Biomarkers for detecting PARP1 function and application thereof

Abstract

The invention relates to the field of biological medicine, and discloses a biomarker for detecting a PARP1 function and application of the biomarker, and the biomarker comprises CBX5, CKAP2, NKTR, PTCH1, ZNF318, CENPF, ARID4B, CDK19, SRCAP, CELF2, CBFA2T3, TIAM1, SARDH, IDI1, BIRC6, PAN3, PRSS23, ITGA2, PROCR, S100A2, GRN, IFIT1, MMP1, RBPMS, JAG1, IFI27, OASL, HMGA2, SPHK1, KLF10, MYO10, ARRDC4, PLAUR, IER3, PTGS2, ANKRD29 and The biomarker is a regulatory gene of the PARP1, and has higher specificity than that of the PARP1.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a set of biomarkers for detecting PARP1 function and their applications. Background Technology

[0002] Poly-ADP-ribose polymerase (PARP) is a key regulatory molecule in DNA damage repair, playing a crucial role in maintaining genome stability. PARP1 activity is closely related to the occurrence and development of various cancers, including breast cancer and ovarian cancer. With the gradual improvement of basic research on DNA damage repair mechanisms and related molecular markers, novel anti-tumor drugs targeting PARP1, such as PARP inhibitors, have been widely used in clinical practice. However, individual differences in tumors are significant, and the importance of PARP1 in the DNA damage response determines the sensitivity of tumors to PARP inhibitors.

[0003] In laboratory studies, PARP1 activation is typically detected by analyzing the level of poly-ADP-ribose (PAR) molecules produced by its catalysis, commonly using Western blot and immunofluorescence (IF) to detect PAR expression levels. Although PAR is a catalytic product of PARP1, it can also be degraded by genes such as PARG, making it difficult for PAR detection to directly reflect PARP1 enzymatic activity. Furthermore, these detection methods often rely on complex procedures, demanding experimental conditions, and challenging sample collection. In clinical applications, PARP1 activity detection is primarily used to assess tumor DNA damage response and sensitivity to PARP inhibitors (such as olaparib), and it also has reference value in assessing neurodegenerative diseases and aging. However, because PARP1 activity is influenced by multiple factors such as cellular state, therapeutic intervention, and the tumor microenvironment, its specificity and standardization are low, and the detection methods require sample collection, which is difficult and invasive for patients. Currently, there is a lack of clinical methods for detecting PARP1, necessitating the development of more targeted approaches.

[0004] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a set of biomarkers for detecting PARP1 function and their applications, aiming to solve the problem of low specificity of existing PARP1 detection.

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

[0007] In a first aspect, the present invention provides a set of biomarkers for detecting PARP1 function, said biomarkers including CBX5, CKAP2, NKTR, PTCH1, ZNF318, CENPF, ARID4B, CDK19, SRCAP, CELF2, CBFA2T3, TIAM1, SARDH, IDI1, BIRC6, PAN3, PRSS23, ITGA2, PROCR, S100A2, GRN, IFIT1, MMP1, RBPMS, JAG1, IFI27, OASL, HMGA2, SPHK1, KLF10, MYO10, ARRDC4, PLAUR, IER3, PTGS2, ANKRD29, and AKR1C3.

[0008] In a second aspect, the invention provides the use of the above-described biomarkers in the preparation of products for detecting PARP1 function in tumor patients.

[0009] A third aspect of the present invention provides the use of reagents for detecting the above-mentioned biomarkers in the preparation of products for detecting PARP1 function in tumor patients.

[0010] Optionally, the tumor is one of breast cancer, ovarian cancer, or head and neck cancer.

[0011] In a fourth aspect, the present invention provides a product comprising reagents for detecting the aforementioned biomarkers.

[0012] Optionally, the product is a reagent kit, reagent, or test strip.

[0013] The present invention has the following beneficial effects:

[0014] This invention proposes a novel set of PARP1-related genes, and the screened functional marker genes are:

[0015] The regulatory genes of PARP1 exhibit higher specificity than PARP1 itself. This broadens the approach to PARP1-related treatments in diseases, providing new targeted technologies with lower risk of side effects. Attached Figure Description

[0016] Figure 1 The mechanism of action of PARP1 functional biomarker provided in the embodiments of the present invention;

[0017] Figure 2 A score map of enrichment analysis of functional marker gene sets in sequencing data samples;

[0018] Figure 3 A correlation diagram between the functional marker gene set of GDSC2 data and the IC50 of drugs;

[0019] Figure 4 Correlation plot of functional marker gene set with PARP1 in GDSC2 data;

[0020] Figure 5 Correlation plot of functional marker gene set with PARP1 in TCGA data;

[0021] Figure 6 A plot showing drug sensitivity analysis of the functional marker gene set from GDSC2 data;

[0022] Figure 7 A graph comparing the functional marker gene set of GDSC2 data with HRD drug sensitivity. Detailed Implementation

[0023] This invention provides a set of biomarkers for detecting PARP1 function and their applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0025] Ten years have passed since the global launch of the first PARP inhibitor, olaparib. As an anti-tumor drug targeting DNA damage repair pathways, PARP inhibitors have achieved great success in the treatment of breast and ovarian cancer. However, their application in other cancers remains limited. On the one hand, the "synthetic lethality" effect based on BRCA mutations is only common in breast and ovarian cancer, which is the main reason for the limited use of PARP inhibitors. On the other hand, the hematologic toxicity caused by combining PARP inhibitors with other treatments such as chemotherapy and radiotherapy, as well as the damage to normal tissues caused by PARP-1-DNA capture ability, are other factors limiting their application. Drug resistance to PARP inhibitors also poses certain limitations to their clinical application. Therefore, there is an urgent need to find new PARP1-related functional biomarkers to improve diagnosis, prognosis prediction, and treatment strategies. Furthermore, a deeper understanding of the mechanisms of action, resistance mechanisms, and combination therapy mechanisms of PARP inhibitors through novel PARP1 biomarkers is crucial for expanding their application in cancer treatment in the future.

[0026] Based on in-depth research on the PARP1 signaling pathway, this invention provides a set of biomarkers for detecting PARP1 function. These biomarkers include CBX5, CKAP2, NKTR, PTCH1, ZNF318, CENPF, ARID4B, CDK19, SRCAP, CELF2, CBFA2T3, TIAM1, SARDH, IDI1, BIRC6, PAN3, PRSS23, ITGA2, PROCR, S100A2, GRN, IFIT1, MMP1, RBPMS, JAG1, IFI27, OASL, HMGA2, SPHK1, KLF10, MYO10, ARRDC4, PLAUR, IER3, PTGS2, ANKRD29, and AKR1C3.

[0027] Besides breast and ovarian cancer, these biomarker genes can also serve as markers for tumor diagnosis and prognostic prediction in other cancers, such as head and neck cancer. They can be used to determine PARP1 activation status; when the selected functional marker genes are highly expressed, PARP1 is activated. Detecting the expression levels of these functional marker genes allows for the determination of PARP1 activation status, thus enabling the selection of combination therapies targeting PARP1. This provides a basis for subsequent experimental research and clinical treatment, offering new intervention methods for targeted therapy, and possesses significant research and clinical application value.

[0028] The mechanism of action of the biomarker for detecting PARP1 function provided in this embodiment of the invention is as follows: Figure 1 As shown, targeting and downregulating functional marker genes of PARP1 can overcome tumor resistance to PARP1 and improve the therapeutic effect of PARP1 inhibitors.

[0029] For example, in patients with BRCA1 / 2 mutations, knocking out the PARP1 functional marker gene accelerates DNA damage accumulation, thereby inducing lethal synthesis in cancer cells. Inhibiting the PARP1 functional marker gene weakens the DNA damage repair function of cancer cells, increasing the effectiveness of radiotherapy and chemotherapy. Silencing the PARP1 functional marker gene via RNAi significantly enhances the efficacy of PARP inhibitors.

[0030] The following detailed description uses specific examples.

[0031] Example 1 Sample Processing

[0032] CRISPR-CAS9 technology was used to construct PARP1 hyperexpression and knockout CAL33 cell lines. Normal CAL33 cell lines, PARP1 hyperexpression CAL33 cell lines, and PARP1 knockout CAL33 cells were cultured normally in T25 culture flasks. When the cells reached 80% density, they were digested with TRIZOL, collected in centrifuge tubes, and sent to a sequencing company for RNA-seq sequencing to obtain sequencing data.

[0033] Example 2: Screening of PARP1 functional marker genes

[0034] Sequencing data were analyzed, and gene expression data of CAL33 cell lines with high PARP1 expression and those with PARP1 knockout were compared. 493 upregulated functional marker genes and 329 downregulated functional marker genes were identified. Further analysis of GDSC2 data revealed the correlation between these upregulated and downregulated functional marker genes and PARP1. 122 upregulated functional marker genes with a positive correlation to PARP1 (P < 0.05) and 141 downregulated functional marker genes with a negative correlation to PARP1 (P < 0.05) were identified. These upregulated and downregulated functional marker genes were then sorted according to their P-values ​​from smallest to largest. The top 20% of genes in each group were selected as the final functional marker genes. Based on the previous analysis, a PARP1 functional marker gene model was constructed. The functional marker genes are divided into 16 upregulated functional marker genes, including CBX5, CKAP2, NKTR, PTCH1, ZNF318, CENPF, ARID4B, CDK19, SRCAP, CELF2, CBFA2T3, TIAM1, SARDH, IDI1, BIRC6, and PAN3, and 21 downregulated functional marker genes, including PRSS23, ITGA2, PROCR, S100A2, GRN, IFIT1, MMP1, RBPMS, JAG1, IFI27, OASL, HMGA2, SPHK1, KLF10, MYO10, ARRDC4, PLAUR, IER3, PTGS2, ANKRD29, and AKR1C3, for a total of 37 functional marker genes.

[0035] Example 3: Functional Marker Gene Analysis

[0036] Figure 2Using sequencing data, ssGSEA scores were calculated for 37 genes, and the 'gene score' package (available at https: / / github.com / pujana-lab / genScore / ) was used to weight downregulated genes into upregulated genes. This score reflects the direction and abundance of mRNA enrichment of these 37 genes in each sample (tumor cell line). A higher score indicates a higher degree of total mRNA enrichment of the PARP1 functional marker gene set in each sample. Under this standard, ssGSEA scores were calculated in CAL33 cell lines with high PARP1 expression and CAL33 cell line samples with PARP1 knockout, respectively.

[0037] Figure 3 Using GDSC data, ssGSEA scores were calculated for 37 genes. The 'gene score' R package was used to weight downregulated genes into upregulated genes. Correlation analysis was performed between ssGSEA scores and the IC50 values ​​of PARP1 / PARP2 (PARP) inhibitors (Niraparib, Olaparib, Rucaparib, Talazoparib, Veliparib). CellTiter-Glo is a high-throughput cell viability assay based on ATP bioluminescence. This technique provides the half-maximal inhibitory concentration (IC50) of a cell line for a drug; a higher IC50 value indicates greater cell sensitivity to the drug. The ssGSEA scores of the 37 genes were negatively correlated with the IC50 of PARP1 / PARP2 (PARP) inhibitors. The R-value (correlation coefficient) measures the linear correlation between two variables; a lower R-value indicates greater sensitivity of the functional marker gene set composed of the 37 genes to the drug.

[0038] Figure 4 Using TCGA data, ssGSEA scores were calculated for 37 genes across 7 cancer types. The 'genescore' R package was used to weight downregulated genes within upregulated genes, and correlation analysis was performed between ssGSEA scores and PARP1 expression levels. Higher R values ​​indicate a stronger correlation between the functional marker gene set composed of the 37 genes and PARP1.

[0039] Figure 5 Using GDSC data, ssGSEA scores were calculated for 37 genes in cell lines. The 'genescore' R package was used to weight downregulated genes into upregulated genes, and correlation analysis was performed between ssGSEA scores and PARP1 expression levels. A higher R value indicates a stronger correlation between the functional marker gene set composed of the 37 genes and PARP1.

[0040] Figure 6Using GDSC data, 37 genes were scored using the ssGSEA method. The ssGSEA scores were ranked from highest to lowest, and the top 100 and bottom 100 genes were used to divide cell line samples into high-scoring and low-scoring PARP1 functional gene sets. Sensitivity analysis was then performed on PARP1 / PARP2 (PARP) inhibitors (Niraparib, Olaparib, Rucaparib, Talazoparib, and Veliparib). The results showed that the PARP1 functional gene set effectively distinguished between PARP1 inhibitor-resistant and sensitive cell lines, and the high-scoring group was more sensitive to PARP inhibitors.

[0041] Figure 7 To further evaluate the performance of the PARP1 functional gene set as a novel biomarker in PARP inhibitor-targeted therapy compared to the existing biomarker, the homologous recombination defect score (scarHRD), the HRD gene set was analyzed using the same method. The results showed that, with an equal number of cell lines, the high PARP1 functional gene set score group exhibited significantly higher sensitivity to PARP than the high HRD score group. These results indicate that the PARP1 functional gene set, as a novel biomarker, is significantly superior to the existing biomarker HRD in a single-factor cancer cell environment.

[0042] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A set of biomarkers for detecting PARP1 function, characterized in that, The biomarkers include CBX5, CKAP2, NKTR, PTCH1, ZNF318, CENPF, ARID4B, CDK19, SRCAP, CELF2, CBFA2T3, TIAM1, SARDH, IDI1, BIRC6, PAN3, PRSS23, ITGA2, PROCR, S100A2, GRN, IFIT1, MMP1, RBPMS, JAG1, IFI27, OASL, HMGA2, SPHK1, KLF10, MYO10, ARRDC4, PLAUR, IER3, PTGS2, ANKRD29, and AKR1C3.

2. The use of the biomarker of claim 1 in the preparation of products for detecting PARP1 function in tumor patients.

3. The use of the reagent for detecting the biomarker of claim 1 in the preparation of a product for detecting PARP1 function in tumor patients.

4. The application according to claim 2 or 3, characterized in that, The tumor is one of the following: breast cancer, ovarian cancer, or head and neck cancer.

5. A product characterized in that, The product includes reagents for detecting the biomarkers of claim 1.

6. The product according to claim 5, characterized in that, The products mentioned are reagent kits, reagents, and test strips.

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