Prevention, early diagnosis and treatment strategy method for breast cancer

Abstract

The invention relates to the field of biomedicine, and discloses a method for prevention, early diagnosis and treatment strategy of breast cancer, which comprises the following steps: step 1, detecting the utilization rate and the protein expression level of a far-end polyadenylation site of CDCA7L gene in a breast tissue sample or a peripheral blood sample of a high-risk group; 2, according to the abnormal change that the utilization rate of the far-end polyadenylation site is reduced and the protein expression level is up-regulated, performing prevention intervention or early diagnosis judgment on an individual with the abnormal change; and 3, constructing an intervention vector of a long 3'untranslated region of the targeted CDCA7L gene. By taking an abnormal variable polyadenylation mode of CDCA7L gene as a molecular mechanism, high-risk population intervention of a prevention link, molecular feature detection of early diagnosis and targeted intervention of a treatment link are linked, so that the three links play a synergistic role on the basis of the same biological basis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for the prevention, early diagnosis, and treatment of breast cancer. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women, and its incidence is increasing globally, with the age of onset becoming increasingly younger. Statistics show that more than 2 million new cases of breast cancer are diagnosed globally each year, with China accounting for approximately 18% of new cases and 12% of deaths annually, posing a serious threat to women's health. With the development of molecular biology techniques, researchers have discovered that abnormal post-transcriptional regulation of genes plays a crucial role in the development and progression of breast cancer, with variable polyadenylation (APA) being one of the key regulatory mechanisms.

[0003] In current technologies, breast cancer prevention focuses on controlling known risk factors, while early diagnosis relies on imaging examinations such as breast ultrasound, mammography, and magnetic resonance imaging. Treatment includes surgical resection, chemotherapy, radiotherapy, endocrine therapy, and targeted therapy, which mainly target malignant phenotypes such as tumor cell proliferation and invasion or known driver genes. This results in each step being independent of the others, lacking an integrated strategy based on the same molecular mechanism, leading to insufficient prevention and treatment effects. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for the prevention, early diagnosis, and treatment of breast cancer. This method solves the problem that in existing technologies, the various stages of breast cancer prevention, early diagnosis, and treatment are independent of each other and lack an integrated strategy based on the same molecular mechanism, resulting in insufficient prevention and treatment effects.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for the prevention, early diagnosis, and treatment of breast cancer, comprising the following steps:

[0006] Step 1: Detect the utilization rate of distal polyadenylation sites and protein expression levels of the CDCA7L gene in breast tissue samples or peripheral blood samples from high-risk individuals;

[0007] Step 2: Based on the abnormal changes of decreased utilization of distal polyadenylation sites and upregulated protein expression levels, preventive interventions or early diagnosis are carried out for individuals with such abnormal changes.

[0008] Step 3: Construct an intervention vector targeting the long 3′ untranslated region of the CDCA7L gene. By using the intervention vector to inhibit the function of the long 3′ untranslated region of the CDCA7L gene, the malignant phenotype of breast cancer cells can be blocked.

[0009] By adopting the above technical solution, and using the abnormal variable polyadenylation pattern of the CDCA7L gene as the molecular mechanism, the intervention of high-risk groups in the prevention stage, the molecular feature detection of early diagnosis, and the targeted intervention in the treatment stage are linked together. This allows the three stages to play a synergistic role based on the same biological basis, avoiding the disconnect between prevention and treatment caused by the dispersed mechanisms in traditional technologies, and improving the overall prevention and treatment effect.

[0010] Preferably, in step one, the high-risk group is women with a family history of breast cancer or a history of atypical breast hyperplasia. The peripheral blood sample is subjected to density gradient centrifugation to separate peripheral blood mononuclear cells and then total RNA is extracted. The breast tissue sample includes tumor tissue and adjacent normal tissue. After collection, the sample is flash-frozen in liquid nitrogen and stored at -80°C.

[0011] Preferably, in step one, when detecting the utilization rate of distal polyadenylation sites of the CDCA7L gene, total RNA is extracted using the TRIzol method, and RNA integrity meets the requirement of RIN≥7. Total protein is extracted using RIPA lysis buffer containing protease inhibitors, and the protein concentration is 50-1000 μg / mL.

[0012] Preferably, in step one, detecting the utilization rate of the distal polyadenylation site of the CDCA7L gene specifically includes the following steps:

[0013] After Poly(A)+ screening of total RNA, sequencing libraries were constructed, and sequencing data were obtained by paired-end sequencing.

[0014] Sequencing data were filtered and genome alignment was performed. Bioinformatics tools were used to calculate the utilization rate of distal polyadenylation sites.

[0015] Preferably, the paired-end sequencing platform is BGIDNBSEQ-T7, the sequencing read length is PE150, and the sequencing volume of each sample is ≥40M reads. The filtering is performed using Trim Galore, and the genome alignment is performed using STAR software to align the filtered data to the human genome GRCh38. The bioinformatics tools include DaPars2 and QAPA.

[0016] Preferably, the detection of the distal polyadenylation site utilization rate of the CDCA7L gene further includes verification using qRT-PCR. The primers used in the qRT-PCR include the primer pair CDCA7L-Total-Fwd / Rev for detecting total transcripts and the primer pair CDCA7L-Long-Fwd / Rev for detecting long 3′ untranslated region transcripts, with 18S rRNA as the internal reference gene.

[0017] Preferably, in step one, the detection of CDCA7L protein expression level involves digesting breast tissue samples or peripheral blood samples from high-risk individuals with trypsin, sequencing them using LC-MS / MS, and quantifying them using iBAQ software with MaxQuant software.

[0018] Preferably, in step two, the preventive intervention is to use an APA regulatory factor inhibitor to intervene in individuals in the high-risk population whose distal polyadenylation site utilization rate is lower than the average of healthy individuals and whose protein expression level is upregulated. The early diagnosis is determined by the determination that the distal polyadenylation site utilization rate in breast tumor tissue is lower than that in adjacent normal tissue and whose protein expression level is upregulated.

[0019] Preferably, in step three, the construction of the intervention vector includes cloning the coding region of the CDCA7L gene and the long 3′ untranslated region or the short 3′ untranslated region into the pLVX lentiviral vector, wherein the nucleotide sequence of the long 3′ untranslated region is SEQ ID NO:1 and the nucleotide sequence of the short 3′ untranslated region is SEQ ID NO:2.

[0020] Preferably, in step three, the intervention vector is packaged with lentivirus and then infects breast cancer cells. A stable expression cell line is established by screening with puromycin. The lentivirus packaging is performed using the HEK293T cell three-plasmid system. The breast cancer cells are MDA-MB-231 cells or BT-549 cells.

[0021] This invention provides a method for the prevention, early diagnosis, and treatment of breast cancer. It has the following beneficial effects:

[0022] 1. This invention uses the abnormal variable polyadenylation pattern of the CDCA7L gene as a molecular mechanism to connect high-risk population intervention in the prevention stage, molecular feature detection in early diagnosis, and targeted intervention in the treatment stage. This allows the three stages to work synergistically based on the same biological basis, avoiding the disconnect between prevention and treatment caused by the dispersed mechanisms in traditional technologies, and improving the overall prevention and treatment effect.

[0023] 2. This invention directly identifies molecular-level characteristics of tumor development by detecting abnormal changes in the utilization rate of distal polyadenylation sites and protein expression levels of the CDCA7L gene. It can detect abnormalities in the early stages of disease or even in the precancerous stage. Moreover, this feature is highly specific in breast cancer tissue, effectively reducing the rate of misdiagnosis and missed diagnosis, and buying time for early intervention.

[0024] 3. This invention screens individuals with genuine disease risk by detecting abnormal APA patterns in the CDCA7L gene in high-risk populations, and then uses APA regulatory factor inhibitors for targeted intervention, which can directly correct abnormal gene expression regulation and block the early process of tumor development at the molecular level, making preventive measures more targeted and effective.

[0025] 4. This invention constructs an intervention vector targeting the long 3′ untranslated region of the CDCA7L gene, which specifically inhibits the function of the tumor-promoting long 3′UTR isoform. It works only against the malignant phenotype of tumor cells and has little impact on normal cells. While blocking the proliferation, migration and tumorigenesis of breast cancer cells, it reduces interference with the body's normal physiological functions and improves the safety of treatment. Attached Figure Description

[0026] Figure 1 This is a diagram showing the variable polyadenylation analysis of the CDCA7L gene in breast cancer tissue according to the present invention.

[0027] Figure 2 This is a graph showing the correlation between CDCA7L expression and survival in breast cancer patients according to the present invention.

[0028] Figure 3 This is a functional verification diagram of different 3′UTR isomers of CDCA7L according to the present invention;

[0029] Figure 4 This is a flowchart of a method for the prevention, early diagnosis, and treatment of breast cancer proposed in this invention. Detailed Implementation

[0030] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Please see the appendix Figure 1 - Appendix Figure 4 This invention provides a method for the prevention, early diagnosis, and treatment of breast cancer, comprising the following steps:

[0032] Step 1: Detect the utilization rate of distal polyadenylation sites and protein expression levels of the CDCA7L gene in breast tissue samples or peripheral blood samples from high-risk individuals;

[0033] Furthermore, in step one, the high-risk group consists of women with a family history of breast cancer or a history of atypical breast hyperplasia. Peripheral blood samples are separated into peripheral blood mononuclear cells by density gradient centrifugation, and total RNA is extracted. Breast tissue samples include tumor tissue and adjacent normal tissue. After collection, the samples are flash-frozen in liquid nitrogen and stored at -80°C.

[0034] Furthermore, in step one, when detecting the utilization rate of the distal polyadenylation site of the CDCA7L gene, total RNA was extracted using the TRIzol method, and RNA integrity met the requirement of RIN≥7. Total protein was extracted using RIPA lysis buffer containing protease inhibitors, with a protein concentration of 50-1000 μg / mL.

[0035] Furthermore, in step one, detecting the utilization rate of the distal polyadenylation site of the CDCA7L gene specifically includes the following steps:

[0036] After Poly(A)+ screening of total RNA, sequencing libraries were constructed, and sequencing data were obtained by paired-end sequencing.

[0037] Sequencing data were filtered and genome alignment was performed. Bioinformatics tools were used to calculate the utilization rate of distal polyadenylation sites.

[0038] Furthermore, the paired-end sequencing platform was BGIDNBSEQ-T7, the sequencing read length was PE150, and the sequencing volume of each sample was ≥40M reads. Trim Galore was used for filtering, and the genome alignment was performed using STAR software to align the filtered data to the human genome GRCh38. Bioinformatics tools included DaPars2 and QAPA.

[0039] Furthermore, the utilization rate of the distal polyadenylation site of the CDCA7L gene was also verified by qRT-PCR. The primers used for qRT-PCR included the primer pair CDCA7L-Total-Fwd / Rev for detecting total transcripts and the primer pair CDCA7L-Long-Fwd / Rev for detecting long 3′ untranslated region transcripts. The internal reference gene was 18S rRNA.

[0040] Furthermore, in step one, the expression level of CDCA7L protein was detected by digesting breast tissue samples or peripheral blood samples from high-risk individuals with trypsin, followed by LC-MS / MS sequencing, and quantification using iBAQ software with MaxQuant software.

[0041] Specifically, step one involves testing specific samples to obtain the utilization rate of the distal polyadenylation site of the CDCA7L gene and the protein expression level, providing a basis for subsequent preventive interventions or early diagnostic judgments.

[0042] Generally, high-risk groups refer to women with a family history of breast cancer or a history of atypical breast hyperplasia. For this group, peripheral blood samples are typically collected for testing. After collection, the peripheral blood sample undergoes density gradient centrifugation to separate peripheral blood mononuclear cells, followed by extraction of total RNA for subsequent genetic testing. Alternatively, breast tissue samples include tumor tissue and adjacent normal tissue, typically referring to tissue located at a certain distance from the tumor margin. To ensure sample stability and the accuracy of subsequent testing, these tissue samples are immediately flash-frozen in liquid nitrogen and stored at -80 degrees Celsius to minimize the impact of sample degradation on the test results.

[0043] When detecting the utilization rate of distal polyadenylation sites in the CDCA7L gene, total RNA was extracted using the TRIzol method. This method is a well-established RNA extraction technique in molecular biology, capable of effectively isolating high-quality RNA. To ensure the reliability of subsequent experiments, the extracted RNA must meet the integrity requirement, i.e., an RNA integrity value (RIN) ≥ 7. For total protein extraction, RIPA lysis buffer containing protease inhibitors was used. This buffer effectively lyses cells or tissues, releasing proteins, and the protease inhibitors inhibit protein degradation. The total protein concentration obtained was controlled within the range of 50-1000 μg / mL to meet the requirements of subsequent detection.

[0044] The detection of the utilization rate of distal polyadenylation sites in the CDCA7L gene involves the following steps: First, the extracted total RNA is screened using Poly(A)+, which enriches mRNAs with polyadenylation tails. These mRNAs are typically functional coding RNAs and can more accurately reflect gene expression and processing. The screened RNA is then used to construct sequencing libraries. The library construction process follows standard library construction procedures to ensure library quality and uniformity.

[0045] Generally, paired-end sequencing is used to obtain sequencing data. As an option, the paired-end sequencing platform is BGIDNBSEQ-T7, the sequencing read length is set to PE150, and the sequencing volume of each sample is ≥40M reads. This sequencing depth and read length can ensure that sufficient sequence information is obtained to meet the needs of subsequent data analysis.

[0046] After sequencing, the raw sequencing data needs to be filtered and aligned to the genome. Specifically, the Trim Galore tool is used for filtering to remove low-quality sequences and adapter sequences from the sequencing data, thereby improving data accuracy. The filtered clean data is then aligned to the human genome GRCh38 using STAR software. After obtaining the aligned sequence data, bioinformatics tools are used to calculate the utilization rate of distal polyadenylation sites. Alternatively, bioinformatics tools such as DaPars2 and QAPA are used, which can effectively calculate the utilization rate of distal polyadenylation sites in the target gene.

[0047] To further verify the reliability of the sequencing and analysis results, the utilization rate of distal polyadenylation sites in the CDCA7L gene was also verified using qRT-PCR. Generally, qRT-PCR uses primers including the primer pair CDCA7L-Total-Fwd / Rev for detecting total transcripts and the primer pair CDCA7L-Long-Fwd / Rev for detecting long 3′ untranslated transcripts, with 18S rRNA as the internal reference gene. The selection of the internal reference gene can correct for errors in sample processing, ensuring the accuracy and reproducibility of the detection results. The qRT-PCR procedure follows standard procedures, including reverse transcription of RNA, preparation of the reaction system, and PCR cycling. By comparing the expression levels of the target gene and the internal reference gene in different samples, the relative utilization rate of distal polyadenylation sites is calculated, thus corroborating the sequencing analysis results.

[0048] In addition, step one also requires detecting the expression level of CDCA7L protein. One possible approach is to digest breast tissue samples or peripheral blood samples from high-risk individuals with trypsin, followed by LC-MS / MS sequencing for detection. Trypsin can cleave proteins into specific peptides, facilitating subsequent separation and detection. LC-MS / MS technology combines the separation capabilities of liquid chromatography with the qualitative and quantitative capabilities of mass spectrometry, enabling the identification and quantification of proteins in the sample. Typically, the data obtained from LC-MS / MS are quantitatively analyzed using iBAQ with MaxQuant software. The iBAQ quantification method, based on the number and intensity of peptide detections, can relatively accurately reflect protein expression levels, providing reliable protein-level data for subsequent analysis. This lays the foundation for preventative intervention or early diagnostic assessment in step two.

[0049] Step 2: Based on the abnormal changes of decreased utilization of distal polyadenylation sites and upregulated protein expression levels, preventive interventions or early diagnosis are carried out for individuals with such abnormal changes.

[0050] Furthermore, in step two, the preventive intervention targets individuals in the high-risk population whose distal polyadenylation site utilization rate is lower than the average of healthy individuals and whose protein expression level is upregulated. This intervention uses APA regulatory factor inhibitors. Early diagnosis is made based on the determination that the distal polyadenylation site utilization rate in breast tumor tissue is lower than that in adjacent normal tissue and whose protein expression level is upregulated.

[0051] Specifically, step two, based on the utilization rate of the distal polyadenylation site of the CDCA7L gene and the protein expression level data obtained in step one, analyzes the abnormal changes in both and carries out targeted preventive interventions or early diagnosis to achieve the prevention and control of breast cancer.

[0052] Generally, preventive interventions primarily target individuals within high-risk groups exhibiting abnormal changes. As mentioned earlier, high-risk individuals are women with a family history of breast cancer or a history of dysplasia of the breast. When identifying abnormalities, the test results of this group must be compared to the mean in the healthy population. The mean in the healthy population is typically obtained by statistically analyzing a sample of healthy women without breast cancer-related risk factors to ensure representativeness and reliability. When the utilization rate of the distal polyadenylation site of the CDCA7L gene in an individual within the high-risk group is lower than the mean in the healthy population, and this is accompanied by an upregulation of CDCA7L protein expression, it is considered an abnormal change requiring preventive intervention.

[0053] As an alternative, preventative intervention employs APA regulatory factor inhibitors, which act on regulatory factors involved in the variable polyadenylation process, thereby modulating the polyadenylation pattern of the CDCA7L gene. Specifically, the intervention method can be chosen based on individual circumstances, such as oral or subcutaneous injection, and the frequency and dosage need to be adjusted according to the type of inhibitor and the individual's response to the drug.

[0054] Early diagnosis primarily targets breast tissue samples. In this case, it's necessary to compare the test results of tumor tissue with those of adjacent normal tissue. Using adjacent normal tissue as a control allows for a more accurate reflection of abnormal changes in the tumor tissue. When the test reveals a decreased utilization rate of the distal polyadenylation site of the CDCA7L gene in breast tumor tissue compared to adjacent normal tissue, and an upregulated expression level of CDCA7L protein compared to adjacent normal tissue, the individual can be identified as a suspected case of breast cancer.

[0055] In one possible implementation, to improve the accuracy of the diagnosis, multiple test results from step one are combined for comprehensive analysis. For example, RNA-seq sequencing data, qRT-PCR validation results, and LC-MS / MS protein quantification data are combined to ensure the consistency of abnormal changes. Generally, when multiple detection methods show the above-mentioned abnormalities in tumor tissue, clinical symptoms and other examination results can be further combined to finally diagnose breast cancer.

[0056] By utilizing the aforementioned preventive interventions and early diagnostic assessments, the abnormal changes in the CDCA7L gene can be used to manage the risks of high-risk populations and identify breast cancer early, thus buying time for subsequent treatment and helping to improve patient prognosis.

[0057] Step 3: Construct an intervention vector targeting the long 3′ untranslated region of the CDCA7L gene. By using the intervention vector to inhibit the function of the long 3′ untranslated region of the CDCA7L gene, the malignant phenotype of breast cancer cells can be blocked.

[0058] Furthermore, in step three, the construction of the intervention vector includes cloning the coding region of the CDCA7L gene and either the long 3′ untranslated region or the short 3′ untranslated region into the pLVX lentiviral vector, wherein the nucleotide sequence of the long 3′ untranslated region is SEQ ID NO:1 and the nucleotide sequence of the short 3′ untranslated region is SEQ ID NO:2.

[0059] Furthermore, in step three, the intervention vector is packaged with lentivirus and then infects breast cancer cells. Stable expression cell lines are established through puromycin screening. Lentiviral packaging is performed using the HEK293T cell three-plasmid system, and the breast cancer cells are MDA-MB-231 cells or BT-549 cells.

[0060] Specifically, step three involves constructing a specific intervention vector and applying it to breast cancer cells to inhibit the function of the 3′ untranslated region of the CDCA7L gene, thereby blocking the malignant phenotype of breast cancer cells.

[0061] The intervention vector was constructed based on the pLVX lentiviral vector. Specifically, the coding region of the CDCA7L gene was cloned and ligated with either a long 3′ untranslated region (UTR) or a short 3′ UTR. The nucleotide sequence of the long 3′ UTR is SEQ ID NO:1, and the nucleotide sequence of the short 3′ UTR is SEQ ID NO:2. During the cloning process, the coding region and the corresponding 3′ UTR fragment of the CDCA7L gene were first obtained by PCR amplification. The primers used for amplification needed to contain restriction enzyme sites that matched the pLVX vector.

[0062] Alternatively, the amplified target fragment and the pLVX vector are double-digested using the aforementioned restriction endonucleases. The digestion reaction is typically performed at 37°C, with the reaction time adjusted according to enzyme activity, generally around 2 hours. After digestion, the target fragment and vector backbone are separated and recovered by agarose gel electrophoresis. Then, they are ligated overnight at 16°C using T4 DNA ligase to form the recombinant vector. The ligation product is transformed into DH5α competent cells, and after culturing, single colonies are picked and sequenced to verify the correctness of the recombinant vector.

[0063] In one possible implementation, the intervention vector needs to be packaged with lentivirus to effectively infect breast cancer cells. Lentiviral packaging utilizes the HEK293T cell three-plasmid system, which contains the recombinant pLVX intervention vector, the psPAX2 packaging plasmid, and the pMD2.G envelope plasmid. Specifically, HEK293T cells in logarithmic growth phase are seeded in 6-well plates and transfected when cell confluence reaches 70%-80%. After transfection, cell supernatants containing packaged lentivirus particles are typically collected at 48 and 72 hours. The collected supernatants are filtered through a 0.45 μm filter to remove cell debris and other impurities. The resulting viral solution can be used directly for infection or aliquoted and stored at -80°C for later use.

[0064] The breast cancer cells used for infection were MDA-MB-231 cells or BT-549 cells. These cells were typically cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody at 37°C in a 5% CO2 incubator. One day before infection, cells were seeded into 6-well plates to achieve 30%-50% confluence at infection. During infection, an appropriate amount of virus solution was added to each well, along with Polybrene at a final concentration of 8 μg / mL to enhance infection efficiency. After 24 hours of incubation, the medium was replaced with fresh medium.

[0065] After infection, stable expression cell lines need to be established through puromycin selection. Generally, 48 hours after infection, puromycin is added to the culture medium at a final concentration of 2 μg / mL, and selection continues for about 2 weeks. During this period, the puromycin-containing culture medium is replaced every 2-3 days to remove uninfected cells. After selection, the mRNA expression level of the CDCA7L gene is detected by qRT-PCR, and its protein expression level is detected by Western blot to verify the effectiveness of stable cell line construction and ensure stable expression of the intervention vector in cells.

[0066] By intervening in the construction, lentiviral packaging, cell infection, and screening processes of the intervention vector, it is possible to enable the intervention vector to function in breast cancer cells, inhibiting the function of the 3′ untranslated region of the CDCA7L gene, thereby blocking the malignant phenotype of breast cancer cells and providing an effective experimental basis and potential means for the treatment of breast cancer.

[0067] By leveraging the abnormal molecular characteristics of reduced utilization and upregulated protein expression at the distal polyadenylation site of the CDCA7L gene, an integrated technical solution encompassing prevention, early diagnosis, and treatment is constructed. In prevention, individuals in high-risk populations exhibiting this abnormality are intervened with an APA regulatory factor inhibitor to maintain the normal APA pattern of the CDCA7L gene. In early diagnosis, this abnormal characteristic is detected in breast tissue samples or peripheral blood samples from high-risk individuals to achieve early identification of breast cancer. In treatment, an intervention vector targeting the long 3′ untranslated region of the CDCA7L gene is constructed to inhibit its function and block the malignant phenotype of breast cancer cells. This approach ensures that prevention, early diagnosis, and treatment all work synergistically around the unified molecular mechanism of abnormal APA in the CDCA7L gene, forming a complete chain of management from risk warning to early identification and intervention. This achieves synergy among all aspects of breast cancer prevention and treatment, addressing the problem in existing technologies where prevention, early diagnosis, and treatment are independent and lack an integrated strategy based on the same molecular mechanism, leading to insufficient prevention and treatment effectiveness.

[0068] The following is a further description with reference to specific embodiments:

[0069] Example 1

[0070] CDCA7L is a nuclear DNA-binding protein that plays a crucial role in transcriptional regulation by binding to specific promoter regions. CDCA7L can act as a target gene of c-Myc and its interacting protein, significantly supporting the complete transformation activity of c-Myc (i.e., participating in tumor transformation promotion). During transcription, it primarily functions as a repressor, influencing gene activity and cell fate by regulating the expression of genes such as MAOA (monoamine oxidase A). Furthermore, CDCA7L plays a role in multiple cell signaling pathways, upstream regulating key apoptosis and cell cycle regulators such as caspase-3, Cyclin D1, and E2F1. In summary, CDCA7L exerts core functions in both physiological and pathological contexts by affecting chromatin conformation, enhancing transcription factor binding efficiency, and regulating key cell cycle and apoptosis pathways. Its aberrant expression in various tumors and its involvement in cell proliferation, apoptosis, and invasion suggest its potential value as a biomarker and a possible new target for precision medicine.

[0071] Thirty-nine pairs of paired breast cancer and adjacent normal tissue (NAT) samples were collected. Total RNA was extracted using the TRIzol method, and quality was assessed using the Agilent Bioanalyzer. Samples with RIN ≥ 7 were used for library construction. RNA was selected using Poly(A)+ and then sequenced at both ends on the BGIDNBSEQ-T7 platform (PE150, ≥ 40 Mreads / sample). After removing low-quality sequences and adapters using Trim Galore, the sequencing data were aligned to the human genome GRCh38 using STAR, and the 3′UTR variable polyadenylation (APA) pattern of CDCA7L was evaluated using DaPars2 and QAPA, respectively.

[0072] To validate the APA results, specific primers covering the entire CDCA7L transcript and the distinct poly(A)site region were designed. SYBR Green qRT-PCR was used to analyze the relative distinct PAS utilization rate, and total CDCA7L transcript expression was simultaneously detected. The 18S gene was used as the internal reference gene. For protein level detection, tissue samples were lysed, digested with trypsin, and then sequenced using LC-MS / MS. Quantification was performed using MaxQuant software with iBAQ. All data were analyzed using paired statistical methods (Wilcoxon or paired t-test), with significance levels at a certain level. Figure 1 The annotation is in Chinese.

[0073] As attached Figure 1 As shown, (a) RNA-seq signals show the difference in 3′UTR usage of CDCA7L in breast cancer and adjacent normal tissue.

[0074] (bc) represents the percentage of distal poly(A) sites used in CDCA7L analyzed using DaPars2(b) and QAPA(c). The results show that the use of distal poly(A) sites was significantly reduced in BCs.

[0075] (d) shows the proximal and distal poly(A) sites of CDCA7L mRNA at the 3′UTR, and marks the primer positions used to detect the whole transcript and the long transcript.

[0076] (e) qRT-PCR was used to detect the use of CDCA7L distal poly(A)site in breast cancer and adjacent normal tissues. The results showed that the use of distal poly(A)site was significantly reduced in BCs.

[0077] (f) shows the total expression level of CDCA7L mRNA in breast cancer and adjacent normal tissues analyzed by qRT-PCR, and there was no statistically significant difference between the two.

[0078] (g) The results of proteomics detection (iBAQ quantification) show that the protein level of CDCA7L is significantly upregulated in breast cancer.

[0079] Data are presented as box plots and violin plots, with significance levels expressed as *P<0.05; **P<0.01; **P<0.001.

[0080] Primer design and qRT-PCR validation (for PAS):

[0081] Design principles: Total primers (for detecting all CDCA7L transcripts): forward primers located at the end of the CDS or near the 5′ end of the 3′ UTR;

[0082] Long-3′UTR primer (specifically recognizes long transcripts upstream of the distal PAS): The reverse primer spans a unique segment upstream of the distal PAS;

[0083] Product length 80-150bp, Tm about 60℃, GC 40-60%.

[0084] Reverse transcription: 1 μg RNA, PrimeScript RT or equivalent, random hexamer + oligo-dT, 42℃ for 30 min; inactivated at 95℃ for 3 min.

[0085] qPCR: SYBR Green system, 10 μL reaction (2×Master Mix 5 μL, 10 μM primers 0.3 μL each, cDNA 1 μL, water added to 10 μL).

[0086] Cycles: 95℃ for 30s; 40 cycles (95℃ for 5s, 60℃ for 30s); melting curves confirmed specificity.

[0087] Internal reference: 18S rRNA.

[0088] Calculation: Use =2^(-ΔCt_Long) / 2^(-ΔCt_Total) relative to the distinct PAS, and normalize the sample intrinsic parameters;

[0089] Paired Wilcoxon was used between groups; effect sizes were reported as Hedges'g or Cliff's delta. Figure e corresponds to this result.

[0090] The following table shows the primer sequences:

[0091] Table 1

[0092]

[0093]

[0094] As attached Figure 2 As shown, a and b represent the Kaplan-Meier survival analysis results of CDCA7L expression based on the KMploter database (https: / / kmplot.com / analysis / ), respectively showing the survival difference between breast cancer patients in the "low expression" and "high expression" CDCA7L groups in the RNASeq cohort (a) and the MicroArray cohort (b). a RNASeq cohort: hazard ratio (HR) = 1.76, logrank P < 0.0001; b MicroArray cohort: HR = 1.47, logrank P < 0.0001.

[0095] c represents the Kaplan-Meier survival analysis results of CDCA7L expression in the TCGA cohort (HR = 1.4, logrank P < 0.05), also compared by expression level.

[0096] Figure 2 In this context, Probability represents the probability of survival, Time (months) represents the follow-up period (in months), and HR represents the hazard ratio.

[0097] Plasmid construction and stable transfection:

[0098] To investigate the function of different 3′UTR isoforms of CDCA7L, the CDS region of CDCA7L and either a long (3′UTRL) or a short (3′UTRS) 3′UTR were cloned into the pLVX lentiviral vector. An empty vector (Vec) was used as the control group. Virus packaging was performed using the HEK293T cell triplasmid system (psPAX2 and pMD2.G), and the supernatant was collected and filtered through a 0.45 μm filter. MDA-MB-231 and BT-549 cells were infected with viral supernatant containing 8 μg / mL polybrene for 48 h, and then selected using 2 μg / mL puromycin to establish stable expression cell lines.

[0099] qRT-PCR and Western blot

[0100] Total RNA (TRIzol, Invitrogen) was extracted from cells, reverse transcribed, and then qRT-PCR was performed to detect CDCA7L expression. GAPDH was used as the internal control gene. Protein expression was detected by extracting total protein using RIPA lysis buffer, separating it by SDS-PAGE, transferring it to a membrane, and detecting it with a CDCA7L-specific antibody (Abcam). β-actin was used as an internal control.

[0101] Cell proliferation assay (CCK-8)

[0102] Cells with different treatments were seeded at 2000 cells / well in 96-well plates. CCK-8 solution was added at 0, 24, 48, 72 and 96 h, respectively. After incubation for 2 h, the absorbance was read at 450 nm and cell growth curves were plotted.

[0103] Transwell migration experiments:

[0104] An 8 μm pore size membrane was added to a 24-well Transwell chamber. 1 × 10⁵ cells were seeded in the upper chamber (serum-free medium), and medium containing 10% FBS was added to the lower chamber as a chemokine. After 24 h, cells were fixed (4% paraformaldehyde) and stained with crystal violet. The number of migrating cells was counted in five random fields of view.

[0105] Mouse subcutaneous tumor formation experiment

[0106] Different stably transfected MDA-MB-231 cells (5 × 10⁶ cells / mouse) were suspended in 100 μL PBS and subcutaneously injected into 4-6 week old female NCG nude mice (n = 6 per group). Tumor volume (volume = length × width × width / 2) was measured every 3 days. At the end of the experiment, the animals were sacrificed and the tumor weight was measured. All animal experiments were approved by the institution's animal ethics committee.

[0107] Statistical analysis: All experiments were repeated three times, and data are expressed as mean ± standard deviation (SD). Student's t-test or one-way ANOVA was used for comparisons between groups, and p < 0.05 was considered statistically significant.

[0108] As attached Figure 3As shown, the constructed CDCA7L long 3′UTR (3′UTRL) and short 3′UTR (3′UTRS) isoforms (a) overexpression vectors are illustrated. qRT-PCR results showed that overexpression of both the long and short isoforms significantly upregulated CDCA7L mRNA levels in MDA-MB-231 and BT-549 cells (b). Western blot analysis further confirmed that overexpression of 3′UTRL significantly increased CDCA7L protein expression, while overexpression of 3′UTRS did not change CDCA7L protein expression levels (c). CCK-8 assays showed that overexpression of 3′UTRL significantly enhanced the proliferation of breast cancer cells, while overexpression of 3′UTRS had little effect on malignant cell proliferation (de). Transwell migration assays showed that overexpression of 3′UTRL significantly enhanced the migration ability of breast cancer cells, while overexpression of 3′UTRS had little effect on cell migration (fg). In in vivo experiments, lentiviral systems were used to construct stable cell lines overexpressing 3′UTRL and 3′UTRS. MDA-MB-231 cells were subcutaneously injected to establish a mouse subcutaneous tumorigenesis model. The results showed that the proliferation capacity of breast cancer cells overexpressing 3′UTRL was significantly enhanced, while the isoform overexpressing 3′UTRS had no significant effect on the growth of breast cancer cells (hj).

[0109] sequence list

[0110] SEQ ID NO:1(CDCA7L-Long-UTR):

[0111]

[0112] SEQ ID NO:2(CDCA7L-Short-UTR):

[0113]

[0114] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for the prevention, early diagnosis, and treatment of breast cancer, characterized in that, Includes the following steps: Step 1: Detect the utilization rate of distal polyadenylation sites and protein expression levels of the CDCA7L gene in breast tissue samples or peripheral blood samples from high-risk individuals; Step 2: Based on the abnormal changes of decreased utilization of distal polyadenylation sites and upregulated protein expression levels, preventive interventions or early diagnosis are carried out for individuals with such abnormal changes. Step 3: Construct an intervention vector targeting the long 3′ untranslated region of the CDCA7L gene. By using the intervention vector to inhibit the function of the long 3′ untranslated region of the CDCA7L gene, the malignant phenotype of breast cancer cells can be blocked.

2. The method for prevention, early diagnosis, and treatment of breast cancer according to claim 1, characterized in that: In step one, the high-risk group refers to women with a family history of breast cancer or a history of atypical breast hyperplasia. The peripheral blood sample is subjected to density gradient centrifugation to separate peripheral blood mononuclear cells and then total RNA is extracted. The breast tissue sample includes tumor tissue and adjacent normal tissue. After collection, the samples are flash-frozen in liquid nitrogen and stored at -80°C.

3. The method for prevention, early diagnosis, and treatment of breast cancer according to claim 1, characterized in that: In step one, when detecting the utilization rate of distal polyadenylation sites of the CDCA7L gene, total RNA was extracted using the TRIzol method, and RNA integrity was required to meet RIN≥7. Total protein was extracted using RIPA lysis buffer containing protease inhibitors, with a protein concentration of 50-1000 μg / mL.

4. A method for the prevention, early diagnosis, and treatment of breast cancer according to claim 1, characterized in that: Step one, detecting the utilization rate of the distal polyadenylation site of the CDCA7L gene, specifically includes the following steps: After Poly(A)+ screening of total RNA, sequencing libraries were constructed, and sequencing data were obtained by paired-end sequencing. Sequencing data were filtered and genome alignment was performed. Bioinformatics tools were used to calculate the utilization rate of distal polyadenylation sites.

5. A method for the prevention, early diagnosis, and treatment of breast cancer according to claim 4, characterized in that: The paired-end sequencing platform is BGIDNBSEQ-T7, the sequencing read length is PE150, and the sequencing volume of each sample is ≥40Mreads. The filtering is performed using Trim Galore. The genome alignment is performed using STAR software to align the filtered data to the human genome GRCh38. The bioinformatics tools include DaPars2 and QAPA.

6. A method for the prevention, early diagnosis, and treatment of breast cancer according to claim 4, characterized in that: The detection of the utilization rate of the distal polyadenylation site of the CDCA7L gene also includes verification by qRT-PCR. The primers used in the qRT-PCR include the primer pair CDCA7L-Total-Fwd / Rev for detecting total transcripts and the primer pair CDCA7L-Long-Fwd / Rev for detecting long 3′ untranslated region transcripts. The internal reference gene is 18S rRNA.

7. A method for the prevention, early diagnosis, and treatment of breast cancer according to claim 1, characterized in that: In step one, the detection of CDCA7L protein expression level involves digesting breast tissue samples or peripheral blood samples from high-risk individuals with trypsin, followed by LC-MS / MS sequencing, and then quantifying using iBAQ software with MaxQuant software.

8. A method for the prevention, early diagnosis, and treatment of breast cancer according to claim 1, characterized in that: In step two, the preventive intervention is to use APA regulatory factor inhibitors to intervene in individuals in the high-risk population whose distal polyadenylation site utilization rate is lower than the average of healthy individuals and whose protein expression level is upregulated. The early diagnosis is determined by the fact that the distal polyadenylation site utilization rate in breast tumor tissue is lower than that in adjacent normal tissue and whose protein expression level is upregulated.

9. A method for the prevention, early diagnosis, and treatment of breast cancer according to claim 1, characterized in that: In step three, the construction of the intervention vector includes cloning the coding region of the CDCA7L gene and the long 3′ untranslated region or the short 3′ untranslated region into the pLVX lentiviral vector, wherein the nucleotide sequence of the long 3′ untranslated region is SEQ ID NO:1 and the nucleotide sequence of the short 3′ untranslated region is SEQ ID NO:

2.

10. A method for the prevention, early diagnosis, and treatment of breast cancer according to claim 1, characterized in that: In step three, the intervention vector is packaged with lentivirus and then infects breast cancer cells. Stable expression cell lines are established by screening with puromycin. The lentivirus packaging is performed using the HEK293T cell three-plasmid system. The breast cancer cells are MDA-MB-231 cells or BT-549 cells.

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