Use of pcbp1 gene l100q site mutation in colorectal cancer drug resistance

Abstract

The application belongs to the technical field of biological medicine, and discloses a use of PCBP1 gene L100Q site mutation in colorectal cancer drug resistance. The application provides a use of the PCBP1 gene L100Q site mutation in preparation of a biomarker for evaluating colorectal cancer drug resistance risk, as a target in preparation of a drug for treating and / or preventing colorectal cancer drug resistance, as a screening marker of an anti-colorectal cancer drug resistance drug, and as a target in preparation of a tumor drug resistance diagnostic reagent. The PCBP1 gene L100Q site mutation can significantly promote colorectal cancer tumor cell stemness, and causes colorectal cancer to be resistant to oxaliplatin and 5-FU, and can be used as a potential treatment site for a colorectal cancer oxaliplatin or 5-FU resistant patient. The potential target can be used for guiding personalized treatment of CRC patients, and has a wide application prospect and important clinical value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the use of a PCBP1 gene L100Q site mutation in drug resistance in colorectal cancer. Background Technology

[0002] Colorectal cancer (CRC) is the third most common solid tumor worldwide. Adjuvant chemotherapy is recommended for stage II colon cancer patients with high-risk factors (poor histological differentiation with normal mismatch repair or microsatellite stability, T4, angiolymphatic invasion, preoperative bowel obstruction, bowel perforation, insufficient or fewer than 12 lymph nodes detected in the specimen, nerve invasion, positive or indeterminate surgical margins) and stage III CRC patients. Recommended chemotherapy regimens include oxaliplatin-based CapeOx or FOLFOX regimens, or single-agent 5-FU / LV, or capecitabine. For advanced or metastatic CRC patients, chemotherapy drugs selected are also based on oxaliplatin or 5-FU.

[0003] CRC patients exhibit clinical resistance to oxaliplatin or 5-FU. Current research has revealed some mechanisms of oxaliplatin or 5-FU resistance, including alterations in DNA repair mechanisms, changes in metabolic pathways within tumor cells, and reduced drug-DNA binding capacity. Exploring these mechanisms can provide new targets and strategies for overcoming oxaliplatin or 5-FU resistance. In recent years, with a deeper understanding of the molecular mechanisms of tumor drug resistance, CRC treatment has become increasingly personalized. Screening and targeted therapy based on tumor molecular characteristics are becoming research hotspots in CRC treatment. These studies mainly focus on the following aspects: Immunotherapy has become an important component of CRC treatment. Clinical trials have shown that immunotherapeutic drugs such as PD-1 and PD-L1 inhibitors may be effective in some oxaliplatin-resistant CRC patients; targeted therapy against specific CRC variants is also an important direction for personalized treatment. For example, targeted drugs against RAS and BRAF mutations have shown some efficacy in clinical trials; through in-depth studies of the genome and transcriptome of CRC patients, researchers are searching for new biomarkers associated with oxaliplatin or 5-FU resistance to better predict patient response to treatment and develop new treatment strategies. Overall, research into personalized treatment for CRC to overcome oxaliplatin or 5-FU resistance is ongoing and in-depth.

[0004] However, despite some progress in personalized treatment for CRC to overcome oxaliplatin or 5-FU resistance, several technical limitations and challenges remain: 1) Insufficient accuracy in predicting treatment outcomes: Current predictive models and biomarkers are not yet able to accurately predict patient responses to treatment; 2) Complexity of resistance mechanisms: Oxaliplatin resistance mechanisms are highly complex, involving multiple molecular pathways and biological processes, and current research is insufficient, limiting the development of treatment strategies targeting resistance; 3) Cost and accessibility: Some personalized treatment technologies and drugs are expensive and may not be easily available to all regions and patients. This limits the widespread adoption and application of these treatment strategies; 4) Low degree of personalization in treatment regimens: Although the concept of personalized treatment is widely accepted, a degree of standardized treatment still exists in clinical practice. Differences in individual patients' genotypes, phenotypes, and environmental factors are not fully considered.

[0005] In summary, while personalized treatment holds potential advantages in overcoming oxaliplatin or 5-FU resistance in CRC, several technical limitations remain. Future research should focus on tumor molecular profiling and developing individualized treatment strategies targeting specific tumor markers. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide the use of a PCBP1 gene L100Q site mutation (L100 site T point mutation to A) in colorectal cancer drug resistance.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides the use of a mutation at the L100Q site of the PCBP1 gene in the preparation of a biomarker for assessing the risk of drug resistance in colorectal cancer.

[0009] Secondly, the present invention provides the use of the PCBP1 gene L100Q site mutation as a target in the preparation of a medicament for the treatment and / or prevention of drug resistance in colorectal cancer.

[0010] Thirdly, the present invention provides the use of the PCBP1 gene L100Q site mutation as a screening marker for drugs resistant to colorectal cancer.

[0011] Fourthly, the present invention provides the use of the PCBP1 gene L100Q site mutation as a target in the preparation of diagnostic reagents for tumor drug resistance.

[0012] Fifthly, the present invention provides the use of an inhibitor targeting the L100Q site mutation of the PCBP1 gene in the preparation of a drug for treating colorectal cancer resistance.

[0013] This invention discovers that the L100Q mutation (a T-point mutation at L100 site to A) in the RNA or DNA conjugate protein 1 (PCBP1) gene can promote stemness in CRC tumor cells and lead to resistance to oxaliplatin and 5-FU. Compared to wild-type PCBP1, PCBP1 L100P (a T-point mutation at L100 site to C) and PCBP1 L100R (a T-point mutation at L100 site to G) significantly promote CRC resistance to oxaliplatin or 5-FU at the cellular level. This invention provides a target (PCBP1 gene L100Q mutation) leading to CRC resistance to oxaliplatin and 5-FU and can be used to guide personalized treatment for CRC patients.

[0014] In a preferred embodiment of the use described in this invention, the drug resistance is resistance to chemotherapy drugs.

[0015] As a further preferred embodiment of the use described in this invention, the chemotherapy drug is a platinum-based chemotherapy drug and / or 5-FU.

[0016] As a further preferred embodiment of the use described in this invention, the platinum-based chemotherapy drug is cisplatin or oxaliplatin.

[0017] In a sixth aspect, the present invention provides a kit for assessing the risk of drug resistance in colorectal cancer, the kit comprising a reagent for detecting mutations at the L100Q site of the PCBP1 gene.

[0018] In a preferred embodiment of the kit described in this invention, the drug resistance is resistance to chemotherapy drugs.

[0019] Furthermore, the chemotherapy drug is a platinum-based chemotherapy drug and / or 5-FU.

[0020] Furthermore, the platinum-based chemotherapy drug is cisplatin or oxaliplatin.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] This invention is the first to discover that mutations at the L100Q site of the PCBP1 gene can promote stemness in CRC tumor cells and lead to resistance to oxaliplatin and 5-FU in CRC. Compared to wild-type PCBP1, mutations at the L100Q site of the PCBP1 gene significantly promote the formation of tumor spheroids in CRC, while mutations at the L100P or L100R sites of the PCBP1 gene have no effect on the formation of tumor spheroids in CRC. Mutations at the L100Q site of the PCBP1 gene can significantly affect the expression of stemness-related proteins (EPCAM / OCT4 / SOX2 / NUMB) in CRC tumor cells. Mutations at the L100Q site of the PCBP1 gene can significantly increase the IC50 of oxaliplatin in CRC tumor cell lines (RKO), thereby promoting resistance to oxaliplatin in CRC tumor cell lines. Mutations at the L100Q site of the PCBP1 gene can significantly increase the IC50 of 5-FU in CRC tumor cell lines (RKO), thereby promoting resistance to 5-FU in CRC tumor cell lines. Mutations at the L100Q site of the PCBP1 gene can significantly promote the stemness of CRC tumor cells and lead to resistance to oxaliplatin and 5-FU in CRC. This potential target can be used to guide personalized treatment for CRC patients and has broad application prospects and important clinical value. Attached Figure Description

[0023] Figure 1 Mutations at the L100Q site of the PCBP1 gene significantly promoted tumor spheroid formation in CRC tumor cells, while mutations at the L100P or L100R sites had no effect on this ability. In vitro spheroid formation assays were used to detect the size and number of tumor spheroids formed by different types of mutations at the L100 site of the PCBP1 gene in the RKO CRC tumor cell line. Figure 1 A shows the morphology of tumor spheres formed by each group of cells under a light microscope. Mutations at the L100Q site of the PCBP1 gene can significantly increase the volume of tumor spheres. Figure 1 B statistically analyzed the number of tumor spheres formed in each group of cells, and the mutation at the L100Q site of the PCBP1 gene significantly increased the number of tumor spheres formed. (ns: P≥0.05; ****: P<0.0001)

[0024] Figure 2 Mutations at the L100Q site of the PCBP1 gene can significantly affect the expression of stemness-related proteins in CRC. Western blotting was used to detect the expression of stemness-related proteins (EPCAM / OCT4 / SOX2 / NUMB) in the RKO CRC tumor cell line after different types of mutations at the L100 site of the PCBP1 gene.

[0025] Figure 3Mutations at the L100Q site of the PCBP1 gene significantly increase the IC50 of oxaliplatin in CRC tumor cell lines, thereby promoting oxaliplatin resistance in these cell lines. CRC cell lines overexpressing the wild-type PCBP1 gene and those with the PCBP1 gene L100 mutation were treated with oxaliplatin at specific concentration gradients. The IC50 of oxaliplatin in different groups was calculated to determine the degree of oxaliplatin resistance. Figure 3 A showed that the L100Q mutation in the PCBP1 gene promoted the cell viability of RKO cells treated with oxaliplatin. The MTS method was used to detect the cell viability of RKO cell lines after 48 hours of treatment with different concentrations of oxaliplatin (0.02 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, and 160 μM). "----" indicates a viability of 50%. Figure 3 B showed that the L100Q mutation in the PCBP1 gene significantly increased the IC50 of oxaliplatin, thereby promoting the resistance level of RKO cell lines to oxaliplatin.

[0026] Figure 4 Mutations at the L100Q site of the PCBP1 gene significantly increase the IC50 of 5-FU in CRC tumor cell lines, thereby promoting 5-FU resistance in these cell lines. RKO cell lines overexpressing wild-type PCBP1 and those with the PCBP1 L100 mutation were treated with 5-FU at specific concentration gradients. The IC50 of 5-FU in different groups was calculated to determine the degree of 5-FU resistance. Figure 4 A showed that the L100Q mutation in the PCBP1 gene promoted the cell viability of RKO cells treated with 5-FU. The MTS method was used to detect the cell viability of RKO cell lines after 48 hours of treatment with different concentrations of 5-FU (0.02 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, and 160 μM). "----" indicates a viability of 50%. Figure 4 B showed that the L100Q mutation in the PCBP1 gene significantly increased the IC50 of 5-FU, thereby promoting the resistance level of RKO cell lines to 5-FU. Detailed Implementation

[0027] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0028] The terms “comprising” or “including” mean that the stated elements, integers, or steps are included, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations consisting of the stated elements, integers, or steps.

[0029] Point mutation, also known as a single-base substitution, refers to a mutation caused by a change in a single base. It is divided into two categories: conversion and transversion. Conversion involves substitution between purines or between pyrimidines; transversion involves substitution between purines and pyrimidines. Point mutations can produce four different effects: synonymous mutations, missense mutations, nonsense mutations, and stop codon mutations. In this invention, the mutation at the L100 site of the PCBP1 gene results in a missense mutation, which is a base substitution mutation that leads to changes in the amino acid sequence of the polypeptide product or the base sequence of the functional RNA.

[0030] "Tumor spheroid formation ability": refers to the ability of tumor cells (specifically the CRC tumor cell line RKO) to form tumor spheroids when cultured in vitro in a medium containing EGF and bFGF. It is the gold standard for measuring the stemness of tumor cells.

[0031] "Tumor cell stemness" refers to the self-renewal and differentiation capabilities of tumor cells, similar to those of stem cells. This stemness characteristic allows tumor cells to proliferate indefinitely, forming tumors and making them resistant to conventional treatments. It is regulated by multiple signaling pathways, including Notch, Wnt, and Hedgehog. Abnormal activation or inhibition of these pathways can lead to an increase or decrease in tumor cell stemness. Tumor cell stemness is considered one of the important causes of tumor recurrence, metastasis, and drug resistance.

[0032] "IC50": Also known as half-inhibitory concentration, it is a commonly used indicator in biology and pharmacology to measure the level of inhibition or activity of a compound or drug on a biological system. The IC50 value represents the concentration at which a compound or drug inhibits a biological process or activity by 50% under specific conditions. In this invention, a higher IC50 indicates greater resistance of the tumor cell line to oxaliplatin or 5-FU, and stronger drug resistance.

[0033] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0034] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified. The nucleotide sequence of PCBP1 has the NCBI accession number NG_029956.1.

[0035] Example 1:

[0036] CRC cell lines stably overexpressing wild-type PCBP1 and mutant PCBP1 (L100P / L100Q / L100R) were constructed.

[0037] (1) Confirmation that the wild-type CRC cell line RKO (purchased from ATCC, USA) has no PCBP1 gene L100 site mutation: The culture medium of the well-grown adherent RKO cells was discarded, and the cells were washed twice with PBS and the supernatant was aspirated; Genomic DNA of the RKO cell line was extracted according to the instructions of the Genomic DNA Purification Kit (EZ Bioscience, Cat. No.: B0007), and PCR amplification was performed with the corresponding primers and then sequenced to confirm that the PCBP1 gene L100 site of the RKO cell line had no mutation.

[0038] (2) Following the instructions of the ViaFect™ Transfection reagent product, the human PCBP1 sgRNA plasmid (vector pLenti-U6-hPCBP1 sgRNA-EFS-NS-SpCas9-2A-Puro, lentiviral backbone, puromycin resistance, purchased from Shanghai Lianfeng / Guangzhou Youming Biotechnology Co., Ltd.) was transfected into the RKO cell line. After 36 hours of transfection, the cells were collected and re-coated into 96-well plates using a serial dilution method. After the cells were cultured statically for 7-10 days, the cell clone formation was observed under a microscope. When a clone with a single cell was observed in the well, the well was marked. When the clone grew to a suitable size, two copies of the 96-well plate were prepared for single clone formation. One copy was used for further culture, and the other copy was used to extract genomic DNA for detection until the PCBP1 knockout RKO cell line was finally obtained through screening.

[0039] (3) Obtain plasmids (pLenti-U6-EFS-NS-SpCas9-2A-Blast, lentiviral backbone, with 3Flag tag, Blast resistance) that stably overexpress wild-type PCBP1 (WT-OE) and mutant PCBP1 (L100P / L100Q / L100R): purchased from Shanghai Lianfeng Biotechnology Co., Ltd. and Guangzhou Youming Biotechnology Co., Ltd.

[0040] (4) Virus packaging: Seed healthy 293T cells into 10cm cell culture dishes. When the cell density reaches 80%, virus packaging can be performed. Prepare virus packaging plasmids and target plasmids (plasmids stably overexpressing wild-type PCBP1 (WT-OE) and mutant PCBP1 (L100P / L100Q / L100R)) according to the following ratios: Opti-MEM 200μL + psPAX2 6μg + pMD2.G 2μg + target plasmid 8μg + PEI (0.5mg / mL) 100μL. After adding PEI, vortex thoroughly and let stand at room temperature for 15 minutes. Then, add the PEI solution dropwise to the 293T cell culture medium. After 8 hours of cell transfection, replace the medium with fresh medium and continue culturing. At the same time, add 1:500 sodium phosphate solution. Butyrate; after culturing for 48 hours, collect the cell supernatant containing the virus, centrifuge the virus supernatant at 3000 rpm for 5 minutes, discard the cell debris at the bottom of the tube and collect the supernatant; filter the supernatant with a 0.45 μm pore size filter to obtain the virus solution, aliquot and store at -80℃ to avoid repeated freeze-thaw cycles.

[0041] (5) Eukaryotic cell lentivirus infection: PCBP1 knockout RKO cell line was seeded in 6-well plates in advance. The next day, the culture medium was discarded, and 1 mL of fresh culture medium and 1 mL of the above filtered virus solution were added to each well. At the same time, 2 μL of Polybrene at a concentration of 2 mg / mL was added, mixed well and placed in a cell culture incubator for culture. After 10 hours of infection, the culture medium was discarded, and 2 mL of fresh complete culture medium was added for continued culture. After 24 hours, 10 μm Blast antibiotic screening was started.

[0042] (5) Finally, Western blotting was used to verify that the CRC cell line RKO, which stably overexpressed wild-type PCBP1 (WT-OE) and mutant PCBP1 (L100P / L100Q / L100R), was obtained.

[0043] Example 2:

[0044] The size and number of tumor spheres formed by different types of mutations at the L100 site of the PCBP1 gene in the CRC cell line RKO were detected by in vitro spheroidization experiments.

[0045] (1) Prepare tumor cell spheroidization medium: Add 20 μL / mL B27, 20 ng / mL LEGF, 20 ng / mL bFGF, 10 μg / mL heparin and 10 μL / mL P / S to DMEM / F12 medium.

[0046] (2) The RKO cell lines that were stably overexpressing wild-type PCBP1 (WT-OE) and mutant PCBP1 (L100P / L100Q / L100R) and were in good growth condition as obtained in Example 1 were digested and centrifuged to remove the serum-containing culture medium. The cells were washed twice with PBS and then resuspended in the above-mentioned spheroidizing culture medium to form a single-cell suspension for cell counting. The cells were then seeded in 96-well cell culture plates. Each group of cells was seeded in 3 replicates, with 500 cells / well and 100 μL of culture medium per well.

[0047] (3) Cultivate at 37℃ and 5% CO2, and gently add cell culture medium every 2-3 days. Observe the morphology and growth of cell spheres under a microscope.

[0048] (4) On day 6 of culture, the morphology of tumor spheres in each treatment group was observed under a microscope, and the number of cell spheres with a diameter greater than 50 μm in each well in each treatment group was counted.

[0049] See results Figure 1 Compared with the WT-OE group, mutations at the L100Q site of the PCBP1 gene significantly promoted the ability of CRC tumor spheroids to form, while mutations at the L100P or L100R sites of the PCBP1 gene had no effect on the ability of CRC tumor spheroids to form.

[0050] Example 3:

[0051] The expression of stemness-related proteins (EPCAM / OCT4 / SOX2 / NUMB) at different mutation sites of the PCBP1 gene in the CRC tumor cell line RKO was detected by Western blotting.

[0052] (1) Protein extraction from adherent cells: Cells were cultured in 10cm diameter dishes until the cell density reached 80%, at which point cell proteins were collected. Cells were washed three times with pre-chilled PBS. RIPA Lysis buffer (500 mL) was prepared: 11.7g of 400mM NaCl, 3g of 25mM HEPES, 0.2mM EDTA, 0.037g of NA2EDTA2H2O, 0.007g of 1.5mM MgCl2, and 1% NP40. Protease inhibitors were added to the RIPA Lysis buffer at a ratio of 1:100. The culture dish was placed on ice, and approximately 500μL of pre-chilled RIPA Lysis buffer containing protease inhibitors was added. The culture dish was shaken to allow the protease inhibitors to dissolve. The buffer was evenly distributed in the culture dish to fully lyse the cells. The cells were scraped off using a cell scraper that had been rinsed with pure water and transferred to a new pre-chilled EP tube. The tube was placed on ice for 20 minutes. The tube was then centrifuged at 13,000 rpm at 4°C for 15 minutes to collect cell debris at the bottom of the tube. The supernatant was collected and transferred to a new pre-chilled EP tube. The tube was then mixed with 5×SDS loading buffer and RIPALysis buffer and boiled in a 95°C metal bath for 10 minutes.

[0053] (2) The protein samples after the above treatment were subjected to protein electrophoresis, membrane transfer, antibody incubation (EPCAM: abcam, ab32392; OCT4: abcam, ab181557; SOX2: Proteintech, 10006239; NUMB: abcam, ab14140) and development in sequence to finally obtain the required image.

[0054] EPCAM is a surface marker of tumor stem cells, a type I transmembrane polypeptide associated with cell adhesion, proliferation, differentiation, and migration. OCT4, an embryonic stem cell transcription factor, indicates the presence of stem cell-like tumor cells in tumors, and its expression can lead to early metastasis, invasion, radiotherapy and chemotherapy resistance, and recurrence after treatment. SOX2, a key transcription factor for stem cells, not only has the potential to maintain stem cell self-renewal and multi-lineage differentiation, but its high expression on tumor cells can promote multiple aspects of tumor proliferation, differentiation, invasion, metastasis, and drug resistance. NUMB is primarily considered a tumor suppressor, and its tumor-suppressive mechanism is related not only to cell polarity, cell division direction, and epithelial-mesenchymal transition, but also closely linked to important pathways such as Notch, p53, and Hh. Results are shown in […]. Figure 2 Mutations at the L100Q site of the PCBP1 gene can significantly affect the expression of CRC stemness-related proteins.

[0055] Example 4:

[0056] CRC cell lines overexpressing the wild-type PCBP1 gene and CRC cell lines with the PCBP1 gene L100 mutation were treated with oxaliplatin at certain drug concentration gradients. The IC50 of oxaliplatin in different groups was calculated to determine their tolerance to oxaliplatin.

[0057] (1) The RKO cell lines overexpressing wild-type PCBP1 and PCBP1 gene L100 mutant in the logarithmic growth phase were digested, resuspended and counted, and seeded in 96-well plates at 2000 cells / well.

[0058] (2) Each group was treated with the following oxaliplatin concentration gradients: 0 μM, 0.02 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM and 160 μM;

[0059] (3) Cell viability was detected using the MTS method after 48 hours: Prepare the appropriate volume of MTS detection solution as needed, add serum-free culture medium at a ratio of detection solution: culture medium = 1:5, and incubate at 37°C with 5% CO2 for 2 hours; based on the absorbance spectrum of the formazan product generated after MTS reduction, read the data at the absorption peak of 490nm, and subtract the data read at 630nm to reduce noise values ​​caused by cell debris and other non-specific absorbance values; calculate the cell viability based on the absorbance value A, cell viability = (experimental group A / control group) × 100%. Calculate the IC50 value and plot the dose-response curve.

[0060] See results Figure 3 Compared with the WT-OE group, the L100Q mutation of the PCBP1 gene significantly increased the IC50 of oxaliplatin in CRC tumor cell lines, thereby promoting the resistance of CRC tumor cell lines to oxaliplatin.

[0061] Example 5:

[0062] CRC cell lines overexpressing the wild-type PCBP1 gene and CRC cell lines with the PCBP1 gene L100 mutation were treated with 5-FU at certain drug concentration gradients. The 5-FU IC50 of different groups was calculated to determine their tolerance to 5-FU.

[0063] (1) The RKO cell lines overexpressing wild-type PCBP1 and PCBP1 gene L100 mutant in the logarithmic growth phase were digested, resuspended and counted, and seeded in 96-well plates at 2000 cells / well.

[0064] (2) Each group was treated with the following 5-FU concentration gradients: 0 μM, 0.02 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM and 160 μM;

[0065] (3) Cell viability was detected using the MTS method after 48 hours: Prepare the appropriate volume of MTS detection solution as needed, add serum-free culture medium at a ratio of detection solution: culture medium = 1:5, and incubate at 37°C with 5% CO2 for 2 hours; based on the absorbance spectrum of the formazan product generated after MTS reduction, read the data at the absorption peak of 490nm, and subtract the data read at 630nm to reduce noise values ​​caused by cell debris and other non-specific absorbance values; calculate the cell viability based on the absorbance value A, cell viability = (experimental group A / control group) × 100%. Calculate the IC50 value and plot the dose-response curve.

[0066] See results Figure 4 Compared with the WT-OE group, the L100Q mutation of the PCBP1 gene significantly increased the IC50 of 5-FU in CRC tumor cell lines, thereby promoting the resistance of CRC tumor cell lines to 5-FU.

[0067] In summary, this invention reveals that, compared to wild-type PCBP1, mutations at the L100Q site of the PCBP1 gene significantly promote tumor spheroid formation in CRC and affect the expression of stemness-related proteins. In vitro treatment of corresponding cell lines with gradient drug concentrations confirmed that mutations at the L100Q site of the PCBP1 gene significantly promote CRC resistance to oxaliplatin and 5-FU. This potential target can be used to guide personalized treatment for CRC patients, such as assessing and predicting the efficacy of chemotherapy and tailoring individualized treatment plans. Furthermore, this target holds promise as a potential therapeutic site for CRC patients resistant to oxaliplatin or 5-FU, possessing significant clinical value.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. The use of a reagent for detecting PCBP1 gene L100Q site mutation in the preparation of a kit for detecting the sensitivity of colorectal cancer to chemotherapeutic drugs, characterized in that, The chemotherapeutic drug is oxaliplatin and / or 5-FU. The chemotherapeutic drug is oxaliplatin and / or 5-FU. The chemotherapeutic drug is

Images