Breast cancer screening biomarker combination as well as detection kit and application thereof

Abstract

The invention discloses a breast cancer screening biomarker combination as well as a detection kit and application thereof, and relates to the technical field of breast cancer screening biomarkers. Two kinds of circular RNAs (hsacirc0006174 and hsacirc0059914) are found and verified as breast cancer specific biomarkers, so that the breast cancer screening biomarker combination can be used for screening breast cancer; expression of the biomarker in plasma samples of a breast cancer patient and a healthy control person is remarkably different, after the biomarker is combined with a traditional tumor marker, the diagnosis AUC value can be increased to 0.912 (0.878-0.945) and is far higher than the detection effect of a single marker, diagnosis sensitivity and specificity can be greatly improved, the early screening efficiency of breast cancer can be improved, and the detection effect of the biomarker is greatly improved. And the kit also has high clinical application potential, and can provide a more accurate basis for early diagnosis and individualized treatment of breast cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of breast cancer screening biomarkers, and in particular to a breast cancer screening biomarker combination, a detection kit and an application thereof. Background Art

[0002] Breast cancer is the most common malignant tumor in women worldwide and one of the leading causes of cancer-related death in women. Early screening and diagnosis are key to improving the prognosis and survival of breast cancer patients, but existing screening methods still have significant limitations. Currently, commonly used breast cancer screening methods in clinical practice include imaging examinations such as breast ultrasound and mammography, as well as traditional tumor marker tests such as carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125), and carbohydrate antigen 153 (CA153). However, imaging examinations have limited sensitivity for detecting small early lesions and carry radiation risks or lack diagnostic accuracy for dense breast tissue. Traditional tumor markers, due to their low specificity and sensitivity, are prone to false-positive or false-negative results, making them difficult to meet clinical needs, especially in the early stages of the disease.

[0003] In recent years, circular RNA (circRNA), a class of non-coding RNA molecules with covalently closed loop structures, has become a hot topic in tumor biomarker research due to its high stability, strong tissue specificity, and significant disease relevance. CircRNAs are not easily degraded by RNases within cells and play a vital role in tumorigenesis and progression through multiple mechanisms, including regulation of miRNAs, RNA-binding proteins, and translational regulation. They often exhibit abnormal expression patterns during tumorigenesis and progression, providing new potential targets for early tumor detection. Although studies have demonstrated the diagnostic value of circRNAs in malignant tumors such as gastric and lung cancer, specific circRNA markers for breast cancer and their combined detection strategies have not been fully validated, and efficient, non-invasive circRNA detection technologies are still lacking in clinical practice.

[0004] Therefore, developing a highly specific and sensitive breast cancer screening method based on circRNAs to address the shortcomings of existing technologies has important clinical and social value for achieving early detection of breast cancer and improving diagnostic accuracy. This invention aims to identify and validate specific, highly expressed circRNA molecules in the plasma of breast cancer patients, construct a combined detection model, and provide a stable, non-invasive, and efficient technical solution for early breast cancer screening. Summary of the Invention

[0005] The present invention discloses a breast cancer screening biomarker combination, a detection kit and an application thereof. A high-specificity and high-sensitivity breast cancer screening biomarker combination based on circRNA is developed, and a detection kit is designed based on the biomarker combination to solve the problem that existing breast cancer early screening is prone to false positive or false negative results due to low marker specificity and sensitivity, and is therefore difficult to meet clinical needs in the early stages of the disease.

[0006] The technical solution adopted in the present invention is as follows:

[0007] A breast cancer screening biomarker combination comprises two circular RNAs, hsa_circ_0006174 and hsa_circ_0059914. The hsa_circ_0006174 is a circular structure with its 3' and 5' ends connected end to end formed by reverse splicing of the nucleotide sequence shown in SEQ ID NO: 1. The hsa_circ_0059914 is a circular structure with its 3' and 5' ends connected end to end formed by reverse splicing of the nucleotide sequence shown in SEQ ID NO: 2.

[0008]

[0009] Furthermore, the hsa_circ_0006174 is formed by reverse splicing of the 3rd and 4th exons of the gene RAD23B; the hsa_circ_0059914 is formed by reverse splicing of the 3rd to 8th exons of the gene AHCY.

[0010] The application of the breast cancer screening biomarker combination in the preparation of a breast cancer screening reagent.

[0011] A detection kit for breast cancer screening comprises reagents for detecting the expression levels of hsa_circ_0006174 and hsa_circ_0059914 in plasma, wherein the reagents comprise a primer pair for specifically amplifying hsa_circ_0006174 and a primer pair for specifically amplifying hsa_circ_0059914.

[0012] Preferably, the primer pair sequences for specifically amplifying hsa_circ_0006174 are shown as SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 7, respectively, wherein SEQ ID NO: 3 is the upstream primer, and either SEQ ID NO: 4 or SEQ ID NO: 7 is the downstream primer.

[0013]

[0014] Preferably, the primer pair sequences for specifically amplifying hsa_circ_0059914 are shown as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9, respectively, wherein any one of SEQ ID NO: 5 and SEQ ID NO: 8 is an upstream primer, and any one of SEQ ID NO: 6 and SEQ ID NO: 9 is a downstream primer.

[0015]

[0016] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0017] 1. The present invention mainly solves the problem of insufficient sensitivity and specificity of existing biomarkers in early breast cancer screening. Traditional breast cancer screening methods (such as imaging examinations, tumor marker detection such as CEA and CA153) often face low sensitivity and high false positive rates in the diagnosis of early breast cancer, which limits their clinical application. To overcome this limitation, the present invention discovered and verified two circular RNAs (hsa_circ_0006174 and hsa_circ_0059914) as breast cancer-specific biomarkers, proposed a combined detection method, and significantly improved the accuracy of early diagnosis of breast cancer.

[0018] 2. The experimental results of the present invention show that the combined detection of hsa_circ_0006174 and hsa_circ_0059914 showed significant differences in plasma samples from breast cancer patients and healthy controls, and the AUC value of this combination in diagnosing breast cancer was 0.849 (0.804-0.893), which was significantly higher than the AUC value of a single circRNA (such as the AUC of hsa_circ_0006174 was 0.820, and the AUC of hsa_circ_0059914 was 0.740). Furthermore, the AUC value of the model using circRNA and traditional tumor markers was increased to 0.912 (0.878-0.945), greatly improving the diagnostic sensitivity and specificity. This method effectively reduces the false positive and false negative rates, not only improves the efficiency of early screening for breast cancer, but also has high clinical application potential, and can provide a more accurate basis for early diagnosis and personalized treatment of breast cancer;

[0019] 3. The technical solution of the present invention can overcome the limitations of existing breast cancer screening methods and provide a highly sensitive, highly specific, non-invasive screening method with significant social, economic, and technical benefits. It promotes the development of early breast cancer screening technology and further facilitates personalized diagnosis and treatment and precision medicine.

[0020] 4. The present invention is based on peripheral blood plasma samples for detection, without the need for invasive procedures such as tissue biopsy. The collection process is simple and safe, with high patient compliance, and is suitable for large-scale population screening;

[0021] 5. The circRNA in the present invention has a covalently closed loop structure, strong tolerance to RNAse degradation, a long half-life, and can be stably present in plasma, ensuring the repeatability and reliability of the test results, and solving the technical pain point of easy degradation of traditional RNA markers;

[0022] 6. This invention uses a combination of highly specific biomarkers to accurately detect breast cancer in its early stages, helping to increase early diagnosis rates and provide a time window for clinical intervention and treatment plan development, thereby improving patient prognosis.

[0023] 7. The corresponding detection method of the present invention is based on mature qPCR technology, which is simple to operate, cost-effective, and compatible with existing clinical testing processes. The kit can integrate a joint detection module for circRNA and traditional markers, providing medical institutions with an efficient and integrated screening tool, promoting the standardization and popularization of early breast cancer screening technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the formation and structure of circular RNA hsa_circ_0006174;

[0025] Figure 2 Schematic diagram of the formation and structure of circular RNA hsa_circ_0059914;

[0026] Figure 3 This is the Sanger sequencing result of the reverse splicing site fragment of circular RNA hsa_circ_0006174;

[0027] Figure 4 This is the Sanger sequencing result of the reverse splicing site fragment of circular RNA hsa_circ_0059914;

[0028] Figure 5 Figure 2 shows the differential expression of hsa_circ_0006174 and hsa_circ_0059914 in breast tumor cells MCF-7 and MDA-MB-231 and normal breast cells MCF-10A (*p<0.05, **p<0.01);

[0029] Figure 6 Figure 2 shows the differential expression of hsa_circ_0006174 and hsa_circ_0059914 in the plasma of breast cancer patients and healthy controls (***p<0.001);

[0030] Figure 7 This is the ROC curve of the joint detection of hsa_circ_0006174 and hsa_circ_0059914;

[0031] Figure 8 ROC curves for hsa_circ_0006174 and hsa_circ_0059914 respectively;

[0032] Figure 9 This is the ROC curve of the combined detection model of circRNA combination and traditional tumor markers;

[0033] Figure 10 The ROC curves for the three traditional tumor markers CEA, CA125 and CA153 are shown respectively;

[0034] Figure 11 This is the clinical decision curve (DCA) of the model. DETAILED DESCRIPTION

[0035] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0036] Throughout this specification, unless otherwise specified, the terms used herein should be understood to have the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0037] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0038] Example 1

[0039] The purpose of this study is to explore the structures of hsa_circ_0006174 and hsa_circ_0059914. The experimental materials used in this example include:

[0040] Breast tumor cell line MCF-7 (purchased from Shanghai Cell Bank, Chinese Academy of Sciences);

[0041] Total RNA extraction kit (brand: Omega, product number: R6834-01);

[0042] Reverse transcription kit (brand: Exongen, product number: A502-02);

[0043] Agarose gel extraction kit (brand: Transgene, product number: EG101-01).

[0044] Hsa_circ_0006174 (chr9: 110,059,146-110,080,883) is formed by reverse splicing of the 3rd and 4th exons of the RAD23B gene (nucleic acid sequence see SEQ No. 1) and is 349 nucleotides long. Its formation and structure are shown in the following figure. Figure 1 Hsa_circ_0059914 (chr20: 32,868,074-32,891,161) is formed by reverse splicing of exons 3 to 8 of the AHCY gene (see SEQ No. 2 for the nucleic acid sequence), with a length of 753 nucleotides. Its formation and structure are shown in the following figure. Figure 2 shown.

[0045] In order to further verify the structures of these two circRNAs, the following specific experimental steps were taken:

[0046] 1. Experimental steps:

[0047] (1) Total RNA extraction: MCF-7 cells in the logarithmic growth phase were used to extract total RNA according to the instructions of the total RNA extraction kit, and the RNA purity (OD260 / 280 = 1.8-2.1) and concentration were determined;

[0048] (2) cDNA synthesis: Take 1 μg of total RNA, add 1 μL RNase-free water to 13 μL, then add 4 μL 5× reverse transcription reaction mixture and 3 μL reverse transcriptase mixture, incubate at 25°C for 10 minutes, incubate at 55°C for 15 minutes, and inactivate at 85°C for 5 minutes to obtain the cDNA template.

[0049] (3) Specific primer design: Divergent primers were designed for the reverse splicing sites of hsa_circ_0006174 and hsa_circ_0059914 (sequences shown in Table 1 ), and the amplified product fragment sizes were 185 bp and 232 bp, respectively.

[0050] Table 1 Primer sequences for circRNA reverse splicing site verification

[0051]

[0052] (4) PCR amplification and sequencing verification: PCR reaction system (20 μL): 10 μL qPCR SYBR Green Fast TaqMix, 1 μL cDNA template, 1 μL upstream and downstream primers (10 μM), and RNase-free water to 20 μL. Reaction procedure: 95°C pre-denaturation for 3 minutes; 40 cycles (95°C denaturation for 10 seconds, 60°C annealing and extension for 30 seconds). The amplified products were separated by 2% agarose gel electrophoresis, and the target fragments were recovered and subjected to Sanger sequencing (sequencing results are shown in Figure 3 、 Figure 4 ).

[0053] 2. Experimental results

[0054] Sequencing results showed that the amplified fragment of hsa_circ_0006174 contained the reverse splicing site of exons 3-4 of the RAD23B gene (the sequence was consistent with the reverse splicing site formed by SEQ ID NO: 1), and the amplified fragment of hsa_circ_0059914 contained the reverse splicing site of exons 3-8 of the AHCY gene (the sequence was consistent with the reverse splicing site formed by SEQ ID NO: 2), confirming that the two RNA molecules were circular structures.

[0055] Example 2

[0056] This example explores the differential expression of hsa_circ_0006174 and hsa_circ_0059914 in breast tumor cells. The experimental materials used include:

[0057] Breast cancer cell lines MCF-7 and MDA-MB-231, and normal breast epithelial cell line MCF-10A (all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences);

[0058] qPCR kit (brand: Exongen, cat. no.: A403);

[0059] Primers for the internal reference gene β-actin (upstream: 5'-GTGGCCGAGGACTTTGATTG-3' (SEQ ID NO: 10), downstream: 5'-CCTGTAACAACGCATCTCATATT-3' (SEQ ID NO: 11)).

[0060] The experimental steps and results are as follows:

[0061] 1. Experimental procedures (1) Cell culture and RNA extraction: MCF-7, MDA-MB-231 and MCF-10A cells were cultured in DMEM medium containing 10% fetal bovine serum, and total RNA was extracted and cDNA was synthesized (the method was the same as in Example 1).

[0062] qPCR assay: A 20 μL qPCR kit was used. The reaction system consisted of 10 μL qPCR SYBR Green Fast TaqMix, 1 μL upstream and downstream primers (10 μM, sequences shown in Table 2), 1 μL cDNA template, and RNase-free water to a final volume of 20 μL. Reaction conditions included initial denaturation at 95°C for 3 minutes followed by 40 cycles of denaturation at 95°C for 10 seconds and annealing and extension at 60°C for 30 seconds. Relative expression levels were calculated using the 2-ΔΔCt method, using β-actin as an internal reference.

[0063] Table 2 qPCR detection primer sequences

[0064]

[0065] 2. Experimental results

[0066] like Figure 5 As shown in the results, the experimental results showed that the expression levels of hsa_circ_0006174 and hsa_circ_0059914 in breast cancer cells (MCF-7 and MDA-MB-231) were significantly higher than those in normal breast cells (MCF-10A). The expression levels of hsa_circ_0006174 in MCF-7 and MDA-MB-231 were 2.09 times and 3.31 times that of MCF-10A, respectively; the expression levels of hsa_circ_0059914 in MCF-7 and MDA-MB-231 were 1.47 times and 1.41 times that of MCF-10A, respectively. This suggests that these two circRNAs may serve as specific biomarkers for breast cancer screening and provides data support for subsequent clinical applications.

[0067] Example 3

[0068] This example explores the differences in expression levels of hsa_circ_0006174 and hsa_circ_0059914 in the plasma of breast cancer patients and healthy controls, thereby constructing a breast cancer screening model based on plasma samples. The experimental materials used include:

[0069] Plasma samples: Peripheral blood from 142 breast cancer patients (confirmed by pathology) and 122 healthy controls (collected from Sichuan Cancer Hospital, ethics approval number: SCCHEC-02-2024-195);

[0070] TRNzol Universal Total RNA Extraction Kit (brand: TIANGEN, product number: DP424);

[0071] Traditional tumor marker detection kits (CEA, CA125 and CA153, brand: Mindray, approval numbers are Guangdong Medical Device Registration No. 20212401027, Guangdong Medical Device Registration No. 20212401030 and Guangdong Medical Device Registration No. 20212401028 respectively).

[0072] The experimental steps and results are as follows:

[0073] 1. Experimental steps (1) Plasma RNA extraction: Take 200 μL of plasma, add 1 mL of TRIzol reagent, extract total RNA according to the instructions, and reverse transcribe to synthesize cDNA (the method is the same as Example 1).

[0074] (2) Detection of circRNA and traditional markers: The expression levels of hsa_circ_0006174 and hsa_circ_0059914 were detected by the qPCR method of Example 2; the concentrations of CEA, CA125, and CA153 were detected by electrochemiluminescence immunoassay.

[0075] (3) Diagnostic model construction: The ROC curve and clinical decision curve (DCA) were used to analyze the effect of the combined model. The ROC curve was drawn using the pROC package in R software (v4.2.0) and the AUC value was calculated. The caret, ggplot2, and dplyr packages in R software were used to process the data and draw the DCA curve.

[0076] 2. Experimental results

[0077] like Figure 6 As shown in the results, the experimental results showed that hsa_circ_0006174 and hsa_circ_0059914 were significantly higher in the plasma of breast cancer patients than in the healthy control group (p<0.001). This result is consistent with the expression trend in cell experiments, indicating that these two circRNAs are upregulated in the plasma of breast cancer patients, suggesting that they have the potential to serve as potential biomarkers for breast cancer.

[0078] The data were processed using the pROC and ggplot2 packages in R software, and 1000 bootstrap resamplings were performed to draw the ROC curve and calculate the AUC value. The results showed that the AUC value of the circRNA panel composed of hsa_circ_0006174 and hsa_circ_0059914 was 0.849 (0.804-0.893) (as shown in Figure 2). Figure 7 ), which was significantly higher than hsa_circ_0006174 (AUC was 0.820, 0.770-0.871) or hsa_circ_0059914 (AUC was 0.740, 0.690-0.799) alone (as shown in Figure 8These results indicate that the combined detection of these two circRNAs has a high discriminative ability in the diagnosis of breast cancer and can effectively improve the accuracy of early screening.

[0079] When the circRNA combination (hsa_circ_0006174 and hsa_circ_0059914) was combined with traditional tumor markers (CEA, CA125, and CA153), the AUC value of the model was further significantly improved, reaching 0.912 (0.878-0.945) (e.g. Figure 9 The AUC of CEA alone was 0.619 (0.551-0.686), the AUC of CA125 was 0.748 (0.690-0.806), and the AUC of CA153 was 0.648 (0.582-0.714), which were significantly lower than the effect of combining with circRNA (as shown in Figure 2). Figure 10 In addition, DCA analysis (as shown in Figure 11 The results (shown in Figure 2) show that within most threshold probability ranges, the combined model can significantly improve the net benefit of breast cancer diagnosis compared to single circRNA combinations or traditional tumor marker combinations. This result shows that the combined detection method of circRNA and traditional tumor markers has significant advantages in early breast cancer screening and can effectively improve the diagnostic sensitivity and specificity of breast cancer.

[0080] Finally, it should be noted that the terms "comprises," "comprising," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the invention.

[0081] The above-described embodiments merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the technical concept of the present application, and all such variations and improvements fall within the scope of protection of the present application.

Claims

1. A breast cancer screening biomarker combination, characterized in that: The invention comprises two circular RNAs, hsa_circ_0006174 and hsa_circ_0059914. The hsa_circ_0006174 is a circular structure whose 3' and 5' ends are connected head to tail, formed by reverse splicing of the nucleotide sequence shown in SEQ ID NO:

1. The hsa_circ_0059914 is a circular structure whose 3' and 5' ends are connected head to tail, formed by reverse splicing of the nucleotide sequence shown in SEQ ID NO:

2.

2. The breast cancer screening biomarker combination according to claim 1, wherein: The hsa_circ_0006174 is formed by reverse splicing of the 3rd and 4th exons of the RAD23B gene; the hsa_circ_0059914 is formed by reverse splicing of the 3rd to 8th exons of the AHCY gene.

3. Use of the breast cancer screening biomarker combination according to claim 1 or 2 in the preparation of a breast cancer screening reagent.

4. A detection kit for breast cancer screening, characterized in that: The invention comprises a reagent for detecting the expression levels of hsa_circ_0006174 and hsa_circ_0059914 in plasma, wherein the reagent comprises a primer pair for specifically amplifying hsa_circ_0006174 and a primer pair for specifically amplifying hsa_circ_0059914.

5. The detection kit according to claim 4, wherein The primer pair sequences for specifically amplifying hsa_circ_0006174 are shown as SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 7, respectively, wherein SEQ ID NO: 3 is an upstream primer, and either SEQ ID NO: 4 or SEQ ID NO: 7 is a downstream primer.

6. The detection kit according to claim 4, wherein The primer pair sequences for specifically amplifying hsa_circ_0059914 are shown as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 9, respectively, wherein any one of SEQ ID NO: 5 and SEQ ID NO: 8 is an upstream primer, and any one of SEQ ID NO: 6 and SEQ ID NO: 9 is a downstream primer.

Images