A novel broad-spectrum infection and non-infection tumor drug resistance marker and application thereof

Abstract

The present application relates to the biomedical technology field, specifically to a novel broad-spectrum infection and non-infection tumor drug resistance marker and application thereof, wherein the tumor drug resistance marker is a D132 site mutation of AURKA or a mutation in AURKA 129 ALED 132 Cutting site mutation. The present application discloses a new target point of the tumor cell killing activity of a chemotherapy drug, i.e. AURKA D132 , which is highly conserved in different species; it is found by cBioPortal database analysis that AURKA D132 mutation really exists in clinical tumor samples; further experiments prove that AURKA D132 mutation can inhibit cell apoptosis and reduce the sensitivity of cells to apoptosis stimulation, and experiments on in-vivo tumor-bearing models prove that AURKA D132 mutation can reduce the treatment effect of paclitaxel on cervical cancer cells, and thus AURKA D132 related mutation can be used as a diagnosis and treatment index of paclitaxel resistance.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a novel, broad-spectrum biomarker for drug resistance in both infectious and non-infectious tumors and its applications. Background Technology

[0002] Malignant tumors are a common and serious disease that severely endangers human life and health. Chemotherapy is currently one of the main treatments for malignant tumors, killing cancer cells by inducing apoptosis. Among current chemotherapy drugs, paclitaxel is one of the best natural anti-tumor drugs. It is an antimitotic agent that targets and binds to microtubules, inhibiting microtubule depolymerization, interfering with spindle assembly and chromosome separation, causing mitotic arrest and thus inducing apoptosis. As one of the most ideal natural anti-tumor drugs, paclitaxel is a first-line chemotherapy drug in clinical practice for treating various infectious and non-infectious cancers, including ovarian cancer, breast cancer, Kaposi's sarcoma, cervical cancer, and endometrioid carcinoma. However, tumor resistance developed during chemotherapy is a major challenge for the clinical application of paclitaxel, greatly limiting its effectiveness. Paclitaxel resistance is a cumulative effect. Although several mechanisms of paclitaxel resistance have been reported, such as overexpression of the multidrug resistance gene (MDR1), mutation of β-tubulin, and mutation of pro-apoptotic genes, treatments targeting common resistance mechanisms have not achieved the expected results. Therefore, it is extremely important to explore key detection and treatment targets for paclitaxel resistance and guide targeted therapy to solve tumor drug resistance. Existing technologies still need to be improved and developed. Summary of the Invention

[0003] In view of the shortcomings of the prior art, the purpose of this invention is to provide a novel, broad-spectrum tumor drug resistance biomarker and its application, aiming to solve the problem that the lack of detection and treatment targets for anti-tumor drug resistance in the prior art makes targeted therapy for tumor drug resistance difficult.

[0004] The technical solution of the present invention is as follows:

[0005] A tumor drug resistance marker, wherein the tumor drug resistance marker is a D132 site mutation in AURKA or a mutation in AURKA. 129 ALED 132 Cleavage site mutations. Among them, the D132 site in AURKA is highly conserved in humans, mice, rats, cattle, pigs, and even Xenopus; AURKA's... 129 ALED 132 The cleavage site is highly conserved in eukaryotes such as humans, cattle, and pigs.

[0006] The tumor drug resistance markers mentioned above are used to identify AURKA. 129 ALED132 Caspase-3, Caspase-7, and Caspase-8 sequences are mutated, deleted, or inactivated.

[0007] The tumor drug resistance markers mentioned herein, wherein the tumor is one of the following: infected and non-infectious related lymphoma, Kaposi's sarcoma, cervical cancer, breast cancer, liver cancer, and endometrioid carcinoma.

[0008] The application of the tumor drug resistance markers, wherein the tumor drug resistance markers are used to predict antitumor drug resistance.

[0009] The application of the tumor drug resistance marker, wherein the step of using the tumor drug resistance marker to predict antitumor drug resistance includes:

[0010] After treating tumor cell lines with antitumor drugs, AURKA 35 The expression levels of protein-cleaved peptides were detected.

[0011] If AURKA 35 If the expression level of the protein cleavage peptide is lower than a preset value, the antitumor drug is considered to have high resistance; if AURKA 35 If the expression level of the protein cleavage peptide is higher than a preset value, the antitumor drug is determined to have low resistance.

[0012] The application of the tumor drug resistance marker, wherein the antitumor drug is paclitaxel.

[0013] The application of the tumor drug resistance markers includes using the tumor drug resistance markers to prepare a detection kit for predicting antitumor drug resistance.

[0014] The application of the tumor drug resistance markers includes using the tumor drug resistance markers to prepare tumor diagnosis and screening kits.

[0015] The application of the tumor drug resistance markers includes using the tumor drug resistance markers to prepare targets for the design of anti-drug-resistant tumor drugs and anti-drug-resistant tumor drugs designed for the targets.

[0016] Beneficial effects: This invention discovers AURKA D132 and AURKA 129 ALED 132 It is a key target for anti-tumor drugs (such as paclitaxel) to kill tumor cells. The study also found that AURKA D132 Cleavage significantly upregulates the expression of pro-apoptotic effector molecules FOS, FOSB, Jun, and EGR1, interfering with centrosome formation and spindle assembly, and promoting apoptosis. This invention reveals a novel target for the tumor-killing activity of chemotherapeutic drugs, namely AURKA. D132This target is highly conserved across different species. Analysis of the cBioPortal database revealed AURKA. D132 The mutation actually exists in clinical tumor samples. Further experiments demonstrated that AURKA D132 Mutations can reduce the sensitivity of cells to apoptotic stimuli, and in vivo tumor-bearing model experiments have demonstrated that AURKA D132 Mutations can reduce the effectiveness of paclitaxel in treating cervical cancer cells, therefore AURKA D132 Mutations can serve as diagnostic and therapeutic indicators of paclitaxel resistance. This invention provides a novel, broad-spectrum key protein target for drug resistance in both infectious and non-infectious tumors, and elucidates a new molecular mechanism of tumor drug resistance. It holds promise for predicting and evaluating the efficacy of chemotherapy in cancer patients and for providing more feasible and effective strategic tools for the subsequent design or screening of anti-resistant tumor drugs. Attached Figure Description

[0017] Figure 1 The diagram shows the results of AURKA's specific cleavage of a 35 kDa peptide in apoptosis of various types of tumor cells. (A) Constructing GFP-tagged N-terminal and C-terminal truncated versions of the AURKA protein and identifying antibodies that recognize the C-terminal 350-403 amino acid epitopes of the AURKA protein; (B) In γ-herpesvirus-negative / positive cells, AURKA is cleaved in apoptosis induced by the anticancer compound STS to produce a 35 kDa peptide (i.e., AURKA). 35 (C) In cervical cancer, breast cancer, and liver cancer cell lines, AURKA is cleaved during STS-induced apoptosis to produce AURKA. 35 (D) In ​​PEL cells, cervical cancer cells, and breast cancer cells, AURKA is cleaved during paclitaxel-induced apoptosis to produce AURKA. 35 .

[0018] Figure 2 Caspase-3, 7, and 8 and the antitumor drug STS promote apoptosis in AURKA 35 The relevant results were plotted. Among them, (A) broad-spectrum caspase inhibitor Z-VAD-FMK and caspase-3 inhibitor Z-DEVD-FMK inhibited AURKA in the apoptosis process of HEK293T cells. 35 (B) Broad-spectrum caspase inhibitor Z-VAD-FMK inhibits AURKA in the apoptosis process of BJAB cells. 35 (C) Construction of HEK293T cell lines with different Caspase gene knockouts; (D) Caspase-3, Caspase-7, or Caspase-8 knockout significantly inhibits STS and paclitaxel-induced AURKA. 35 Cut the product.

[0019] Figure 3 The diagram shows that aspartate D132 is a key site for AURKA protein cleavage during apoptosis. (A) Bioinformatics software MEROPS and CaspDB predict potential protease cleavage sites for the AURKA peptide; (B) Mutations at the D132 site block STS-induced AURKA peptide cleavage; (C) The Site Prediction website assesses the cleavage of AURKA by caspases -3, -7, and -8.

[0020] 129 ALEDFE 134 Sequence site probability; (D)AURKA 129 ALEDFE 134 Conservation of sequence sites in sequence alignment across different species.

[0021] Figure 4 The results of analyzing the mutation frequency and distribution of AURKA with Caspase-3, Caspase-7 and Caspase-8 in clinical tumor samples using the cBioPortal database are shown in the figure.

[0022] Figure 5 For AURKA 133-403 A diagram showing the transcriptional and apoptosis results of AP-1 family genes, which are peptide-induced apoptosis-promoting effector molecules. Among them, (A)AURKA D132 Mutations inhibit STS-mediated apoptosis. (B)AURKA WT ,AURKA 1-132 ,AURKA 133-403 Clustering diagram of differentially expressed genes, AURKA WT vs AURKA 133-403 Top 10 upregulated gene expression heatmap; (C)AURKA WT vs AURKA 1-132 and AURKA WT vs AURKA 133-403 Volcano plot of differentially expressed genes; (D)AURKA WT vs AURKA 133-403 GO enrichment analysis of differentially expressed genes; (E)AURKA 133-403 Upregulates the transcriptional levels of key pro-apoptotic signaling molecules such as AP-1-related genes Jun, FOS, FOSB, and their downstream EGR1; (F)AURKA WT vs AURKA 133-403 KEGG pathway enrichment analysis of differentially expressed genes.

[0023] Figure 6 For AURKAD132 The results of site mutations and cleavage products interfering with cell cycle progression are shown in the figure. Among them, (A)AURKA D132 Localization of cleavage products in HEK293T cells; (B) AURKA D132 The effects of site mutations and cleavage products on mitosis in HEK293T cells; (C) Quantitative analysis of the time elapsed from prophase to cytokinesis in mitotic cells expressing different AURKA mutants.

[0024] Figure 7 For AURKA D132 A schematic diagram illustrating how cleavage products interfere with the formation of centrosomes and spindles during cell mitosis. (A) shows the number and morphology of centrosomes in cells expressing different AURKA mutants during interphase and metaphase of mitosis; (B) shows the spindle morphology in cells expressing different AURKA mutants during metaphase and anaphase of mitosis.

[0025] Figure 8 To verify AURKA D132 The results diagram shows the potential targets for paclitaxel resistance. Among them, (A)AURKA D132 Site cleavage products inhibit cell clone generation; (B)AURKA D132 Mutations reduced the efficacy of paclitaxel in the in vitro treatment of cervical cancer cells; (C)AURKA D132 The mutation reduced the in vivo therapeutic effect of paclitaxel on cervical cancer cells. Detailed Implementation

[0026] This invention provides a novel, broad-spectrum biomarker for drug resistance in both infectious and non-infectious tumors and its 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 merely illustrative and not intended to limit the scope of the invention.

[0027] Example 1

[0028] Aurora kinase A (AURKA) undergoes D132 site-specific cleavage during apoptosis.

[0029] 1. Identification of AURKA antibodies that specifically recognize the C-terminal amino acid region of the AURKA protein:

[0030] HEK293T cells were transfected with AURKA mutants of different segments tagged with GFP for expression. Cells were collected 24 hours after transfection, lysed, and proteins were extracted for Western blotting. Anti-GFP and anti-AURKA protein C-terminal antibodies were used to detect the expression of different AURKA protein segments. Results showed that the anti-AURKA protein antibody mA01 specifically recognized the 350-403 amino acid segment, such as… Figure 1 As shown in Figure A.

[0031] 2. AURKA undergoes specific cleavage during apoptosis:

[0032] Staurosporine (STS) is a precursor compound for anticancer research and plays a crucial role in the field of antitumor therapy. For example, Glivec, the first molecularly targeted anticancer drug for treating chronic myeloid leukemia, is derived from STS. We used STS as an apoptosis inducer to induce apoptosis in various virus-associated tumor cell lines, and Western blotting was used to detect PARP1 cleavage as an indicator of apoptosis. The results showed that in STS-treated cells, including KSHV-latently infected negative and positive B lymphoma cells (BJAB, BCBL1, and BC3) and endothelial cells (iSLK, iLSK-16); EBV-negative and positive B cells (DG75, B95.8), PARP1 was specifically cleaved in a time-dependent manner, demonstrating successful apoptosis induction.

[0033] In this case, we used a validated antibody that specifically recognizes the AURKA C-terminus to detect AURKA protein expression and found that AURKA was specifically cleaved, producing a 35 kDa AURKA cleavage fragment (e.g., Figure 1 (As shown in Figure B). To determine the prevalence of AURKA protein cleavage, we further induced apoptosis in various cancer cells, including human cervical cancer cells (HeLa), breast cancer cells (MDA-MB-231), and liver cancer cells (SMMC-7721 and HLE), using STS treatment, and then detected AURKA protein cleavage. The results showed that endogenous AURKA cleavage (e.g., as shown in Figure B) was detected in all of these different tumor cell types. Figure 1 As shown in Figure C, this indicates that the cleavage of AURKA protein in STS-induced apoptosis is a common phenomenon.

[0034] To further investigate whether AURKA cleavage can be triggered by other apoptosis inducers, we induced apoptosis in B lymphocytes before and after EBV and KSHV infection, HeLa cervical cancer cells, and MDA-MB-231 human breast cancer cells using paclitaxel (Taxol), a commonly used chemotherapeutic drug, and then detected AURKA protein cleavage. The results showed that stable AURKA cleavage bands (e.g., [missing information]) could be detected in paclitaxel-induced apoptosis in these various cancer cell types. Figure 1 (As shown in D). The above results strongly suggest that AURKA cleavage is a common phenomenon in antitumor drug-mediated apoptosis, indicating that AURKA cleavage is a key target for antitumor drugs to kill target cells.

[0035] 3. Caspase-3, -7, and -8 mediate AURKA peptide cleavage during apoptosis:

[0036] Given that caspase activation is a common characteristic of apoptotic cells, and activated caspases can cleave a large number of substrate proteins, to confirm whether caspases are involved in AURKA cleavage, we first pretreated HEK293T cells for 1 h with a broad-spectrum caspase inhibitor (Z-VAD-FMK, 25 μM) and a caspase-3 inhibitor (Z-DEVD-FMK, 25 μM), respectively, and then co-treated them with STS for 6 h. The results showed that both Z-VAD-FMK and Z-DEVD-FMK significantly inhibited STS-mediated AURKA cleavage. Figure 2 (As shown in Figure A). Simultaneously, pretreatment with the broad-spectrum caspase inhibitor (Z-VAD-FMK) also inhibited STS-mediated AURKA lysis in BJAB cells (as shown in Figure A). Figure 2 (As shown in B).

[0037] The above results indicate that caspases are involved in the cleavage of AURKA during apoptosis, and it is speculated that caspase-3 may be a key protease mediating AURKA cleavage during apoptosis. Since Z-DEVD-FMK is not only a caspase-3 inhibitor but also effectively inhibits caspase-6, caspase-7, and caspase-8, to further determine which caspase mediates AURKA-specific cleavage, we successfully constructed stable cell lines in HEK293T cells with the knockout of caspase-3, -6, -7, and -8 using CRISPR-Cas9 technology. Figure 2As shown in Figure C), after treatment with the apoptosis inducers STS and Taxol, AURKA protein cleavage was examined. The results showed that AURKA cleavage bands were almost undetectable in Caspase-3 and Caspase-8 knockout cell lines; Caspase-7 knockout significantly inhibited AURKA cleavage during apoptosis; however, Caspase-6 knockout did not affect AURKA cleavage. These results indicate that Caspase-3, Caspase-7, and Caspase-8 are key proteases mediating AURKA cleavage during apoptosis. Figure 2 (As shown in D).

[0038] 4. Identification of AURKA-specific cleavage sites:

[0039] Based on the molecular weight of the C-terminal cleavage band of AURKA protein detected by Western blotting, we can infer that the main cleavage site of the AURKA peptide is likely located at the N-terminus. To determine the specific cleavage site of AURKA, we used two biological software programs, MEROPS and CaspDB, to predict potential protease cleavage sites of AURKA protein. The results showed that MEROPS software predicted two potential cleavage sites for AURKA, namely D132 and K141. Figure 3 As shown in Figure A); CaspDB software predicts that AURKA has multiple potential cleavage sites. In addition to aspartic acid at position 132, which is also a potential cleavage site, there are two sites with high C-terminal peptide scores: D350 (0.91) and D294 (0.77). Figure 3 (As shown in A). In view of AURKA D132 The site is a key node in both the catalytic and regulatory domains of AURKA, and D132 is predicted as a cleavage site by two databases. We... Figure 1 The molecular weight of the AURKA cleavage product observed at the C-terminus was approximately 35 kDa, suggesting that D132 may be the cleavage site of AURKA during apoptosis.

[0040] To determine the specific cleavage site of AURKA, we constructed AURKA cleavage patterns with myc tags at the C-terminus. D132A AURKA K141A AURKA D294A and AURKA D350A The mutant was subjected to STS treatment to induce apoptosis. Immunoblot analysis showed that AURKA... D132A The mutation significantly blocks STS-induced AURKA protein cleavage. Figure 3As shown in Figure B, D132 is the cleavage site of AURKA during apoptosis. According to the Caspase substrate recognition principle, Caspase recognizes at least four consecutive amino acids in the substrate, from the amino terminus to the cleavage site, which are P4-P3-P2-P1. The classic recognition sequences of Caspase-3, Caspase-7, and Caspase-8 are DXXD, XEXD, and (I / L / V)EXD, respectively. Analysis of the AURKA sequence revealed that P4... 129 ALED 132 P1 is not a classic Caspase recognition sequence. We used Site Prediction software to analyze the cleavage of AURKA by Caspase-3, Caspase-7, and Caspase-8. 129 ALED 132 Possibility ( Figure 3 (as shown in C), and for AURKA protein 129 ALED 132 Interestingly, after performing conservation analysis on the four sites, we discovered... 130 led 132 The 129A site is highly conserved across all six species, but it is highly conserved only in humans, pigs, and cattle. In mice and rats, the 129A site is mutated to T-tyrosine. Figure 3 (As shown in D).

[0041] 5. cBioPortal analysis of the mutation probability and distribution of AURKA with Caspase-3, Caspase-7, and Caspase-8 in different tumors:

[0042] The TCGA PanCancer Atlas Studies module was selected in the cBioPortal database to analyze the mutation frequency and distribution of AURKA, Caspase-3, Caspase-7, and Caspase-8 in 10,967 clinical samples across 30 tumor types. We found high-frequency mutations of AURKA in colon cancer, endometrial cancer, esophageal cancer, cervical cancer, melanoma, bladder cancer, and head and neck tumors. High-frequency mutations of Caspase-3, Caspase-7, and Caspase-8 were also observed, suggesting a potential correlation between the two. Figure 4 (As shown).

[0043] The experimental steps involved in Example 1 are as follows:

[0044] Cell sample lysis preparation:

[0045] 1) Collect an appropriate amount of cells, centrifuge at 2000 rpm for 3 min at room temperature, and discard the culture medium.

[0046] 2) Wash twice with pre-cooled PBS, centrifuge at 2000 rpm for 3 min at room temperature, and discard the PBS.

[0047] 3) Add an appropriate amount of RIPA protein lysis buffer containing protease inhibitors and lyse on ice for 30 minutes.

[0048] 4) Shake once every 5 minutes to ensure complete decomposition.

[0049] 5) Centrifuge the fully lysed cells at 14,500 rpm for 5 min in a centrifuge at 4°C.

[0050] 6) Transfer the supernatant after centrifugation to a new EP tube, which is the total protein solution. Measure the protein concentration according to the Coomassie Brilliant Blue G-250 method.

[0051] 7) Aliquot the protein samples according to the required loading amount and add 6X loading.

[0052] 8) Boil in a 100℃ metal bath for 5 minutes until the protein denatures.

[0053] 9) Store at -80℃ for later use or proceed directly to Western blotting.

[0054] Western blot assay steps:

[0055] 1) SDS-PAGE electrophoresis: The denatured protein samples were added to a 9% SDS-PAGE gel for gel electrophoresis separation (80V, 30min; 100V, 90min).

[0056] 2) Transfer: The 0.45μm NC membrane was immersed in 1X NC membrane transfer equilibration buffer and transferred using GenScript eBlot. TM The L1 rapid wet transfer apparatus uses a standard procedure for film transfer, which takes approximately 10 minutes.

[0057] 3) Blocking: Place the NC membrane in 5% skim milk powder (prepared with PBS) and incubate at room temperature for 1 hour.

[0058] 4) Primary antibody incubation: Wash 3 times with TBST at room temperature for 5 minutes each time. Add the primary antibody diluted in an appropriate ratio and incubate overnight at 4°C.

[0059] 5) Secondary antibody incubation: Wash 3 times with TBST at room temperature, 5 min each time. Use the corresponding species' fluorescent secondary antibody 800.

[0060] 6) Incubate at room temperature in the dark for 1 hour; wash 3 times with TBST at room temperature, 5 minutes each time.

[0061] 7) Development: Develop using an Odyssey dual-color imager.

[0062] cBioPortal database data collection:

[0063] The cBioPortal database can be accessed at https: / / www.cbioportal.org / . Enter the gene name in "Quick search" and click "search". In "Cacncer Type Summary", select "CancerType" to download the genomic variations of this gene in 10,967 samples from 30 tumor types in the TCGAPanCancerAtlasStudies, including amplifications, mutations, and complex alterations.

[0064] Reagent preparation:

[0065] RIPA lysis buffer: 1% NP-40, 2mM EDTA, 150mM NaCl, 50mM Tris (pH 7.6), (1mM PMSF, 1g / mL Aprotinin, 1g / mL leupeptin and 1g / mL pepstatin).

[0066] Example 2

[0067] AURKA 133-403 Fragment upregulates the expression of AP-1 family genes, which are pro-apoptotic effector molecules, and cell apoptosis.

[0068] 1. AURKA D132A Mutation inhibits STS-induced apoptosis activity:

[0069] To investigate AURKA D132 To investigate whether cleavage is involved in regulating the apoptosis process, we constructed wild-type AURKA. WT With the non-cuttable mutant AURKA D132A The HEK293 stable cell line was used. Apoptosis was induced by STS at concentrations of 0, 0.5, and 1 μM. After 6 h of induction, Annexin-PE / 7-VAD dual-labeled flow cytometry was used to detect the response of AURKA wild-type and mutant cells to the apoptotic drug. The results showed that after apoptosis was induced by different concentrations of STS, the response of wild-type and mutant AURKA cells to the apoptotic drug was significantly reduced. WT In comparison, AURKA D132A All mutants significantly inhibited apoptosis. Figure 5 (As shown in Figure A). The above results indicate that AURKA D132 The mutation reduced the cell's sensitivity to apoptosis-inducing drugs, suggesting that the drugs work by promoting AURKA. D132 Site cleavage induces apoptosis.

[0070] 2. AURKAD132 The cleavage products promote cell death:

[0071] To further clarify AURKA D132 To investigate the effect of lysis products on apoptosis, we constructed cells stably expressing AURKA. WT and its D132 site cleavage product mutant (AURKA) 1-132 and AURKA 133-403 HEK293 cell line was used, and apoptosis was detected by Annexin V-PE / 7-AAD double-label flow cytometry. The results showed that the total apoptosis rate was significantly higher in AURKA cells compared to the control group (1.93%). WT Overexpression did not significantly promote apoptosis (3.39%). However, AURKA... 133-403 The total apoptosis rate of the group cells (8.82%) was significantly increased. Notably, compared with AURKA... WT Compared to the group with a cell necrosis rate of 1.10%, AURKA 133-403 Group cell necrosis rate (6.64%) and AURKA 1-132 The cell necrosis rate (5.92%) in all groups was significantly increased. These results indicate that AURKA... D132 The cleavage products promote cell death. Figure 5 (As shown in A).

[0072] To gain a deeper understanding of AURKA D132 The molecular mechanism by which the C-terminal cleavage peptide promotes apoptosis is investigated in this invention regarding the molecular mechanism of AURKA overexpression. WT AURKA 1-132 and AURKA 133-end We conducted mRNA sequencing analysis on stable HeLa cell lines to explore their potential mechanisms of action and function. Through RNA sequencing, we performed hierarchical cluster analysis of global gene expression in HeLa cells expressing the full-length AURKA cell line with the YFP marker, as well as its N-cut (1-132) or C-cut (133-end) mutants, and mapped the AURKA... 133-403 Compared to AURKA WT Heatmap of the top 10 upregulated gene expressions ( Figure 5 (As shown in B). After performing statistical analysis of gene expression differences in the sequencing results, we found that AURKA... 133-403 Compared to AURKA WT There were 84 differentially expressed genes, of which 58 were upregulated and 26 were downregulated. Figure 5 (As shown in C). AURKA WT vs. AURKA 133-403GO enrichment analysis of differentially expressed genes showed that the differentially expressed genes were mainly enriched in signaling pathways such as positive regulation of cell death, positive regulation of apoptosis, and positive regulation of programmed cell death. Figure 5 (As shown in D). Activator protein 1 (AP-1) is a key signaling protein molecule downstream of JNK that mediates pro-apoptotic effects, mainly composed of the Jun and Fos subfamilies. Quantitative real-time PCR detection of gene expression confirmed that AURKA... D132 The C-terminal cleavage peptide significantly upregulated the transcriptional levels of the AP-1 constituent subunits FOS and FOSB, and also significantly upregulated the transcriptional level of EGR1 downstream of Jun. Figure 5 (As shown in E). Further analysis of AURKA was performed using KEGG enrichment analysis. WT vs. AURKA 133-403 Differentially expressed genes were identified, and the results showed that these genes were significantly enriched in pathways including human T-cell leukemia virus (HTLV) infection, Kaposi's sarcoma-associated herpesvirus (KSHV) infection, viral carcinogenesis, amphetamine addiction, and rheumatoid arthritis. Figure 5 (As shown in F). The above results are from AURKA. D132 The theory behind the claim that cleavage promotes apoptosis is supported by cleavage.

[0073] The experimental steps involved in Example 2 above are as follows:

[0074] Annexin V-PE / 7-AAD dual-label flow cytometry detection of cell apoptosis

[0075] 1) Induce apoptosis according to experimental requirements.

[0076] 2) Cell Collection: Collect 1 million treated cells. Handle adherent cells carefully. First, collect the floating cells after induced apoptosis. Digest the cells with trypsin without EDTA, and collect the cells after stopping digestion. Centrifuge at 300g, 4℃ for 3 minutes, and discard the culture medium.

[0077] 3) Washing cells: Wash cells twice with cooled PBS, centrifuging at 300g and 4℃ for 3 minutes each time, and discard the supernatant.

[0078] 4) Cell resuspension: Add 100 μL of 1X Binding Buffer and gently mix until a single-cell suspension is formed.

[0079] 5) Cell staining: Add 5 μL Annexin V-PE and 5 μL 7-AAD Staining Solution, mix gently; incubate at room temperature in the dark for 10 min; add 400 μL 1X Binding Buffer, mix gently. Analyze the stained samples by flow cytometry 1 h later.

[0080] 6) Sample Analysis: Flow cytometry was performed with an excitation wavelength of 488 nm. PE fluorescence was detected in channel FL2, and 7-AAD fluorescence was detected in channel FL3. 10,000 events were collected for each sample. Data analysis was performed using FlowJ software, with FL2 on the x-axis and FL3 on the y-axis. The positive and negative boundaries of the two fluorescence parameters were determined based on the PE and 7-AAD fluorescence values, and a cross-shaped threshold was established.

[0081] RNA extraction and quantitative RT-PCR

[0082] 1) RNA extraction: Total RNA was extracted from cells using TRIzol (Invitrogen), and the RNA concentration was quantified using Nanodrop_2000.

[0083] 2) Reverse transcription: 1 μg of total RNA was reverse transcribed using a reverse transcription reagent (Yeasen, 11141ES60) to obtain cDNA that could be used for subsequent quantitative PCR.

[0084] 3) Real-time quantitative PCR: Using the cDNA obtained above as a template, real-time quantitative PCR was performed on a BIO-RAD detection system using SYBR Green Premix (Yeasen, 11201ES03), with human β-actin as an internal reference gene. －△△CT The method detects the relative expression level of the target gene in the sample.

[0085] Primers for detecting the target gene and the human β-actin gene are shown in Table 1.

[0086] Table 1.

[0087]

[0088] Example 3

[0089] AURKA D132 Cutting disrupts the processes of cell mitosis, including centrosome formation and spindle assembly.

[0090] 1. AURKA D132 The intracellular localization of the cleavage product changes:

[0091] The precise localization of proteins in subcellular compartments is closely related to their function. We used GFP-labeled AURKAWT N-terminal truncated body AURKA 1-132 and C-terminal truncated body AURKA 133-403 Transiently transfected HEK293T cells, the intracellular localization of AURKA and its lysis products was observed. We found that: with AURKA... WT Diffuse and uniform expression differs between the cytoplasm and nucleus, AURKA 1-132 AURKA is clearly aggregated in the cell nucleus; while overexpression of AURKA 133-403 AURKA was observed in the cells. 133-403 The translocation to the cytoplasm, or the aggregation and expression in the cytoplasm exhibiting punctate fluorescence, suggests that this phenomenon may be related to AURKA kinase activity. Figure 6 (As shown in A).

[0092] AURKA is a cell cycle-related protein that plays a crucial role in mitosis. To investigate AURKA... D132 To investigate the effect of cleavage on the cell cycle, we used live-cell imaging to analyze wild-type and mutant AURKA cells carrying the GFP marker, tracking the process of cell mitosis. The results showed that, compared to GFP-AURKA, AURKA... D132A Mutation, N-terminal truncated form AURKA 1-132 and C-terminal truncated body AURKA 133-403 Both prolonged the total time of mitosis from metaphase to telophase. We also found that AURKA... D132 The N-terminal polypeptide AURKA generated after cleavage 1-132 Unable to form centrosome-like punctate fluorescence, with a high proportion of AURKA expression. 133-403 The cells exhibit aggregated punctate fluorescence in the cytoplasm, but unfortunately, the dynamic mitotic process of these cells cannot be captured because the brightness of these punctate fluorescence exceeds the light intensity threshold of the high-resolution live-cell imaging system Delta Vision. Figure 6 (As shown in Figure B). We quantitatively analyzed the time taken from prophase to cytokinesis in mitotic cells expressing different AURKA mutants. Figure 6 C).

[0093] 2. AURKA protein cleavage interferes with centrosome formation and spindle assembly:

[0094] AURKA, a cell cycle protein, is primarily involved in centrosome formation and maturation, and bipolar spindle assembly during mitosis. Located in the centrosome, AURKA is essential for centrosome maturation and division, as well as spindle formation during mitosis. We further investigated AURKA... D132Does cleavage affect centrosome and spindle formation? HEK293T cells were transfected with a GFP fusion expression plasmid related to AURKA, and immunostaining was performed using antibodies specific to the centrosome marker γ-tubulin and the spindle marker α-tubulin. Results showed that during interphase, cleavage affects centrosome and spindle formation. WT In comparison, AURKA 133-403 It forms numerous noncentrosome punctate fluorescences. During metaphase of mitosis, it interacts with AURKA. WT In comparison, AURKA 1-132 and AURKA 133-403 Centrosome formation defects result in only one of the two formed centrosomes being completely colocalized with endogenous γ-tubulin, while the other centrosome, being smaller, cannot be completely colocalized with endogenous γ-tubulin. This illustrates the AURKA... D132 Cutting disrupts normal centrosome formation ( Figure 7 (As shown in A).

[0095] Secondly, the results of spindle staining showed that, compared with AURKA WT In comparison, AURKA 1-132 and AURKA 133-403 This causes abnormal spindle morphology. During the mid and late stages of filamentation, we observed similarities to AURKA. WT In comparison, AURKA 1-132 and AURKA 133-403 Formation of weak, sparse, or short stellar microtubules ( Figure 7 (As shown in Figure B). In summary, these data demonstrate that AURKA D132 Cutting disrupts the normal formation of the centrosome and spindle, while Caspases mediates AURKA. D132 Cutting is essential for the formation of apoptotic microtubule networks.

[0096] The experimental steps involved in Example 3 above are as follows:

[0097] Cell transfection steps:

[0098] 1) Cells in good growth condition plated 24 hours before transfection.

[0099] 2) After the cells have adhered to the wall for about 18 hours and the cell confluence reaches 70%, transfection can begin.

[0100] 3) Discard the complete culture medium in the culture dish, wash once with PBS, and then replace with blood-free and antibiotic-free culture medium.

[0101] 4) Prepare the PEI transfection system configuration (taking a 6-well plate as an example).

[0102] Solution A: Dilute 2 μg of plasmid with 100 μL of blood-free and antibiotic-free DMEM;

[0103] Solution B: Dilute 6 μL of PEI with 100 μL of blood-free and antibiotic-free DMEM;

[0104] Gently mix solutions A and B separately and let stand at room temperature for 5 minutes.

[0105] Add solution B dropwise to solution A, mix gently, and let stand at room temperature for 15 minutes.

[0106] 5) Add 200 μL of plasmid and PEI mixture dropwise into a 6-well plate, gently mix from front to back and side to side, and then put it back into the incubator.

[0107] 6) Replace with fresh complete culture medium 6 hours after transfection.

[0108] 7) Continue culturing, and observe the transfection efficiency under a fluorescence microscope 24 hours after transfection.

[0109] Immunofluorescence assay procedure (using a 14mm slide as an example):

[0110] 1) Place the 14mm cell spread sheet in a 12-well plate and wash three times with PBS.

[0111] 2) Plate 0.5 million healthy cells in a 12-well plate and allow them to adhere for about 18 hours before transfection.

[0112] 3) Observe the cell transfection efficiency under a fluorescence microscope 24 hours after transfection. Carefully discard the culture medium and wash twice with PBS.

[0113] 4) Add 1 mL of 4% paraformaldehyde and mix gently. Fix at room temperature in the dark for 15 min.

[0114] 5) Add 1 mL of Blocking Buffer (0.02% FSG + 0.02% Tritonx + PBS) to immerse the slide for 10 min.

[0115] 6) Primary antibody dilution: Dilute the primary antibody in Blocking Buffer at an appropriate ratio, vortex to mix, centrifuge at 14000 rpm for 1 min, and let stand on ice until ready for use.

[0116] 7) Drop 30 μL of the diluted primary antibody onto a Parafilm membrane, and press the slide face down onto the droplet. Incubate at room temperature for 1 hour.

[0117] 8) Transfer the slide face up to a 12-well plate and wash three times with 1 mL of Blocking Buffer.

[0118] All the following operations must be performed under light-protected conditions.

[0119] 9) Secondary antibody dilution: Dilute the secondary antibody 1:500 in Blocking Buffer and vortex to mix. Centrifuge at 14000 rpm for 1 min and let stand on ice until ready to use.

[0120] 10) Add 30 μL of diluted secondary antibody to the Parafilm membrane, press the slide face down onto the droplet, and incubate at room temperature in the dark for 1 h.

[0121] 11) Transfer the slide face up to a six-well plate, add 1 mL of Blocking Buffer and wash twice, then wash once with PBS.

[0122] 12) Dilute DAPI in Mounting Media at a ratio of 1:1000. Using a 10μL pipette tip, add one drop of the DAPI-containing Mounting Media onto a glass slide, placing the slide face down on the drop. Seal the slide with nail polish and allow it to dry.

[0123] Example 4

[0124] AURKA D132 Mutations reduce the efficacy of paclitaxel against cervical cancer cells.

[0125] To clarify the effects of AURKA protein mutations and cleavage products on tumor cell growth and tumorigenicity, based on the AURKA protein cleavage sites identified in Example 1, we constructed stably AURKA-expressing structures. WT Mutant AURKA D132A and its cleavage product mutant (AURKA) 1-132 and AURKA 133-403 The HeLa cell line was used. Cloning experiments showed that, compared to wild-type AURKA, AURKA... D132A Mutant and cleavage product AURKA 1-132 and AURKA 133-403 All inhibited the in vitro clonal growth of cells, among which AURKA 133-403 The inhibitory effect on clonogenesis was the most significant. Figure 8 (As shown in Figure A). Subsequently, we will stably express the unloaded YFP, YFP-AURKA. WT or irreversible mutant YFP-AURKA D132A The same number of HeLa cells were seeded for 10 days, and then treated with different concentrations (0, 25 nM, 50 nM) of Taxol for 48 hours to detect AURKA. D132 Does the mutation affect the sensitivity of cervical cancer HeLa cells to paclitaxel? Results showed that after paclitaxel treatment, compared with the vector group and AURKA... WT Compared to the group, the mutant AURKA D132AThe cell clone number decreased by the lowest fold in this group ( Figure 8 (As shown in Figure B). We further evaluated AURKA using a nude mouse tumor-bearing assay. D132 The impact of mutations on sensitivity to chemotherapy drugs in vivo. We will use a stable expression vector, AURKA... WT and AURKA D132A A tumorigenesis experiment was conducted using a stable HeLa cell line of human cervical cancer, and the effects on tumor growth were observed by intraperitoneal injection of physiological saline or paclitaxel (10 mg / kg, once every 2 days, for a total of 5 times). The results showed that wild-type AURKA significantly promoted tumor growth in vivo. D132A It inhibits tumor growth. In contrast, paclitaxel treatment significantly inhibits the overexpression of Vector and AURKA. WT The tumor volume and weight were reduced, but AURKA was not significantly inhibited. D132A Tumor growth in the mutant group ( Figure 8 (As shown in C). This illustrates AURKA. D132A It reduced the sensitivity of cervical cancer cells to paclitaxel, suggesting that AURKA D132 Site mutations can reduce the therapeutic efficacy of chemotherapy drugs on tumor cells and are a potential indicator of drug resistance targets.

[0126] The experimental steps involved in Example 4 above are as follows:

[0127] Plate colony formation experiment

[0128] 1) Seed 10,000 or 3,000 cells into a 100cm² culture dish, add 10mL of DMEM medium, mix well from all sides, and then incubate in an incubator.

[0129] 2) Replace the culture medium with fresh culture medium every 3 days.

[0130] 3) After 10 days of culture, obvious clonal colonies were observed, and preparations were made for crystal violet staining.

[0131] 4) Discard the culture medium and wash once with PBS.

[0132] 5) Add 10 mL of 4% paraformaldehyde and fix at room temperature in the dark for 20 min.

[0133] 6) Wash twice with PBS; add 10 mL of 0.1% crystal violet dye and incubate at room temperature in the dark for 20 min.

[0134] 7) Wash twice with PBS, air dry, and use a Canon scanner to capture images.

[0135] Nude mouse tumor-bearing experiment

[0136] 1) Purchase 4-week-old female BALB / c Nude mice and acclimatize them for one week in a clean-grade mouse house.

[0137] 2) Well-cultured Vector, AURKAWT, and AURKAD132 HeLa stable cell lines were counted using a live cell counter. 10 x 10⁶ cells from each tumor were resuspended in 200 μL of a solution (blood-free, antibiotic-free DMEM mixed with Matrigel 1:1). Cells were then injected subcutaneously into nude mice using a 1 mL insulin syringe.

[0138] 3) Tumor formation was observed in nude mice 5 days after subcutaneous injection, and drug administration began the following day.

[0139] 4) The control group was given normal saline (1 mL / kg), while the experimental group was given paclitaxel (10 mg / kg; intraperitoneal injection; once every 2 days, for a total of 5 times).

[0140] 5) On the day following the end of drug administration, nude mice were euthanized by cervical dislocation, the subcutaneous tumor was removed, and the tumor was photographed and its weight measured.

[0141] Reagent preparation

[0142] 1) In vivo paclitaxel stock solution: Add each solvent in sequence: 10% DMSO → 40% PEG300 → 5% Tween-80 → 45% saline. For example, to prepare a 1 mg / mL paclitaxel stock solution: 20 mg paclitaxel + 2 mL DMSO → 8 mL PEG300 → 1 mL Tween-80 → 9 mL saline.

[0143] 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. The application of a reagent for detecting a biomarker in the preparation of a reagent for evaluating tumor drug resistance, wherein the tumor is cervical cancer, the biomarker is a D mutation at position 132 of AURKA changing to A; and the tumor drug is a paclitaxel-based drug.

2. The application of the reagent for detecting biomarkers according to claim 1 in the preparation of reagents for evaluating tumor drug resistance, characterized in that, The steps for evaluating tumor drug resistance include: The sensitivity of cancer patients to paclitaxel chemotherapy is evaluated by detecting whether there is a D-to-A mutation at position 132 of AURKA.

3. The application of a reagent for detecting biomarkers in the preparation of a kit for evaluating tumor drug resistance, characterized in that, The kit includes reagents for detecting biomarkers; the biomarker is a D mutation at position 132 of AURKA that is changed to A; the tumor is cervical cancer; and the tumor drug is a paclitaxel-based drug.

Images