A colorectal cancer marker and its use

Abstract

The application belongs to the technical field of biological detection and biological medicine, and particularly relates to a colorectal cancer marker and application thereof. The application has been confirmed through research that LIMP2 is highly expressed in colorectal cancer patients, is lowly expressed in normal tissues, and the expression of LIMP2 is closely related to the prognosis of colorectal cancer, has the potential to become an auxiliary diagnostic marker of colorectal cancer, and can also be used for prognosis evaluation through the expression level. In addition, knocking down the expression level of LIMP2 can inhibit the migration and invasion ability of colorectal cancer, and is expected to be used as a target for preventing and treating colorectal cancer. It can be seen that the LIMP2 provided by the application can be used not only as a biomarker for diagnosing or / and evaluating the prognosis of colorectal cancer, but also as a target for developing drugs for preventing and treating colorectal cancer, has important potential application value, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of biological detection and biomedicine, specifically relating to a colorectal cancer biomarker and its application. Background Technology

[0002] Colorectal cancer is one of the most common malignant cancers worldwide, with high incidence and mortality rates. It is reported that in 2020, there were 19.3 million new cancer cases and nearly 10 million cancer deaths globally, with colorectal cancer accounting for 10% of the incidence and 9.4% of the mortality.

[0003] Currently, the main treatments for colorectal cancer include surgical resection, radiotherapy, chemotherapy, and immunotherapy. Despite significant advancements in diagnostic methods and treatment techniques for colorectal cancer in recent years, the five-year survival rate remains low, especially for patients with metastatic colorectal cancer. Most patients initially diagnosed with metastatic colorectal cancer eventually develop metastatic malignancies, with up to 30-50% of colorectal cancer patients experiencing liver metastases during the course of the disease. Therefore, understanding the molecular mechanisms of liver metastasis in colorectal cancer can help provide diagnostic, prognostic, and therapeutic targets for patients with liver metastases.

[0004] LIMP2 is a member of the class B scavenger receptor protein family and a major receptor protein for EV71 virus. LIMP2 plays a crucial role in regulating cellular immunity and lysosomal cholesterol homeostasis; its dysfunction can induce various diseases, including cardiomyopathy, muscular dystrophy, and epilepsy. LIMP2 also plays an important role in cancer regulation. Studies have shown that LIMP2 is not only a potential diagnostic target for gastric cancer but also a biomarker for hepatocellular carcinoma stem cells. However, there are currently no reports on LIMP2 serving as a diagnostic biomarker for colorectal cancer. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention provides a colorectal cancer biomarker LIMP2. LIMP2 can not only be used as a biomarker for the diagnosis and / or prognostic assessment of colorectal cancer, but also as a target for the development of drugs to prevent and treat colorectal cancer, and has important potential application value.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] The first aspect of this invention provides the application of a reagent for detecting LIMP2 expression levels in the preparation of products for diagnosing colorectal cancer.

[0008] The second aspect of this invention provides the application of a reagent for detecting LIMP2 expression levels in the preparation of colorectal cancer prognostic assessment products.

[0009] Preferably, the expression level of LIMP2 includes the expression levels of mRNA and / or protein.

[0010] This study found that LIMP2 exhibits high mRNA and protein expression levels in colorectal cancer cells SW620, HCT116, RKO, DLD1, HT29, and CACO-2. Furthermore, its expression level in colorectal cancer tissues is significantly higher than in normal tissues. The overall survival rate of colorectal cancer patients with high LIMP2 expression is significantly lower than that of the low LIMP2 expression group, indicating a poorer prognosis for those with high LIMP2 expression. These results suggest that LIMP2 is highly expressed in colorectal cancer, and that high expression is associated with poor prognosis.

[0011] A third aspect of this invention provides the use of a reagent that inhibits LIMP2 in the preparation of drugs for the prevention and treatment of colorectal cancer.

[0012] Preferably, inhibiting LIMP2 inhibits the migration and invasion of colorectal cancer cells.

[0013] This invention has revealed that, through in vitro and in vivo experiments, knocking down LIMP2 significantly inhibits the migration and invasion of colorectal cancer cells. Simultaneously, it significantly reduces the number and size of liver metastatic tumor nodules in mice. These results suggest that LIMP2 may also be used to prevent and treat colorectal cancer, and it could be used as a target for the development of drugs to combat colorectal cancer.

[0014] Preferably, the reagents for detecting LIMP2 expression include Primer-F and Primer-R as shown in SEQ ID NO:1 and 2.

[0015] Preferably, the product includes a reagent kit.

[0016] Preferably, the reagents for inhibiting LIMP2 include shRNAs as shown in SEQ ID NO:3 and 4.

[0017] A fourth aspect of the present invention provides a kit for the diagnosis and / or prognostic assessment of colorectal cancer, the kit comprising a reagent for detecting LIMP2 expression levels.

[0018] The fifth aspect of the present invention provides a medicament for the prevention and treatment of colorectal cancer, wherein the medicament uses a reagent that inhibits LIMP2 as the main active ingredient.

[0019] Preferably, the drug further includes pharmaceutically acceptable excipients. These excipients may be diluents, binders, lubricants, disintegrants, solubilizers, stabilizers, and some pharmaceutical matrices that are available in the pharmaceutical field; they may also be functional pharmaceutical excipients available in the pharmaceutical field, including surfactants, suspending agents, emulsifiers, and some novel pharmaceutical polymers, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid-polylactic acid copolymer (PLGA), hyaluronic acid, etc.

[0020] Preferably, the dosage form of the drug includes injections, powders for injection, tablets, granules, capsules, pills, sustained-release preparations, and oral liquid preparations.

[0021] The dosage forms mentioned above refer to those commonly used in clinical practice. Drug preparations can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or locally), allowing for flexible administration via various routes such as oral, subcutaneous, intramuscular, and intravenous injection. If certain drugs are unstable under gastric conditions, they can be formulated into enteric-coated tablets.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] This invention reveals that LIMP2 is highly expressed in colorectal cancer patients and lowly expressed in normal tissues. Furthermore, LIMP2 expression is closely related to the prognosis of colorectal cancer, suggesting that LIMP2 not only has the potential to serve as an auxiliary diagnostic biomarker for colorectal cancer but can also be used for prognostic assessment through its expression level. In addition, knocking down LIMP2 expression inhibits the migration and invasion abilities of colorectal cancer, suggesting that LIMP2 may also be a potential target for the prevention and treatment of colorectal cancer. These results demonstrate that LIMP2 can not only be used as a biomarker for the diagnosis and / or prognostic assessment of colorectal cancer but also as a target for the development of drugs to prevent and treat colorectal cancer, possessing significant potential application value and broad application prospects. Attached Figure Description

[0024] Figure 1 The expression of LIMP2 mRNA and protein in colorectal cancer cells;

[0025] Figure 2 This study investigates the expression of LIMP2 in human colorectal cancer tissue microarrays and its relationship with clinical prognosis. Figure A shows the representative field of view of LIMP2 expression in colorectal cancer tissue microarrays via immunohistochemistry; Figure B shows a statistical graph of LIMP2 expression in tumor and non-tumor tissues in tissue microarrays; and Figure C shows the relationship between LIMP2 expression and clinical prognosis.

[0026] Figure 3The results validated the knockdown of LIMP2 in stable colorectal cancer cells; A represents the validation of protein and RNA levels after knockdown in HCT116 cells; B represents the validation of protein and RNA levels after knockdown in RKO cells.

[0027] Figure 4 The effect of LIMP2 knockdown on cell scratch healing is shown in Figure 1. A represents the results of the scratch healing experiment on HCT116 cells after LIMP2 knockdown at 24 and 48 hours; B represents the data quantification graph of HCT116 cell scratch healing at 24 and 48 hours; C represents the results of the scratch healing experiment on RKO cells after LIMP2 knockdown at 24 and 48 hours; and D represents the data quantification graph of RKO cell scratch healing at 24 and 48 hours. All figures were generated using GraphPadprism 8.0 (*p<0.05, **p<0.001, ***p<0.0001, ****p<0.00001).

[0028] Figure 5 The effect of LIMP2 knockdown on cell migration is shown in Figure A. A represents the cell migration results of HCT116 cells after LIMP2 knockdown; B represents the data quantification of HCT116 cell migration after 36 hours; C represents the cell migration results of RKO cells after LIMP2 knockdown; and D represents the data quantification of RKO cell migration after 48 hours. All figures were generated using GraphPadprism 8.0 (*p<0.05, **p<0.001, ***p<0.0001, ****p<0.00001).

[0029] Figure 6 To illustrate the effect of LIMP2 knockdown on cell invasion; where A represents the experimental results of HCT116 cells after LIMP2 knockdown and 48 h of cell invasion; B represents the data quantification graph of HCT116 cells after 48 h of invasion; C represents the experimental results of RKO cells after LIMP2 knockdown and 48 h of cell invasion; D represents the data quantification graph of RKO cells after 48 h of invasion. The graphs were generated using GraphPadprism 8.0 (*p<0.05, **p<0.001, ***p<0.0001, ****p<0.00001).

[0030] Figure 7 The results show the effect of LIMP2 knockdown on a mouse model of colorectal cancer liver metastasis. Figure A shows the method for constructing the nude mouse model of colorectal cancer liver metastasis; Figure B shows the anatomical morphology of the liver tissue after sacrifice in the nude mouse model of colorectal cancer liver metastasis; Figure C shows HE staining of liver tissue sections; Figure D shows the statistical difference analysis of the number of nodules. Statistical analysis was performed using GraphPadprism 8.0, p < 0.00001. Detailed Implementation

[0031] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0032] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0033] Example 1: Correlation Study of LIMP2 with Colorectal Cancer

[0034] 1. Real-time quantitative polymerase chain reaction (RT-PCR) and Western blot assay were used to detect the mRNA and protein levels of LIMP2 (Gene ID: 950) in the colorectal cancer cell line.

[0035] (1) Experimental methods: The mRNA level of LIMP2 in normal human intestinal epithelial cells HIEC6 and colorectal cancer cells SW620, HCT116, RKO, DLD1, HT29, and CACO-2 was detected by RT-PCR, and the protein level of LIMP2 in these cells was detected by Western blot. The specific methods are as follows:

[0036] a. Cell Culture: HIEC6, SW620, HCT116, DLD1, and CACO-2 were cultured in RPMI 1640 medium containing 1% penicillin-streptomycin and 10% fetal bovine serum, while RKO and HT29 were cultured in DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum. All cell lines were placed in a 37°C incubator with 5% CO2. Cell status was observed daily, and cell passages were performed when the cells reached approximately 80% confluence to ensure optimal cell condition.

[0037] b. RT-PCR steps: Using TB Premix Ex Taq TM II (TaKaRa, catalog number RR820A) was used to detect mRNA expression levels. The specific preparation system is as follows:

[0038]

[0039] The reaction program was set as follows: pre-denaturation (95℃, 30s) - denaturation (95℃, 5s) - annealing (65℃, 30s), for a total of 40 cycles. After the reaction was completed, the specificity of the melting curve was determined, and the obtained data were processed according to... ΔΔ The Ct method is used to calculate the relative mRNA expression level of a sample.

[0040] c. Western blot steps:

[0041] ① Collecting cellular proteins: Take cells in the logarithmic growth phase with a cell density of 80-90%, wash the cells twice with PBS, add an appropriate amount of RIPA lysis buffer, lyse on ice for 10 min, scrape off the cells with clean cells, collect the cell-containing lysate and transfer it to a 1.5 mL EP tube. Sonicate at 4℃ for 10 min, centrifuge at 12000 rpm for 10 min, and transfer the supernatant to a new EP tube for later use.

[0042] ② Protein quantification: The protein concentration of each sample was detected using BCA protein quantification reagent. The sample with the lowest concentration was used as the standard, and the concentrations of the remaining samples were adjusted to be consistent. After mixing the protein sample with 5× loading buffer, the sample was heated in a 100℃ metal bath for 5 minutes to denature, and then immediately placed on ice or stored in a -20℃ freezer.

[0043] ③ Gel preparation: Since the molecular weight of the target protein LIMP2 is 75 kDa, a 10% separating gel and a 5% stacking gel are prepared, with the following volumes:

[0044]

[0045] ④ Electrophoresis: Add the pre-stained marker and protein sample (20 μg) sequentially to the SDS-PAGE gel lanes, electrophore at a constant voltage of 80V for 30 min, and then electrophore at 120V for 60-90 min.

[0046] ⑤ Transfer: After completion, gently pry open the glass plate and cut off the top layer of concentrated gel to prepare a 0.2μm PVDF membrane. Activate the membrane by soaking it in methanol for 5 minutes. Using the "sandwich" method, sandwich the gel and PVDF membrane between two sheets of transfer filter paper. Transfer the membrane at a constant voltage of 120V for 1 hour, avoiding overheating. The entire transfer process should be carried out on ice.

[0047] ⑥ Blocking and Antibody Incubation: The membrane was placed on a shaker and washed three times with 0.1% TBST buffer for 10 min each time. 5% skim milk was added for blocking at room temperature for 1 h. The membrane was then washed three times with 0.1% TBST buffer for 10 min each time. Primary antibody (LIMP2 antibody, Abcam, 1:2000) was added and the membrane was incubated overnight at 4°C on a shaker. After primary antibody incubation, the membrane was washed three times with 0.1% TBST for 10 min each time. Secondary antibody (anti-rabbit secondary antibody, Earthox, both at a ratio of 1:5000) was added, and the membrane was incubated on a shaker at room temperature for 1 h. The membrane was then washed three times with 0.1% TBST for 10 min each time.

[0048] ⑦ Development: Prepare the ECL luminescence solution according to the instructions (ECL luminescence solution purchased from Santa / USA), drain the PVDF membrane with absorbent paper, add an appropriate amount of luminescence solution, and collect the protein blot signal using a chemiluminescence analyzer.

[0049] (2) Experimental results: RT-PCR and Western blot results ( Figure 1 The results showed that, compared with normal intestinal epithelial cells HIEC6, LIMP2 had higher mRNA and protein expression levels in colorectal cancer cells SW620, HCT116, RKO, DLD1, HT29, and CACO-2.

[0050] 2. Immunohistochemical detection of LIMP2 protein levels in colorectal cancer tissues

[0051] (1) Experimental Methods: Immunohistochemical staining with LIMP2 was performed on a microarray (human colorectal cancer tissue microarray (Cat#HColA180Su21), purchased from Shanghai Xinchao Biotechnology Co., Ltd.) containing 94 cases of colorectal adenocarcinoma tissue and 86 cases of non-tumor tissue. The staining intensity was then quantified. The specific method is as follows:

[0052] ① Baking the slides: Place the tissue microarray in a 60℃ oven for 2 hours to liquefy the paraffin on the tissue slides;

[0053] ② Dewaxing: The tissue chip was immersed in xylene I for 10 min → xylene II for 10 min → anhydrous ethanol for 3 min → 95% ethanol for 3 min → 85% ethanol for 3 min → 70% ethanol for 3 min → ddH2O for 3 min in sequence.

[0054] ③ Blocking endogenous peroxidase: Prepare a 0.3% H2O2 solution in the dark, soak for 10 min, and then wash with ddH2O for 3 min × 4 times;

[0055] ④ Antigen retrieval: Place the tissue slide in 10mM sodium citrate retrieval solution (pH 6.0), heat on high for 5 minutes and on medium-low for 25 minutes in a microwave oven, then remove and allow to cool naturally at room temperature.

[0056] ⑤ Serum blocking: After absorbing the water around the tissue slice, mark the anti-permeability zone with an immunohistochemical pen (PAP PEN), and add immunohistochemical blocking solution to block for 1 hour.

[0057] ⑥ Incubation of primary and secondary antibodies: Dilute LIMP2 antibody (Proteintech Group, 1:500) with antibody dilution buffer at a ratio of 1:800 and incubate overnight in a humidified chamber at 4°C; the next day, remove the humidified chamber from 4°C and equilibrate at room temperature for 1 hour, then wash in PBS for 5 minutes × 4 times, add DAKO secondary antibody, and incubate at 37°C for 30 minutes.

[0058] ⑦ DAB staining: After removing the tissue slides from the 37℃ incubator, wash them in PBS for 5 min × 4 times. Prepare DAB staining solution at a ratio of 1:50. Start timing after adding the staining solution and observe the staining of the tissue slides under a microscope. When the tissue shows a brown positive color, stop the staining by placing it in ddH2O.

[0059] ⑧ Hematoxylin counterstaining: Wash in ddH2O for 5 min × 4 times, add hematoxylin staining solution to counterstain cell nuclei for 30 s, immediately wash in ddH2O for 5 min × 4 times, then soak in PBS for 5 min to restore color, and rinse with tap water for 3 min; ⑩ Mounting and photography: Place tissue slides in an oven to dry, add mounting medium to mount, and scan with a slide scanner after the tissue slides have dried.

[0060] (2) Experimental results: Immunohistochemical results ( Figure 2 AB) showed that the expression level of LIMP2 in colorectal cancer tissues was significantly higher than that in normal tissues.

[0061] 3. High LIMP2 expression levels are associated with poor prognosis.

[0062] (1) Experimental method: The Kaplan-Meier method was used to plot the survival analysis curve.

[0063] Kaplan-Meier survival curves: The relationship between clinical information and LIMP2 expression levels in 94 colorectal cancer patients (human colorectal cancer tissue microarray (Cat#HColA180Su21, purchased from Shanghai Xinchao Biotechnology Co., Ltd., containing tissue slices and clinical information of 94 colorectal cancer patients) was analyzed using R software. The patients were grouped according to the level of LIMP2 expression (based on the median) and Kaplan-Meier survival curves were plotted.

[0064] (2) Experimental results: Based on the Kaplan-Meier survival curve ( Figure 2 C) It can be seen that the overall survival rate of colorectal cancer patients with high LIMP2 expression is significantly lower than that of the low LIMP2 expression group, indicating that the prognosis of high LIMP2 expression is poor.

[0065] As can be seen from Example 1, LIMP2 is highly expressed in colorectal cancer, and high expression is associated with poor prognosis.

[0066] Example 2: Stable knockdown of LIMP2 by small hairpin RNA (shRNA) can inhibit the malignant manifestations of colorectal cancer.

[0067] 1. Constructing a colorectal cancer cell line with stable LIMP2 knockdown

[0068] (1) Experimental Methods: HCT116 cells and RKO cells were treated with negative control sh-NC (empty vector of plcok.1-copGFP-2A-PURO plasmid) and shRNAs that knocked down LIMP2 (including sh1 and sh2, plcok.1-copGFP-2A-PURO plasmid loaded with shRNA), respectively. Proteins and RNA were extracted from both cell types, and the effect of LIMP2 knockdown was verified by Western blot and RT-PCR experiments. The target sequence of sh1 was 5'-CCTCAATGAGAGTGTTCACAT-3' (SEQ ID NO:3), and the target sequence of sh2 was 5'-CCTCAATGAGAGTGTTCACAT-3' (SEQ ID NO:4). The specific methods are as follows:

[0069] 1) Transformation

[0070] ①Rewarming competent cells: After the competent DH5α cells are taken out of the -80℃ freezer, they are immediately thawed on ice for 10 minutes. They are then gently tapped with a finger to mix (do not blow or suck), and 50 μL is transferred into a clean, enzyme-free EP tube for later use.

[0071] ② Thermal activation: Add 1 μL / tube of the target plasmid to be packaged (sh-NC, sh1 and sh2 plasmids), incubate on ice for 30 min, heat shock at 42℃ for 45 s, and stand on ice for 2 min;

[0072] ③ Shaking: Add 500 μL / tube of SOC medium (antibiotic-free), shake at 220 rpm for 45 min in a shaker at 37°C;

[0073] ④ Plating: Spread 50 μL of bacterial culture onto an LB agar plate containing Amp+ and air dry at room temperature for 10 min. To obtain as many single clones as possible, use the streak plate method to perform four-zone streak (streak 20 μL of bacterial culture in the first zone, then streak the second, third, and fourth zones).

[0074] ⑤ Bacterial culture: Invert the plates and incubate them overnight (12-18h) in a 37℃ incubator;

[0075] ⑥ Picking single clones and shaking: Pick 2 colonies from each dish into 5 mL of LB medium (containing Amp) + In this process, the shaking in a 37℃ shaker at 220 rpm is performed for 16-18 hours.

[0076] ⑦ Plasmid mini-extraction: Take 1 mL / tube of bacterial culture for plasmid mini-extraction (Tiangen endotoxin-free plasmid mini-extraction kit cat.#DP118-02), and store the remaining bacterial culture at 4℃ for later detection;

[0077] ⑧ Enzyme digestion identification: After agarose gel electrophoresis identification, select bacterial cultures for incubation in 200 mL LB medium (containing Amp) + Shake overnight at 37℃ on a shaker at 220 rpm;

[0078] ⑨ Large-scale plasmid extraction: Centrifuge the bacterial culture at 3500g / min for 20min, discard the supernatant, and use the precipitate to extract plasmids using a plasmid extraction kit (Tiangen endotoxin-free plasmid small-scale extraction kit, cat.#DP118-02). Detect the plasmid DNA concentration using Nanodrop and store at -80℃.

[0079] 2) Lentiviral packaging

[0080] Expression plasmids (sh-NC, sh1, and sh2, purchased from Guangzhou Qingke / China), packaging plasmids (psPAX 2, purchased from Guangzhou Qingke / China), and lentiviral capsid plasmids (pMD2.G, purchased from Guangzhou Qingke / China) were simultaneously transfected into 293T cells to obtain lentiviral supernatant. The specific methods are as follows:

[0081] ① 293T cell seeding: Select 293T cells with low passage number (3-5 generations) and good growth status, and seed the cells in 5 × 10⁶ wells of a six-well plate. 5 / hole;

[0082] ② Co-transfection with expression plasmids, packaging plasmids, and lentiviral capsid plasmids:

[0083] Prepare two 1.5mL centrifuge tubes, A and B, and prepare the following system:

[0084]

[0085] Mix systems A and B thoroughly, let stand at room temperature for 15-20 minutes, then evenly add the mixture dropwise to a culture plate containing complete culture medium. Gently shake to mix (given the weak adhesion of 293T cells, this step does not require medium replacement). Return the plate to the cell culture incubator and replace with fresh complete culture medium after 8-12 hours. Collect the virus suspension at 24 hours and 48 hours, centrifuge at 2000 rpm for 10 minutes, collect the cell supernatant, and filter again using a 0.45 μm cell filter. Transfer the filtered virus suspension to a new 50 mL centrifuge tube and store at 4°C (the virus suspension can be stored at 4°C for one week without significant decrease in titer; for long-term storage, freeze at -80°C).

[0086] 3) Viral infection and validation knockdown efficacy

[0087] The packaged viral solution was added to target cells (HCT116 cells and RKO cells) for infection to obtain stably transfected cell lines. The collected lentivirus supernatant was thawed on ice beforehand for cell infection. Cells were seeded into 5 × 10⁶ wells. 5 Add 2 mL of virus solution to each well, along with polybrene diluted 1:1000 (to enhance infection efficiency). Replace with fresh virus solution and infect cells for 24 hours until the cell density reaches 70-80%. Then add a certain concentration of puromycin to screen for successfully infected cells. The commonly used concentration of puromycin is 10 μg / mL, but the specific concentration depends on the cell's tolerance. Finally, perform Western blotting / RT-PCR verification. After successful verification, stable transfected cells can be passaged and cryopreserved at -80°C for six months. For long-term storage, liquid nitrogen is required.

[0088] (2) Experimental results: Western blot and RT-PCR results ( Figure 3 The results showed that, compared with the sh-NC group, the protein and mRNA levels of sh-LIMP2 (sh1 and sh2) in both groups of HCT116 and RKO cells were significantly reduced (p<0.05). These results demonstrate that Example 2 successfully constructed stable LIMP2 knockdown colorectal cancer cell lines HCT116 and RKO.

[0089] 2. Knocking down LIMP2 inhibits the migration and invasion of colorectal cancer cells.

[0090] (1) Experimental methods: The cell scratch healing assay was used to detect the migration ability of LIMP2 knockdown colorectal cancer cells, and the Transwell assay was used to detect the migration and invasion abilities of LIMP2 knockdown colorectal cancer cells. The specific method of the cell scratch healing assay is as follows:

[0091] ① Prepare a cell scratching device. Attach the adhesive side of the four-compartment scratching device firmly to the cell culture plate until it does not fall off when shaken.

[0092] ② Cell plating: Digest cells in the logarithmic growth phase, resuspend the cells in an appropriate amount of culture medium, and count the cells to 3 × 10⁻⁶. 5 Add an appropriate amount of culture medium to prepare a cell suspension with a total volume of 400 μL. Add 100 μL of cell suspension to each cell and place the culture plate in a 5% CO2, 37°C incubator for culture.

[0093] ③ Scratch recording: After all the cells have adhered to the wall, gently remove the scratcher, wash the cells twice with sterile PBS, take pictures and record them under an inverted microscope;

[0094] ④ Healing record: At regular intervals, take pictures using an inverted microscope and record the cell healing process;

[0095] ⑤ Image processing and analysis: ImageJ software was used to calculate the cell scratch area, and GraphPad Prism 8.0 software was used for statistical analysis.

[0096] The specific method for the Transwell cell migration assay is as follows:

[0097] First, remove the Transwell chamber, rinse it with sterile PBS, digest the cells in the logarithmic growth phase, and resuspend the cells in basal medium free of fetal bovine serum. The cell count should be 2 × 10⁶ cells / mL. 5 Cells were evenly seeded per well in the upper layer of the chamber. 200 μL of serum-free culture medium was added to the upper layer, and 700 μL of complete culture medium containing 10% fetal bovine serum and 1% penicillin / streptomycin was added to the lower layer. The culture plate was placed in a 5% CO2 incubator at 37°C and incubated for 24 hours. After 24 hours, the chambers were collected, and the culture medium from both layers was aspirated. The chambers were gently washed with PBS. Cells were fixed with 4% paraformaldehyde for 15 minutes, stained with crystal violet for 10 minutes, and excess staining was rinsed with tap water. Cells inside and outside the chambers were wiped clean with cotton swabs. After drying, the chambers were observed and photographed under an inverted microscope. The number of stained cells was counted using ImageJ software for image analysis, and statistical analysis was performed using GraphPad Prism 8.0 software.

[0098] The specific method for the Transwell cell invasion assay is as follows:

[0099] Melt the matrix gel (Corning, catalog number 356234) at 4°C in advance. Dilute the matrix gel with serum-free basal medium at a ratio of 1:8, mix thoroughly, and place on ice. Remove the Transwell chambers, rinse them with sterile PBS, and add 50 μL of diluted matrix gel to each well. To ensure a smooth and even spread of the matrix gel, place the cell plate stably in a 4°C refrigerator for 1 hour. Place the cell plate containing the chambers in a 37°C cell culture incubator for 1 hour to allow the matrix gel to solidify. Aspirate the supernatant from the chambers, digest the cells in the logarithmic growth phase, and resuspend the cells in serum-free basal medium. Count the cells at 2 × 10⁶ cells / well. 5 Each well contains 1 sample, evenly spread on the upper layer of the chamber. Add 200 μL of preheated (37°C) fetal bovine serum-free medium to the upper layer, and 700 μL of preheated (37°C) complete medium containing 10% fetal bovine serum and 1% penicillin / streptomycin to the lower layer. Subsequent steps are the same as for the migration assay.

[0100] (2) Experimental results: Results of scratch healing experiment ( Figure 4 The results showed that in HCT116 and RKO cells with stable LIMP2 knockdown, compared with sh-NC, the migration and healing abilities of both sh-LIMP2 cell groups were significantly inhibited at both 24h and 48h time points. (Transwell cell migration assay results) Figure 5 The results showed that, compared with sh-NC, the migration ability of sh-LIMP2 cells in both groups was significantly inhibited. Transwell cell invasion assay results ( Figure 6 The results showed that, compared with sh-NC, the invasive ability of both sh-LIMP2 cell groups was significantly inhibited. These results indicate that LIMP2 knockdown can significantly inhibit the migration and invasion of colorectal cancer cells.

[0101] 3. In vivo experiments of a mouse model of colorectal cancer liver metastasis

[0102] (1) Experimental Methods: A mouse model of colorectal cancer liver metastasis was established to investigate the effect of LIMP2 knockdown on colorectal cancer metastasis. Three groups were set up: sh-NC group, sh1 group, and sh2 group (sequences identical to SEQ ID NO: 3 and 4), with five male BALB / c nude mice in each group. The specific methods are as follows:

[0103] 1) Establishment of a liver metastasis model: Male BALB / c nude mice aged 4-6 weeks were purchased from Zhejiang Vital River Laboratory Animal Co., Ltd. All mice were provided with free access to food and water and underwent a 12-hour light / dark cycle under standard pathogen-free conditions. To investigate the effect of LIMP2 deficiency on in vivo metastasis, 1×10⁻⁶ mice were used... 6One HCT116 cell (shRNA-NC or sh-LIMP2) was diluted in 25 μL of sterile PBS and matrix gel (dilution ratio 1:1) and then injected into the spleen of 4-week-old male mice to establish a liver metastasis model.

[0104] 2) Observation of the effect of LIMP2 knockdown on colorectal cancer metastasis: During the tumor formation process, the growth status of mice and tumor growth were observed every 3 days. After 4 weeks, nude mice were euthanized using CO2 to obtain tumor tissue. The intact liver tissue from the dissected mice was used for subsequent paraffin embedding and HE staining. First, the number of tumor nodules on the surface of the liver tissue was counted. One lobe of the liver tissue was fixed in 4% paraformaldehyde and embedded in paraffin. Then, HE staining was performed on the paraffin sections, and the slides were scanned and uploaded using a high-throughput slide scanning image analysis system after being mounted with cedar oil.

[0105] (2) Experimental results: Animal experiment results ( Figure 7 The results showed that, compared with the sh-NC group, the number of liver metastatic tumor nodules in the sh1 and sh2 groups was significantly reduced, and the size of the tumor nodules was also visibly reduced (P<0.05).

[0106] In conclusion, LIMP2 can be used not only as a biomarker for the diagnosis and / or prognostic assessment of colorectal cancer, but also as a target for the development of drugs to prevent and treat colorectal cancer, thus possessing significant potential application value.

[0107] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. Application of reagents for detecting LIMP2 expression levels in the preparation of products for diagnosing colorectal cancer.

2. Application of reagents for detecting LIMP2 expression levels in the preparation of colorectal cancer prognostic assessment products.

3. The application of reagents that inhibit LIMP2 in the preparation of drugs for the prevention and treatment of colorectal cancer, characterized in that, The drug is used to inhibit the migration and invasion of colorectal cancer cells. The reagent that inhibits LIMP2 is shRNA, and the target sequence of the shRNA is SEQ ID NO:

3.

4. The application according to claim 1 or 2, characterized in that, LIMP2 expression levels include mRNA and / or protein expression levels.

5. The application according to claim 1 or 2, characterized in that, The reagents for detecting LIMP2 expression include Primer-F and Primer-R, as shown in SEQ ID NO: 1 and 2.

6. The application according to claim 1 or 2, characterized in that, The product includes a reagent kit.

Images