Marker for breast cancer diagnosis, metastasis risk assessment and prognosis assessment and application thereof

Abstract

The invention relates to the technical field of biomedicine, and particularly discloses a marker for breast cancer diagnosis, metastasis risk assessment and prognosis assessment and application of the marker. The marker comprises a full-length isomer ZNF132-L of the ZNF132 gene, a truncated isomer ZNF132-S of the ZNF132 gene, and the methylation level of a promoter CpG island of the ZNF132 gene. Wherein ZNF132-L and ZNF-S show opposite biological functions in breast cancer, ZNF132-F / ZNF132-S is used for breast cancer diagnosis, metastasis risk assessment and prognosis assessment, the diagnosis sensitivity reaches 88.06%, the specificity reaches 78.69%, the area under an ROC curve is 0.8566, MSP methylation detection and the ratio of ZNF132 L / S are combined, the diagnosis sensitivity can reach 96.88%, and the specificity is 81.25%. The invention further provides a specific primer pair and a kit for detecting the marker, breast cancer diagnosis and metastasis risk and prognosis evaluation can be sensitively and accurately realized, and a new strategy is provided for clinic.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to biomarkers for breast cancer diagnosis, metastasis risk assessment, and prognostic evaluation, and their applications. Background Technology

[0002] Breast cancer is the most common malignant tumor among women worldwide, with approximately 90% of breast cancer-related deaths attributed to metastasis. Due to the occult growth characteristics of metastatic lesions, by the time clinical imaging can identify metastases, patients are often already in advanced stages of the disease, resulting in a five-year survival rate of less than 30% for metastatic breast cancer. Finding molecular markers capable of detecting early occult metastasis in breast cancer has become a pressing clinical challenge. In recent years, epigenetic research has provided new insights into this problem. DNA methylation, as a core mechanism of reversible epigenetic modification, has shown that abnormal changes (such as hypermethylation of CpG islands in tumor suppressor gene promoter regions) are significantly associated with the metastatic potential of breast cancer, and its dynamic imbalance has been shown to occur earlier than imaging changes. Therefore, it is necessary to screen for methylation markers with early warning potential for occult metastasis to improve patient survival rates and quality of life, and reduce treatment costs and socioeconomic burden. Notably, DNA methylation can alter the openness of chromatin, thereby affecting the accessibility of splicing factors to DNA, thus influencing gene splicing processes to produce different isoforms that exert different biological functions in tumors. This "epigenetic-splicing regulation" cross-interaction mechanism provides a new approach for developing combined diagnostic biomarkers.

[0003] ZNF132 is a member of the KRAB-type zinc finger protein family. A search of the Uniprot online database revealed two isoforms: the full-length ZNF132-Long (ZNF132-L) and the truncated ZNF132-Short (ZNF132-S). This isoform functional differentiation is a novel feature of tumor markers (e.g., the dual role of the TP53 isoform in lung cancer, and the opposite role of the CD44 isoform in breast cancer). However, the differential expression of ZNF132 isoforms in breast cancer and their predictive value for the disease have not yet been investigated. Summary of the Invention

[0004] This invention utilizes the online database TCGA and laboratory-collected breast cancer and paired adjacent normal tissue samples to detect and analyze the expression of ZNF132 gene isoforms in breast cancer. The results showed that ZNF132-L expression in breast cancer was significantly lower than in adjacent normal tissue, while ZNF132-S expression in breast cancer was significantly higher than in the corresponding adjacent normal tissue. Further analysis of the expression of ZNF132-L and ZNF132-S using more samples from a tissue bank revealed that the ratio obtained from the combined detection of ZNF132-L and ZNF132-S had a sensitivity of 88.06%, a specificity of 78.69%, and an area under the ROC curve of 0.8566, making it a valuable diagnostic indicator. Subsequently, the correlation between ZNF132 expression and methylation was further analyzed. The results showed that the ZNF132 promoter was significantly hypermethylated in breast cancer cell lines. Demethylation intervention could significantly restore the mRNA expression level of ZNF132-L and inhibit the expression of ZNF132-S. The results of combined MSP detection of ZNF132 promoter methylation and ZNF132L / S ratio improved the diagnostic sensitivity for breast cancer to 96.88% and the specificity to 81.25%. Stable breast cancer cell lines overexpressing ZNF132-L and ZNF132-S were constructed. Experimental results confirmed that overexpression of ZNF132-L inhibited breast cancer cell proliferation, while ZNF132-S had the opposite effect, promoting breast cancer cell proliferation. Overexpression of ZNF132-L promoted breast cancer cell apoptosis, while overexpression of ZNF132-S inhibited apoptosis. Overexpression of ZNF132-L inhibited angiogenesis, while overexpression of ZNF132-S promoted angiogenesis. Overexpression of ZNF132-L inhibited the in vivo proliferation and metastasis of breast cancer cells, while overexpression of ZNF132-S promoted the in vivo proliferation and metastasis of breast cancer cells.

[0005] In view of this, the purpose of the present invention is to provide a biomarker for breast cancer diagnosis, metastasis risk assessment, and prognostic assessment, and its application.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides biomarkers for breast cancer diagnosis, metastasis risk assessment, and prognostic evaluation. The biomarkers include ZNF132 gene isoforms, specifically the full-length ZNF132-L isoform and the truncated ZNF132-S isoform. Specifically, ZNF132-L is expressed in breast cancer tissue at a lower level than in adjacent normal tissue, while ZNF132-S is expressed in breast cancer tissue at a higher level than in adjacent normal tissue. Overexpression of ZNF132-L inhibits breast cancer cell proliferation, while overexpression of ZNF132-S... ZNF132-L and ZNF132-S have opposite effects, promoting breast cancer cell proliferation. Overexpression of ZNF132-L promotes breast cancer cell apoptosis, while overexpression of ZNF132-S inhibits apoptosis. Overexpression of ZNF132-L inhibits angiogenesis, while overexpression of ZNF132-S promotes angiogenesis. Overexpression of ZNF132-L inhibits the proliferation and metastasis of breast cancer cells in vivo, while overexpression of ZNF132-S promotes the proliferation and metastasis of breast cancer cells in vivo. Breast cancer patients with high ZNF132-L expression have longer overall survival, recurrence-free survival, and disease-free survival.

[0008] Preferably, the marker is the ratio of the expression levels of ZNF132-L and ZNF132-S.

[0009] Furthermore, the biomarkers also include the methylation level of the CpG islands of the ZNF132 gene promoter, wherein the ZNF132 promoter is significantly hypermethylated in breast cancer cell lines.

[0010] The present invention also provides qPCR specific primer pairs for detecting the expression level of ZNF132 gene isoforms, the primer pairs including ZNF132-L specific primer pairs and ZNF132-S specific primer pairs;

[0011] The ZNF132-L specific primer pair includes:

[0012] Upstream primer SEQ ID No. 1: 5'-ACCTTGAGCTGTGACCTCA-3';

[0013] Downstream primer SEQ ID No. 2: 5'-ACTGTGTTCCCTGATGCTCA-3';

[0014] The ZNF132-S specific primer pair includes:

[0015] Upstream primer SEQ ID No. 3: 5'-TGAAGTCAACCATTCCCACC-3';

[0016] Downstream primer SEQ ID No. 4: 5'-ACTGTGTTCCCTGATGCTCA-3'.

[0017] This invention also provides MSP-specific primers for detecting the methylation status of CpG islands in the ZNF132 gene promoter, wherein the MSP-specific primers for detecting the methylation status of CpG islands in the ZNF132 gene promoter comprise:

[0018] Methylation primer pairs:

[0019] Upstream primer SEQ ID No. 5: 5'-TATCGCGCGGAGGTGTTCGC-3';

[0020] Downstream primer SEQ ID No. 6: 5'-CTTTTCTTAACCTCGACCTCG-3';

[0021] Unmethylated primer pairs:

[0022] Upstream primer SEQ ID No. 7: 5'-TTGTATTGTGTGGAGGTGTTTGT-3';

[0023] Downstream primer SEQ ID No. 8: 5'-ACTTTTCTTAACCTCAACCTCA-3'.

[0024] The present invention also provides the application of the biomarkers described in any of the above claims, the qPCR-specific primer pairs described above, and the MSP-specific primer pairs described above in the preparation of products for breast cancer diagnosis, metastasis risk assessment, and prognostic assessment.

[0025] Furthermore, the product mentioned in the breast cancer diagnosis, metastasis risk assessment, and prognostic assessment is a breast cancer diagnosis, metastasis risk assessment, and prognostic assessment kit.

[0026] The present invention also provides a detection kit for breast cancer diagnosis, metastasis risk assessment, and prognosis assessment. The kit includes reagents for detecting the expression level of the full-length ZNF132-L isoform of the ZNF132 gene and reagents for detecting the expression level of the truncated ZNF132-S isoform of the ZNF132 gene.

[0027] Preferably, the reagents used to detect the expression level of the full-length ZNF132-L isoform of the ZNF132 gene include a characteristic primer pair for detecting ZNF132-L expression by real-time quantitative PCR, comprising:

[0028] Upstream primer SEQ ID No. 1: 5'-ACCTTGAGCTGTGACCTCA-3';

[0029] Downstream primer SEQ ID No. 2: 5'-ACTGTGTTCCCTGATGCTCA-3';

[0030] Reagents for detecting the expression level of the truncated ZNF132-S isoform of the ZNF132 gene include characteristic primer pairs for detecting ZNF132-S expression using real-time quantitative PCR, including:

[0031] Upstream primer SEQ ID No. 3: 5'-TGAAGTCAACCATTCCCACC-3';

[0032] Downstream primer SEQ ID No. 4: 5'-ACTGTGTTCCCTGATGCTCA-3'.

[0033] Furthermore, the kit also includes MSP-specific primers for detecting the methylation status of the CpG islands in the ZNF132 gene promoter.

[0034] Preferably, the MSP-specific primers used to detect the methylation status of the CpG islands in the ZNF132 gene promoter include:

[0035] Methylation primer pairs:

[0036] Upstream primer SEQ ID No. 5: 5'-TATCGCGCGGAGGTGTTCGC-3';

[0037] Downstream primer SEQ ID No. 6: 5'-CTTTTCTTAACCTCGACCTCG-3';

[0038] Unmethylated primer pairs:

[0039] Upstream primer SEQ ID No. 7: 5'-TTGTATTGTGTGGAGGTGTTTGT-3';

[0040] Downstream primer SEQ ID No. 8: 5'-ACTTTTCTTAACCTCAACCTCA-3'.

[0041] The beneficial effects of this invention: This invention discloses the use of ZNF132 gene isoforms as biomarkers for breast cancer diagnosis and metastasis risk assessment, and their application in the preparation of products for breast cancer diagnosis and metastasis risk prediction. Through online database TCGA analysis and validation on clinical tissue samples, this invention confirms that ZNF132-L expression in breast cancer is significantly lower than in adjacent normal tissue, and its expression level is positively correlated with the prognosis of breast cancer patients and negatively correlated with the risk of breast cancer metastasis; ZNF132-S expression in breast cancer is significantly higher than in adjacent normal tissue, and it can enhance the malignant biological behavior of breast cancer cells. Combined detection of the expression levels of ZNF132-L and ZNF132-S in the same sample and analysis of the ratio showed high sensitivity (88.06%) and good specificity (78.69%) for breast cancer diagnosis. Furthermore, the ZNF132 promoter is significantly hypermethylated in breast cancer cell lines; demethylation intervention can significantly restore the mRNA expression level of ZNF132-L while inhibiting the expression of ZNF132-S. By detecting the expression levels of the two isoforms of the ZNF132 gene and the promoter methylation status, breast cancer can be diagnosed and metastatic risk assessed, providing a new strategy for early diagnosis and dynamic prognostic monitoring of breast cancer. The results of combining MSP detection of ZNF132 promoter methylation and the ZNF132L / S ratio improved the diagnostic sensitivity of breast cancer to 96.88% and the specificity to 81.25%. This invention also discloses specific primers for detecting the methylation level of the promoter CpG aggregation region of the ZNF132 gene using the MSP method, including: specific primers for promoter methylation status: upstream primer SEQ ID No. 5, downstream primer SEQ ID No. 6; and specific primers for promoter unmethylation status: upstream primer SEQ ID No. 7, downstream primer SEQ ID No. 8. These primers can specifically bind to the methylated and unmethylated site sequences of the CpG islands in the ZNF132 gene promoter region, enabling sensitive, rapid, and accurate detection of the methylation level of the CpG islands in the ZNF132 promoter region. This allows for the sensitive, rapid, and accurate detection of the methylation level of the CpG islands in the ZNF132 promoter region in a sample, and can accurately identify breast cancer patients. This invention also discloses specific primers for detecting the expression of ZNF132 gene isoforms using real-time quantitative PCR. The primers are: upstream primer SEQ ID No. 1 and downstream primer SEQ ID No. 2, which can specifically bind to ZNF132-L; upstream primer SEQ ID No. 3 and downstream primer SEQ ID No. 4, which can specifically bind to ZNF132-S. This method can sensitively, rapidly, and accurately detect the expression levels of different ZNF132 isoforms, effectively distinguish between breast cancer tissue and normal breast tissue, and indicate the risk of metastasis in breast cancer patients.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 from the following description and claims. Attached Figure Description

[0042] Figure 1 The results of ZNF132 expression level analysis in normal breast tissue and various molecular subtypes of breast cancer based on the TCGA database are as follows: A is a comparison between normal tissue and breast cancer tissue; B is the analysis of specific molecular subtypes.

[0043] Figure 2 The mRNA expression level of ZNF132 in breast cancer cell lines and normal breast epithelial cells was detected by RT-PCR; where A represents the expression of ZNF132-L and B represents the expression of ZNF132-S.

[0044] Figure 3 To detect the mRNA expression level of ZNF132 in breast cancer cell lines and normal breast epithelial cells by real-time quantitative PCR; where A represents the expression of ZNF132-L and B represents the expression of ZNF132-S, with MCF10A as the control.

[0045] Figure 4 To analyze the differential expression of ZNF132-L and ZNF132-S in paired breast cancer tissue samples (n=59) by real-time quantitative PCR; where A represents the expression of ZNF132-L in the paired samples; and B represents the expression of ZNF132-S in the paired samples.

[0046] Figure 5 The sensitivity and specificity of ZNF132-L and ZNF132-S expression in breast cancer, adjacent normal, or normal samples (n=128) were calculated by real-time quantitative PCR. The standard for determining a positive result was a normalized value of ZNF132-L / ZNF132-S < 1, and the standard for determining a negative result was a normalized value of ZNF132-L / ZNF132-S ≥ 1.

[0047] Figure 6 The ROC curves were obtained after detecting and analyzing the expression of ZNF132-L and ZNF132-S in breast cancer, adjacent normal or normal samples (n=128) by real-time quantitative PCR.

[0048] Figure 7This study analyzed the relationship between ZNF132-L expression and prognosis in breast cancer. A represents the correlation between ZNF132-L expression and overall survival in breast cancer patients; B represents the correlation between ZNF132-L expression and recurrence-free survival in breast cancer patients; and C represents the correlation between ZNF132-L expression and disease-free survival in breast cancer patients.

[0049] Figure 8 This study analyzed the promoter methylation of ZNF132 in normal breast tissue and various molecular subtypes of breast cancer based on the TCGA database. A represents a comparison between normal and breast cancer tissues; B represents the analysis of specific molecular subtypes.

[0050] Figure 9 A shows the experimental results of detecting the methylation status of the ZNF132 promoter in breast cancer cell lines using the MSP method; B shows the results of detecting the mRNA expression levels of ZNF132-L and ZNF132-S in different breast cancer cells after treatment with 5-Aza using real-time quantitative PCR.

[0051] Figure 10 The results of MSP and real-time quantitative PCR were analyzed in 32 pairs of paired breast cancer and adjacent normal tissue samples. Figure A shows the MSP results; Figure B shows the sensitivity and specificity of the MSP method; Figure C shows the sensitivity and specificity of ZNF132-L and ZNF132-S expression; and Figure D shows the combined analysis of MSP and the ZNF132-L / S expression ratio. The criteria for combined detection were: MSP detection or ZNF132-L / S expression <1 was considered positive, and the rest were negative.

[0052] Figure 11 The results of the experiments used to verify the construction of ZNF132-L stable transgenic cells were obtained by real-time quantitative PCR and Western blot experiments. Among them, A is the result of the real-time quantitative PCR verification experiment; B is the result of the Western blot verification experiment.

[0053] Figure 12 The experimental results were used to verify the effectiveness of ZNF132-S stable cell construction in Western blot experiments.

[0054] Figure 13 The results show the effect of ZNF132 overexpression on breast cancer cell proliferation; A and B are the CCK8 experimental results of ZNF132-L; C and D are the CCK8 experimental results of ZNF132-S.

[0055] Figure 14The results show the effects of ZNF132 overexpression on the migration and invasion of MB231 cells. Among them, A is the image result of the Transwell migration and invasion experiment; B is the statistical result of the Transwell migration experiment; and C is the statistical result of the Transwell invasion experiment.

[0056] Figure 15 The experimental results show the effect of ZNF132 overexpression on apoptosis in MB231 cells; where A is a schematic diagram of the control group; B is a schematic diagram of the ZNF132-L group; C is a schematic diagram of the ZNF132-S group; and D is the experimental statistical analysis results.

[0057] Figure 16 The results show the effects of ZNF132 overexpression on the angiogenesis capacity of MB231 cells; A is the photographic result of the tubule formation experiment; B is the statistical diagram of luminal branching nodes; and C is the statistical diagram of luminal formation length.

[0058] Figure 17 The results show the effects of ZNF132 overexpression on the angiogenesis capacity of SKBR3 cells; A is the image of the tubule formation experiment; B is a statistical diagram of the branching nodes of the lumen; and C is a statistical diagram of the lumen formation length.

[0059] Figure 18 The results of experiments on the effects of ZNF132 overexpression on the growth and metastasis of breast cancer cells in vivo are shown. Among them, A is a schematic diagram of mice in the subcutaneous tumor-bearing model experiment; B is the tumor volume growth curve; C is the tumor weight statistics; D is a schematic diagram of mice in the intracardiac injection metastasis model; E is the fluorescence intensity value of the metastasis site. Detailed Implementation

[0060] To make the technical problems, technical solutions, and beneficial effects solved by this invention clearer, the technical solutions of this invention will be further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of this invention and should not be construed as limiting the scope of protection of this invention. All technologies implemented based on the above content of this invention are covered within the scope of protection intended by this invention. It should be noted that, unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified. It should be noted that all breast cancer tissue and paired adjacent normal tissue samples involved in this invention were obtained from Chongqing University Cancer Hospital, strictly adhering to the principle of informed consent from patients, and have been reviewed and approved by the Ethics Review Committee of Chongqing University Cancer Hospital.

[0061] Example 1: Expression detection and analysis of ZNF132 isomers

[0062] The expression of the ZNF132 gene (ZNF132-L in this case, as ZNF132-S is not included in the database) in breast cancer was analyzed using an online database (https: / / www.uab.edu / home / ). The results showed that compared to normal breast tissue, the expression of ZNF132-L was significantly reduced in breast cancer and its various subtypes. Figure 1 As shown in A and B.

[0063] RT-PCR was used to detect the expression of ZNF132 isoforms in human breast cancer cell lines (BT-549, MDA-MB-231, MDA-MB-468, YCC-B1, HCC1937, SK-BR-3, JIMT-1, MCF7, T47D, ZR-75-1) and normal breast epithelial cell lines (HMEC and MCF10A). The results showed that ZNF132-L expression was downregulated or absent in most breast cancer cell lines. Figure 2 As shown in Figure A; compared to HMEC and MCF10A cell lines, ZNF132-S expression was significantly increased in breast cancer cell lines, such as... Figure 2 As shown in B.

[0064] The expression of ZNF132 isoforms in human breast cancer cell lines and normal breast epithelial cell line (MCF10A) was detected by real-time quantitative PCR. The results showed that, compared to MCF10A, ZNF132-L expression was mostly downregulated or absent in breast cancer cell lines. Figure 3 As shown in Figure A; compared to MCF10A, ZNF132-S showed significantly increased expression in breast cancer cell lines, such as... Figure 3 As shown in B.

[0065] The expression of ZNF132 isoforms in 59 pairs of breast cancer tissues and corresponding adjacent normal tissues was detected by real-time quantitative PCR. The results showed that the expression of ZNF132-L in breast cancer tissues was lower than that in the corresponding adjacent normal tissues. Figure 4 As shown in Figure A, ZNF132-S expression in breast cancer tissues is higher than that in the corresponding adjacent normal tissues, such as... Figure 4 As shown in B.

[0066] Among them, 59 pairs of breast cancer tissue and corresponding adjacent normal tissue samples were obtained from Chongqing University Cancer Hospital, strictly following the principle of informed consent from patients, and have been reviewed and approved by the Ethics Review Committee of Chongqing University Cancer Hospital.

[0067] The primers for real-time quantitative PCR and RT-PCR are:

[0068] ZNF132-L specific primer pair:

[0069] Upstream primer SEQ ID No. 1: 5'-ACCTTGAGCTGTGACCTCA-3';

[0070] Downstream primer SEQ ID No. 2: 5'-ACTGTGTTCCCTGATGCTCA-3';

[0071] ZNF132-S specific primer pair:

[0072] Upstream primer SEQ ID No. 3: 5'-TGAAGTCAACCATTCCCACC-3';

[0073] Downstream primer SEQ ID No. 4: 5'-ACTGTGTTCCCTGATGCTCA-3'.

[0074] The experimental methods and procedures for real-time quantitative PCR and RT-PCR are as follows:

[0075] 1. RNA extraction from cells and tissues

[0076] 1) After digesting the cells, centrifuge them, resuspend the cell pellet in 1 mL PBS, transfer it to a 1.5 mL enzyme-free EP tube, centrifuge at 2000 rpm for 5 minutes, aspirate the supernatant with negative pressure, add 1 mL Trizol to resuspend the cell pellet, and grind the tissue in liquid nitrogen. Add 1 mL Trizol for every 50-100 mg of tissue and homogenize it using a homogenizer.

[0077] 2) Let stand for 20-30 minutes. To further improve the RNA yield, it can be placed at -80℃ for sedimentation before subsequent operations. To make the lysis more complete, after standing, it can be centrifuged at 12000rpm for 5 minutes at 4℃. Take the supernatant and transfer it into a new enzyme-free EP tube.

[0078] 3) Add 0.2 mL of chloroform, shake vigorously, let stand on ice for 3 minutes, then place in a centrifuge at 4°C and centrifuge at 12000 rpm for 15 minutes. After centrifugation, the solution can be observed to be divided into three layers, from top to bottom: aqueous phase (RNA), intermediate layer and organic phase.

[0079] 4) Transfer the upper layer of the aqueous phase to a new enzyme-free EP tube (about 0.4-0.5 mL), add 1.5 times the volume of isopropanol, mix by inverting, let stand on ice for 10 minutes, and centrifuge at 12000 rpm for 10 minutes at 4°C.

[0080] 5) Discard the supernatant, add 1 mL of pre-cooled 75% ethanol, shake the precipitate and soak it in the ethanol to wash it, centrifuge at 7500 rpm for 5 minutes at 4°C, slowly pour off the ethanol, and centrifuge again at 7500 rpm for 2 minutes at 4°C.

[0081] 6) Absorb the remaining ethanol under negative pressure, observe the amount of precipitate, determine the amount of enzyme-free water to be added and record it, open the EP tube and put it in a fume hood to dry it thoroughly. After it is fully dried, add enzyme-free water to the sample and resuspend the precipitate.

[0082] 7) RNA concentration was determined using a Nanodrop spectrophotometer. Before detection, 1 μL of enzyme-free water was used as a blank for calibration. After quantification, qualified samples were labeled with their name, concentration, purity, and extraction date and stored at -80℃.

[0083] 2. Reverse transcription

[0084] 1) Based on the different concentrations of RNA measured, calculate the required volumes of RNA and enzyme-free water, and prepare reaction system A with a total volume of 12.5 μL:

[0085]

[0086] After the prepared sample system was vortexed and mixed, it was incubated at 70°C for 5 min, and then immediately placed in an ice bath to terminate the reaction for 5 min, thus completing the treatment.

[0087] 2) Prepare reaction system B with a total volume of 37.5 μL;

[0088]

[0089] 3) Shake well with a vortex mixer, then centrifuge quickly with a handheld centrifuge until all the liquid settles to the bottom of the tube. Then place it in a PCR instrument for reaction at 25°C for 5 minutes, 42°C for 1 hour, 70°C for 15 minutes, and 4°C for 10 minutes. The entire operation is carried out in an enzyme-free environment to prevent RNA degradation.

[0090] 4) After the reaction is complete, the resulting cDNA product should be stored at -20℃.

[0091] 3. Real-time quantitative PCR

[0092] 1) Dilute the cDNA with DEPC water at a ratio of 7:1;

[0093] 2) Prepare the reaction system, with a total volume of 10 μL, and operate in the dark throughout the process:

[0094]

[0095]

[0096] After preparation, mix well, centrifuge briefly, and then place in a PCR instrument;

[0097] 3) The PCR reaction program is set as follows: First, perform initial pre-denaturation at 95℃ for 10 minutes, followed by the amplification cycle stage, which is run for a total of 40 cycles. Each cycle includes high-temperature denaturation at 95℃ for 15 seconds and annealing / extension at 60℃ for 1 minute. After amplification, enter the melting curve stage, maintain 95℃ for 5 seconds, maintain 65℃ for 5 seconds, and slowly increase the temperature from 65℃ to 95℃ (0.5℃ per second). Monitor the fluorescence decay value continuously at this rate.

[0098] 4) Export the data for processing.

[0099] 4. RT-PCR

[0100] 1) Prepare the reaction system, with a total volume of 10 μL:

[0101]

[0102] 2) After thoroughly mixing the prepared mixture by vortexing, centrifuge briefly, place it in a PCR instrument, and set the reaction program: initial 95℃ pre-denaturation for 2 minutes, followed by amplification reactions of the internal control gene (23 cycles) and the target gene (32 cycles), each cycle including 95℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 30 seconds, and finally 72℃ final extension for 3 minutes. Collect the products for agarose gel electrophoresis.

[0103] 3) Prepare a 2% agarose gel;

[0104] 4) After electrophoresis at 120V for 30 minutes, the gel was removed and imaged using a gel imaging system.

[0105] Example 2: Sensitivity, specificity, and ROC curves of combined detection using ZNF132-L and ZNF132-S

[0106] The expression levels of ZNF132 isoforms in 128 breast cancer tissues, adjacent normal tissues, and normal tissues were detected using real-time quantitative PCR. Of these, 67 were cancerous tissues and 61 were adjacent or normal tissues. Subsequently, the expression levels of ZNF132-L and ZNF132-S were detected in the same samples, and the ratios were calculated and analyzed. A positive result was defined as a normalized ratio ZNF132-L / ZNF132-S < 1, and a negative result was defined as a normalized ratio ZNF132-L / ZNF132-S > 1. The results showed that, based on the established criteria, the sensitivity of this diagnostic method was 88.06%, and the specificity was 78.69%. Figure 5As shown in the figure. Subsequently, using this analytical data, an ROC curve was plotted using Prism10. The calculated area under the curve (AUC) was 0.856688, with a 95% confidence interval of 0.7874–0.9259, p < 0.0001.

[0107] Figure 6 As shown.

[0108] Example 3: Survival curve analysis of ZNF132 in breast cancer

[0109] The prognostic information of ZNF132 (ZNF132-L in this case, as the TCGA database does not yet include survival prognostic information for ZNF132-S) in the TCGA-BRCA dataset was analyzed using the Kaplan-Meier Plotter online database (https: / / kmplot.com / analysis / ). The results showed that breast cancer patients with high ZNF132-L expression had longer overall survival (OS), recurrence-free survival (RFS), and disease-free survival (DFS). Figure 7 As shown in AC.

[0110] Example 4: Superiority of ZNF132 Methylation Analysis and Combined Detection

[0111] The promoter methylation level of the ZNF132 gene (ZNF132-L in this case, as ZNF132-S is not included in the database) in breast cancer tissues was analyzed using the UALCAN online database (https: / / www.uab.edu / home / ). The results showed that the promoter methylation of ZNF132-L was significantly higher in all subtypes of breast cancer tissues than in normal breast tissues. Figure 8 As shown in A and B.

[0112] The methylation status of the ZNF132 promoter in human breast cancer cell lines (BT-549, MDA-MB-231, MDA-MB-468, YCC-B1, HCC1937, SK-BR-3, JIMT-1, MCF7, T47D, ZR-75-1) was detected using the MSP method. The results showed that the ZNF132 promoter was significantly hypermethylated in breast cancer cell lines. Figure 9 As shown in Figure A.

[0113] Treatment of breast cancer cell lines with the DNA methyltransferase inhibitor 5-Aza showed that demethylation intervention significantly restored the mRNA expression level of ZNF132-L and inhibited the expression of ZNF132-S. Figure 9 As shown in B.

[0114] The methylation status of the ZNF132 promoter in paired cancer and adjacent normal tissue DNA from 32 breast cancer patients was detected using the MSP method. The results showed that methylation was detected in more than half of the cancer tissues. Figure 10 As shown in Figure A. Analysis of these results shows that the sensitivity of the MSP method for diagnosing breast cancer is 68.75%, and the specificity is 84.38%. Figure 10 As shown in B. The ZNF132-L / S ratio was measured in these 32 pairs of samples. The sensitivity for diagnosis using the ZNF132-L / S ratio was 84.38%, and the specificity was 87.5%. Figure 10 As shown in C. The combined detection of MSP and ZNF132L / S ratios improved the sensitivity of the diagnosis of these 32 pairs of samples to 96.88% and the specificity to 81.25%. Figure 10 As shown in D.

[0115] The primers for the MSP method are:

[0116] Methylation primer pairs:

[0117] Upstream primer SEQ ID No. 5: 5'-TATCGCGCGGAGGTGTTCGC-3';

[0118] Downstream primer SEQ ID No. 6: 5'-CTTTTCTTAACCTCGACCTCG-3';

[0119] Unmethylated primer pairs:

[0120] Upstream primer SEQ ID No. 7: 5'-TTGTATTGTGTGGAGGTGTTTGT-3';

[0121] Downstream primer SEQ ID No. 8: 5'-ACTTTTCTTAACCTCAACCTCA-3'.

[0122] The primers for real-time quantitative PCR are:

[0123] ZNF132-L specific primer pair:

[0124] Upstream primer SEQ ID No. 1: 5'-ACCTTGAGCTGTGACCTCA-3';

[0125] Downstream primer SEQ ID No. 2: 5'-ACTGTGTTCCCTGATGCTCA-3';

[0126] ZNF132-S specific primer pair:

[0127] Upstream primer SEQ ID No. 3: 5'-TGAAGTCAACCATTCCCACC-3';

[0128] Downstream primer SEQ ID No. 4: 5'-ACTGTGTTCCCTGATGCTCA-3'.

[0129] The experimental methods and procedures for MSP detection and 5-Aza cell treatment are as follows:

[0130] 1. DNA extraction

[0131] 1) Remove the original culture medium, wash 3 times with PBS, remove all PBS, add 1 ml DNAzol, mix the cells by pipetting, and transfer to enzyme-free EP tubes for later use.

[0132] 2) Centrifuge at 10,000 rpm for 10 minutes using a 4°C centrifuge, and transfer the supernatant to a clean, enzyme-free EP tube;

[0133] 3) Add anhydrous ethanol equivalent to twice the volume of DNA, gently invert and mix 10 times, and let stand at room temperature for 5 minutes.

[0134] 4) Centrifuge at 400 rpm for 90 seconds using a 4℃ centrifuge, remove the supernatant and retain the precipitate;

[0135] 5) Wash twice with 800 μl of 75% ethanol to remove impurities;

[0136] 6) Add 400 μl of 8 mmol sodium hydroxide solution to dissolve the genomic DNA, invert and mix 10 times, or use a 200 μl pipette tip to mix thoroughly.

[0137] 7) Add 0.5 μl of proteinase K, shake for 30 seconds, and then heat in a 37°C metal bath for 1 hour (until the solution becomes clear).

[0138] 8) Add 400 μl of phenol-chloroform, shake for 30 seconds, let stand at room temperature for 5 minutes, then centrifuge at 14000 rpm for 10 minutes, and transfer the supernatant to a clean, enzyme-free EP tube (perform in a fume hood).

[0139] 9) Add 30 μl of 3 mol sodium acetate and mix by inverting the container 10 times;

[0140] 10) Add 2 times the volume of anhydrous ethanol to the mixture, mix thoroughly, and let stand at -20℃ for 2 hours to allow precipitation;

[0141] 11) Centrifuge at 14000 rpm for 20 minutes using a 4℃ centrifuge, remove the supernatant and retain the precipitate;

[0142] 12) Add 800 μl of 75% ethanol to wash (invert the container, no need to blow or swirl), let stand at room temperature for 5 minutes, then centrifuge at 14000 rpm for 10 minutes and remove the supernatant;

[0143] 13) Open the EP tube cap, place it in a fume hood to dry the residual alcohol completely, and add an appropriate amount of TE buffer to dissolve the DNA;

[0144] 14) Use NanoDrop to measure DNA concentration and purity, record the results, and store in a -80℃ freezer for long-term preservation.

[0145] 2. DNA bisulfite modification

[0146] The specific procedures are as follows, based on the EZ DNA methylation-Gold Kit method:

[0147] 1) Configure CT Conversion Reagent

[0148]

[0149]

[0150] 2) Add 24 mL of anhydrous ethanol to M-Wash Buffer for later use.

[0151] 3) Add 2ug of DNA to 130uL CT Conversion Reagent.

[0152] 4) Incubate according to the following procedure:

[0153]

[0154] 5) Add 600 μL of M-Binding Buffer to the above mixture, transfer it to a centrifuge column, and centrifuge at 12000 rpm for 30 s.

[0155] 6) Continue with the following steps:

[0156]

[0157] 7) Transfer the centrifuge column to a new EP tube, add 10 μL of M-Elution Buffer, centrifuge at 12000 rpm for 30 seconds.

[0158] 3. MSP

[0159] 1) Prepare the reaction system (total volume 12.5 μl)

[0160]

[0161] 2) Place the prepared mixture into the PCR instrument and run it according to the following program: 95℃ for 10 minutes, followed by 40 cycles of amplification. Each cycle is performed sequentially at 95℃ for 30 seconds, 58℃ (primer U) / 60℃ (primer M) for 30 seconds, and 72℃ for 30 seconds, and finally at 72℃ for 5 minutes to complete the reaction.

[0162] 3) Perform agarose gel electrophoresis on the PCR products.

[0163] 4. Demethylation treatment

[0164] 1) Seed cells in 6-well plates and add drugs when the cell confluence reaches 60%-70%;

[0165] 2) Dissolve 5-Aza in DMSO to prepare a stock solution (final concentration 5mM), and store at -20℃ protected from light;

[0166] 3) Dilute the stock solution to the required working concentration using complete culture medium, then discard the original culture medium. Add fresh culture medium containing 5-Aza to the experimental group (1 μL 5-Aza / 1 mL culture medium), and add culture medium containing an equal volume of DMSO to the control group.

[0167] 4) Continuous drug administration for 72 hours, during which fresh drug-containing culture medium should be replaced every 24 hours to maintain drug concentration.

[0168] 5) Extract cellular RNA, reverse transcribe it, and then use real-time quantitative PCR to detect gene expression.

[0169] Example 5: Construction and validation of stable overexpression cell lines of ZNF132-L and ZNF132-S

[0170] 1. Construction and validation of human breast cancer cell lines overexpressing ZNF132-L

[0171] Using the pLVX-IRES-Neo-3xFlag plasmid as a template, the full-length ZNF132-L sequence was inserted to construct the ZNF132-L lentiviral overexpression vector (pLVX-ZNF132-L-IRES-Neo-3xFlag), which was then verified by sequencing. Using the constructed target plasmid and corresponding control vector, lentiviral packaging was performed on 293T cells in logarithmic growth phase. The lentiviral fluid was collected and used to infect breast cancer cell lines (MB231 and SKBR-3) to establish stable transfected cell lines. Neomycin was used to screen for stable ZNF132-L overexpression. Overexpression efficiency was verified by qRT-PCR and Western blot experiments. The results showed that both transcriptional and translational levels indicated successful construction of ZNF132-L overexpression. Figure 11 As shown in AB.

[0172] 2. Construction and validation of human breast cancer cell lines overexpressing ZNF132-S

[0173] Using the pLVX-IRES-Puro-3xFlag plasmid as a template, the complete ZNF132-S sequence was inserted to construct the ZNF132-S lentiviral overexpression vector (pLVX-ZNF132-S-IRES-Puro-3xFlag), which was then sequenced for verification. Using the constructed target plasmid and corresponding control vector, lentiviral packaging was performed on 293T cells in logarithmic growth phase. The lentiviral fluid was collected and used to infect breast cancer cell lines (MB231 and SKBR-3) to establish stable transfected cell lines. Purine selection was used to establish stable ZNF132-S overexpression cell lines. Western blot analysis verified the overexpression efficiency, confirming the successful construction of stable ZNF132-S overexpression cell lines. Results are shown below. Figure 12 As shown.

[0174] The methods and procedures for screening human breast cancer cell lines that stably express ZNF132-L and ZNF132-S include the following:

[0175] 1. Cell infection

[0176] 1) Breast cancer cells are pre-seeded evenly on a six-well plate, with a confluence of 60%-70%, and then infected with a virus.

[0177] 2) Mix the virus solution and complete culture medium at a ratio of 1:1, add polybrene to the mixed culture medium at a ratio of 1 μL / mL, and replace the cell culture medium with the mixed culture medium for infection;

[0178] 3) Add G418 or Puro to screen for stable strains 48 hours after infection;

[0179] 4) After continuous administration for 14 days, once the cells are stable and resistant to the drug, cellular RNA and protein are extracted to verify the overexpression efficiency.

[0180] The experimental methods and steps for Western blot validation include the following:

[0181] 1. Protein extraction

[0182] 1) Select cells with a confluence of about 80%-90%, wash the cells with PBS 2-3 times and then dry them completely. Add cell lysis buffer (RIPA:PSMF:Cocktail = 100:1:1), add 200-300μL of lysis buffer to each 10cm dish, place on ice and shake for 5-10 minutes to allow them to lyse fully.

[0183] 2) Use a cell scraper to scrape the cells evenly in one direction and collect them into an EP tube. Mark the tube and use ultrasound to lyse the cells. Sonicate for 3 seconds, 3-4 times, with a 3-5 second interval between each time. The ultrasound process will generate heat. To prevent protein degradation in the sample, place the sample on ice for several tens of seconds after each ultrasound. You can stop the ultrasound when there are no large patches of unlysed cells visible to the naked eye in the EP tube.

[0184] 3) Place the ultrasonicated sample in a centrifuge at 4°C and centrifuge at 12,000 rpm for 10 minutes. Transfer the supernatant to a new EP tube.

[0185] 2. Protein concentration determination (BCA method)

[0186] 1) Preparation of working solution: Mix reagent A and reagent B in a ratio of 50:1 and store in the dark;

[0187] 2) Dilute the standard BSA solution 2 mg / mL with DEPC water to 1 mg / mL. Select 7 wells in a 96-well plate and add BSA 0, 0.5, 2.5, 5, 10, 15, 20 (μL) and DEPC water 20, 19.5, 17.5, 15, 10, 5, 0 (μL) in sequence, so that the final volume of each well is 20 μL.

[0188] 3) Dilute the protein sample by adding 18 μL of DEPC water to every 2 μL of protein. After dilution, add 20 μL of the protein dilution to a 96-well plate. Each sample needs to be added to 3 replicates.

[0189] 4) Finally, add 200 μL of working solution to each well, mix gently, and incubate in a 37°C oven for 30 minutes.

[0190] 5) Measure the absorbance at 562 nm using an ELISA reader, record the value, plot a standard curve, and calculate the sample concentration based on the standard curve.

[0191] 3. Protein Sample Preparation

[0192] Calculate the required protein sample volume based on the measured protein sample concentration. For example, with a protein sample of 30 μg and a loading volume of 10 μL: protein sample (μL) = 30 μg / measured concentration (mg / mL), 5× Loading buffer 2 μL, and finally make up the total volume to 10 μL with protein lysis buffer. Place the prepared sample in a metal bath at 100℃ for denaturation for 10 minutes. Store the denatured protein at -20℃. If longer storage is required, it can be stored at -80℃.

[0193] 4. Western Blot

[0194] 1) Carefully clean the glass plate with dish soap and filter it with pure water. After drying, install it and add pure water between the two glass plates. Let it stand for 10 minutes and observe for any leakage.

[0195] 2) If there is no leakage, prepare the gel according to the instructions of the YARN-C Rapid Gel Reagent Kit (taking a 10% separating gel as an example):

[0196]

[0197]

[0198] Add the lower gel solution, buffer solution, and coagulant to a centrifuge tube, gently shake to mix, being careful not to create air bubbles, and do not shake for too long to avoid solidification. Pour the mixed solution between two glass plates, and then add anhydrous ethanol to press the line, adding 200 μL each time, for a total of 3 times. After the lower gel solidifies, pour off the anhydrous ethanol and wash away the residual ethanol with pure water.

[0199] 3) Add the upper gel solution, buffer solution and coagulant to the centrifuge tube, shake and mix in the same way, then add it between the two glass plates. Immediately after adding, insert the comb. After the upper gel solidifies, remove the prepared gel. If storage is required, it can be soaked in pure water at 4°C.

[0200] 4) Install the gel plate onto the power supply frame, with the concave side of the gel plate facing the power supply frame. Fix the gel plate and the power supply frame in the power supply tank and add 1× electrophoresis solution to submerge the gel plate.

[0201] 5) Remove the comb and check for air bubbles in the wells. If there are no air bubbles, you can proceed with the sample loading. Add 10 μL of protein sample (30 μg protein) to the sample wells and 4 μL of protein marker to the marker wells. Use 1×Loading buffer to bring the total volume to 10 μL to ensure that the sample volume in each well is the same. If there are any remaining samples in the wells, use 1×Loading buffer to bring the total volume to 10 μL.

[0202] 6) After running the upper gel at 60V, switch to 120V for the lower gel, or run the entire process at 80V. Stop electrophoresis when the bromophenol blue indicator line reaches near the bottom.

[0203] 7) Cut the PVDF membrane according to the size of the adhesive. Put the cut PVDF membrane into anhydrous ethanol for 2 minutes to activate it, and then put it into 1× electrotransfer solution. Place the sponge pad and filter paper on the transfer clamp and put it into 1× electrotransfer solution with the positive electrode facing upward.

[0204] 8) Pry open the glass plate, remove the top layer of adhesive and the bromophenol blue indicator line, place the PVDF membrane on the gel, remove the membrane and gel from the glass plate, and place them on the negative electrode side of the transfer clip. The order from top to bottom is membrane, gel, filter paper, sponge pad, transfer clip negative electrode. Gently remove the excess air between the membrane and gel and then clamp the transfer clip.

[0205] 9) Place the assembled transfer clip into the transfer tank, along with an ice box. Pour in pre-cooled 1× transfer buffer, place the transfer tank on ice, and connect the power supply to the positive and negative terminals correctly before performing the transfer. The standard transfer conditions are constant current 200mA for 90 minutes. The transfer conditions can be adjusted according to the protein size and exposure results.

[0206] 10) After the transfer is complete, remove the PVDF membrane, put it in 5% milk, and seal it in a shaker at 37°C for 1-2 hours. After sealing, wash the membrane with TBST 3 times, 5 minutes each time.

[0207] 11) Dilute the antibody with primary antibody dilution buffer. Select the dilution ratio according to the antibody instructions. Cut the appropriate band and add it to the diluted primary antibody. Incubate overnight on a shaker at 4°C.

[0208] 12) After the primary antibody incubation is completed, wash the membrane 3 times with TBST for 5 minutes each time. Prepare the corresponding secondary antibody according to the primary antibody properties. The secondary antibody is usually diluted with 5% milk at a ratio of 1:5000. Then, place the band in the secondary antibody and incubate at room temperature for 1 hour.

[0209] 13) After the secondary antibody incubation is completed, wash the membrane 3 times with TBST for 10 minutes each time. Prepare the developing solution according to the ratio of solution A to solution B = 1:1. Gently wipe the strip dry, immerse it in the developing solution for 1-2 minutes, and then expose it.

[0210] Example 6: Effects of ZNF132-L and ZNF132-S overexpression on breast cancer cell function

[0211] 1. Effects on the proliferation ability of breast cancer cells

[0212] The CCK8 assay was used to examine the effects of ZNF132-L and ZNF132-S overexpression on breast cancer cell proliferation. The results showed that cells overexpressing ZNF132-L had reduced proliferative capacity. Figure 13 As shown in A and B, cells overexpressing ZNF132-S exhibit enhanced proliferation ability, such as... Figure 13 As shown in C and D, overexpression of ZNF132-L inhibits breast cancer cell proliferation, while ZNF132-S has the opposite effect, promoting breast cancer cell proliferation.

[0213] The experimental methods and steps for CCK8 detection include the following:

[0214] 1) Digest cells with trypsin, centrifuge and resuspend, count the cells, and seed them into 96-well plates, 2500 cells / well (100μL cell suspension);

[0215] 2) After 6 hours, change the culture medium and add 100 μL of complete culture medium and 10 μL of CCK8 solution to each well. Incubate at 37°C in the dark for 1 hour, and then measure the absorbance at 450 nm using a microplate reader.

[0216] 3) Cell viability was detected at specific time points;

[0217] 4) Record the absorbance values ​​at different time points, plot the cell proliferation curve, and perform statistical analysis.

[0218] 2. Effects on breast cancer cell migration and invasion

[0219] The effects of ZNF132-L and ZNF132-S overexpression on the migration and invasion abilities of breast cancer cells were investigated using Transwell assays. The results showed that ZNF132-L overexpression inhibited the migration and invasion abilities of the MB231 cell line, while ZNF132-S overexpression promoted both. Figure 14 As shown in AC.

[0220] The methods and steps for the cell migration experiment are as follows:

[0221] 1) After the cells are digested by trypsin, centrifuge at 800 rpm for 5 minutes, discard the supernatant, add the appropriate volume of serum-free culture medium to resuspend the cell pellet according to the amount of cell pellet, and then add 20 μL of cell suspension to a cell counting chamber for counting.

[0222] 2) Take a 24-well plate, add 600 μL of culture medium containing 20% ​​FBS to each well, and then place the chamber into the well;

[0223] 3) Based on the total cell concentration, prepare 2 × 10⁻⁶ cells per chamber. 4 Calculate the required cell suspension volume for each cell, and calculate the total required volume based on a final volume of 100 μL per well. After replenishing with serum-free culture medium and mixing well, seed the cells evenly into the upper layer of the well at a rate of 100 μL / well. After seeding, temporarily place the 24-well plate on the operating table and let it stand for 30 minutes, then transfer it to the incubator for 24 hours of culture.

[0224] 4) Remove the chamber, rinse twice with PBS, fix with 4% paraformaldehyde at room temperature for 30 minutes, stain directly with crystal violet for 30 minutes after fixation, then wash away excess crystal violet dye. Be careful to wipe away any remaining cells in the upper chamber with a cotton swab and then air dry.

[0225] 5) Take photos under a fluorescence inverted microscope, and then perform cell counting and statistical analysis.

[0226] The methods and procedures for the cell invasion experiment are as follows:

[0227] 1) Before the experiment, the pipette tip was pre-cooled at -20℃ and transferred to ice just before use. The Matrigel was slowly melted in an ice bath at 4℃ and mixed with the pre-cooled serum-free culture medium at a ratio of 1:6. Low temperature operation was maintained throughout the process.

[0228] 2) Place the 24-well plate into the chamber, inject 600 μL of culture medium containing 20% ​​FBS into the lower chamber, and spread 80 μL of diluted matrix gel evenly on the bottom of the upper chamber. Place the plate back into the 37°C curing oven and let it stand for 2 hours.

[0229] 3. Effects on the apoptosis capacity of breast cancer cells

[0230] Flow cytometry was used to detect cell apoptosis and evaluate the effects of ZNF132-L and ZNF132-S overexpression on the apoptotic capacity of breast cancer cells. The results showed that ZNF132-L overexpression promoted apoptosis in breast cancer cells, while ZNF132-S overexpression inhibited apoptosis. Figure 15 As shown in AD.

[0231] The methods and steps for detecting apoptosis using flow cytometry are as follows:

[0232] 1) Taking T25 flask as an example, when the cell density is about 90%, collect the old culture in centrifuge tubes;

[0233] 2) After washing the cells with 2-3 mL of PBS, collect the PBS into the same centrifuge tube;

[0234] 3) Digest the cells. After digestion is stopped, collect the cell suspension in the same centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells in 4 mL PBS, centrifuge again and discard the supernatant.

[0235] 4) After resuspending the cells in the experimental group and the control group in 200 μL of binding buffer, they were transferred into EP tubes. Then, a group of empty cells was used as the quality control group. After resuspending the cells in 150 μL of binding buffer, the cells were divided into 50 μL tubes and named blank group, FITC single staining group, and PI single staining group, respectively.

[0236] 5) 10 μL of FITC and 10 μL of PI were added to the experimental group and the control group, respectively. No treatment was given to the blank group. 2.5 μL of the corresponding reagent was added to the single staining group.

[0237] 6) After incubating in the dark for 15 minutes, place the cells in a flow cytometer for detection. Statistical analysis will be performed after the detection is completed.

[0238] 3) After the matrix gel solidifies, perform cell digestion, cell counting, and seeding, following the same steps as described in the migration experiment. The seeding cell quantity is 3 × 10⁶ cells / year. 4 indivual.

[0239] 4. Effects on the angiogenesis ability of breast cancer cells

[0240] The effects of ZNF132-L and ZNF132-S overexpression on tumor-associated angiogenesis were evaluated using an endothelial cell tubule formation assay. The results showed that ZNF132-L overexpression inhibited angiogenesis, while ZNF132-S overexpression promoted angiogenesis. (Results are as follows...) Figure 16 As shown in AC and 17A-C.

[0241] The experimental methods and steps for endothelial cell tubule formation are as follows:

[0242] 1) Collect conditioned medium for different cells, filter it through a 0.22 μm filter, and culture human umbilical vein endothelial cells (HUVEC) for 24 hours;

[0243] 2) Pre-cool the 24-well plate and pipette tip at -20℃, and dissolve the Matrigel on ice;

[0244] 3) Add 80 μL of matrix adhesive to each well (avoid air bubbles when adding), gently shake the plate to evenly cover the bottom of the well with adhesive, and incubate at 37°C for 60 minutes to allow the matrix adhesive to solidify;

[0245] 4) After digestion and conditioned medium treatment, HUVECs were centrifuged and resuspended in ECM basal medium containing 50% conditioned medium. After cell counting, cells were injected at a rate of 3 × 10⁻⁶ cells / year. 4 Cells were seeded into each well of a 24-well plate, with a total volume of 500 μL per well;

[0246] 5) After incubating in the incubator for 6-8 hours, carefully remove the culture medium from the wells, being careful not to touch the gel or cell network. Gently wash 1-2 times with PBS, add 500 μL of Calcein-AM working solution (2 μM) to each well, and incubate at 37°C in the dark for 30 minutes.

[0247] 6) Discard the staining solution, wash twice with PBS for 5 minutes each time, and avoid light during the operation;

[0248] 7) Add 500 μL of serum-free culture medium, use a fluorescence microscope for imaging, and record and analyze the morphology and characteristics of the vascular network.

[0249] Example 7: Effects of ZNF132-L and ZNF132-S overexpression on the in vivo proliferation and metastasis of breast cancer cells.

[0250] A subcutaneous tumor-bearing model in nude mice was established to examine the effects of ZNF132-L and ZNF132-S overexpression on the in vivo proliferation of breast cancer cells. The results showed that ZNF132-L overexpression inhibited the in vivo proliferation and metastasis of breast cancer cells, while ZNF132-S overexpression promoted the in vivo proliferation and metastasis of breast cancer cells. Figure 18 As shown in AE.

[0251] SPF-grade BALB / c nude mice (4-6 weeks old) were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., and bred according to SPF requirements. Animal experiments were conducted in accordance with the principles of laboratory animal welfare, and all experiments were approved by the Scientific Research Ethics Committee of Chongqing University Cancer Hospital.

[0252] The methods and steps for constructing a subcutaneous tumor-bearing model in nude mice are as follows:

[0253] 1) Select 5-week-old female mice for subcutaneous tumor formation;

[0254] 2) Select cells in good condition. When the confluence reaches 80%-90%, digest and centrifuge them. Resuspend the cell pellet in PBS, count the cells, and then add 5 × 10⁶ cells per 100 μL of PBS. 6 For each cell, take the corresponding cell suspension and PBS, aliquot them into new EP tubes, and place them on ice;

[0255] 3) Fix the nude mouse, disinfect the area near the thigh on its back, and then inject 100 μL of cell suspension subcutaneously into the nude mouse;

[0256] 4) About 6 days after the injection, observe the growth of the tumor, measure the weight of the nude mouse, and measure the length and width of the tumor with calipers. Record the volume and observe every 3 days thereafter.

[0257] 5) At 30 days, the mice were euthanized under anesthesia, dissected to remove the intact tumor, and the weight of the tumor was measured and photographed.

[0258] 6) Draw the tumor growth curve.

[0259] The methods and steps for constructing the intracardiac injection mouse transfer model are as follows:

[0260] 1) Select 6-week-old female mice for intracardiac injection;

[0261] 2) The Luc tag was transferred into breast cancer cells, and drug screening was conducted for 14 days;

[0262] 3) Select cells in good condition. When the confluence reaches 80%-90%, digest and centrifuge them. Resuspend the cell pellet in PBS, count the cells, and then add 1×10⁶ cells per 100 μL of PBS. 6For each cell, take the corresponding cell suspension and PBS, aliquot them into new EP tubes, and place them on ice;

[0263] 4) Nude mice were anesthetized with azithromycin. After disinfecting the area of ​​the second intercostal space on the left sternum, 100 μL of cell suspension was injected into the mouse heart.

[0264] 5) Seven days after injection, measure the weight of nude mice, inject luciferase substrate to observe the transfer, and count the fluorescence intensity.

[0265] Observe every 7 days thereafter;

[0266] 6) Mice were euthanized under anesthesia at day 21, and samples were collected, transferred, and embedded in paraffin for further testing.

[0267] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Biomarkers for breast cancer diagnosis, metastasis risk assessment, and prognostic evaluation, characterized in that, The biomarkers include ZNF132 gene isoforms, which include the full-length ZNF132-L isoform and the truncated ZNF132-S isoform. ZNF132-L is expressed in breast cancer tissue at a lower level than in adjacent normal tissue, while ZNF132-S is expressed in breast cancer tissue at a higher level than in adjacent normal tissue. Overexpression of ZNF132-L inhibits breast cancer cell proliferation, while ZNF132-S has the opposite effect, promoting breast cancer cell proliferation. Overexpression of ZNF132-L can promote apoptosis in breast cancer cells, while overexpression of ZNF132-S can inhibit apoptosis; overexpression of ZNF132-L can inhibit angiogenesis, while overexpression of ZNF132-S can promote angiogenesis; overexpression of ZNF132-L can inhibit the proliferation and metastasis of breast cancer cells in vivo, while overexpression of ZNF132-S can promote the proliferation and metastasis of breast cancer cells in vivo; breast cancer patients with high expression of ZNF132-L have longer overall survival, recurrence-free survival, and disease-free survival.

2. The marker according to claim 1, characterized in that, The marker is the ratio of ZNF132-L to ZNF132-S expression levels.

3. The marker according to any one of claims 1 to 2, characterized in that, The biomarkers also include the methylation level of the CpG islands in the promoter of the ZNF132 gene, in which the promoter of ZNF132 is significantly hypermethylated in breast cancer cell lines.

4. A qPCR-specific primer pair for detecting the expression level of ZNF132 gene isoforms, characterized in that, The primer pairs include ZNF132-L specific primer pairs and ZNF132-S specific primer pairs; The ZNF132-L specific primer pair includes: Upstream primer SEQ ID No. 1: 5'-ACCTTGAGCTGTGACCTCA-3'; Downstream primer SEQ ID No. 2: 5'-ACTGTGTTCCCTGATGCTCA-3'; The ZNF132-S specific primer pair includes: Upstream primer SEQ ID No. 3: 5'-TGAAGTCAACCATTCCCACC-3'; Downstream primer SEQ ID No. 4: 5'-ACTGTGTTCCCTGATGCTCA-3'.

5. MSP-specific primers for detecting the methylation status of CpG islands in the ZNF132 gene promoter, characterized in that, The MSP-specific primers used to detect the methylation status of the CpG island in the ZNF132 gene promoter include: Methylation primer pairs: Upstream primer SEQ ID No. 5: 5'-TATCGCGCGGAGGTGTTCGC-3'; Downstream primer SEQ ID No. 6: 5'-CTTTTCTTAACCTCGACCTCG-3'; Unmethylated primer pairs: Upstream primer SEQ ID No. 7: 5'-TTGTATTGTGTGGAGGTGTTTGT-3'; Downstream primer SEQ ID No. 8: 5'-ACTTTTCTTAACCTCAACCTCA-3'.

6. The use of the biomarker according to any one of claims 1 to 3, the qPCR-specific primer pair according to claim 4, and the MSP-specific primer pair according to claim 5 in the preparation of products for breast cancer diagnosis, metastasis risk assessment, and prognostic assessment.

7. A diagnostic kit for breast cancer diagnosis, metastasis risk assessment, and prognostic evaluation, characterized in that, The kit includes reagents for detecting the expression level of the full-length ZNF132-L isoform of the ZNF132 gene and reagents for detecting the expression level of the truncated ZNF132-S isoform of the ZNF132 gene.

8. The reagent kit according to claim 7, characterized in that, Reagents for detecting the expression level of the full-length ZNF132-L isoform of the ZNF132 gene include characteristic primer pairs for detecting ZNF132-L expression using real-time quantitative PCR, including: Upstream primer SEQ ID No. 1: 5'-ACCTTGAGCTGTGACCTCA-3'; Downstream primer SEQ ID No. 2: 5'-ACTGTGTTCCCTGATGCTCA-3'; Reagents for detecting the expression level of the truncated ZNF132-S isoform of the ZNF132 gene include characteristic primer pairs for detecting ZNF132-S expression using real-time quantitative PCR, including: Upstream primer SEQ ID No. 3: 5'-TGAAGTCAACCATTCCCACC-3'; Downstream primer SEQ ID No. 4: 5'-ACTGTGTTCCCTGATGCTCA-3'.

9. The reagent kit according to any one of claims 7-8, characterized in that, The kit also includes MSP-specific primers for detecting the methylation status of the CpG islands in the ZNF132 gene promoter.

10. The reagent kit according to claim 9, characterized in that, MSP-specific primers used to detect the methylation status of CpG islands in the ZNF132 gene promoter include: Methylation primer pairs: Upstream primer SEQ ID No. 5: 5'-TATCGCGCGGAGGTGTTCGC-3'; Downstream primer SEQ ID No. 6: 5'-CTTTTCTTAACCTCGACCTCG-3'; Unmethylated primer pairs: Upstream primer SEQ ID No. 7: 5'-TTGTATTGTGTGGAGGTGTTTGT-3'; Downstream primer SEQ ID No. 8: 5'-ACTTTTCTTAACCTCAACCTCA-3'.

Images