Breast cancer plasma transporter miRNA marker and application thereof

Abstract

The invention relates to a breast cancer plasma transporter miRNA marker and application thereof, and belongs to the technical field of biological detection. In the prior art, biomarkers for breast cancer liquid biopsy have the problems of low content, high heterogeneity, insufficient time resolution and the like, so that the detection reliability is limited. According to the invention, a specific miRNA (micro Ribonucleic Acid) marker (including one or more of let-7d-5p, miR-103a-3p and miR-22-3p) of the plasma transporter is provided. The marker can be used for preparing a breast cancer detection reagent or kit. Clinical verifications show that the miRNAs are remarkably and highly expressed in plasma migrators of breast cancer patients, the AUC value reaches 0.763-0.808, the sensitivity is 66.7%-86.7%, the specificity is 66.7%-88.3%, the miRNAs have the advantages of high signal-to-noise ratio and dynamic monitoring, and the accuracy and practicability of breast cancer detection are remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology and relates to miRNA biomarkers of breast cancer plasma transporters and their applications. Background Technology

[0002] Breast cancer (BC) remains the most common type of cancer among women worldwide and the leading cause of cancer-related deaths. It is projected that by 2040, the number of new cases globally will exceed 3 million annually, and the death toll will surpass 1 million. Encouragingly, mortality rates continue to decline in regions with widespread early screening and advanced treatments, highlighting the crucial role of timely screening in reducing breast cancer mortality. While tissue biopsy remains the gold standard for diagnosis, its invasiveness limits the practicality of repeat monitoring. Against this backdrop, liquid biopsy has become a powerful tool for early detection, prognostic assessment, and disease monitoring of breast cancer. However, current biomarkers for liquid biopsy—including circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and exosomes—are scarce and highly heterogeneous in complex backgrounds, and lack sufficient temporal resolution for real-time monitoring of dynamic disease progression, thus limiting their reliability and clinical application.

[0003] Migratory bodies, organelles dependent on cell migration, are emerging as important mediators in liquid biopsies, exhibiting unique advantages over traditional biomarkers. These vesicular structures form specifically along contractile fibers during cell movement, imparting spatiotemporal specificity and allowing direct quantification of in vivo migration activities—such as tumor spread or immune cell migration. Crucially, migratory bodies selectively enrich functional substances such as chemokines, calcium ions, and nucleic acids, rather than randomly packaging cellular contents. This active enrichment mechanism significantly enhances disease specificity and signal strength. Furthermore, migratory bodies fill a key gap in liquid biopsies by capturing real-time dynamic cellular behavior, as evidenced by their role in reshaping the tumor microenvironment and promoting metastasis in various cancer types. The diagnostic potential of migratory bodies is already emerging, for example, using urinary podocyte migratory bodies to diagnose kidney disease. With their behaviorally correlated origins, selectively packaged substances, and dynamic functional insights, migratory bodies represent a new generation of liquid biopsy biomarkers, breaking through static molecular profiling analysis and providing a window into active disease processes.

[0004] MicroRNAs (miRNAs) are small non-coding RNAs, approximately 22 nucleotides in length, that regulate gene expression by inhibiting mRNA translation and / or inducing degradation, playing a crucial role in cancer progression. Compared to extracellular vesicles or free-living miRNAs, miRNAs exhibit selective enrichment in migratory bodies, giving them unique advantages as biomarkers for liquid biopsies. First, migratory body miRNAs provide behavior-specific detection by directly reflecting the active migration behavior of specific cell populations. Detection of tumor-associated miRNAs in migratory bodies not only indicates the presence of a tumor but also specifically reveals the ongoing invasion and metastasis process, significantly enhancing clinical diagnostic value. Second, the selective loading mechanism ensures a high signal-to-noise ratio by centrally encapsulating specific miRNAs. This enrichment effect offers a dual advantage: enhancing disease specificity through the accumulation of migration-related regulatory miRNAs, while reducing background interference through local amplification, thus improving detection sensitivity. Third, migratory body miRNAs provide dynamic functional insights unavailable with conventional miRNA analysis. While conventional methods only provide static molecular snapshots, migratory body miRNAs can capture cellular behavior in real time, potentially revealing the molecular programs driving key processes such as epithelial-mesenchymal transition and matrix remodeling. Therefore, miRNAs not only have diagnostic value, but also serve as a functional window for observing disease activity mechanisms, making them a next-generation biomarker for tracking dynamic pathological processes. Summary of the Invention

[0005] In view of this, one objective of the present invention is to provide a breast cancer plasma transporter miRNA marker, and another objective is to provide an application of the breast cancer plasma transporter miRNA marker.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a miRNA biomarker for breast cancer plasma transporters, wherein the biomarker is one or more of let-7d-5p, miR-103a-3p and miR-22-3p; Preferably, the miRNA is derived from plasma transporters; Preferably, the nucleic acid sequence of let-7d-5p is shown in SEQ ID NO:1, the nucleic acid sequence of miR-103a-3p is shown in SEQ ID NO:3, and the nucleic acid sequence of miR-22-3p is shown in SEQ ID NO:7; Application of the breast cancer plasma transporter miRNA marker in the preparation of breast cancer detection reagents; Application of the aforementioned breast cancer plasma transporter miRNA markers in the preparation of breast cancer detection kits; Furthermore, the present invention also provides a breast cancer detection kit, comprising any one of the aforementioned breast cancer plasma transporter miRNA markers.

[0007] The beneficial effects of this invention are as follows: 1. Provides novel diagnostic biomarkers for breast cancer with high specificity and high sensitivity. This invention is the first to discover and validate that let-7d-5p, miR-103a-3p, and miR-22-3p can serve as specific miRNA biomarkers for breast cancer plasma transporters. These three biomarkers exhibit excellent diagnostic performance in distinguishing breast cancer patients from healthy controls / non-breast cancer patients. The AUC of let-7d-5p was 0.763, and the specificity was 73.3%. The AUC of miR-103a-3p was 0.801, with a specificity as high as 88.3%. The miR-22-3p has an AUC of 0.808 and a sensitivity of 86.7%.

[0008] 2. By leveraging the unique advantages of transporters, the signal-to-noise ratio and specificity of detection are improved. Compared with traditional liquid biopsy biomarkers (such as ctDNA and exosomes), this invention, based on migration bodies, has core advantages: Behavioral correlation: The formation of the miRNA is directly related to cell migration activities. Therefore, the miRNA it carries can not only indicate the presence of tumors, but also specifically reflect the dynamic behaviors of tumor cells such as invasion and metastasis, providing functional information that goes beyond static molecular snapshots. Selective enrichment: Instead of randomly packaging cellular contents, the mitosome actively enriches specific miRNAs associated with migration. This mechanism significantly enhances the intensity of disease-related signals and reduces background noise, thereby achieving higher detection sensitivity and specificity.

[0009] 3. It provides a new and effective tool for the early detection and dynamic monitoring of breast cancer. The biomarkers provided by this invention offer a new solution for the clinical management of breast cancer: Non-invasive and convenient: Based on plasma samples, it avoids the invasive pain of traditional tissue biopsies, and facilitates repeated sampling and dynamic monitoring of disease progression or treatment response; Potential for early screening: Its high sensitivity and specificity make it a potential candidate for early screening of breast cancer, which could help achieve early detection and treatment, ultimately reducing mortality.

[0010] This invention utilizes the unique biological characteristics of the migratory body, a novel organelle, to successfully screen for a high-performance combination of plasma miRNA biomarkers for breast cancer. This technique not only provides a high-precision, non-invasive diagnostic method but also demonstrates significant potential application value in dynamic disease monitoring and prognostic assessment due to its ability to reveal tumor migration behavior.

[0011] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0012] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1 For the purpose of this study, A. Isolation, sequencing, and differential expression analysis of cell milosomes; B. Validation of cell milosome miRNAs; C. Validation of plasma milosome miRNAs. Figure 2 Image of cell migration bodies: A. WGA staining, B. Scanning electron microscopy, C. Transmission electron microscopy; Figure 3 The sequencing results are shown in Figure A. Venn diagram and Figure B. heatmap. Figure 4 For differential expression volcano plots, A. MDA-MB-231 migmatid vs. MCF-10A migmatid, B. Hs578T migmatid vs. MCF-10A migmatid; Figure 5 The images are from RT-qPCR studies. A. MDA-MB-231 miraculosome vs. MCF-10A miraculosome; B. Hs578T miraculosome vs. MCF-10A miraculosome. Figure 6 Scanning electron micrographs of other breast cancer cell migration bodies; Figure 7 Figures from RT-qPCR: A. MCF-7 miraculosome vs. MCF-10A miraculosome; B. BT474 miraculosome vs. MCF-10A miraculosome. Figure 8 The images show the characterization of plasma transporters, A. Transmitted electron microscopy, B. Western blot. Figure 9 These are images used for clinical validation; A. let-7d-5p, B. miR-103a-3p, C. miR-22-3p; Figure 10This is a graph showing the ROC analysis. Detailed Implementation

[0013] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0014] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual pictures. They should not be construed as limiting the invention. To better illustrate the embodiments of the invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0015] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "front," and "rear" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present invention. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0016] Example 1: Extraction and Analysis of Cell Migrations Cell migration bodies were extracted using a modified protocol from the literature. Cells were seeded in 150 mm culture dishes pre-coated with fibronectin (1 µg / mL) and cultured for 24–48 hours according to the cell growth rate. The culture medium was then discarded, and the cells were washed once with PBS. Cell suspension was then prepared by trypsin-EDTA digestion, and all centrifugation was performed at 4°C. Debris was removed by centrifugation at 600 g for 10 min, followed by centrifugation at 2000 g for 20 min. The supernatant was centrifuged at 18,000 g for 30 min to separate the crude migration bodies. The crude migration bodies were dissolved in extraction buffer and mixed with 10% OptiPrep™ at a 1:1 volume ratio (total volume 2 mL). The mixture was loaded onto a pre-packed iodixanol density gradient (concentration gradient from bottom to top: 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1 mL per layer). After centrifugation at 150,000 g for 4 hours, the gradient layers were gently collected from top to bottom (1 mL per layer). Layers 4-6 were mixed with 1000 µL PBS and centrifuged at 18,000 g for 30 minutes. The precipitate was washed with PBS and centrifuged again at 18,000 g for 30 minutes. The resulting precipitate was used directly for Western blotting (WB), transmission electron microscopy (TEM), and RT-qPCR analysis.

[0017] Confocal culture dishes (35 mm) were coated with 10 µg / mL fibronectin at 37°C for at least 30 minutes. Cells were seeded and cultured under standard conditions (37°C, 5% CO2) for 16–20 hours. After fixation with 4% paraformaldehyde (PFA) at room temperature for 10 minutes, cells were washed 2–3 times with phosphate-buffered saline (PBS). Subsequently, samples were stained in the dark with 1 µg / mL WGA-Alexa Fluor™ 488 for 10 minutes, washed with PBS, and observed (e.g., ...). Figure 2 A).

[0018] Using a superfiltration method, 25 mm cell slides were placed in six-well plates and coated with 10 µg / mL fibronectin at 37°C for at least 30 minutes. Cells were then seeded onto slides and cultured under standard conditions for 16–20 hours. Next, 1 mL of 2.5% glutaraldehyde solution was added, and the plates were fixed at room temperature for 2 hours. After removing the fixative, the samples were washed three times with PBS buffer for 15 minutes each time. Subsequently, the samples were fixed with 1% osmium tetroxide solution for 1–2 hours, and after carefully removing the osmium tetroxide, washed three more times with PBS buffer (15 minutes each time). A gradient ethanol dehydration method was used (30%, 50%, 70%, 80%, 90%, 95% concentrations), with each concentration treated for 15 minutes. After treatment with 100% ethanol for 20 minutes, the samples were transferred to fresh 100% ethanol. Drying was completed using a critical point dryer. The samples were fixed onto a carbon-coated stage with conductive adhesive and sputtered with platinum using an ion sputtering instrument for approximately 120 seconds. Finally, images were captured using a Hitachi Regulus 8100 scanning electron microscope (Hitachi, Japan). Images were also captured using a high-resolution laser scanning confocal microscope (e.g., Figure 2 B).

[0019] The purified migrations were negatively stained for ultrastructural observation. The specific procedure was as follows: 10 μL of the purified migrations were added to a copper grid and allowed to stand for 1 minute. Excess liquid was blotted out with filter paper, followed by the addition of 10 μL of uranium acetate solution, and the mixture was allowed to stand for another minute. Excess liquid was blotted out again with filter paper, and the mixture was allowed to dry at room temperature for several minutes. Images were then captured using a Hitachi HT7800 transmission electron microscope (Hitachi, Japan) at 80-120 kV (e.g., ...). Figure 2 C).

[0020] Example 2: Cell Migratory Body Analysis miRNA expression profiling was performed on migratory cells derived from MDA-MB-231, Hs578T, and MCF-10A cell lines. The results showed that migratory cells from these three cell lines shared 168 overlapping miRNAs (e.g., ...). Figure 3 A), of which 166 are known miRNAs (such as... Figure 3 B).

[0021] Using the MCF-10A cell migration complex as a control, a screening criterion for differential expression between the experimental and control groups was established (|log2 fold change| ≥ 1, P ≤ 0.01). The results showed that, compared with MCF-10A cells, the miRNAs of MDA-MB-231 and Hs578T cells showed significant co-upregulation of seven miRNAs: let-7d-5p, miR-100-5p, miR-103a-3p, miR-146a-5p, miR-21-3p, miR-210-3p, and miR-22-3p (e.g., ...). Figure 4The miRNA sequence listing is shown in Table 1. The target genes of these upregulated miRNAs are primarily enriched in cancer pathways, with secondary enrichment observed in breast cancer. These findings suggest that these seven miRNAs may serve as biomarkers for breast cancer diagnosis.

[0022] Total RNA was extracted from cells and migratory organisms using TRIzol™ reagents. cDNA synthesis was then performed using a polyadenylated miRNA first-strand cDNA synthesis kit. Quantitative PCR was conducted in 20 μL volumes using 2×SGFast qPCR Master Mix premixed buffer. The thermal cycling conditions were: 95°C pre-denaturation for 3 min, followed by 40 cycles (95°C for 5 s → 60°C for 30 s), and finally, melting curve analysis was performed. The temperature was increased at a rate of 0.5°C / s from 65°C to 95°C. To target the expression level of miRNAs in cell migratory organisms, 2... -△△Ct The cDNA assay was performed using U6 as an internal control and MCF-10A cells and migratory controls as calibration samples (normalized to 1.0-fold). U6 and the universal reverse primer are included in the cDNA kit.

[0023] Next, we validated the differential expression of these seven miRNAs using RT-qPCR. MCF-10A cell migration bodies were used as the control group in the experiment. The results showed (e.g.) Figure 5 The expression levels of miRNAs in MDA-MB-231 cell migratory cells ranged from 1.81-fold (miR-103a-3p) to 485.64-fold (miR-146a-5p), while those in Hs578T cell migratory cells ranged from 4.95-fold (miR-21-3p) to 699.51-fold (miR-146a-5p). RT-qPCR results were highly consistent with sequencing data, confirming that the expression levels of these miRNAs were significantly upregulated in both MDA-MB-231 and Hs578T cell migratory cells compared to MCF-10A cell migratory cells.

[0024] To investigate the widespread presence of migratory bodies in breast cancer cells with different invasive potentials, we selected two representative cell lines, MCF-7 and BT-474, for validation. The purified migratory bodies, observed under negative staining transmission electron microscopy, exhibited characteristic vesicle morphologies ranging from 0.5 to 3 μm in diameter (e.g., ...). Figure 6 Subsequently, we used real-time quantitative PCR to compare the expression differences of seven miRNAs in MCF-7, BT-474, and MCF-10A cell migration bodies, confirming that five miRNAs (let-7d-5p, miR-103a-3p, miR-21-3p, miR-210-3p, and miR-22-3p) were upregulated in MCF-7 and BT-474 cell migration bodies (e.g., let-7d-5p, miR-103a-3p, miR-21-3p, miR-210-3p, and miR-22-3p). Figure 7By comparing and analyzing the migratory variants of four breast cancer cell lines (MDA-MB-231, Hs578T, MCF-7, and BT-474) with the MCF-10A cell line, five continuously upregulated miRNAs (let-7d-5p, miR-100-5p, miR-21-3p, miR-210-3p, and miR-22-3p) were identified and used for subsequent clinical analysis.

[0025]

[0026] Example 3 Clinical Validation This invention included 60 plasma samples. All samples were collected from the First Affiliated Hospital of Army Medical University, and collection was completed before the intervention was implemented. To isolate the migratory bodies from the plasma, we modified the previously published protocol as follows: In short, 2 ml of blood was first collected using an EDTA anticoagulant tube and centrifuged at 800g for 10 minutes at room temperature to remove blood cells. The plasma was then transferred to a low-absorption 15 ml centrifuge tube and centrifuged at 1000g for 5 minutes at 4°C to further remove residual blood cells. The supernatant was collected into a low-absorption EP tube and diluted with EDTA-containing PBS buffer. Next, it was centrifuged at 20,000g for 60-90 minutes at 4°C, and 90% of the supernatant was aspirated. Finally, it was centrifuged at 20,000g for 10 minutes at 4°C, and the remaining supernatant was aspirated. The resulting precipitate and plasma migratory bodies were used for subsequent experiments.

[0027] Migrations isolated from human plasma were characterized. Transmission electron microscopy imaging revealed that typical migratory structures consist of vesicles interconnected by contractile filaments, and some migratory structures also contained migratory-derived nanoparticles (MDNPs) (e.g., ...). Figure 8 A). Western blot analysis confirmed the presence of migration-specific marker proteins (such as...) in all plasma migrations. Figure 8 B).

[0028] After isolating the mitozosomes from plasma, miRNAs were extracted using TRIzol reagent, and U6 and five candidate miRNAs were quantified by RT-qPCR. Results showed no significant difference in U6 expression between breast cancer (BC) and non-breast cancer (N-BC) patients. P =0.074), but the expression of all five target miRNAs showed significant differences (P<0.05). Notably, miR-21-3p and miR-210-3p had excessively high Ct values ​​(>35), indicating insufficient abundance in plasma transporters for reliable detection, and were therefore not included in subsequent clinical validation. Previous studies have shown that U6 is unsuitable as an internal reference gene for circulating miRNA quantification and should be standardized by calculating the global mean ΔCt value. -ΔCtDefined as the normalized level of miRNA. Finally, we compared the expression levels of let-7d-5p, miR-103a-3p, and miR-22-3p in healthy controls (n=18), non-breast cancer patients (BBD, n=12), and breast cancer patients (n=30) (2). -ΔCt Of particular note is that these three migration miRNAs showed significant differences between non-breast cancer patients (including healthy controls and non-breast cancer patients, n=30) and breast cancer patients (n=30). P <0.001), (e.g. Figure 9 ).

[0029] ROC analysis indicates (e.g.) Figure 10 These three miRNAs have diagnostic value for breast cancer detection. They performed well in distinguishing breast cancer (BC) from healthy controls (HC) and breast-derived breast cancer (BBD). Specifically, let-7d-5p had an AUC of 0.763 (95% CI, 0.644–0.883), with a sensitivity of 66.7% and a specificity of 73.3%. miR-103a-3p had an AUC of 0.801 (95% CI, 0.690–0.912), also with a sensitivity of 66.7% and a specificity as high as 88.3%. Similarly, miR-22-3p had an AUC of 0.808 (95% CI, 0.699–0.917), with a sensitivity of 86.7% and a specificity of 66.7%.

[0030] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Plasma migrator miRNA markers of breast cancer, characterized in that: The miRNA is one or more of let-7d-5p, miR-103a-3p and miR-22-3p.

2. The breast cancer plasma-migrating body miRNA marker according to claim 1, characterized by: The miRNA is derived from plasma migratory bodies. 3.The breast cancer plasma-migrating body miRNA marker of claim 2, characterized in that: The nucleic acid sequence of the let-7d-5p is shown as SEQ ID NO: 1, the nucleic acid sequence of the miR-103a-3p is shown as SEQ ID NO: 3, and the nucleic acid sequence of the miR-22-3p is shown as SEQ ID NO:

7.

4. The breast cancer plasma migratory body miRNA marker according to claim 3.

5. Use of the breast cancer plasma migratory body miRNA marker according to any of claims 1-4 in the preparation of a breast cancer detection reagent.

6. Use of the breast cancer plasma migratory body miRNA marker according to any of claims 1-4 in the preparation of a breast cancer detection kit.

7. A breast cancer detection kit characterized by: A breast cancer detection reagent or kit comprising any of the breast cancer plasma migratory body miRNA markers according to claims 1-4.

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