Application of serum piRNA-2572505 as breast cancer early diagnosis marker

Abstract

The invention relates to application of serum piRNA-2572505 as a breast cancer early diagnosis marker, and belongs to the technical field of medical treatment and public health. The invention firstly provides a marker for early breast cancer diagnosis, wherein the marker is serum piRNA-2572505; meanwhile, the marker is prepared into a kit for early breast cancer diagnosis. Compared with healthy donors, the expression of piRNA-2572505 in early breast cancer patients is remarkably improved, and the good early diagnosis capability is achieved; meanwhile, the expression level of the piRNA-2572505 is related to the age, so that the piRNA-2572505 can be used for screening indexes of women aged less than 45 years old.

Description

Technical Field

[0001] The present invention relates to the application of serum piRNA-2572505 as an early diagnostic marker for breast cancer, and belongs to the technical field of medical and health care. Background Art

[0002] Cancer statistics reports show that the relative survival rate of patients diagnosed with early breast cancer is much higher than that of patients diagnosed with advanced breast cancer. The 5-year survival rate of patients diagnosed with stage 0 and I breast cancer is close to 100%, 93% for stage II, and 72% for stage III. Therefore, early detection, early diagnosis, and early treatment are the keys to reducing the mortality rate of breast cancer and improving the prognosis.

[0003] Non-coding RNA (ncRNA) is a class of small RNA molecules with special functions, which play a key role in development and homeostasis. In particular, the frequent dysregulation of ncRNA in cancer can lead to the occurrence, development, and metastasis of tumors. ncRNA can be divided into housekeeping and regulatory ncRNA. Regulatory ncRNA is further divided into small ncRNA and long ncRNA with a boundary of 200 nucleotides. Small molecule ncRNA is further divided into small interfering RNA (siRNA), microRNA (miRNA), and piRNA. piRNA is a novel small ncRNA with a length of 26-30 nucleotides. It was first discovered in mammalian testes in 2006 and can bind to the highly conserved RNA-binding protein PIWI and utilize this epigenetic mechanism to play its function in mammalian germline cells.

[0004] However, with continuous in-depth research, it has been found that the abnormal differential expression of piRNA in various malignant tumors can change the development process of tumors and is expected to become an attractive clinical target for regulating the clinical outcomes of cancer patients. As a short RNA fragment with a length of only 26-30 nt, piRNA has a long lifespan in cells and is distributed in almost all human genomes, indicating that piRNA is not as easily degraded as long RNA fragments. At the same time, it can easily cross the cell membrane and exist in the nucleus and cytoplasm for a longer time to play various roles. Therefore, piRNA has certain potential in cancer diagnosis, treatment, and prognosis as a potential non-invasive method. Summary of the Invention

[0005] In view of the above problems and current situation, the present invention provides a kit for early diagnosis of breast cancer that is convenient, fast, and highly specific.

[0006] The technical solution of the present application is as follows:

[0007] The first object of the present application is to protect the application of serum piRNA-2572505 as an early breast cancer diagnosis marker.

[0008] The second object of the present application is to protect a kit for diagnosing early breast cancer, including: the above-mentioned marker, reverse transcription reaction system, fluorescence quantitative PCR reaction system, and internal reference system.

[0009] According to a specific embodiment of the present application, the fluorescence quantitative PCR reaction system includes a forward primer and a reverse primer for piRNA-2572505, SYBR Green mixture, and water;

[0010] According to a specific embodiment of the present application, the forward primer sequence for piRNA-2572505 is shown in SEQ ID No.1, specifically 5’-AAAAGCTGGGTTGAGAGGGCGCGG-3’.

[0011] According to a specific embodiment of the present application, the internal reference system includes forward and reverse primers for internal reference U6;

[0012] According to a specific embodiment of the present application, the forward primer sequence of U6 is shown in SEQ ID No.2, specifically 5’-CTCGCTTCGGCAGCACA-3’;

[0013] According to a specific embodiment of the present application, the reverse primer sequence of U6 is shown in SEQ ID No.3, specifically 5’-AACGCTTCACGAATTTGCGT-3’.

[0014] According to a specific embodiment of the present application, the reagents of the reverse transcription reaction system include polyadenylate polymerase, reverse transcriptase mixture, reverse transcription buffer, and nuclease-free double-distilled water.

[0015] The third object of the present application is to protect a kit for diagnosing young breast cancer prepared using the above-mentioned marker, including: the marker described in claim 1, reverse transcription reaction system, fluorescence quantitative PCR reaction system, and internal reference system.

[0016] According to a specific embodiment of the present application, the fluorescence quantitative PCR reaction system includes a forward primer and a reverse universal primer for piRNA-2572505, SYBR Green mixture, and pure water.

[0017] According to a specific embodiment of the present application, the internal reference system includes forward and reverse primers for internal reference U6.

[0018] According to the specific embodiments of the present application, the reagents in the reverse transcription reaction system include polyadenylate polymerase, reverse transcriptase mixture, reverse transcription buffer, and nuclease-free double-distilled water.

[0019] Advantages of the present invention

[0020] 1. The expression level of piRNA-2572505 in the serum of breast cancer patients is significantly increased

[0021] The present invention first proposes that compared with healthy donors, the expression level of piRNA-2572505 in the serum of breast cancer patients is significantly increased. Diagnostic ROC curve analysis shows that the AUC is 0.7894, the sensitivity is 68.94%, and the specificity is 82.17%.

[0022] 2. The expression of serum piRNA-2572505 is significantly increased in early breast cancer patients

[0023] Compared with healthy donors, the expression of piRNA-2572505 is significantly increased in early breast cancer patients, and it has good early diagnostic ability. Diagnostic ROC curve analysis shows that the AUC is 0.7773, the sensitivity is 69.72%, and the specificity is 82.17%.

[0024] 3. The expression of serum piRNA-2572505 is significantly increased in young breast cancer (≤45 years old) patients

[0025] Compared with healthy donors (≤45 years old), the expression of piRNA-2572505 is significantly increased in young breast cancer patients, and it has good early warning ability. Combined diagnostic ROC curve analysis shows that the AUC is 0.8303, the sensitivity is 76.09%, and the specificity is 84.62%. Description of the drawings

[0026] Figure 1 Shows the expression difference of serum piRNA-2572505 between breast cancer patients and healthy people; where HD: healthy people, BC: breast cancer;

[0027] Figure 2 Shows the expression difference of serum piRNA-2572505 between early breast cancer patients and healthy people; where HD: healthy people, early-BC: early breast cancer;

[0028] Figure 3 Shows the expression difference of serum piRNA-2572505 between young breast cancer patients and healthy people; where HD: healthy people, BC: breast cancer;

[0029] Figure 4For the diagnostic efficacy of piRNA-2572505 in early breast cancer; where Sensitivity: sensitivity, Speccificity: specificity, AUC: diagnostic efficacy;

[0030] Figure 5 For the diagnostic efficacy of piRNA-2572505 in young breast cancer; where Sensitivity: sensitivity, Speccificity: specificity, AUC: diagnostic efficacy. Detailed implementation mode

[0031] The following implementations are used to illustrate the present invention, but are not used to limit the scope of the present invention. Unless otherwise specified, the technical means used in the implementation cases are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

[0032] The following implementations are used to illustrate the present invention, but are not used to limit the scope of the present invention. Unless otherwise specified, the technical means used in the implementation cases are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

[0033] Implementation case 1: Application of piRNA-2572505 in the diagnosis of breast cancer

[0034] 1. Experimental design

[0035] The experimental design was to collect serum samples from newly diagnosed breast cancer patients and healthy volunteers in the Affiliated Tumor Hospital of Shandong First Medical University to obtain serum RNA. The expression of piRNA-2572505 was detected by qPCR, and the differences in its expression among healthy volunteers, breast cancer, early breast cancer, and young breast cancer were statistically analyzed, and its diagnostic efficacy was analyzed.

[0036] 2. Patients and samples involved in the experiment

[0037] 129 healthy controls and 161 breast cancer patients admitted to the Affiliated Tumor Hospital of Shandong First Medical University from March 2023 to March 2024 were included, and TNM classification was performed based on postoperative pathological examination. The clinical data included in this study mainly included age, AJCC tumor stage (8th edition), estrogen receptor, progesterone receptor, HER-2, etc. Approximately 2 ml of serum samples from the subjects were collected before chemotherapy, radiotherapy, and surgical treatment, centrifuged at 3000 g for 10 minutes, the supernatant was taken, placed in a centrifuge tube and stored at -80 °C for later use.

[0038] 3. Determination of the level of piRNA-2572505 in serum

[0039] The qPCR method was used to analyze the expression levels of piRNA-2572505 in the sera of breast cancer patients and healthy volunteers. The specific steps are as follows:

[0040] 3.1, Total RNA Extraction

[0041] (1) The collected serum samples were centrifuged at 3000 g for 10 minutes at 4°C to remove any possible impurities, and then centrifuged at 12000 g for 10 minutes. The serum was divided into 250 μL aliquots and transferred to 1.5 ml EP tubes and labeled. 750 μL of TRIzol LS reagent was added, and the tube was vigorously vortexed until well mixed;

[0042] (2) After allowing the TRIzol LS to fully lyse the serum for 15 minutes, 200 μL of chloroform was added to the EP tube. The tube lid was tightened, and the tube was vortexed on the vortex oscillator for 10 seconds and then allowed to stand for 3 - 5 minutes;

[0043] (3) The EP tube was placed in a 4°C refrigerated centrifuge and centrifuged at 12000 g for 15 minutes. After centrifugation, the EP tube was carefully removed. At this time, the liquid in the tube was divided into three liquid phases. The upper layer was a colorless aqueous phase containing dissolved RNA, the middle layer was a white flocculent precipitate, and the bottom layer was a phenol - chloroform phase. The upper colorless aqueous phase was aspirated into a new EP tube, taking care not to aspirate the white floc;

[0044] (4) Isopropanol equal in volume to the solution volume in the new EP tube was added. The tube lid was tightened and the tube was inverted up and down for 10 - 15 seconds and then allowed to stand for 10 minutes;

[0045] (5) After standing, the EP tube was placed in a 4°C centrifuge and centrifuged at 12000 g for 10 minutes. After centrifugation, the EP tube was carefully removed. At this time, the RNA precipitate adhered to the bottom of the tube wall. After discarding the supernatant, 1 ml of 75% ethanol was added. The tube lid was tightened and the tube wall was gently flicked to wash the RNA precipitate;

[0046] (6) The EP tube was placed in a 4°C centrifuge and centrifuged at 7500 g for 5 minutes;

[0047] (7) After centrifugation, the supernatant was carefully discarded. The RNA sank to the bottom of the tube. The EP tube was placed on a clean filter paper and allowed to air dry, taking care not to dry completely. Then, an appropriate amount of enzyme - free water was added to dissolve the RNA. After standing at room temperature for 10 minutes, the corresponding experiment was carried out and the remaining RNA solution was stored in a - 80°C refrigerator.

[0048] 3.2, Reverse Transcription Reaction and qPCR

[0049] (1) Reverse transcription reaction system: 1.25 μL miRNA RT Enzyme Mix, 5 μL 2X miRNA RT Reaction Solution, 3.75 μL total RNA. Reaction conditions: 37 °C for 60 min, 85 °C for 5 min, hold at 4 °C. According to the number of samples, prepare the mixed reaction solution according to the reaction system in the following table, and shake well.

[0050] (2) qPCR reaction system: Target gene reaction system: 10 μL 2X SYBR Green Pro Taq HS Premix, 0.4 μL piRNA2572505 forward primer (the sequence is shown in SEQ ID No. 1, specifically 5’-AAAAGCTGGGTTGAGAGGGCGCGG-3’), 0.4 μL universal reverse primer, 2 μL DNA template, 7.2 μL enzyme-free water;

[0051] Internal reference reaction: 10 μL 2X SYBR Green Pro Taq HS Premix, 0.4 μL forward primer of U6 (the sequence is shown in SEQ ID No. 2, specifically 5’-CTCGCTTCGGCAGCACA-3’), 0.4 μL reverse primer of U6 (the sequence is shown in SEQ ID No. 3, specifically 5’-AACGCTTCACGAATT TGCGT-3’), 2 μL DNA template, 7.2 μL enzyme-free water.

[0052] (3) PCR reaction conditions

[0053] In the biosafety cabinet, add the above-prepared reaction solutions into the corresponding wells of the PCR amplification plate respectively, pay attention to avoid contamination, and then attach a transparent film. Note that each sample is prepared with 1 duplicate well. Centrifuge carefully and mix well. Perform the operation on the machine, and use the LightCycler 480 quantitative PCR instrument for detection, and react according to the program set in Table 1.

[0054] Table 1 PCR reaction conditions

[0055] Step Temperature Time Number of cycles Pre-denaturation 95℃ 30 sec 1 Denaturation 95℃ 5 sec 40 Annealing 60℃ 30 sec

[0056] 4. Mathematical statistical analysis

[0057] Statistical analysis was performed using SPSS 22.0 and GraphPad Prism 6.0 software. For the comparison of measurement data, if it follows a normal distribution, parametric tests were used and expressed as mean ± standard deviation; if it does not follow a normal distribution, non-parametric tests were used, the Mann-Whitney test was used and expressed as median and interquartile range. The comparison among three groups was performed using the Kruskal-Wallis H test. The chi-square test was used for the comparison of frequencies. Binary logistic regression was used to establish a diagnostic combination, and the differentially expressed mRNAs were determined by the receiver operating characteristic curve (ROC curve) and the area under the curve (AUC). A p < 0.05 was considered statistically significant.

[0058] 5. Summary of the case characteristics of breast cancer patients and the relationship with the expression level of piRNA-2572505

[0059] Table 2 analyzed the clinical characteristics of breast cancer patients, including age, T stage, lymph node metastasis, distant metastasis, TNM stage, ER level, PR level, and HER2 level. The expression level of piRNA-2572505 was not related to the above factors (P > 0.05), and the difference was not statistically significant.

[0060] Table 2 Summary of the case characteristics of breast cancer and the relationship with the expression level of piRNA-2572505

[0061]

[0062]

[0063] 6. The relationship between the case characteristics of early breast cancer patients and the expression level of piRNA-2572505

[0064] Table 3 analyzed the clinical characteristics of early breast cancer patients, including age, T stage, lymph node metastasis, TNM stage, ER level, PR level, and HER2 level. The expression level of piRNA-2572505 was not related to the above factors (P > 0.05), and the difference was not statistically significant.

[0065] Table 3 Summary of the case characteristics of early breast cancer and the relationship with the expression level of piRNA-2572505

[0066]

[0067] 7. Difference in the level of serum piRNA-2572505 between healthy people and breast cancer patients

[0068] The levels of piRNA-2572505 in the sera of breast cancer patients and healthy individuals were quantified using qPCR to determine whether piRNA-2572505 could serve as a biomarker for breast cancer diagnosis. The results showed that the expression levels of piRNA-2572505 were significantly elevated in breast cancer patients, with a statistically significant difference (p < 0.0001), as shown in Figure 1 Similarly, the levels of piRNA-2572505 in the sera of early breast cancer patients and healthy individuals were quantified using qPCR to determine whether piRNA-2572505 could serve as a biomarker for early breast cancer diagnosis. The results showed that the expression levels of piRNA-2572505 were significantly elevated in early breast cancer patients, with a statistically significant difference (p < 0.0001). As shown in Figure 2 .

[0069] 8. Difference in the levels of serum piRNA-2572505 between young breast cancer patients and healthy individuals

[0070] The levels of piRNA-2572505 in the sera of young breast cancer patients and healthy individuals were quantified using qPCR to determine whether piRNA-2572505 could serve as a biomarker for young breast cancer diagnosis. The results showed that the expression levels of piRNA-2572505 were significantly elevated in young breast cancer patients, with a statistically significant difference (p < 0.0001). As shown in Figure 3 .

[0071] 9. Diagnostic efficacy of serum piRNA-2572505 for early breast cancer

[0072] Furthermore, the present invention utilized ROC curve analysis to detect the diagnostic efficacy of piRNA-2572505 for early breast cancer. The ROC curve analysis showed that the AUC was 0.7773, the sensitivity was 69.72%, and the specificity was 82.17%. As shown in Figure 4 .

[0073] 10. Diagnostic efficacy of serum piRNA-2572505 for young breast cancer

[0074] Furthermore, the present invention utilized ROC curve analysis to detect the diagnostic efficacy of piRNA-2572505 for young breast cancer. The ROC curve analysis showed that the AUC was 0.8303, the sensitivity was 76.09%, and the specificity was 84.62%. As shown in Figure 5 .

Claims

1. Application of serum piRNA-2572505 as an early diagnostic marker for breast cancer.

2. A kit for diagnosing early breast cancer, characterized in that, Including: The marker, reverse transcription reaction system, fluorescence quantitative PCR reaction system, and internal reference system described in claim 1.

3. The kit according to claim 2, wherein The fluorescence quantitative PCR reaction system includes a forward primer and a reverse primer for piRNA-2572505, SYBR Green mixture, and water; Preferably, the forward primer sequence for piRNA-2572505 is as shown in SEQ ID No.1, specifically 5’-AAAAGCTGGGTTGAGAGGGCGCGG-3’.

4. The kit according to claim 2, characterized in that, The internal reference system includes forward and reverse primers for internal reference U6; Preferably, the forward primer sequence of U6 is as shown in SEQ ID No.2, specifically 5’-CTCGCTTCGGCAGCACA-3’; Preferably, the reverse primer sequence of U6 is as shown in SEQ ID No.3, specifically 5’-AACGCTTCACGAATTTGCGT-3’.

5. The kit according to claim 2, characterized in that, The reagents of the reverse transcription reaction system include polyadenylate polymerase, reverse transcriptase mixture, reverse transcription buffer, and nuclease-free double-distilled water.

6. A kit for diagnosing young breast cancer prepared with the marker described in claim 1, comprising: The marker, reverse transcription reaction system, fluorescence quantitative PCR reaction system, and internal reference system described in claim 1.

7. The kit according to claim 6, wherein The fluorescence quantitative PCR reaction system includes a forward primer and a reverse universal primer for piRNA-2572505, SYBR Green mixture, and pure water.

8. The kit according to claim 6, wherein The internal reference system includes forward and reverse primers for internal reference U6.

9. The kit according to claim 6, wherein The reagents of the reverse transcription reaction system include polyadenylate polymerase, reverse transcriptase mixture, reverse transcription buffer, and nuclease-free double-distilled water.

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