A molecular marker for detecting microsatellite instability and application thereof

Abstract

The application discloses a new molecular marker for detecting microsatellite instability of tumor cells and application thereof. The molecular marker is SirT7, a member of Sirtuin family of histone deacetylase. The application finds that SirT7 in human tumor tissues is negatively correlated with protein expression of MSH2, a protein of DNA damage repair system, so as to determine that SirT7 is a new molecular marker for showing microsatellite instability of tumor tissues. On the other hand, microsatellite instability of tumor cells indicates tumor drug resistance. Therefore, the application further finds that SirT7 in tumor cells can cause microsatellite instability under treatment of chemotherapeutic drugs, and tumor cells with SirT7 knocked down show sensitivity to various chemotherapeutic drugs. Therefore, the application takes SirT7 as a new molecular marker for microsatellite instability of tumors, predicts tumor drug resistance, provides possibility for targeted selection of chemotherapeutic drugs for tumors, and provides a new idea for precise treatment of tumors.

Description

Technical Field

[0001] This invention relates to the field of tumor drug resistance prediction and detection. Specifically, this invention relates to a molecular marker for detecting microsatellite instability and its application. Background Technology

[0002] Cell death induced by gene-damaging drugs is central to anti-tumor therapy. While significant progress has been made in anti-tumor treatment, the emergence of resistance to gene-damaging drugs severely limits its effectiveness. Therefore, in-depth research into the causes of tumor drug resistance and how to promptly detect drug sensitivity are crucial for tumor drug therapy. DNA damage stress is considered a factor affecting the effectiveness of DNA-damaging drugs. DNA damage stress leads to double-strand or single-strand damage in DNA, thereby activating the intracellular DNA damage repair system to repair DNA damage, inhibit cell death, and thus lead to tumor drug resistance. However, a high-fidelity DNA mismatch repair (MMR) system exists within cells to repair mismatches between bases and mismatches in base insertion / deletion, maintaining genomic stability. Furthermore, MMR plays a crucial initiation role in apoptosis caused by DNA damage. In tumor cells lacking or inactivated by MMR, the deletion of single or double nucleotide repeat sequences results in repeats of varying lengths, causing genomic instability, also known as microsatellite instability (MSI), which leads to tumor drug resistance. Numerous studies have demonstrated that MSI is a marker of tumor drug resistance. Therefore, effectively and rapidly detecting microsatellite instability (MSI) is crucial for predicting tumor drug sensitivity and tolerance. Numerous studies have shown a close correlation between MSI and the development of colorectal cancer, gastric cancer, and endometrial cancer; further research indicates that many tumors, such as lung cancer, esophageal cancer, bladder cancer, and thyroid cancer, are also closely related to MSI. MSI detection has multiple clinicopathological significances. Approximately 15% of colorectal cancers exhibit MSI, with over 90% of patients with typical hereditary nonpolyposis colorectal cancer (HNPCC) having MSI tumors. Compared to colorectal cancer without MSI, colorectal cancer with MSI has a better prognosis, and the drug responses differ between the two groups.

[0003] Microsatellite instabilities in existing technologies can generally be detected using the following two methods, with the detection items being MLH1, MSH2, MSH6, and PMS2:

[0004] 1) PCR method: Select specific primers, use normal tissue as a control, perform PCR amplification on microsatellite loci in vitro, and analyze the amplified products by polyacrylamide gel electrophoresis and various display systems (autoradiography, silver staining, etc.) to see if there are any changes in migration rate.

[0005] 2) Genetic testing, which involves directly sequencing the relevant DNA. However, due to the high cost and demanding experimental conditions, genetic testing is not yet widely used in clinical practice.

[0006] PCR has become a commonly used detection method and has been proven to be the most effective primary screening tool. However, the conventional PCR method, which involves detecting five microsatellite genes in separate tubes and performing gel electrophoresis on the products, has many drawbacks, such as cumbersome operation and high cost. Multiplex PCR for microsatellite gene detection is more complex due to the competition and interference factors between primers, making it difficult for amplified products to be misaligned, which directly leads to uncertainty in the detection results. Therefore, multiplex PCR detection products have very high requirements for the specificity of the primer sequences and the concentration of primers in the reaction system.

[0007] Currently, clinical methods for detecting microsatellite instability primarily rely on the testing standards established by the American Cancer Institute, which detect the stability of five genomic microsatellite loci: two single nucleotide repeat sites (BAT-25, BAT-26) and three dinucleotide repeat sites (D2S123, D5S346, D17S250). This method determines microsatellite locus stability by comparing the copy number of the target repeat region in tumor samples and normal control samples using PCR amplification and electrophoresis. Based on the proportion of unstable sites in the total number of detected sites, the microsatellite instability status of a sample can be classified into three states: highly unstable (MSI-H), low unstable (MSI-L), and stable (MSS). This detection method detects too few sites, has a complex experimental protocol, and is time-consuming. Recent studies on genomic sequencing data of cancer samples have shown no significant difference between samples with MSI-L and MSS phenotypes, and the PCR electrophoresis method cannot provide this conclusion. While some patents and literature suggest increasing MSI detection sites and designing experiments to improve detection accuracy, these PCR and electrophoresis-based detection methods cannot be automated at high throughput. Furthermore, increasing the number of detection sites increases experimental complexity and cost, failing to fundamentally solve the problem. In addition, these PCR and electrophoresis-based detection methods involve complex and expensive experimental procedures and require both normal and tumor tissues as controls.

[0008] Besides detecting microsatellite loci to determine microsatellite instability, another mainstream method is immunohistochemistry, which uses antigen-antibody hybridization to detect the expression of genes related to the mismatch repair system in tumors to determine the state of microsatellite instability. While this method has high sensitivity, like the methods mentioned above, it is experimentally complex, has low reproducibility, and requires normal samples as controls.

[0009] The Sirtuin family of histone deacetylases is a crucial class of proteins regulating chemotherapy resistance in tumors. These are enzymes homologous to yeast Sir2. The Sirtuin family consists of seven members, SirT1-7, all of which possess a conserved NAD-dependent catalytic core region, promoting NAD-dependent deacetylase and ADP-ribotransferase activities. Sirtuins, dependent on their deacetylation activity, participate in regulating cellular metabolism, aging, proliferation, stress resistance, and inflammatory oxidative stress responses. Sirtuins exhibit complex pro- and anti-cancer effects. SirT7 is the only Sirtuin protein located in the nucleolus. Extensive cancer research classifies SirT7 as a proto-oncogene. On one hand, SirT7 maintains cancer characteristics by deacetyling H3K18; on the other hand, SirT7's function of promoting rDNA transcription aligns perfectly with the rapid proliferation of cancer cells requiring ribosome production. SirT7 itself does not exhibit proto-oncogene characteristics, as overexpression of SirT7 does not lead to carcinogenesis in fibroblasts. Therefore, it is believed that SirT7 overexpression in cancer cells may be to meet the needs of rapidly proliferating cancer cells for ribosome production. Recent reports indicate that SirT7 promotes chromosome condensation and participates in double-stranded DNA damage (DSB) repair through desuccinylation of H3K22. These studies reveal that SirT7 may be a hot topic in tumor research. Investigating the relationship and regulation of SirT7 with tumor drug resistance will become extremely meaningful, facilitating the search for predictive biomarkers of tumor chemotherapy drug resistance, which can be practically used in clinical tumor prognosis prediction and chemotherapy drug guidance.

[0010] This invention will investigate the relationship between SirT7 and tumor microsatellite instability, with the aim of proposing a new molecular marker for detecting tumor microsatellite instability and predicting tumor drug resistance. Summary of the Invention

[0011] The purpose of this application is to provide a novel molecular biomarker with high sensitivity and specificity, which can be practically applied to the detection of tumor microsatellite instability and its application in tumor drug resistance. To achieve the above objective, this invention is implemented through the following technical solution:

[0012] As a first aspect of the invention, it is to provide a molecular marker for detecting microsatellite instability, said molecular marker being the SirT7 level.

[0013] Preferably, the application of SirT7 level as a molecular marker is defined as the DNA level or protein level of SirT7 in cells.

[0014] Preferably, the method for detecting the SirT7 protein level in cells is to detect the SirT7 protein level in tumor tissues or cells by immunohistochemistry, Western blot, or ELISA, thereby predicting tumor microsatellite stability. High SirT7 protein expression levels indicate tumor microsatellite instability, while low SirT7 protein expression levels indicate tumor microsatellite stability.

[0015] Preferably, the tumor tissue includes, but is not limited to, breast cancer tissue, and the cell type includes, but is not limited to, the MCF-7 cell line. The tumor tissue may also be colorectal cancer, gastric cancer, endometrial cancer, lung cancer, esophageal cancer, bladder cancer, or thyroid cancer.

[0016] Preferably, the SirT7 protein level reflects the SirT7 protein expression status, which includes high expression, moderate expression, and low expression.

[0017] As a second aspect of the present invention, a biomarker for predicting resistance to chemotherapy drugs in tumors is provided, characterized in that the biomarker is SirT7.

[0018] Preferably, high SirT7 expression predicts tumor drug resistance.

[0019] Preferably, the tumor chemotherapy drug includes, but is not limited to, 5-fluorouracil (5-FU) and paclitaxel (Taxol).

[0020] As a third aspect of the invention, it is to provide an inhibitory drug targeting SirT7 that prevents tumor drug resistance by reducing SirT7 levels.

[0021] In a typical embodiment, the protein levels of SirT7 and MSH2 in tumor tissue microarrays were analyzed by immunohistochemical experiments. It was found that SirT7 was highly expressed while MSH2 was lowly expressed in breast cancer tumor tissues, and SirT7 and MSH2 were negatively correlated, indicating microsatellite instability.

[0022] In another typical embodiment, ShRNA targeting SirT7 is further designed and chemically synthesized according to ShRNA design principles, virally packaged, and a SirT7 knockdown stable tumor cell line is constructed. Furthermore, the stability of microsatellites in cells under chemotherapy drug treatment is verified, and SirT7 determines the instability of microsatellites under chemotherapy drug treatment.

[0023] SirT7 knockdown stable tumor cell lines were treated with a variety of chemotherapy drugs, and cell growth was observed. Changes in cell death were detected by MTT assay. The results showed that SirT7 knockdown tumor cells determine the tumor's sensitivity to drugs.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] (1) This invention has experimentally demonstrated that SirT7 and MSH2 are negatively correlated in tumor tissue, SirT7 determines the stability of cell microsatellites under chemotherapy drug treatment, and cells with SirT7 knockdown are more sensitive to drugs. This provides a new direction for the development of tumor drugs and provides a new drug target for the treatment of tumors, including inhibitory drugs, namely, using SirT7 protein as the target of inhibitory action.

[0026] (2) Given that numerous studies have shown that breast cancer, colorectal cancer, gastric cancer, endometrial cancer, lung cancer, esophageal cancer, bladder cancer, thyroid cancer, etc. are all closely related to microsatellite instability, the SirT7 microsatellite instability detection molecular marker provided by this invention can be applied to drug resistance detection of various tumors including those mentioned above.

[0027] (3) Compared with PCR detection methods, conventional PCR detection of microsatellite instability requires the detection of more alleles and often uses multiplex PCR combined with automated sequencing, which is complex and not very operable. This invention only detects the DNA or protein level of SirT7, and the detection method is simple and highly operable. Attached Figure Description

[0028] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0029] Figure 1 Example 1: An experiment showing a negative correlation between SirT7 and MSH2 in tumor tissue: Figure a shows the protein levels of SirT7 and MSH2 in breast cancer tumor tissue analyzed by immunohistochemistry; Figure b shows the correlation analysis obtained in Figure a.

[0030] Figure 2 In Example 2, multiplex PCR analysis showed that SirT7 knockdown tumor cells exhibited microsatellite stability under chemotherapy drug stimulation.

[0031] Figure 3 In Example 3, the sensitivity test of SirT7 knockdown cells to various chemotherapy drugs was conducted: Figure a shows the survival rate of SirT7 knockdown cells treated with 5-FU; Figure b shows the survival rate of SirT7 knockdown cells treated with Taxol. Detailed Implementation

[0032] Microsatellite instability (MSI) refers to the loss of mismatch repair genes due to DNA methylation or gene mutation, which leads to changes in the length of microsatellite repeat sequences. This manifests as differences in the number of repeat units of the same microsatellite locus between different individuals and between normal tissues and certain abnormal tissues of the same individual. As a result, the microsatellite locus cannot play its normal regulatory role, leading to abnormal cell proliferation and differentiation, and promoting the formation of malignant tumors.

[0033] Mismatch repair proteins in immunohistochemistry include four molecules: MLH1, PMS2, MSH2, and MSH6. This group of proteins is commonly used in colorectal cancer to determine whether its development is due to a microsatellite instability mechanism.

[0034] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the reagents and equipment used in the present invention are conventional reagents and equipment in this technical field. Of course, the instruments and materials used in the embodiments are not limited to the examples listed herein, but are based on their ability to solve the technical problems of the present invention and achieve the corresponding technical effects. In addition, the molecular biology methods not described in detail in the embodiments are all conventional methods in the art, and specific operations can be found in molecular biology guides or product instructions.

[0035] Example 1: SirT7 and MSH2 are negatively correlated in tumor tissue.

[0036] The protein detection methods are as follows:

[0037] Western blot steps

[0038] (1) Preparation of modified polyacrylamide gel: Clean the gel plate and place it on the gel making rack in the correct order; check for leaks; prepare the separating gel and seal with ddH2O; after the separating gel solidifies, prepare the concentrating gel, then insert the comb, and it can be used after the concentrating gel has completely solidified.

[0039] (2) Electrophoresis: Place the prepared gel and electrophoresis tank; add the running buffer into the electrophoresis tank; load the sample; adjust the voltage of the electrophoresis apparatus to 80V to start electrophoresis. When the loading is completely in the separating gel, increase the voltage to 120V until the loading reaches the bottom of the separating gel and end the electrophoresis.

[0040] (3) Transfer: Soak the PVDF membrane in methanol for 30 seconds and then put it into the transfer buffer for later use; add the transfer buffer into the transfer tank and cool it with an ice-water mixture beforehand; place the sponge, filter paper, gel and PVDF membrane in the correct order and then place them in the slots of the transfer tank; start the electrophoresis apparatus and use 80V voltage for transfer, which generally takes 90 minutes.

[0041] (4) Blocking: Place the transferred PVDF membrane into 5% skim milk prepared with PBST and incubate on a shaker at room temperature for 1-1.5 hours.

[0042] (5) Add primary antibody: Dilute the specific primary antibody to an appropriate concentration with 5% skim milk and incubate on a shaker at room temperature for 2 hours or on a shaker at 4°C overnight. Then wash with PBST 3-4 times at room temperature, 10 min each time.

[0043] (6) Add secondary antibody: Dilute the corresponding secondary antibody (conjugated with HRP) to the required concentration with 5% skim milk, incubate at room temperature for 2-2.5 hours, and then wash with PBST at room temperature 4-5 times, 10 minutes each time.

[0044] (7) Development and fixing: In the darkroom, remove the liquid from the PVDF membrane and drop the mixed luminescent reagents A and B onto the membrane; place the PVDF membrane in a light-proof box and cover it with an X-ray film; develop and fix the X-ray film in sequence; rinse the X-ray film clean and dry it in an oven to analyze the results.

[0045] 10×Running buffer (electrophoresis buffer)

[0046]

[0047] 10× Transfer buffer

[0048] Tris 165g

[0049] Glycine 720g

[0050] ddH2O to 5L

[0051] Immunohistochemical detection

[0052] (1) Dewax sections to water using standard procedures. Antigen retrieval can be performed after this step if necessary.

[0053] (2) Wash with PBS for 3 min / 2 times.

[0054] (3) In order to reduce non-specific background staining caused by endogenous peroxidase, the slides were incubated in HydrogenPeroxide Block for 10-15 minutes.

[0055] (4) Wash with PBS for 5 min / 2 times.

[0056] (5) Add Ultra V Block and incubate at room temperature for 5 minutes to block non-specific background staining. (Note: Do not incubate for more than 10 minutes, otherwise it will lead to a decrease in specific staining. If the primary antibody dilution contains 5-10% normal sheep serum, this step can be omitted.)

[0057] (6) Wash with PBS for 5 min / 2 times.

[0058] (7) Add primary antibody working solution (5% BSA + anti-target protein antibody) and incubate at 37°C for 1-2 hours. (The specific incubation time and temperature are to be determined by the experimenter.)

[0059] (8) Wash with PBS for 5 min / 2 times.

[0060] (9) Add Primary Antibody Enhancer and incubate at room temperature for 20 minutes.

[0061] (10) Wash with PBS for 5 min / 2 times.

[0062] (11) Add HRP Polymer (enzyme-labeled secondary antibody) and incubate at room temperature for 30 minutes. (Note: HRP Polymer is light-sensitive; avoid unnecessary light exposure and store in opaque vials.)

[0063] (12) Wash with PBS for 5 min / 2 times.

[0064] (13) Add 1-2 drops of DAB Plus Chromogen (or AEC Plus Chromogen) to 1 ml of DAB Plus Substrate (or AEC Plus Substrate), mix well, and then add to the slide. Incubate for 3-15 minutes. (The specific time depends on the staining intensity.)

[0065] (14) Rinse thoroughly with tap water, re-dye, dehydrate, clear, and seal.

[0066] ELISA testing steps:

[0067] 1. Coat the SIRT7 antibody with 50 μl of antibody dilution buffer (SIRT7 antibody is diluted with NaHCO3 solution at a ratio of 1:1000) into the ELISA plate using a pipette. Seal the wells with adhesive tape and incubate at room temperature on a shaker for 2 hours.

[0068] 2. Remove the primary antibody and wash four times with 300 μl of PBS each time.

[0069] 3. Block each well with 300 μl of blocking solution (5% milk powder, PBS as solvent) for 1 hour;

[0070] 4. Remove the blocking solution, add 100 μl of standard or sample to each well, and let stand at room temperature for 2 hours;

[0071] 5. Then add 50 μl of anti-rabbit HRP (1:2000 dilution ratio), seal the wells with tape, and incubate on a shaker at room temperature for 2 hours;

[0072] 6. Aspirate the liquid from the well and wash four times with 300 μl of PBS each time.

[0073] 7. Add 200 μl of Substrate Solution (TMB solution) to each well and incubate at room temperature for 30 minutes on the lab bench;

[0074] 5. Add 100 μl of Stop Solution (2 mol / L sulfuric acid solution) to each well; the solution color will change from blue to yellow.

[0075] 6. Read the light absorption value at 450nm within 30 minutes, and read the calibration value at 540nm wavelength.

[0076] Figure 1 a. Purchase breast cancer tissue microarrays, rehydrate the microarrays by permeation with xylene, followed by a series of steps involving ethanol and water, then extinguish endogenous catalase with H2O2, repair antigens with EDTA, administer primary and secondary antibodies, stain with hematoxylin, and finally dehydrate the microarrays by a series of steps involving ethanol and xylene, mount them with neutral resin, and observe the protein expression of SirT7 and MSH2 under a microscope. Figure 1 b. Score and statistically analyze the images captured in Figure a, and analyze the correlation between SirT7 and MSH2.

[0077] Immunohistochemical staining slides from breast cancer were scored, with staining results categorized into 12 levels from 1 to 12. The weakest staining on a slide was assigned a score of 1, and the strongest staining a score of 12. All slides were scored sequentially according to staining intensity. Score 1-4 was defined as low, 5-8 as medium, and 9-12 as high. The number of slides falling into each of these three scoring ranges was then counted to obtain the score. Figure 1 The values ​​in the table in b.

[0078] Results obtained from image acquisition and analysis methods are expressed as mean ± standard deviation (x ± s), while results obtained from manual counting methods are compared using a nonparametric rank-sum test. Statistical data were analyzed using SPSS 11.5 statistical software, with P < 0.05 considered statistically significant.

[0079] The results are as follows Figure 1 As shown, tumor tissues with high SirT7 expression and low MSH2 expression demonstrate a negative correlation between SirT7 and MSH2.

[0080] Example 2: SirT7 determines microsatellite instability under chemotherapy treatment

[0081] First, the SirT7-ShRNA plasmid was constructed. Then, the SirT7 stably knocked-down human breast cancer stable cell line MCF-7 was constructed. The cells were seeded in 6 cm solutions, treated with 5-FU (200 μM) after 12 hours, and collected after 48 hours to extract DNA. Primers for five alleles (BAT-25, BAT-26, NR-24, CAT-25, and SEC-63) to detect intracellular microsatellite stability were designed. Multiplex PCR was performed on the DNA using these primers to identify microsatellite activity.

[0082] BAT-25Forward:5'-TCG CCT CCA AGA ATG TAA GT-3'

[0083] Reverse:5'-TCT GCA TTT TAA CTA TGG CTC-3'

[0084] BAT-26Forward:5'-TGA CTA CTT TTG ACT TCA GCC-3'

[0085] Reverse:5'-AAC CAT TCA ACA TTT TTA ACC C-3'

[0086] CAT-25Forward:5'-CCT AGA AAC CTT TAT CCC TGC TT-3'

[0087] Reverse:5'-GAG CTT GCA GTG AGC TGA GA-3'

[0088] NR-24Forward:5'-GCT GAA TTT TAC CTC CTG AC-3'

[0089] Reverse:5'-ATT GTG CCA TTG CAT TCC AA-3'

[0090] SEC-63Forward:5'-AGT AAA GCA CCC AAG AAA ACT GC-3'

[0091] Reverse:5'-TGC TTT TGT TTC TGT TGC TTT G-3'

[0092] Microsatellite instability PCR detection method:

[0093] PCR: Add the reaction mixture and specific site primers from the PCR kit to each sample group. Reaction steps: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 1 min, 72℃ final extension for 10 min, cycle 30 times.

[0094] Detection of microsatellite locus PCR amplification products using polyacrylamide gel electrophoresis and Exred staining: A 12% polyacrylamide gel was prepared using 30% acrylamide stock solution and 10% ammonium persulfate (APS). 3 μL of each sample was loaded and electrophoresed at 150 V for 2 hours in a vertical electrophoresis tank. A 1‰ Exred chromogenic solution was used for colorimetric reaction. The gel was then photographed and analyzed using a gel imaging system.

[0095] The results are as follows Figure 2 The results showed that 5-FU treatment increased microsatellite instability in the presence of SirT7, while microsatellites remained stable in SirT7 knockdown cells. These findings indicate that SirT7 determines the stability of cellular microsatellites under chemotherapeutic treatment.

[0096] Example 3: SirT7 knockdown tumor cells are sensitive to multiple chemotherapy drugs

[0097] Figure 3 a. The stable cell lines with SirT7 knockdown were treated with 5-FU (200 μM) for 24 hours and 36 hours, and then cell viability was detected by MTT assay. Figure 3 b. The SirT7 knockdown stable cell lines were treated with Taxol (1 μM) for 24 hours and 36 hours, and then cell viability was detected by MTT assay.

[0098] The results are as follows Figure 3 As shown, SirT7 knockdown (SirT7-ShR) cells died more under drug treatment, indicating that SirT7 knockdown cells are more sensitive to drugs.

[0099] This experiment suggests the possibility of developing an inhibitor drug that targets SirT7, thereby increasing the sensitivity of tumor cells to chemotherapy drugs by reducing the protein expression level of SirT7.

[0100] As an application of SirT7 as a molecular marker provided by this invention, the method involves detecting the expression level of SirT7 protein in cancer patients. High SirT7 expression or SirT7 protein levels in tumor tissues significantly higher than in adjacent non-tumor tissues indicate microsatellite instability, meaning chemotherapy drug resistance has developed. This insight can be used to guide chemotherapy drug use.

[0101] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a product for detecting SirT7 expression levels in the preparation of products for predicting resistance to chemotherapy drugs in tumors, characterized in that, The chemotherapy drug is 5-fluorouracil or paclitaxel, and the tumor is breast cancer.

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