Introduction

Breast cancer remains one of the most prevalent cancers among females worldwide, with over 2 million new cases diagnosed in 2021 [1]. Several risk factors contribute to breast cancer, including alcohol consumption, radiation exposure, hormonal imbalance, a sedentary lifestyle, and hereditary genetic factors [1, 2]. Extensive research has linked the expression of specific miRNAs to cancer, indicating their potential as cancer biomarkers [3]. miRNAs are non-coding RNAs (17 to 25 bp) that can suppress mRNA expression by binding to the 3’ or 5’ UTR, regulating various biological activities such as cell cycle control and apoptosis, and playing crucial roles in cancer initiation and progression. These short non-coding single-stranded RNAs act post-transcriptionally to regulate gene expression. During miRNA biogenesis, the pre-miRNA precursor arms can generate either 5p or 3p miRNA species [4,5,6]. The selective maturation or co-existence of the 5p and 3p species is biologically significant as they target different sets of genes [7, 8]. miRNAs, particularly those within the same family, are often clustered in specific chromosomal locations to provide regulatory advantages [9]. One such human miRNA cluster, the chromosome 19 miRNA cluster (C19MC), is mapped on chromosome 19 and comprises 46 highly homologous miRNA genes [9, 10]. C19MC is a primate-specific miRNA cluster that emerged late in primate evolution, with bioinformatics analyses suggesting its crucial roles in primate reproduction, development, and differentiation compared to lower vertebrates [11, 12]. While C19MC is predominantly expressed in reproductive tissues and certain cancers like colorectal cancer, breast cancer, and primitive neuroectodermal brain tumors [13,14,15], its role in EMT gene regulation remains unknown [16,17,18]. Certain miRNAs, such as mir-224 and mir-373, have been shown to correlate with breast cancer [19, 20].

Extracellular RNAs (exRNAs), including miRNAs, present in biofluids are proposed as non-invasive biomarkers for monitoring organ function and can be quantitatively analyzed. In plasma, miRNAs exist as soluble ribonucleoproteins enclosed in exRNA carriers and transported through the vasculature. However, comprehensive studies involving healthy individuals are necessary to gain insights into the abundance and compositional variability of plasma miRNAs [21,22,23].

The aim of this study is to compare the expression of miRNAs from the C19MC cluster in breast cancer tumor and non-tumor samples, as well as in the serum of individuals affected by breast cancer and healthy individuals. The study aims to investigate the potential role of C19MC miRNAs as non-invasive tumor markers and their overall impact on breast cancer detection.

Methods and materials

Samples

In this case–control study, we enrolled 100 patients diagnosed with breast cancer as the case group, and peripheral venous blood samples were collected from healthy individuals as the control group. Tumor tissue samples and adjacent non-tumor tissues were collected from the 100 breast cancer patients, 20 of whom had a family history of cancer. Due to limitations in sample collection and the necessity to minimize test errors and unwanted variables, we collected both tissue and serum samples from the same breast cancer-affected patients.The patients’ ages ranged from 25 to 81, with an average age of 48 years. The study was approved by the Ethics Committee of the Department of Biology at the University of Tabriz (14,000,907-198-7). It followed ethical guidelines outlined in the 1975 Declaration of Helsinki, and full written consent was obtained from all participants.

RNA extraction and Real-time assay (qPCR)

RNA extraction was carried out using a commercially available kit from ZiAViZ company, which utilizes magnetic nanoparticles. Samples were analyzed using the one-step real-time RT-PCR method, which involves four primers: RP1, RP2, P1, and P2. RP1 contains P1 and an 11-base sequence that is complementary to the 3’ end of the target sncRNA, while RP2 contains P2 and an 11-base sequence that is identical to the 5’ end of the target sncRNA. The detection process includes three stages: in Stage 1, reverse transcription occurs, where RP1 hybridizes with the target sncRNA, which is then extended in the presence of Reverse Transcriptase M-MLV (RNase H-) and dNTPs at 37 °C. In Stage 2, RP2 hybridizes with the cDNA of the miRNA at 37 °C, and both sequences are extended in the presence of hot-start Taq polymerase (HS Taq) and dNTPs at 60 °C after denaturation at 95 °C. Stage 3 involves conventional PCR with the primers P1 and P2, and the amplification of cDNA is continuously monitored in real-time using SYBR green I (Table 1).

Table 1 The characteristics of the primers used in this study
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Statistical analyses

Statistical analyses were performed using GraphPad Prism software (v8.04) with a p-value of 0.05 considered significant. Receiver operating characteristic (ROC) curves were constructed to assess the diagnostic accuracy of identified miRNAs as potential biomarkers for breast cancer. The area under the curve (AUC) was calculated to assess diagnostic performance, with a higher AUC value indicating better diagnostic accuracy.

Results

The findings showed that certain C19MC miRNAs, including mir-515–1, mir-515–2, mir-516-A1, mir-516-A2, mir-516-B1, mir-516-B2, mir-517-A were highly expressed in breast cancer tissue and serum samples (Table 2). Additionally, the study revealed that the expression of several of these miRNAs was significantly different between tumor margins and breast cancer tumors, indicating the potential role of these miRNAs in driving the breast cancer phenotype.

Table 2 The P-Value of expression chang in Tomoral tissue and serum in breast cancer patients
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Importantly, ROC curve analysis showed that mir-515–1, mir-515–2, mir-516-A1, mir-516-A2, mir-516-B1, mir-516-B2, and mir-517-A had high sensitivity and specificity and could serve as potential tumor markers for breast cancer detection. The expression of mir-515–2, mir-516-A2, and mir-516-B2 in patient serum was also significantly different from that of the control group, indicating their potential as non-invasive biomarkers for breast cancer detection. In the context of biomarkers, a high AUC (Area Under the Curve) value of these microRNA in the ROC curve indicates the predictive performance of a biomarker in distinguishing between two groups or conditions (Figs. 1 and 2).

Fig. 1
figure 1

The expression of various microRNAs in tumor margin and BC tumor and ROC curves

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Fig. 2
figure 2

The detection of various micro-RNAs in case and control serums and ROC curves

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Discussion

Breast cancer is a prevalent cancer among females, and various risk factors, including non-coding RNAs, have been identified as essential in tumor initiation and progression [24,25,26]. C19MC is a micro-non-coding RNA cluster expressed predominantly in various tissues of the human genome [27]. This study aimed to analyze the expression of C19MC miRNAs in breast cancer tissue and tumor margins, as well as measure their concentration in serum samples of both cases and controls.

The findings revealed significant differences in the expression levels of several C19MC miRNAs, including miR-515–2, miR-516-A2, miR-516-B2, miR-516-B1, and miR-517-A, between breast cancer tumors and tumor margins. Notably, the expression levels of these miRNAs were higher in both breast cancer tissue and serum samples compared to tumor margins. Additionally, when comparing miRNA expression levels with their genomic positions, two different trends emerged: in breast cancer, miRNA expression slightly increased towards the 5ʹ end of the cluster, while in margin tissues, expression levels showed a decreasing tendency [26,27,28,29].

Furthermore, analysis of the data revealed differential expression between the two tissue sample sets. Extracellular RNAs (exRNAs) in biofluids are proposed as non-invasive biomarkers for monitoring organ function, and cell-lineage-specific microRNAs are present in plasma as soluble ribonucleoproteins enclosed in exRNA carriers and transported through the vasculature. However, more extensive studies involving healthy individuals are needed to gain insights into plasma miRNA abundance and composition variability.

The study also highlighted the functional roles of individual C19MC miRNAs in diverse systems. For example, mir-520-h has been found to inhibit migration and invasion of pancreatic cells [28], mir-520-b suppresses the migration of breast cancer cells by targeting hepatitis B X-interacting protein and IL-8 [29, 30], mir-520-c-3p inhibits hepatocellular carcinoma cell invasion by targeting glypican-3 [31] and abrogates breast cancer cell metastasis by suppressing TGFBR2 [32]. However, mir-520-c-3p may promote invasion and metastasis in breast tumor cells by suppressing CD44 [33].

Various studies suggest that miRNAs such as mir-512-3p, mir-512-5p, mir-516a-5p, mir-516b-5p, and mir-498-5p could play pivotal roles in the development of cancer therapies [34,35,36,37]. Future studies should focus on elucidating the molecular processes and pathways regulated by these miRNAs. Although C19MC is the largest human miRNA cluster, its functions are still relatively unknown [38,39,40]. With further understanding, it is reasonable to predict that C19MC could provide a variety of diagnostic and therapeutic targets in the field of cancer [41].

In conclusion, this study demonstrates significant differences in the expression of several C19MC miRNAs between tumor margins and breast cancer tumors, highlighting their potential role in breast cancer detection. C19MC miRNAs have the potential to be utilized as important and non-invasive biomarkers in breast cancer screening. However, many aspects of C19MC still need to be unraveled, posing a challenge for future research in this area. Further studies are necessary to elucidate the molecular processes and pathways involved.