Application of LNPEP as therapeutic target in preparation of medicine for treating or screening colon cancer

Abstract

The invention relates to the technical field of biological medicine, in particular to application of LNPEP as a therapeutic target to preparation of drugs for treating or screening colon cancer. The invention provides application of LNPEP as a therapeutic target in preparation of a medicine for treating or predicting colon cancer. After the LNPEP is knocked down, the drug resistance of the colon cancer to the FOLFOX treatment scheme is obviously improved. Meanwhile, compared with the NC group, the KD group has no influence on cell proliferation after knocking down the LNPEP. After the LNPEP gene is silenced, the autophagy degree of the cell is activated; compared with a medicine adding group, the influence of gene knock-down is greater. After the autophagy inhibitor is used, drug resistance caused by LNPEP knock-down is reversed, which indicates that LNPEP related drug resistance is associated with autophagy. The invention provides a new treatment strategy and drug selection for clinical treatment of colon cancer, and has important clinical application value and wide market prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of LNPEP as a therapeutic target in the preparation of drugs for treating or screening colon cancer. Background Technology

[0002] Colorectal cancer is one of the most common cancers worldwide. Currently, the standard treatment for locally advanced colorectal cancer is neoadjuvant chemoradiotherapy combined with surgery, aiming to preserve organs, improve the complete response rate (CCR), and reduce local recurrence. In the treatment of colorectal cancer, various treatment regimens have been developed to suit different disease stages and individual patient conditions, including comprehensive treatment strategies such as chemotherapy, targeted therapy, and immunotherapy. Among these, the FOLFOX regimen has achieved significant therapeutic effects in some colorectal cancer patients, effectively prolonging survival and improving quality of life.

[0003] However, existing FOLFOX chemotherapy has several problems: 1. The pathological complete response rate is relatively low. 2. Patient responses vary, and some patients do not benefit from treatment, potentially missing the surgical window due to early tumor progression. 3. There are currently no universally accepted efficacy assessment criteria, making it impossible to determine efficacy before surgery and adjust treatment plans accordingly. 4. Currently, there are no tumor molecular markers that can accurately predict chemotherapy response, tumor recurrence, and overall prognosis; tumor markers related to neoadjuvant chemotherapy for colorectal cancer are still in the exploratory stage and are not yet clinically available. This means that patients cannot be accurately stratified before treatment, making individualized treatment difficult.

[0004] Tumor drug resistance is one of the core reasons for chemotherapy failure in colorectal cancer, and abnormal activation of autophagy has been proven to be an important mechanism by which tumor cells develop chemotherapy resistance. Autophagy is a highly conserved cellular homeostasis regulatory mechanism that maintains cellular metabolic balance by degrading damaged organelles and abnormal proteins within cells through lysosomes. Autophagy plays a dual role in tumor development and progression: in the precancerous stage, autophagy exerts a tumor-suppressive effect by clearing abnormal cellular components; in the advanced stage of tumor development and during chemotherapy, autophagy can be induced and activated by chemotherapeutic drugs, providing energy and nutrients to tumor cells, promoting tumor cell survival, thereby reducing the sensitivity of tumor cells to chemotherapeutic drugs and leading to drug resistance.

[0005] Numerous studies have confirmed the close link between autophagy and chemotherapy resistance in colorectal cancer. For example, downregulation of HMBOX1 expression in colorectal cancer promotes reduced transcription of the E3 ubiquitin ligase HACE1, decreased ATG5 K63-ubiquitination, and reduced proteasome-mediated degradation, leading to increased ATG5 levels and enhanced autophagy. Ultimately, this results in colorectal cancer cells developing resistance to 5-fluorouracil (5-FU) chemotherapy. IL-6 activates autophagy through the IL-6 / JAK2 / BECN1 pathway and promotes chemotherapy resistance in colorectal cancer (CRC). CircSEC24B activates autophagy and induces chemotherapy resistance in colorectal cancer through OTUB1-mediated SRPX2 deubiquitination. These studies suggest that targeting the autophagy pathway is a potential strategy to reverse chemotherapy resistance in colorectal cancer, but currently, there is a lack of clearly defined regulatory targets suitable for clinical translation.

[0006] The LNPEP gene encodes a zinc-dependent aminopeptidase, a protein that can be secreted into maternal serum, present in intracellular vesicles containing the insulin-responsive glucose transporter GLUT4, or form a type II transmembrane glycoprotein. Studies have shown that abnormal LNPEP gene expression may contribute to the development and progression of diseases such as autism and psoriasis. Early reports indicated that the androgen receptor (AR) plays an inhibitory role in HCC progression, while overexpression of cyclic LNPEP may reverse the inhibitory effect of AR on hepatocellular carcinoma invasion or tumor metastasis. Hyperglycemia or hyperinsulinemia leads to decreased survival in endometrial cancer. Studies have shown that LNPEP is involved in insulin-mediated increases in the malignant potential of endometrial cancer. LNPEP has been suggested as a novel target for molecular targeted therapy of endometrial cancer. However, to date, the expression characteristics and biological functions of the LNPEP gene in colorectal cancer, especially its association with resistance to FOLFOX chemotherapy and autophagy regulation, have not been reported, and its value in colorectal cancer treatment remains to be explored.

[0007] In summary, the FOLFOX chemotherapy regimen for locally advanced colorectal cancer faces severe challenges, including high drug resistance rates, difficulty in predicting efficacy, and a lack of personalized treatment. Abnormal autophagy is one of the key mechanisms mediating drug resistance. LNPEP, as a gene whose function is not fully understood, has yet to reveal its role in colorectal cancer drug resistance. Therefore, in-depth exploration of the biological function of the LNPEP gene in colorectal cancer, clarifying its association with autophagy regulation and resistance to the FOLFOX chemotherapy regimen, and developing LNPEP-based efficacy prediction biomarkers and drug resistance reversal targets are of significant theoretical and clinical value for solving clinical treatment challenges and improving the treatment outcomes for colorectal cancer patients. Summary of the Invention

[0008] The purpose of this invention is to explore the application of the LNPEP target in the treatment of colorectal cancer with FOLFOX chemotherapy regimen, to predict the resistance of colorectal cancer to FOLFOX chemotherapy regimen, to accurately stratify patients before treatment, to identify patients who may develop resistance to FOLFOX chemotherapy regimen in advance, thereby providing an important basis for individualized clinical treatment plans, improving treatment effects and patient prognosis.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of LNPEP as a therapeutic target in the preparation of drugs for treating or predicting colon cancer.

[0010] This invention also provides the application of LNPEP as a therapeutic target in the preparation of drugs that reverse drug resistance caused by FOLFOX treatment.

[0011] Preferably, the reagent used in the FOLFOX treatment is 5-fluorouracil and / or oxaliplatin.

[0012] The present invention also provides the use of formulations that promote the expression of the LNPEP gene or protein in the preparation of drugs that reverse resistance to FOLFOX treatment.

[0013] Preferably, the reagent used in the FOLFOX treatment is 5-fluorouracil and / or oxaliplatin.

[0014] The present invention also provides a medicament for treating colon cancer, including an agent that promotes LNPEP expression.

[0015] The present invention also provides a kit for predicting resistance to FOLFOX treatment in colorectal cancer, the kit comprising reagents for detecting the expression level of the LNPEP gene or protein.

[0016] The present invention also provides a drug for reversing resistance to the FOLFOX treatment regimen in colorectal cancer, comprising an active ingredient that promotes the expression of the LNPEP gene or protein.

[0017] Preferably, the active ingredient is an autophagy inhibitor.

[0018] Advantages of this invention over existing technologies: This invention utilizes proteomics analysis of colon tumor tissues from patients with locally advanced colorectal cancer who underwent neoadjuvant chemotherapy and radical resection to screen for the differentially expressed molecular marker LNPEP. The impact of LNPEP on resistance to different treatment regimens and the related mechanisms were also investigated. Results showed that knockdown of LNPEP significantly increased resistance to FOLFOX treatment in colon cancer. Furthermore, compared to the NC group, knockdown of LNPEP in the KD group had no effect on cell proliferation. Silencing the LNPEP gene activated autophagy; the effect of gene knockdown was greater compared to the drug-treated group. The drug resistance induced by LNPEP knockdown was reversed after the use of an autophagy inhibitor, indicating a link between LNPEP-related drug resistance and autophagy. This provides a new treatment strategy and drug option for the clinical treatment of colon cancer, possessing significant clinical application value and broad market prospects. Attached Figure Description

[0019] Figure 1 The results of differential gene analysis between the remission group and the non-remission group; Figure 2 The results of downregulating LNPEP to induce resistance in colon cancer cells to FOLFOX treatment (the vertical axis represents the viability of colon cancer cells after drug addition, the horizontal axis NC represents LNPEP control + FOLFOX treatment, and KD represents the LNPEP knockdown + FOLFOX treatment group; the left figure is HT29 cells, and the right figure is HCT116 cells). Figure 3 To illustrate the effect of downregulating LNPEP on the proliferation of colon cancer cells (the vertical axis represents the viability of colon cancer cells, the horizontal axis NC represents the control group, and KD represents the LNPEP knockdown treatment group; the left figure shows HT29 cells, and the right figure shows HCT116 cells). Figure 4 To investigate the effect of siRNA transient transfection technology on LNPEP knockdown on LNPEP expression; Figure 5 To knock down the expression levels of LC3 and Beclin-1 after LNPEP; Figure 6 To investigate the effects of using autophagy inhibitors on drug resistance caused by LNPEP knockdown. Detailed Implementation

[0020] The following detailed description of the solutions provided by the present invention, in conjunction with the embodiments, should not be construed as limiting the scope of protection of the present invention.

[0021] Example 1

[0022] 1. Experimental Procedure

[0023] 1.1 Transient transfection of siRNA

[0024] (1) Dissolving and aliquoting siRNA: Centrifuge LNPEP-siRNA and NC-siRNA powder at 2500 rpm for 2 min to allow them to accumulate at the bottom of the tube. Gently open the tube cap, add RNase-free H2O to dissolve and prepare a 20 μM solution, aliquot it into RNase-free EP tubes, and store at -20℃.

[0025] (2) Colon cancer cells HCT-116 or HT-29 were digested with trypsin and resuspended to dilute the cell concentration to 2 × 10⁻⁶. 5 2 ml of cells / ml was seeded into a six-well plate.

[0026] (3) When the cells have grown to 50%~70% after 24 h, remove the culture medium, wash with serum-free McCoy's 5A medium, and add 1.5 ml of serum-free medium to each well.

[0027] (4) According to the lipofectamine 2000 instructions, dissolve 5 μl of siRNA in 250 μl of opti-mem and gently pipette 3-5 times to mix. Separately, take an RNase-free EP tube, dissolve 5 μl of lipofectamine 2000 in 250 μl of opti-mem and gently pipette 3-5 times to mix. Let stand at room temperature for 5 minutes.

[0028] (5) Mix lipofectamine 2000 and siRNA dilution, gently pipette to mix, and let stand at room temperature for 20 minutes.

[0029] (6) Add the transfection complex to the six-well plate, shake the plate to mix it evenly, incubate for 4 hours, then replace with complete culture medium for subsequent experiments.

[0030] 1.2 CCK-8 Experiment

[0031] (1) After digestion and centrifugation, the cells were resuspended and the cell concentration was adjusted to 3×10⁻⁶ cells / mL using complete culture medium. 3 100 μl of cell suspension was seeded into each well of a 96-well plate, with 5 replicates per group.

[0032] (2) After 12 hours, once the cells have adhered to the cell wall, add 10 μl of CCK-8 solution to each well and incubate at 37°C for 2 hours.

[0033] (3) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of each well at 450 nm and record the OD450.

[0034] 1.3 Drug Resistance Reversal Experiment

[0035] Cells with knocked-down LNPEP gene were treated in combination with an autophagy inhibitor, while a control group (NC) was set up that received chemotherapy alone (FOLFOX treatment regimen: oxaliplatin + 5-fluorouracil). After 24 hours of treatment, cell viability was detected by the CCK-8 assay, with KD representing the treatment group.

[0036] 1.4 Immunoprecipitation assay

[0037] (1) Protein was extracted using lysis buffer and protein concentration was measured using the BCA method.

[0038] (2) Removal of specific binding: Collect 500 μg of protein, add 1 μg of ordinary IgG of the same species as the IgG used in immunoprecipitation and 20 μl of ProA+G Agrarose (protein A / G agarose beads), mix well, and shake slowly at 4°C for 1 h. After centrifuging the sample at 2500 rpm for 5 min, carefully collect the supernatant into a new centrifuge tube.

[0039] (3) Mix the IP antibody with the protein sample at 4°C overnight. Add 1 μg of IP antibody to 500 μg of protein sample. Add 20 μl of ProA+G Agrarose to the incubated sample and incubate for 2 h.

[0040] (4) Wash the sample with 1 ml PBS, centrifuge at 2500 rpm for 5 min and discard the supernatant. Repeat 4 times. After the last wash, add the sample loading buffer and incubate at 100℃ for 5 min.

[0041] 1.5 Western blotting assay

[0042] (1) Prepare glass plates and glue-making frames in advance. The glass plates need to be washed with distilled water and dried.

[0043] (2) Prepare SDS-PAGE: Prepare separating gels of different concentrations according to the molecular weight of the target protein.

[0044] (3) Gel preparation: Prepare the separating gel according to the instructions. Use a pipette to slowly add the gel to the glass plate. Add 2 ml of isopropanol to remove air bubbles and seal the gel. After the lower gel solidifies, pour off the isopropanol and absorb the remaining liquid with absorbent paper. Use a pipette to add the prepared upper gel solution, carefully injecting to avoid air bubbles, and insert the comb teeth.

[0045] (4) Sample loading: After the upper gel solidifies, add electrophoresis buffer to the electrophoresis tank, remove the comb, and add an appropriate amount of sample to the wells. When loading the sample, add markers to both wells of the protein sample.

[0046] (5) Electrophoresis: Select the appropriate voltage and electrophoresis time according to the molecular weight of the sample protein.

[0047] (6) Transfer: Activate the PVDF membrane in methanol solution beforehand. After electrophoresis, open the gel casting plate and cut the gel. Place the PVDF membrane and gel in the transfer clamp in the order of positive and negative electrodes, avoiding air bubbles between the gel and membrane. Place the transfer clamp in the transfer tank, add transfer buffer, and transfer at a constant current of 250 mA for 90 min. The transfer must be performed in an ice bath.

[0048] (7) Sealing: After the transfer is completed, the PVDF membrane is placed in 5% skim milk powder and sealed on a shaker at room temperature for 1.5 h.

[0049] (8) Incubation with primary antibody: After blocking, the PVDF membrane is washed three times with TBST for 8 min each time. Prepare the appropriate concentration of primary antibody according to the instructions, and then put the PVDF membrane into the diluted primary antibody and incubate overnight at 4°C.

[0050] (9) Incubation of secondary antibody: The primary antibody was retrieved the next day and stored in a refrigerator at 4°C. The membrane was washed three times with TBST for 10 minutes each time. Secondary antibody of the corresponding species was prepared using secondary antibody dilution buffer and incubated on a shaker at room temperature for 2 hours.

[0051] (10) Color development: Wash the membrane 3 times with TBST. Take ECL chemiluminescence color development solution, mix solutions A and B in a 1:1 ratio, mix well with a pipette before use. Place the PVDF membrane in a gel imaging system, add an appropriate amount of color development solution, expose, save the image and analyze the grayscale value using ImageJ software.

[0052] 1.6 Electron Microscope

[0053] (1) Sample preparation: Collect 1×10⁻⁶ cells in the logarithmic growth phase. 7 (1 cell), gently wash twice with PBS, and centrifuge to form cell clusters; (2) Fixation: Aspirate the supernatant, gently add 1 ml of electron microscopy fixative along the wall, keep the precipitate unchanged, fix at room temperature for 0.4 h, and store at 4 ℃.

[0054] (3) Gradient dehydration: Soak in 30%→50%→70%→80%→90%→95%→100% ethanol in sequence, 20 min for each step, and repeat 100% ethanol twice; (4) Resin embedding: ① Acetone: resin = 3:1 → 1:1 → 1:3 gradient permeation, 3 hours per step, and finally pure resin overnight at 4℃; ② Add fresh resin to the embedding mold and cure at 37℃ for 12 hours + 60℃ for 24 hours. (5) Ultrathin sectioning: Trim the block into a pyramid shape, cut 60nm thin sections using an ultrathin slicer, and then use a copper mesh to collect and dry the sections; (6) Electronic staining: ① Stain with uranium acetate in the dark for 15 min, rinse with double distilled water; ② Stain with lead citrate in a fume hood for 10 min, rinse with double distilled water again, and air dry; (7) Electron microscopy observation: TEM is adjusted to 100 kV, cells are located under low magnification, target structures (such as autophagosomes) are observed under high magnification, and images are taken.

[0055] 1.7 Data Processing

[0056] Experimental data were replicated three times. SPSS and Graphpad Prism software were used for statistical analysis and graphing. T-tests were used to compare data between two groups; ANOVA one-way ANOVA was used for data from three or more groups. p <0.05 indicates that the data are significantly different.

[0057] 2. Results

[0058] 2.1 Differential Proteomics Analysis

[0059] Patients with locally advanced colorectal cancer who received neoadjuvant chemotherapy and radical surgery at Jinan Central Hospital from January 2012 to December 2023 were selected as the study subjects. Proteomic analysis of the treatment response group and the non-response group revealed that, compared with the non-response group, the LNPEP gene was significantly highly expressed in the response group. Figure 1 Next, we will analyze the impact and mechanism of the LNPEP gene on resistance to FOLFOX treatment.

[0060] 2.2 Screening for potential molecular biomarkers with differential expression

[0061] To investigate the effect of LNPEP on resistance to FOLFOX treatment in colorectal cancer, cell viability at different drug concentrations was measured using a CCK-8 assay. The IC50 values ​​of the treatment regimen against HCT116 and HT-29 colorectal cancer cells were calculated using GraphPad Prism nonlinear regression fitting to determine the optimal drug concentration (Table 1). Next, the effect of LNPEP on resistance to FOLFOX treatment was validated. The results showed that knockdown of LNPEP significantly increased resistance to FOLFOX in colorectal cancer. Figure 2 Meanwhile, it was found that, compared with the NC group, simply knocking down LNPEP in the KD group had no effect on cell proliferation. Figure 3 ).

[0062] Table 1 Drug concentrations for different cell types

[0063] 2.3 Molecular mechanism by which LNPEP affects resistance to FOLFOX treatment regimen

[0064] Decreased tumor drug sensitivity is often accompanied by increased autophagy, leading to autophagy-related drug resistance. The effects of combining chemotherapy drugs with autophagy induction / inhibition have been widely confirmed. To further investigate the effect of LNPEP on cellular autophagy, transient siRNA transfection was performed to knock down LNPEP, and the expression levels of LC3 and Beclin-1 were detected. An autophagy inhibitor was then added for reverse validation.

[0065] The results showed that, compared with the control group, knocking down LNPEP activated autophagy; and the effect of gene knockdown was greater than that of the treatment group. Therefore, downregulating LNPEP in colon cancer cells can activate autophagy. Figures 4-5 (Si-LNPEP represents LNPEP knockdown; LNPEP represents leucyl / cysteyl aminopeptidase; Vinculin represents focal adhesion protein (internal control); the vertical axis of the right figure represents expression level, NC represents control group; KD represents LNPEP knockdown; NC(+) represents LNPEP control + FOLFOX treatment; KD(+) represents LNPEP knockdown + FOLFOX treatment).

[0066] Next, the autophagy inhibitor chloroquine (CQ) was used in combination with the drug to investigate whether LNPEP-related resistance was related to autophagy. Experimental results showed that 5 μM CQ had no lethal effect on colon cancer cells, but Western blot experiments confirmed that 5 μM CQ effectively inhibited autophagy. After the application of the autophagy inhibitor, the resistance previously caused by LNPEP knockdown was reversed, indicating a link between LNPEP-related resistance and autophagy. Figure 6 ).

[0067] As illustrated in the above embodiments, this invention utilizes proteomics analysis of colon tumor tissues from patients with locally advanced colorectal cancer who underwent neoadjuvant chemotherapy and radical resection to screen for the differentially expressed molecular marker LNPEP. The impact of LNPEP on resistance to different treatment regimens and the related mechanisms were also verified. Results showed that knockdown of LNPEP significantly increased resistance to FOLFOX treatment in colon cancer. Furthermore, it was found that, compared to the NC group, knockdown of LNPEP in the KD group had no effect on cell proliferation. When the LNPEP gene was silenced, autophagy was activated; the effect of gene knockdown was greater compared to the drug-treated group. The use of autophagy inhibitors reversed the drug resistance caused by LNPEP knockdown, indicating a link between LNPEP-related drug resistance and autophagy. This provides a new treatment strategy and drug option for the clinical treatment of colon cancer, possessing significant clinical application value and broad market prospects.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of LNPEP as a therapeutic target in the preparation of drugs for the treatment or prediction of colorectal cancer.

2. Application of LNPEP as a therapeutic target in the preparation of drugs that reverse drug resistance caused by FOLFOX treatment.

3. The application according to claim 2, characterized in that, The agents used in the FOLFOX treatment are 5-fluorouracil and / or oxaliplatin.

4. Application of formulations that promote the expression of the LNPEP gene or protein in the preparation of drugs that reverse resistance to FOLFOX treatment.

5. The application according to claim 4, characterized in that, The agents used in the FOLFOX treatment are 5-fluorouracil and / or oxaliplatin.

6. A drug for treating colon cancer, characterized in that, This includes formulations that promote LNPEP expression.

7. A kit for predicting resistance to FOLFOX treatment in colon cancer, characterized in that, The kit includes reagents for detecting the expression level of the LNPEP gene or protein.

8. A drug for reversing resistance to the FOLFOX treatment regimen in colon cancer, characterized in that, Including active ingredients that promote the expression of the LNPEP gene or protein.

9. The medicament according to claim 8, characterized in that, The active ingredient is an autophagy inhibitor.

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