (miR-92a, miR-200b, miR-106b, and miR-100) could be detected and differentially expressed in the urine of patients with OC and endometrial cancer, compared to control patients. Eighteen miRNAs were tested, and the results showed significant differential expression of four miRNAs between OC and control samples. MiR-92a and miR-200b were up-regulated compared to the control samples, and miR-106b and miR-100 were down-regulated in cancer samples, with miR-92a significantly up-regulated (AUC = 1.00) and miR-106b significantly down-regulated (AUC = 0.969) in the OC group. These miRNAs should be further evaluated as promising candidate biomarkers for the early diagnosis of OC. Overall, to convert urine miRNAs into reliable diagnostic biomarkers, extensive research is needed and several issues still need to be addressed. Summary of recent studies on miRNA detection in urine The microRNA-10 (miR-10) family has been found to play an indispensable role in the evolution of many gynecological cancer types [ 96 , 97 ], and a case-control study showed the differential expression of certain miRNAs in the cells, extracellular space, and urine of patients with OC [ 98 ]. In particular, the expression of miR-10a in the urine of OC patients showed a slight downward trend. As per the study findings, changes in miRNA expression within cells, outside cells, and in urine suggested that they may be potential biomarkers for liquid biopsy [ 98 ]. Savolainen et al. found that, compared with malignant cases, benign samples showed low levels of miR-200a, miR-200b, and miR-200c in tissue and plasma samples [ 99 ]. However, in urine samples, the levels in two patients were significantly higher than those in the other samples, indicating greater variability and inconsistent expression patterns in this sample type. Meanwhile, there was a correlation between the expression of all three miRNAs in the plasma and urine samples of patients with malignant tumors. In addition, this preliminary study suggests that the expression levels of miR-200 in the plasma and urine correlate with high-grade serous ovarian cancer. Although these findings have potential value, large scale research is required for further validation. It is also necessary to evaluate the significance and role of urinarymiRNAs in patients with early-stage OC, especially in a larger patient population. Although current research is still in its early stages, some specific urinary miRNAs have been successfully identified, providing promising results for the diagnosis of OC in clinical practice.

Ovarian cancer (OC) is a common malignant gynecological cancer that seriously threatens the lives and health of women [ 1 – 3 ]. The incidence rate of OC is high, ranking second among female malignant tumors globally, with a case fatality rate ranking fourth. Approximately 530,000 cases occur worldwide every year, of which approximately 275,000 die [ 4 ]. Currently, OC is not believed to be a single disease with a high degree of heterogeneity. The most common type of OC, ovarian epithelial cell carcinoma (EOC), accounts for more than 90% of all gynecological malignancies and is the fifth leading cause of cancer-associated deaths in women [ 5 ]. Due to the insidious onset of OC, patients in the early stages (FIGO stages I-II) do not have obvious symptoms, and approximately 75% of patients with OC are often diagnosed in the late stages (FIGO stages III-IV), when the cancer cells have already spread widely in the abdominal cavity. If patients diagnosed at an early stage, the 5-year survival rate reaches 93%. However, there is no effective screening tool for OC, and the low specificity and high false-positive rates of OC screening have always been an issue. Transvaginal ultrasonography (TVS) and pelvic examination are currently effective methods for diagnosing OC, and though the diagnostic accuracy has improved to some extent, the sensitivity is not sufficiently high. On the other hand, despite the use of serological markers for the early diagnosis of OC, most markers have low specificity and sensitivity, such as OC tumor markers extensively studied including CA-125 [ 6 ], human epididymitis protein 4 (HE4) [ 7 ], alpha fetoprotein (AFP) [ 8 ], kallikrein [ 9 ], prostaglandins [ 10 ], and b-human chorionic gonadotropin (β-HCG) [ 11 ]. Currently, CA-125 is used as the main marker for differentiating between benign and malignant OC. The use of CA-125 for detecting OC is more widely accepted than that of other detection markers, suggesting its greater value in various applications. As priority markers for detecting EOC, CA-125 levels are associated with the OC stage [ 12 ]. While 35 U/ml is considered the upper limit of CA-125 levels in most healthy people, the levels of CA-125 are increased in 50%–60% of patients with OC in Stage I [ 13 ]. Although CA-125 has been routinely used as a clinical OC marker, its sensitivity for detecting early cancer is poor, and its specificity for OC is limited [ 14 ]. The levels of CA-125 are observed to be increased to varying degrees in some common cancers, such as breast cancer [ 15 ], uterine cancer [ 16 ], gastric cancer [ 17 ], liver cancer [ 18 ], and pancreatic cancer [ 19 ]. Similarly, elevated levels of CA-125 can also be found in other benign diseases, such as acute pelvic inflammatory disease [ 16 ], adenomyosis [ 20 ], uterine fibroids [ 21 ], and endometriosis [ 22 ], with false positives to varying extents. Despite the discovery of these tumor biomarkers and related detection techniques, the identification of ideal biomarkers with high specificity and sensitivity remains challenging. In addition, identification of specific biomarkers for each OC subtype to achieve effective diagnosis and treatment strategies is necessary. Currently, investigations on miRNA in the detection and monitoring of OC aim to provide a reliable detection method for OC at the early stages, which is considered a promising biomarker with great potential for several human cancers, including OC. For this review, PubMed, Web of Science, and Scopus databases were screened for articles from 2015 to 2025 (88 references, accounting for 88.9%; 11 references on history of miRNA discovery were before 2015) using keywords such as “miRNA”, “signaling pathways”, “biomarkers”, “ovarian cancer”, and “therapeutic”, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, and inclusion criteria focused on English-language studies.

The early detection of cancer as a diagnostic tool for reducing the mortality rate of OC has great prospects. miRNAs are closely related to the occurrence and development of OC. Although the research field of miRNA biomarkers is not yet mature, it is constantly evolving. Their use for detection is simple, convenient, causes less pain to patients, their expression is stable, and miRNAs are expressed and occur in different environments. Therefore, many specific miRNA signatures have demonstrated superior sensitivity and/or specificity to CA-125 in defined study cohorts (Fig. 2 ). The combination of protein biomarkers and miRNA detection has been shown to be superior. Using a set of biomarkers rather than a single biomarker can improve the diagnostic accuracy, leading to earlier detection and better outcomes. Many studies have also attempted to identify protein biomarkers that can be combined for early mRNA diagnosis of OC in clinical applications. Nevertheless, its application in clinical practice faces field-wide challenges, such as assay standardization and biological heterogeneity, which require further investigation and careful consideration.