Application of MIGA2 as a target in screening or preparing drugs for colorectal cancer treatment

Abstract

This invention belongs to the fields of biomedicine and molecular biology, specifically relating to the application of MIGA2 as a target in the screening or preparation of drugs for treating colorectal cancer. This invention utilizes large-cohort bioinformatics analysis to determine the correlation between MIGA2 and colorectal cancer progression; analyzes the effects of MIGA2 on the proliferation, migration, and invasion of colorectal cancer cells using in vitro cell models; elucidates the key molecular mechanisms by which MIGA2 exerts its anti-tumor function; and analyzes the effect of MIGA2 on colorectal cancer tumor growth in in vivo mouse models, elucidating the influence of MIGA2-mediated mitochondrial-endoplasmic reticulum contact on colorectal cancer tumor growth. These multi-dimensional analyses demonstrate that MIGA2 can be an effective therapeutic target for colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and molecular biology, specifically relating to the application of MIGA2 as a target in screening or preparing drugs for treating colorectal cancer. Background Technology

[0002] Colorectal cancer, as one of the most common malignant tumors of the digestive tract, has seen its incidence rate rise continuously in recent years, becoming a major public health problem threatening human health. Currently, systemic treatment regimens based on chemotherapy combined with molecularly targeted drugs are widely used in clinical practice for advanced and metastatic colorectal cancer, and have achieved certain therapeutic effects. At the same time, tumor immunotherapy, represented by immune checkpoint inhibitors, has also shown promising application prospects in some patients.

[0003] However, key challenges such as chemotherapy resistance and lack of response to immunotherapy are still common in clinical treatment, resulting in poor treatment efficacy and poor prognosis for a considerable proportion of patients.

[0004] Therefore, exploring novel treatment strategies and key targets to improve the clinical outcomes of patients with refractory colorectal cancer is a core scientific issue that urgently needs to be addressed in this field. Summary of the Invention

[0005] The technical problem this invention aims to solve is to overcome the key challenges of existing clinical treatment regimens for colorectal cancer, such as widespread chemotherapy resistance and lack of response to immunotherapy, leading to poor treatment outcomes and prognoses in a significant proportion of patients. To address these problems, this invention provides an application of MIGA2 as a target in the screening or preparation of drugs for treating colorectal cancer. This invention utilizes large-cohort bioinformatics analysis to analyze the correlation between MIGA2 and colorectal cancer progression; analyzes the effects of MIGA2 on the proliferation, migration, and invasion of colorectal cancer cells using in vitro cell models; elucidates the key molecular mechanisms by which MIGA2 exerts its tumor-suppressive function; and analyzes the effects of MIGA2 on colorectal cancer tumor growth in in vivo mouse models, elucidating the influence of MIGA2-mediated mitochondrial-endoplasmic reticulum contact on colorectal cancer tumor growth. These multi-dimensional analyses demonstrate that MIGA2 can be an effective therapeutic target for colorectal cancer.

[0006] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0007] This invention provides the application of the MIGA2 gene or protein in the preparation of kits for diagnosing colorectal cancer or determining prognosis.

[0008] This invention provides an application of MIGA2 as a target in screening or preparing drugs for treating colorectal cancer.

[0009] This invention provides the application of a substance that enhances MIGA2 expression in the preparation of drugs for treating colorectal cancer.

[0010] In this invention, the substance that enhances MIGA2 expression is a substance that can increase the expression level or activity of the MIGA2 gene and / or protein.

[0011] In this invention, the substance that can enhance MIGA2 expression is a MIGA2 overexpression vector.

[0012] In this invention, the overexpression vector is a lentiviral vector, a plasmid vector, or an adenovirus vector.

[0013] In this invention, the overexpression vector is a plasmid vector.

[0014] This invention provides a method for screening drugs for the prevention and / or treatment of colorectal cancer, comprising the following steps:

[0015] S1, In the test group, the substance to be detected is added to the detection system;

[0016] S2, detect the expression level of MIGA2 gene and / or protein in the detection system of the test group; and compare it with the negative control group;

[0017] S3, if the substance to be detected can promote an increase in the expression level of the MIGA2 gene and / or protein, then the substance to be detected is a drug for the prevention and / or treatment of colorectal cancer.

[0018] In this invention, the detection system is for colorectal cancer cells.

[0019] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0020] The reagents and raw materials used in this invention are all commercially available.

[0021] Compared with existing technologies, the beneficial effects of this invention are: This invention provides the application of MIGA2 as a target in screening or preparing drugs for the treatment of colorectal cancer. This invention uses large-cohort bioinformatics analysis to analyze the correlation between MIGA2 and colorectal cancer progression; analyzes the effects of MIGA2 on the proliferation, migration, and invasion abilities of colorectal cancer cells using in vitro cell models; elucidates the key molecular mechanisms by which MIGA2 exerts its anti-tumor function; analyzes the effects of MIGA2 on colorectal cancer tumor growth in in vivo mouse models; and elucidates the effect of MIGA2-mediated mitochondrial-endoplasmic reticulum contact on colorectal cancer tumor growth, thus verifying from multiple dimensions that MIGA2 can be an effective therapeutic target for colorectal cancer. Attached Figure Description

[0022] Figure 1The expression of MIGA2 in colorectal cancer and its clinical prognostic manifestations are as follows:

[0023] Figure 1 (A) is the result of a systematic analysis of the expression characteristics of key genes mediating multi-organelle interactions in the TCGA database.

[0024] Figure 1 (B) represents the relative mRNA levels of MIGA2, MFN2, VDAC1, PTPIP51, and IP3R3 genes in cancerous tissue and adjacent normal tissue in clinical colorectal cancer samples.

[0025] Figure 1 (C) shows the results of bioinformatics analysis of MIGA2 mRNA expression levels in cancerous tissues and adjacent normal tissues of colorectal cancer in the TCGA database.

[0026] Figure 1 (D) shows the results of bioinformatics analysis of mRNA expression levels in colorectal cancer tissue and adjacent normal tissue using the GEO database.

[0027] Figure 1 (E) shows the experimental results of MIGA2 protein expression levels in 10 pairs of colorectal cancer tissues (N: adjacent normal tissue; T: cancer tissue) randomly selected from Western blots.

[0028] Figure 1 (F) shows the expression of MIGA2 protein in cancerous tissue and adjacent normal tissue in colorectal cancer using three immunohistochemical staining results from the Protein Atlas database.

[0029] Figure 1 (G) represents the bioinformatics analysis of the correlation between MIGA2 protein expression levels and overall survival and disease-free survival prognosis.

[0030] Figure 2 To inhibit the proliferation and growth of colorectal cancer cells by overexpressing MIGA2, the following were observed:

[0031] Figure 2 (A) The expression level of MIGA2 protein in different cell lines (Lovo, HT29, HCT116, HCT8, SW480, SW620, DLD1, RKO) was detected by Western blotting.

[0032] Figure 2 (B) shows the experimental results of Western blot analysis of the effect of Dox-inducible MIGA2 protein expression in DLD1 cells.

[0033] Figure 2(C) is a plate colony formation assay to detect the effect of Dox-induced MIGA2 expression on the colony formation ability of DLD1 cells.

[0034] Figure 2 (D) is for Figure 2 (C) Quantitative statistical analysis of the number of clones formed.

[0035] Figure 2 (E) is a schematic diagram of a subcutaneous xenograft experiment in nude mice.

[0036] Figure 2 (F) shows the inhibitory effect of MIGA2 overexpression on tumor growth in tumors obtained from in vivo tumorigenesis experiments.

[0037] Figure 2 (G) is for Figure 2 Quantitative statistics on the dynamic changes in tumor volume in each group in (F).

[0038] Figure 2 (H) is for Figure 2 (F) At the end of the experiment, the tumor tissue collected was weighed and the differences in tumor weight among the groups were statistically analyzed.

[0039] Figure 3 Overexpression of MIGA2 was used to inhibit the migration and invasion of DLD1 cells. Specifically:

[0040] Figure 3 (A) To detect the effect of MIGA2 on the migration and invasion ability of DLD1 cells using a Transwell assay.

[0041] Figure 3 (B) is for Figure 3 (A) Quantitative statistical results of the number of migrating and invading cells.

[0042] Figure 4 To enhance the inhibitory effect of endoplasmic reticulum-mitochondrial contact on DLD1 cell colony formation and in vivo tumorigenesis, the following was observed:

[0043] Figure 4 (A) is the result of Western blot detection of protein overexpression of MIGA2.

[0044] Figure 4 (B) For transmission electron microscopy observation and quantitative analysis, when DLD1 cells were induced to express MIGA2 under specific conditions, the number of endoplasmic reticulum-mitochondrial contact structures (ERMCSs) increased significantly.

[0045] Figure 4 (C) is a schematic diagram of the structure of the artificially constructed mitochondrial-endoplasmic reticulum connecting protein Tether.

[0046] Figure 4(D) shows the results of a protein imprinting assay of DLD1 cells induced to express Tether, which showed that the cells expressed mitochondrial-endoplasmic reticulum connecting proteins.

[0047] Figure 4 (E) is a transmission electron microscopy image of DLD1 cells induced to express Tether, showing a significant increase in ERMCSs (red arrows indicate increased ERMCSs).

[0048] Figure 4 (F) is for Figure 4 (E) Quantitative statistical analysis of ERMCSs in Figure 1.

[0049] Figure 4 (G) shows that Tether overexpression significantly inhibits DLD1 cell colony formation in a plate colony formation assay.

[0050] Figure 4 (H) represents the statistical results of the number of clones formed in (G) graph.

[0051] Figure 4 (I) Experiments on subcutaneous xenografts in nude mice showed that inducing Tether overexpression can inhibit tumor growth.

[0052] Figure 4 (J) is a statistical analysis of the dynamic changes in tumor volume in Figure (I).

[0053] Figure 4 (K) represents the statistical comparison of tumor weight in each group at the end of the experiment.

[0054] Figure 4 (L) shows the changes in body weight of nude mice during tumor formation.

[0055] Figure 5 Experimental results show that the tumor-suppressive function of MIGA2 depends on its structural function in mediating endoplasmic reticulum-mitochondrial contact, including:

[0056] Figure 5 (A) is a transmission electron microscope image of DLD1 cells expressing Vector.

[0057] Figure 5 (B) is a transmission electron microscope image of DLD1 cells expressing MIGA2.

[0058] Figure 5 (C) is for expressing MIGA2 FM Transmission electron micrograph of DLD1 cells.

[0059] Figure 5 (D) is for Figure 5 (A) Figure 5 (B) and Figure 5Quantitative statistical results of the number of ERMCSs in (C).

[0060] Figure 5 (E) refers to V5 and MIGA2 FM Results of plate colony formation experiment using DLD1 cells of type V5.

[0061] Figure 5 (F) refers to V5 and MIGA2 FM Experimental results of MIGA2 protein expression in DLD1 cells of type V5.

[0062] Figure 5 (G) is for Figure 5 The statistical analysis results of (F).

[0063] Figure 5 (H) shows the tumor volume experimental results of the nude mouse subcutaneous xenograft model.

[0064] Figure 5 (I) shows the final tumor weight experiment results of the nude mouse subcutaneous xenograft tumor model. Detailed Implementation

[0065] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0066] The main reagents and instruments used in this invention include: protein concentration determination using the BCA protein quantification kit (catalog number P0012) from Beyotime Biotechnology Co., Ltd.; PVDF membranes required for protein immunoblotting experiments purchased from Bio-Rad; protein electrophoresis, membrane transfer, and chemiluminescence signal acquisition using a protein electrophoresis apparatus, membrane transfer apparatus, and chemiluminescence imaging system manufactured by Bio-Rad, respectively; and cell ultrastructure observation and fluorescence imaging performed using a Zeiss laser confocal microscope (model LSM800) and a cryo-transmission electron microscope system. The above equipment and reagents provided reliable assurance for the smooth implementation of the experiments and the accurate acquisition of data.

[0067] (1) Western blotting: After receiving the cell lysate, the protein was mixed with SDS loading buffer and separated by electrophoresis. The protein was then transferred to the membrane using a wet transfer method. Subsequently, the membrane was blocked with 5% skim milk for 1 hour and washed three times with 1×TBST solution for 15 minutes each time. Next, it was incubated overnight with primary antibody at 4°C and washed three more times with 1×TBST for 15 minutes each time. Afterward, it was incubated with secondary antibody at room temperature for 1 hour, and the above washing steps were repeated. Finally, a chemiluminescence detection system was used for signal acquisition and analysis.

[0068] (2) Transmission electron microscopy: After the cell samples were fixed with glutaraldehyde, they were prepared, embedded and sectioned at the Cryo-Electron Microscopy Center of Zhejiang University, and then photographed using a cryo-electron microscope.

[0069] (3) Plate colony formation experiment: In the plate colony formation experiment, we seeded different stable cell lines at low density (500 cells / plate) in culture dishes containing complete culture medium and cultured them at 37℃ and 5% CO2 for 14 days. The medium was changed every 3 days until visible colonies were formed. Then, the cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. The number of colonies with more than 50 cells was counted and the colony formation rate was calculated. Each experiment was repeated three times. The data were expressed as mean ± standard deviation and statistically analyzed.

[0070] (4) Transwell assay: In the Transwell assay, we used 24-well plates with matching chambers for detection. First, the cells of each group were resuspended in serum-free medium and seeded in the upper chamber (for migration assay, the cells were seeded directly; for invasion assay, Matrigel was pre-laid on the matrix gel in the chamber to simulate the basement membrane). Complete medium containing 10% fetal bovine serum was added to the lower chamber as a chemokine. After the cells were cultured at 37°C and 5% CO2 for 48 hours, the cells that had not migrated / invaded in the upper chamber were wiped away with a cotton swab. The cells that had crossed the membrane were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Multiple fields of view were randomly selected under an inverted microscope for counting. Each experiment was repeated three times independently. Finally, the number of cells that migrated / invaded was calculated and statistically analyzed.

[0071] (5) Tumor formation experiment in nude mice: In the tumor formation experiment in nude mice, we selected 4-6 week old female BALB / c nude mice and injected subcutaneously into the right axilla with cell suspensions of the experimental group and the control group in the logarithmic growth phase (5×10^6 cells / 100μL PBS per mouse). We observed and measured the tumor formation periodically. When the tumor volume reached about 1500 mm, 3 Alternatively, when the experiment reaches the preset endpoint, nude mice are euthanized and the tumor is completely dissected, weighed, and photographed. Some tumor tissue is fixed with 4% paraformaldehyde and then embedded in paraffin, sectioned, and subsequently stained with hematoxylin and eosin (HE) or immunohistochemically analyzed. Each group of experiments contains at least 5 nude mice. Data are expressed as mean ± standard deviation and are statistically analyzed.

[0072] (6) RT-PCR experiment: Take 20-30 mg of tumor tissue and grind it rapidly in a pre-cooled mortar with liquid nitrogen, or add it directly to a pre-cooled homogenizing tube containing lysis buffer and homogenize on ice until completely broken. Immediately transfer the lysate and strictly follow the instructions of the RNA extraction kit. Key steps include: chloroform phase separation, alcohol precipitation, column membrane binding and washing, and adding DNase I at room temperature for 15 minutes during the column purification stage to eliminate genomic DNA contamination. After eluting RNA with 30-50 μL of RNase-free water, take 1 μL to determine the concentration and A260 / A280 ratio (acceptable range 1.8-2.0), and take about 500 ng of RNA for 1% agarose gel electrophoresis to verify integrity—high-quality RNA should show clear 28S / 18S rRNA bands with a brightness ratio of about 2:1. After qualified RNA is aliquoted, store at -80℃ or use directly for reverse transcription.

[0073] Reverse transcription was performed in a 20 μL system: 500 ng–1 μg RNA template, 4 μL 5× buffer, 1 μL dNTP mixture, 1 μL random primer and Oligo(dT) primer mixture, 0.5 μL RNase inhibitor, and 1 μL reverse transcriptase were added, and the volume was brought to a final volume with RNase-free water. The reaction program was: annealing at 25°C for 5–10 minutes, extension at 50°C for 30–60 minutes, and inactivation at 85°C for 5 minutes. The resulting cDNA could be used immediately for qPCR or stored at -20°C.

[0074] qPCR reaction systems were prepared on ice, with ≥3 replicates for each sample containing both the target gene and the internal control gene. A 20 μL system contained: 10 μL 2× premix, 0.4 μL each of forward and reverse primers, 2 μL of cDNA template diluted 5–10 times, and RNase-free water to make up the difference. The reaction program employed a three-step method: 95℃ pre-denaturation for 30 seconds; 40 cycles (95℃ denaturation for 5 seconds, 60℃ annealing for 30 seconds, and 72℃ extension with fluorescence signal acquisition for 30 seconds); SYBR Green dye was used. After completion, melting curve analysis was performed (slowly increasing from 60℃ to 95℃, continuously acquiring signals) to confirm the specificity of the amplified products.

[0075] Example 1

[0076] This invention first utilizes the TCGA database, including 457 cases of TCGA colorectal adenocarcinoma (TCGA-COAD) and 51 paired adjacent normal tissue samples, to systematically analyze the expression characteristics of key genes mediating multi-organelle interactions (such as MIGA2, MFN2, and VPS13D).

[0077] The results of a systematic analysis of the expression characteristics of key genes mediating multi-organelle interactions in the TCGA database are as follows: Figure 1As shown in (A), the TCGA data analysis results show that the expression of MFN2, MIGA2, and VPS13D genes all showed a downregulated trend in colorectal cancer tissues. Specifically, the MIGA2 gene, which is involved in mitochondrial-endoplasmic reticulum contact, and genes that widely regulate the interaction between mitochondria and endoplasmic reticulum, lipid droplets, lysosomes, and other organelles, were all expressed at low levels in colorectal cancer tissues, suggesting that the imbalance of multi-organelle interaction may be closely related to the progression of colorectal cancer.

[0078] Furthermore, bioinformatics analysis was performed on colorectal cancer data from the TCGA and GEO databases. The TCGA-COAD database contained 457 cases of colorectal adenocarcinoma and 51 paired adjacent normal tissue samples. GSE39582 contained 566 colorectal cancer tumor samples.

[0079] Bioinformatics analysis of MIGA2 expression levels in colorectal cancer tissues and adjacent normal tissues in the TCGA database yielded the following results: Figure 1 As shown in (C), the T-test value (T-test) is less than 2.2e-16; the results of bioinformatics analysis of mRNA expression levels in colorectal cancer tissues and adjacent normal tissues using the GEO database are as follows: Figure 1 As shown in (D), the T-test value is 1.1e-9.

[0080] according to Figure 1 (C) and Figure 1 (D) indicates that MIGA2 mRNA expression is significantly reduced in colorectal cancer tissues.

[0081] Therefore, the above analysis results indicate that MIGA2 may be associated with colorectal cancer.

[0082] Example 2

[0083] Study on the expression of MIGA2 gene in colorectal cancer samples

[0084] This embodiment aims to verify the expression of the MIGA2 gene in colorectal cancer samples. Specifically, to further clarify the key role of MIGA2 in the occurrence and development of colorectal cancer, this invention collected tumor tissues and adjacent normal tissues from colorectal cancer patients and detected the transcriptional level of the key gene MIGA2. The specific implementation steps are as follows:

[0085] 1) Sample collection: With the consent of the patients and the approval of the ethics committee of Sir Run Run Shaw Hospital affiliated to Zhejiang University, we applied to the tissue sample bank for tumor tissue and adjacent tissue of 40 patients with colorectal cancer.

[0086] 2) Protein extraction from tissue: Quickly remove approximately 20-50 mg of the target tissue block from a -80°C freezer and place it in a pre-chilled sterile culture dish on ice. Rinse briefly with pre-chilled PBS to remove surface impurities. Then, transfer the tissue to a pre-chilled mortar containing liquid nitrogen and rapidly grind it into a fine powder using a grinding stick. Just before the powder is completely thawed, transfer it entirely to a centrifuge tube containing pre-prepared lysis buffer. Add the lysis buffer at a ratio of approximately 1:10 (weight / volume), which already contains protease and phosphatase inhibitors. Place the centrifuge tube on ice for continuous lysis for 30 minutes, vortexing every 10 minutes to promote lysis. After lysis, briefly place the sample in an ice-water bath. Intermittent sonication was performed to reduce viscosity. Next, the sample was centrifuged at 14,000 × g for 15 minutes at 4°C, and the supernatant was carefully transferred to a new centrifuge tube; this was the total protein lysis buffer. The protein concentration in the supernatant was determined using the BCA method, and based on the results, all sample concentrations were standardized to a uniform concentration using the lysis buffer. An appropriate amount of the standardized protein solution was taken, and 5× loading buffer containing a reducing agent was added. After mixing, the mixture was denatured in a 95°C metal bath for 5 minutes. Finally, the denatured protein samples were aliquoted and immediately stored at -80°C for subsequent proteomics analyses such as Western blotting or mass spectrometry. The MIGA2 protein level was detected using the Western blotting experiment described above.

[0087] The results of RT-PCR experiments on the relative mRNA levels of MIGA2, MFN2, VDAC1, PTPIP51, and IP3R3 genes in colorectal cancer tumor tissues and adjacent normal tissues are as follows: Figure 1 As shown in (B). The results showed that the mRNA expression levels of core genes mediating mitochondrial-endoplasmic reticulum contact (e.g., MIGA2, MFN2, VDAC1, PTPIP51, IP3R3) were significantly lower in tumor tissues than in adjacent normal tissues, further confirming the close association between abnormal organelle interactions and colorectal cancer progression. Notably, among the differentially expressed genes, MIGA2 showed the most significant downregulation, which is the focus of this invention.

[0088] In addition, this invention selected 40 pairs of clinical tissue samples of colorectal cancer, and randomly selected 10 pairs of clinical tissue samples of colorectal cancer for Western blot analysis. The experimental results of Western blot analysis of the MIGA2 protein expression level in the 10 pairs of colorectal cancer tissues (N: adjacent normal tissue; T: cancer tissue) are as follows: Figure 1 As shown in (E).

[0089] The experimental results showed that the expression level of MIGA2 protein in cancerous tissue was significantly lower than that in adjacent normal tissue.

[0090] Furthermore, the expression of MIGA2 protein in colorectal cancer tissue and adjacent normal tissue was analyzed using three staining results from the Protein Atlas database. The results are as follows: Figure 1 As shown in (F).

[0091] Experimental results showed that MIGA2 was characterized by low expression in colorectal cancer.

[0092] The above multi-level experimental results jointly confirm that MIGA2 is significantly underexpressed in colorectal cancer tumor tissues.

[0093] Furthermore, the survival prognosis of colorectal cancer patients was analyzed. The specific implementation steps are as follows:

[0094] The bioinformatics workflow for prognostic survival analysis of colorectal cancer patients begins with a clear research objective: exploring the prognostic value of MIGA2. Subsequently, transcriptome expression matrices and accompanying clinical survival data of colorectal cancer patients are obtained from public databases such as TCGA and GEO. The data preprocessing stage is crucial, requiring sample cleaning and matching, standardization of survival time and status formats, and risk stratification of patients based on median expression values ​​or subsequent model results. Core analyses include: for single genes, survival curves are plotted using the Kaplan-Meier method, and inter-group differences are assessed using the Log-rank test, supplemented by univariate Cox regression to quantify hazard ratios.

[0095] To investigate the correlation between MIGA2 protein expression levels and overall survival and disease-free survival using bioinformatics analysis, this invention analyzes survival prognostic data of colorectal cancer patients obtained from public databases such as TCGA and GEO. The study examines the relationship between the number of individuals at risk (high vs. low expression) with MIGA2 protein expression at a specific time point who have not yet experienced death or disease and are still under investigation, and their survival time (in months). The results are as follows: Figure 1 As shown in (G).

[0096] The results showed that colorectal cancer patients with high MIGA2 expression had a better prognosis.

[0097] The findings of the above studies suggest that MIGA2 may play a tumor-suppressive role in colorectal cancer.

[0098] Example 3

[0099] Studies on the inhibition of colorectal cancer cell proliferation and tumor growth by overexpression of MIGA2

[0100] To elucidate the specific function of MIGA2 in the progression of colorectal cancer, this invention used Western blotting experiments to detect the expression level of MIGA2 protein in different cell lines (e.g., Lovo, HT29, HCT116, HCT8, SW480, SW620, DLD1, and RKO). The experimental results are as follows: Figure 2 As shown in (A).

[0101] The results showed that there were significant differences in the expression level of MIGA2 protein; MIGA2 was relatively highly expressed in HT29 and RKO cells, while the expression of MIGA2 was significantly low in DLD1, SW480 and SW620 cells.

[0102] Based on previous findings that MIGA2 is generally downregulated in colorectal cancer tissues, this invention proposes the hypothesis that MIGA2 may play a tumor-suppressive role.

[0103] Furthermore, to verify this hypothesis, this invention selected DLD1 cells with low endogenous MIGA2 expression levels and constructed a stable MIGA2-overexpressing cell line based on the Tet-on system using doxycycline (Dox)-induced expression. The specific implementation steps are as follows:

[0104] Lentiviral packaging production (PEI transfection method in 12-well plates)

[0105] The goal of this phase is to produce lentiviruses carrying the MIGA2 gene in HEK293T cells.

[0106] The night before the experiment, HEK293T cells were loaded at 2.0 × 10⁶ cells per well. 5 Cells were seeded at a density of 70-80% in 12-well plates and cultured overnight in complete culture medium to achieve a cell density of 70-80% at transfection.

[0107] On the day of transfection, prepare the DNA-PEI complex according to the following ratio per well: Mix 1.0 μg of Tet-pLenti-BSD-MIGA2-V5 plasmid, 0.75 μg of psPAX2 packaging plasmid, and 0.25 μg of pMD2.G envelope plasmid in 50 μL of Opti-MEM; in another tube, mix 6.0 μL of PEI MAX (1 mg / mL) with 50 μL of Opti-MEM, let stand at room temperature for 5 minutes, then mix the two and vortex, incubating at room temperature for 15-20 minutes to form the transfection complex. Discard the old cell culture medium, add 500 μL of fresh, antibiotic-free culture medium to each well, and then add 100 μL of the complex dropwise, mixing gently. Replace with 1 mL of fresh complete culture medium 6-8 hours after transfection. Continue culturing until 48 and 72 hours post-transfection, then collect the cell supernatant, combine them, centrifuge at 2000×g and filter through a 0.45μm filter membrane. The resulting clear virus stock solution can be used immediately or aliquoted and frozen at -80℃.

[0108] DLD1 cell infection: This stage aims to stably integrate the viral genome into DLD1 cells.

[0109] First, a preliminary experiment is needed to determine the minimum lethal concentration (e.g., 4 μg / mL) of DLD1 cells against the screening antibiotic blastomycin. In the formal experiment, DLD1 cells are cultured at 8 × 10⁸ cells per well. 4 Cells were seeded in 12-well plates and cultured overnight. The next day, 1 mL of infection mixture was prepared in each well, containing 500 μL of viral stock solution and 500 mL of culture medium, and incubated for 24 hours. 48 hours after infection, cells were digested and passaged into new plates at a 1:3 ratio, and continuous selection was initiated using culture medium containing BSD selection concentration. The selection medium was changed every 2-3 days. After about 5-7 days, all uninfected control cells died, and the resistant cell clones that formed and proliferated in the experimental wells were the initially successfully constructed stable DLD1-overexpressing MIGA2 cell lines, i.e., MIGA2 overexpressing cell lines or MIGA2 overexpressing groups. DLD1 cells transfected with Tet-pLenti-BSD-V5 plasmid (blank control) were Vector cell lines or Vector groups.

[0110] The core of this stage is to verify whether MIGA2 expression is strictly regulated by Dox and to screen for polyclonal cell lines.

[0111] First, the obtained stable DLD1 cells were cultured in standard medium without Dox and BSD for at least 2 days to fully "shut down" the system and deplete basal expression. Induction validation was then performed: cells were seeded in equal volumes in 12-well plates, one group using standard medium (-Dox) and the other group using medium containing 1 μg / mL Dox (+Dox). Cells were collected after 24-48 hours of treatment.

[0112] This invention uses Western blotting to detect the expression effect of MIGA2 protein in Dox-inducible DLD1 cells, and the experimental results are shown in 2(B).

[0113] The experimental results showed that the -Dox group (without Dox) had no or only very weak bands, while the +Dox group (with 1 μg / mL Dox) showed significant MIGA2-V5 protein bands.

[0114] Furthermore, this invention uses a plate colony formation assay to detect the effect of Dox-induced MIGA2 expression in DLD1 cells on the colony-forming ability of DLD1 cells. The experimental results are as follows: Figure 2 As shown in (C). For Figure 2 (C) Quantitative statistical analysis of the relative colony number was performed, and the results are as follows: Figure 2 As shown in (D).

[0115] Figure 2 (C) and Figure 2 (D) The results showed that under the Dox-induced overexpression of MIGA2, the clonogenic ability of DLD1 cells was significantly inhibited, suggesting that overexpression of MIGA2 can effectively inhibit the proliferation of colorectal cancer cells.

[0116] Example 4

[0117] This invention further validates in vivo function through subcutaneous xenograft experiments in nude mice. Stable cell lines from each group were seeded subcutaneously into nude mice, and Dox was continuously administered via drinking water to induce overexpression of MIGA2.

[0118] The specific implementation steps are as follows:

[0119] 1) Preparation and grouping of laboratory animals

[0120] Prepare 5-week-old female BALB / c nude mice and acclimatize them for one week. Set up four groups: ①MIGA2+Dox group, ②Vector+Dox group, ③MIGA2-Dox group, ④Vector-Dox group, with at least 6 mice in each group.

[0121] 2) Pretreatment before cell seeding

[0122] All stable cells were cultured in Dox-free medium for 3–5 days before the experiment to shut down basal expression. Cells in the logarithmic growth phase were collected, resuspended in PBS, counted, and the concentration adjusted to 5 × 10⁻⁶ cells / day. 6 100 μL of cells per 100 μL, keep on ice for later use.

[0123] 3) Subcutaneous tumor inoculation

[0124] 100 μL of cell suspension was drawn using a 1 mL syringe and slowly injected subcutaneously to form a wheal. All groups were inoculated on the same side of the body (e.g., left side), and the inoculation information for each animal was recorded.

[0125] 4) Dox induction scheme activated

[0126] Starting on the day of inoculation, the MIGA2+Dox and Vector+Dox groups were given drinking water containing 1 mg / mL Dox. The MIGA2-Dox group and the parental cell group were given plain sterile water. All Dox-containing drinking water solutions were replaced with fresh solutions every 2-3 days.

[0127] 5) Tumor growth monitoring

[0128] Starting on day 7 post-inoculation, the long diameter (L) and short diameter (W) of the tumor are measured every 2-3 days. The formula is TV = (L × W). 2 Calculate the tumor volume using 1 / 2. Record and plot the tumor growth curves for each group.

[0129] 6) Sample collection at the end of the experiment

[0130] When the tumor volume reaches 1500 mm 3 The tumor may reach its endpoint when ulceration and necrosis occur. Nude mice are euthanized, and the tumor tissue is completely removed.

[0131] After weighing and photographing, the tissue was divided: some was frozen at -80℃ (for protein / RNA analysis), and some was fixed with 4% paraformaldehyde (for histological analysis).

[0132] 7) Sample Analysis and Validation

[0133] Western blotting was used to detect MIGA2-V5 protein expression in frozen tumor tissues to verify in vivo induction efficiency. Fixed tissues were paraffin-embedded, sectioned, and stained with H&E to assess tumor pathological characteristics and perform statistical analysis. A schematic diagram of the subcutaneous xenograft experiment in nude mice is shown below. Figure 2 As shown in (E). In vivo tumorigenesis experiments demonstrated the inhibitory effect of MIGA2 overexpression on tumor growth, as shown in... Figure 2 As shown in (F); for Figure 2 The quantitative statistical results of the dynamic changes in tumor volume in each group in (F) are as follows: Figure 2As shown in (G); for Figure 2 (F) The tumor tissue collected at the end of the experiment was weighed, and the results of the statistical analysis of the differences in tumor weight among the groups are shown below. Figure 2 As shown in (H).

[0134] The experimental results showed that the growth of xenografts in the MIGA2 overexpression group was significantly inhibited, thus the above experiments confirmed at the in vivo level that MIGA2 has the function of inhibiting tumor growth in the progression of colorectal cancer.

[0135] Example 5

[0136] To comprehensively evaluate the impact of MIGA2 on the malignant phenotype of colorectal cancer cells, this invention further investigated cell migration and invasion capabilities.

[0137] This invention uses Transwell assays to detect the effect of MIGA2 on the migration and invasion abilities of DLD1 cells (Vector group and MIGA2 overexpression group). The Vector group consists of DLD1 cell lines transfected with a blank control plasmid, and the MIGA2 overexpression group consists of the above-mentioned stable DLD1 cell lines overexpressing MIGA2.

[0138] The experimental results are as follows Figure 3 As shown in (A); for Figure 3 Quantitative statistical results of the number of migrating and invading cells in (A) are as follows: Figure 3 As shown in (B).

[0139] Experimental results showed that overexpression of MIGA2 significantly inhibited the migration and invasion ability of DLD1 cells.

[0140] Example 6

[0141] To investigate the potential mechanism by which MIGA2 exerts its anti-cancer function, this invention further examines its effect on organelle contact.

[0142] The specific implementation steps are as follows:

[0143] Phase 1: Sample Pretreatment and Fixation

[0144] 1) In-situ rapid fixation

[0145] Immediately remove the cell culture medium and add pre-cooled 2.5% glutaraldehyde fixative (prepared with 0.1 M phosphate buffer, pH 7.4), ensuring the liquid completely covers the cell layer. Place the culture dish in an ice bath for 0.5 hours to achieve in-situ stabilization of the ultrastructure; this step is crucial for maintaining the natural morphology of membrane contact sites.

[0146] Rinsing and Collection

[0147] 2) After fixation, scrape the adherent cells off the wall with a cell scraper, collect them in a centrifuge tube, and centrifuge at low speed to form cell clumps.

[0148] Phase Two: Post-fixation, Dehydration, and Embedding

[0149] Post-fixation with osmium tetroxide

[0150] 3) Carefully aspirate the supernatant, wash three times with ddH2O for one minute each time, then add 1% osmium tetroxide solution and perform post-fixation at 4°C in the dark for 1-2 hours. This step can specifically bind to the biomembrane and significantly enhance the electronic contrast of the membrane structure.

[0151] Gradient dehydration

[0152] 4) Gradual dehydration was performed using 50%, 70%, 90% and 100% ethanol, with each treatment lasting 15 minutes. The 100% ethanol treatment was repeated twice to ensure that the water in the cells was completely replaced.

[0153] Resin infiltration and polymerization

[0154] 5) After passing through propylene oxide, the sample was gradually immersed in a mixture of propylene oxide and epoxy resin (in ratios of 1:1 and 1:3) for infiltration, and finally transferred to pure epoxy resin for overnight infiltration. The sample was then oriented and embedded in a mold and placed in a 60°C oven for polymerization for 48 hours to form a cuttable, hard resin block.

[0155] Phase 3: Ultrathin Sectioning and Staining

[0156] 6) Preparation of ultrathin sections

[0157] The embedding blocks were carefully trimmed on an ultramicrotome to expose the target cell region. Ultrathin sections with a thickness of 60-80 nm were cut using a diamond scalpel and then retrieved onto a copper grid covered with a Formvar support membrane using a metal ring.

[0158] 7) Double compound staining

[0159] First, stain the sections with a saturated uranyl ethanol acetate solution for 15-30 minutes. After rinsing thoroughly with double-distilled water, counterstain with lead citrate solution for 5-10 minutes to further enhance the electronic contrast of cell membranes and organelles.

[0160] Phase Four: Electron Microscopy Observation and Data Analysis

[0161] 8) Transmission electron microscopy observation and image acquisition

[0162] The prepared copper mesh was loaded into the sample holder and inserted into a transmission electron microscope. At an accelerating voltage of 80 kV, regions rich in cytoplasm were first located in low-magnification mode, then switched to high-magnification mode (≥30,000x recommended) to systematically scan and locate tight contact sites between mitochondria and endoplasmic reticulum. Special attention was paid to characteristic regions where the distance between the two membrane structures remained within the 10-30 nm range and they were arranged in a parallel and closely packed manner, and high-resolution digital images were acquired.

[0163] 9) Structural Measurement and Statistical Analysis

[0164] The professional image analysis software ImageJ was used to perform quantitative analysis on the acquired images: the minimum distance between mitochondria and endoplasmic reticulum membranes was accurately measured; the frequency of contact sites per unit area or specific mitochondrial perimeter was statistically analyzed; and descriptive statistics and inter-group comparisons were performed on the morphological characteristics of the contact interface (such as length and continuity).

[0165] This invention utilizes Western blotting to detect proteins in DLD1 cells that promote contact between mitochondria and endoplasmic reticulum. The experimental results are as follows: Figure 4 As shown in (A); and through transmission electron microscopy observation and quantitative analysis, the analysis results are as follows: Figure 4 As shown in (B).

[0166] according to Figure 4 (B) It can be seen that the length of mitochondria in contact with the endoplasmic reticulum (ERMCSs / Mito Perimeter) is increasing, indicating that when DLD1 cells are induced to express MIGA2 under specific conditions, the number of ERMCSs is significantly increased, and overexpression of MIGA2 can significantly promote the contact between mitochondria and the endoplasmic reticulum.

[0167] Furthermore, to clarify whether this structural change is the direct cause of MIGA2 inhibiting DLD1 cell proliferation, this invention constructed an artificial connective protein, Tether, which can specifically pull mitochondria into contact with the endoplasmic reticulum.

[0168] The specific implementation steps are as follows:

[0169] Part 1: Molecular Cloning and Construction of the Tet-pLenti-BSD-Tether-V5 Expression Plasmid

[0170] Step 1.1: Design and synthesize the Tether gene fragment

[0171] Sequence Design: The DNA sequence encoding the Tether fusion protein was designed, with the following structure: N-terminal mitochondrial targeting sequence: The mitochondrial targeting sequence of human AKAP1 protein, namely MAIQFRSLFLPALPGMALLGWWWFFSRKK, was used. Flexible linker peptide: The α-helical linker sequence AEAAAAKEEAAKEAAAAKA was used to ensure spatial freedom of the domain. Endoplasmic reticulum binding domain: The core region of the FFAT motif of human MIGA2 protein (corresponding to amino acids 292-298) and the 15 amino acids before and after it, totaling 37 amino acids, were extracted. This region can specifically bind to the endoplasmic reticulum membrane protein VAPB. C-terminal V5 tag: The short peptide DYKDDDDK was introduced for subsequent immunoassay.

[0172] DNA synthesis: Gene synthesis was performed using the complete sequence (AKAP1-Linker-FFAT-V5), and restriction enzyme sites (such as NheI and BstBI) compatible with the multiple cloning site of the Tet-pLenti-BSD-V5 vector were added to both ends.

[0173] Step 1.2: Vector linearization and fragment cloning

[0174] The empty vector plasmid Tet-pLenti-BSD-V5 was double-digested with restriction endonucleases NheI and BstBI to generate a linearized vector fragment, which was then purified and recovered by agarose gel electrophoresis. The synthesized Tether gene fragment was double-digested with the same restriction endonucleases to obtain an insert fragment with sticky ends. The linearized vector and the insert fragment were ligated overnight at 16°C using T4 DNA ligase.

[0175] Step 1.3: Transformation, Screening, and Plasmid Validation

[0176] The ligation product was transformed into chemically competent *E. coli* (e.g., DH5α), plated on LB agar plates containing ampicillin, and incubated overnight at 37°C. Single colonies were picked for small-scale culture, plasmids were extracted, and initial screening was performed by restriction enzyme digestion and PCR. Sanger sequencing was performed on the positive clones to verify the correctness of the Tether fusion gene sequence and the accuracy of the reading frame, ensuring the absence of frameshift mutations. The validated plasmid was named Tet-pLenti-BSD-Tether-V5.

[0177] Step 1.4: Large-scale preparation of plasmids

[0178] High-purity plasmids were extracted from large-scale bacterial cultures of positive clones using an endotoxin-free plasmid extraction kit for subsequent cell transfection.

[0179] Part Two: Validating the Function of Tether in Cells

[0180] Step 2.1: Cell transfection and selection of stable transfected cells

[0181] HEK293T cells were co-transfected with the Tet-pLenti-BSD-Tether-V5 plasmid and lentiviral packaging plasmids (psPAX2 and pMD2.G) to produce lentivirus (see the aforementioned virus packaging procedure for specific steps). Viral supernatant was collected to infect target cells (e.g., DLD1), and 1 µg / mL Dox was added to the culture medium after infection to initiate transcription of the Tet-pLenti vector. Forty-eight hours after infection, blastcin was added for selection (concentration needs to be determined experimentally, e.g., 5-10 µg / mL), and selection continued for 7-10 days to obtain a stable transfection pool.

[0182] Step 2.2: Validation of Induced Expression

[0183] Stable cell lines were cultured in Dox-free medium for at least 5 days to shut down basal expression. Two groups were set up: -Dox and +Dox (1 µg / mL, 24–48 hours), and cells were collected. Tether protein expression was detected by Western blotting using the V5 antibody to verify whether it was strictly induced by Dox.

[0184] Step 2.3: Ultrastructure verification (transmission electron microscopy)

[0185] +Dox-induced stable cells and control cells were subjected to routine glutaraldehyde-osmium tetroxide double fixation. Ultrathin sections were prepared, stained with lead and uranium, and observed using transmission electron microscopy. Quantitative analysis: The number of contact sites and the contact membrane length between mitochondria and endoplasmic reticulum at distances of 10-30 nm were counted and compared. The contact frequency and contact area in the Tether expression group were significantly higher than those in the control group.

[0186] This plasmid design includes the mitochondrial targeting sequences AKAP1 and MIGA2, and the binding motif FFAT of the endoplasmic reticulum localizing protein VAPB. A schematic diagram of the artificially constructed mitochondrial-endoplasmic reticulum connective protein (Tether-V5) is shown below. Figure 4 As shown in (C).

[0187] This invention utilizes Western blotting to detect the effects of induced Tether expression in DLD1 cells (+), and the experimental results are as follows: Figure 4 As shown in (D), the experimental results show that the DLD1 cells induced to express Tether constructed in this invention can express mitochondrial-endoplasmic reticulum connexin (Tether-V5).

[0188] This invention analyzes DLD1 cells induced to express Tether using transmission electron microscopy, and the transmission electron microscopy images are shown below. Figure 4As shown in (E), DLD1 cells transfected with a blank plasmid (Vector group) and DLD1 cells induced to express Tether (Tether-V5 group) were included. The quantitative statistical analysis results of the length of mitochondria in contact with the endoplasmic reticulum (ERMCS / Mito Perimeter) measured by transmission electron microscopy are as follows: Figure 4 As shown in (F).

[0189] The results showed that, compared with the Vector group, the endoplasmic reticulum-mitochondrial contact structure was significantly increased in DLD1 cells induced to express Tether.

[0190] Furthermore, this invention investigated the colony-forming ability of DLD1 cells transfected with a blank plasmid (Vector group) and DLD1 cells induced to express Tether (Tether-V5 OE group). Images of the colony-forming ability of DLD1 cells in culture medium are shown below. Figure 4 As shown in (G); for Figure 4 (G) Statistical analysis results of the number of clones formed are as follows Figure 4 As shown in (H).

[0191] Experimental results showed that, compared with the Vector group, Tether overexpression could significantly inhibit the clonogenic ability of DLD1 cells.

[0192] Furthermore, this invention investigated the ability of DLD1 cells transfected with blank plasmids (Vector group) and DLD1 cells induced to express Tether (Tether-V5 OE group) to inhibit tumor growth in a nude mouse subcutaneous xenograft model, and performed statistical analysis.

[0193] Among them, nude mouse xenograft experiments, such as Figure 4 As shown in (I), the statistical analysis results of the dynamic changes in tumor volume in Figure 4(I) are as follows: Figure 4 (J) shows the results; the statistical comparison of tumor weight in each group at the end of the experiment is shown in the figure. Figure 4 (K) shows the changes in body weight of nude mice during tumor formation. Figure 4 As shown in (L).

[0194] Experimental results showed that tumor growth could be significantly inhibited in a nude mouse subcutaneous xenograft tumor model.

[0195] The above results confirm that enhancing the contact between the endoplasmic reticulum and mitochondria may be a key mechanism for inhibiting DLD1 cell proliferation and tumor growth.

[0196] Example 7

[0197] MIGA2's tumor suppression depends on its mediating of ERMCS formation.

[0198] To further verify whether the tumor-suppressive function of MIGA2 depends on its structural function in mediating endoplasmic reticulum-mitochondrial contact, this invention expresses FFAT motif mutant MIGA2 (MIGA2) in DLD1 cells. FM The study aimed to disrupt the ability of mitochondria to mediate contact between the endoplasmic reticulum and systematically examine its effects on the formation of ERMCSs and cell proliferation.

[0199] This invention expresses Vector, MIGA2 and MIGA2 respectively. FM Transmission electron microscopy (TEM) images of DLD1 cells were taken, and the corresponding electron microscopy images are as follows: Figure 5 (A) Figure 5 (B) and Figure 5 As shown in (C). For Figure 5 (A) Figure 5 (B) and Figure 5 The quantitative statistical results of the number of ERMCSs in (C) are as follows: Figure 5 As shown in (D).

[0200] Experimental results showed that overexpression of wild-type MIGA2 significantly increased the number of ERMCSs, while MIGA2 FM Then it loses the ability to mediate the formation of ERMCSs.

[0201] This invention relates to V5 and MIGA2 FM The results of the plate colony formation assay of DLD1 cells from -V5 are as follows: Figure 5 As shown in (E); further experiments were conducted on its protein expression, and the results are as follows. Figure 5 As shown in (F), the statistical analysis results are as follows. Figure 5 As shown in (G).

[0202] Figure 5 (E) Figure 5 (F) and Figure 5 The comprehensive experimental results of (G) show that the FFAT mutant completely loses the inhibitory activity of wild-type overexpression of MIGA2 on the proliferation of DLD1 cells.

[0203] Furthermore, this invention further confirms, through in vivo experiments using a nude mouse subcutaneous xenograft tumor model, that the measured tumor volume is as follows... Figure 5 As shown in (H), the tumor weight is as follows: Figure 5 As shown in (I).

[0204] Figure 5 (H) and Figure 5 (I) Experimental results show that: MIGA2 FM Mutants cannot effectively inhibit tumor growth.

[0205] In summary, the antitumor effect of MIGA2 in colorectal cancer depends on its ability to mediate endoplasmic reticulum-mitochondrial contact, suggesting that this organelle interaction structure plays a key role in the tumor-suppressive function of MIGA2.

Claims

1. Use of a substance for increasing MIGA2 expression in the manufacture of a medicament for treating colorectal cancer, wherein the substance for increasing MIGA2 expression is a substance capable of increasing the expression level of MIGA2 gene and / or protein, i.e. a MIGA2 overexpression vector.

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