Application of MUC12 gene in preparation of medicine for reducing rectal cancer liver metastasis

Abstract

The invention discloses application of MUC12 gene in preparation of drugs for reducing rectal cancer liver metastasis, and belongs to the technical field of medical biology. According to the invention, the clear relation between colorectal cancer liver metastasis and fructose metabolism reprogramming is revealed from a molecular mechanism for the first time. The invention provides a brand-new therapeutic target and strategy by determining the brand-new transfer promoting factor MUC12 and the mediated fructose metabolism regulation effect thereof. Different from the traditional therapy only aiming at the strategies of tumor cell proliferation, blood supply and the like, the method disclosed by the invention intervenes aiming at a tumor nutrition metabolic pathway, and can weaken the metabolic adaptation and proliferation capacity of metastatic tumor cells in the liver more specifically. Experimental results show that the functions of targeted inhibition of MUC12 or downstream fructose metabolism key enzymes KHK and ALDOB thereof can significantly reduce the invasion and metastasis ability of tumor cells, and at the same time, normal growth of primary tumors cannot be significantly affected.

Description

Technical Field

[0001] The present invention relates to the field of medical biotechnology, and in particular to application of the MUC12 gene in preparing a drug for reducing liver metastasis of rectal cancer. Background Art

[0002] Colorectal cancer is a malignant tumor with high morbidity and mortality worldwide, with liver metastasis being one of the leading causes of death. Clinical data show that approximately 50% of colorectal cancer patients will develop liver metastases during the course of the disease, and once distant metastases occur, the patient's prognosis significantly deteriorates. Currently, conventional treatments for colorectal cancer liver metastases include surgical resection, systemic chemotherapy, targeted drug therapy, and local treatments such as ablation and embolization. However, these treatments are often difficult to achieve a radical cure for patients with extensive metastasis. Many patients lose the chance of cure due to excessive tumor burden or incomplete resection of metastatic lesions, and the overall 5-year survival rate remains very low, at only around 15%. Even if liver metastases are surgically resected, the postoperative recurrence rate of metastasis is high. Therefore, there is an urgent need to develop new therapeutic strategies and targets to effectively prevent and inhibit the occurrence and development of colorectal cancer liver metastases.

[0003] Colorectal cancer liver metastasis is a complex, multistep biological process involving multiple steps, including the invasion of primary tumor cells, their entry into the bloodstream, and their colonization and proliferation in the liver. Recent studies have revealed that metabolic reprogramming of tumor cells plays a crucial role in cancer metastasis. Tumor cells adapt to the unfavorable microenvironment by enhancing their glucose metabolism through mechanisms such as the "Warburg effect." Increased fructose intake within the liver's unique nutrient-rich microenvironment may provide tumor cells with an additional survival and proliferation advantage. Epidemiological studies have shown that increased intake of a high-fructose diet (e.g., high-fructose corn syrup) is closely associated with an increased incidence of colorectal cancer in young adults. Unlike glucose, fructose's metabolic pathway in the human body is significantly liver-specific. Studies have shown that colorectal cancer liver metastatic cells significantly enhance the expression of key enzymes in the fructose metabolic pathway to provide sufficient energy and building blocks for cellular structure. For example, aldolase B (ALDOB), a key rate-limiting enzyme in hepatic fructose metabolism, is significantly upregulated in colorectal cancer liver metastatic tissue. Experimental studies have demonstrated that inhibiting ALDOB or reducing fructose intake effectively reduces the growth of liver metastatic tumors in animal models. In addition, ketohexokinase (KHK, also known as fructokinase) is the first key enzyme in fructose metabolism, and it has two subtypes: liver-specific KHK-C and widely distributed KHK-A. The latest studies have shown that the expression of the KHK-A subtype in colorectal cancer liver metastasis tissue is significantly increased, promoting tumor cells through epithelial-mesenchymal transition (EMT) and aerobic glycolysis pathways, accelerating the process of liver metastasis. It can be seen that fructose metabolic reprogramming plays an important driving role in the progression of colorectal cancer liver metastasis. However, existing technologies have not yet clarified the specific regulatory mechanism of the fructose metabolic pathway, especially which key molecules regulate and enhance the utilization of fructose by tumor cells.

[0004] At the same time, the role of the mucin (MUC) family in the development and metastasis of gastrointestinal tumors has also garnered increasing attention. Mucins are a class of large glycoproteins located on the cell surface or secreted into mucus, participating in intercellular adhesion, signal transduction, and immune evasion of tumor cells. MUC12 is a transmembrane mucin that is stably expressed in normal colorectal epithelial tissue, but its expression levels vary significantly in colorectal cancer tissue. Previous studies, using gene expression profiling, have shown that MUC12 may be closely associated with the metastasis of colorectal cancer, and its mRNA expression levels are significantly correlated with patient prognosis. Clinical sample testing has revealed that elevated MUC12 expression is detected in over 50% of primary and metastatic colorectal cancer tissues. However, the specific mechanisms by which MUC12 influences the biological behavior of colorectal cancer cells remain unclear, particularly whether MUC12 is involved in reprogramming fructose metabolism and whether it plays a key role in colorectal liver metastasis. Therefore, the existing technology has the following shortcomings: first, there is a lack of in-depth research on the mechanism of fructose metabolism reprogramming during colorectal cancer liver metastasis; second, the specific functions of mucin family members such as MUC12 in the process of colorectal cancer liver metastasis are still unclear; third, effective anti-metastasis treatment methods based on fructose metabolism and MUC12 regulatory pathways have not yet been developed. Summary of the Invention

[0005] The present invention aims to provide the use of the MUC12 gene in the preparation of a drug for reducing liver metastasis from rectal cancer, thereby addressing the aforementioned problems of the prior art. The present invention relates to the role of mucin 12 (MUC12) and fructose metabolizing enzymes (such as ketose kinase KHK and aldolase B ALDOB) in colorectal cancer liver metastasis, as well as the use of these molecular pathways as therapeutic targets.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is the use of the MUC12 gene in the preparation of a drug for reducing liver metastasis of rectal cancer.

[0008] The second technical solution of the present invention is the use of a MUC12 gene inhibitor in the preparation of a drug for reducing liver metastasis of rectal cancer.

[0009] The third technical solution of the present invention is the use of a fructose metabolic pathway inhibitor in the preparation of a drug for reducing liver metastasis of rectal cancer.

[0010] The fourth technical solution of the present invention is the use of a DNMT1 gene overexpression preparation in the preparation of a drug for reducing liver metastasis of rectal cancer, which overexpresses DNMT1, reduces the expression of the MUC12 gene, and reduces the proliferation and invasion ability of rectal cancer cells.

[0011] The fifth technical solution of the present invention is a drug for reducing liver metastasis of rectal cancer, comprising a MUC12 gene inhibitor, a fructose metabolism pathway inhibitor and / or a DNMT1 gene overexpression preparation.

[0012] Based on the above technical solution, the present invention has the following technical effects:

[0013] (1) This study reveals for the first time a clear molecular link between colorectal cancer liver metastasis and fructose metabolic reprogramming. By identifying MUC12 as a novel pro-metastasis factor and its mediated regulation of fructose metabolism, this study provides a novel therapeutic target and strategy.

[0014] (2) Unlike traditional therapies that only target tumor cell proliferation and blood supply, the present invention intervenes in the tumor nutritional metabolic pathway, which can more specifically weaken the metabolic adaptation and proliferation ability of metastatic tumor cells in the liver.

[0015] (3) The experimental results of the present invention demonstrate that targeted inhibition of MUC12 or its downstream key fructose metabolism enzymes, KHK and ALDOB, can significantly reduce the invasive and metastatic capabilities of tumor cells without significantly affecting the normal growth of the primary tumor. This suggests that the therapeutic strategy provided by the present invention may have both good efficacy and safety, avoiding significant damage or side effects to normal tissues.

[0016] (4) In addition, the discovery of the DNMT1-MUC12 axis also provides a new theoretical basis for epigenetic intervention therapy: by regulating the DNA methylation status of the MUC12 gene promoter, the metabolic phenotype of tumor cells can be indirectly controlled. This provides an important scientific basis for the future development of new epigenetic treatment methods.

[0017] In summary, the present invention provides a new theoretical basis and treatment strategy for the prevention and treatment of colorectal cancer liver metastasis from the two perspectives of metabolic reprogramming and epigenetic regulation, and has important clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 Schematic diagram of the molecular mechanism by which the DNMT1–MUC12–KHK / ALDOB signaling pathway regulates colorectal cancer liver metastasis as described in the present invention.

[0020] Figure 2To analyze the expression characteristics of MUC12 in colorectal cancer liver metastasis and its correlation at the clinical and cellular levels.

[0021] Figure 3 To analyze the molecular mechanism by which MUC12 promotes colorectal cancer liver metastasis by regulating fructose metabolism.

[0022] Figure 4 This is the epigenetic regulatory mechanism by which DNMT1 enhances fructose metabolism by regulating MUC12 expression, thereby promoting colorectal cancer liver metastasis.

[0023] Figure 5 Figure 4 shows the construction of a highly metastatic colorectal cancer cell line and its bioluminescence imaging analysis. Figure A shows the construction process of the highly invasive colorectal cancer cell line F3, Figure B shows the in vivo BLI results in mice, Figure C shows the number and volume of liver metastases in the F3 cell line group, Figure D shows H&E staining, Figure E shows the LW / BW ratio (LW / BW is the liver weight to body weight ratio, commonly used to assess liver tumor size or changes in liver size after drug treatment) of the F3 group, Figure F shows IHC (immunohistochemistry) analysis, Figure G shows the Kaplan-Meier survival curve, and Figure H shows the IHC analysis.

[0024] Figure 6 Figure 2. Fructose metabolism reprogramming and metabolic profile analysis in colorectal cancer liver metastasis cell lines. A represents KEGG pathway enrichment analysis, B and C represent Seahorse energy metabolism analysis, and D and E represent the OCR (Oxygen Consumption Rate) of F3 cells after fructose treatment.

[0025] Figure 7 High expression of MUC12 in colorectal cancer tissues is significantly associated with patient prognosis. A is a heatmap of the top 10 differentially expressed genes in transcriptome sequencing of F0 and F3 cells; B and C are RT-qPCR and Western blot results; D is the TCGA pan-cancer data analysis result; E is the bioinformatics analysis result; F is a representative IHC staining image of the TMA; G is the Kaplan-Meier survival analysis result; and H is the chi-square test result.

[0026] Figure 8 This in vivo experiment validates the role of MUC12 in promoting colorectal cancer liver metastasis. Figure A shows the dynamic monitoring of liver tumor burden by BLI after the establishment of the nude mouse spleen injection model; Figure B shows the macroscopic observation of the nude mouse liver at the end of the experiment; Figure C shows the H&E staining of liver tissue; Figure D shows the LW / BW quantification; and Figure E shows the Kaplan-Meier survival curve.

[0027] Figure 9MUC12 enhances fructose metabolism and promotes the proliferation, migration, and invasion of colorectal cancer cells by regulating the transcriptional activity of the fructose metabolizing enzymes KHK and ALDOB. Figures A and B show cell proliferation assays, C shows a Transwell migration and invasion assay, D shows RT-qPCR results, and E shows ChIP-qPCR analysis results. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0034] The embodiment of the present invention provides the use of the MUC12 gene in preparing a drug for reducing liver metastasis of rectal cancer.

[0035] The embodiments of the present invention also provide the use of a MUC12 gene inhibitor in the preparation of a drug for reducing liver metastasis of rectal cancer.

[0036] In some specific embodiments, the MUC12 gene inhibitor comprises at least one of the following:

[0037] siRNA or shRNA targeting MUC12;

[0038] antisense nucleic acid targeting the MUC12 gene;

[0039] Neutralizing antibodies or antigen-binding fragments thereof that can specifically bind to the MUC12 protein and block its function.

[0040] The embodiments of the present invention also provide the use of a fructose metabolic pathway inhibitor in the preparation of a drug for reducing liver metastasis of rectal cancer.

[0041] In some specific embodiments, the fructose metabolism pathway inhibitor is selected from a KHK enzyme inhibitor and / or an ALDOB enzyme inhibitor.

[0042] The present invention also provides an embodiment of the use of a DNMT1 gene overexpression preparation in the preparation of a drug for reducing rectal cancer liver metastasis, overexpressing DNMT1, reducing the expression of the MUC12 gene, and reducing the proliferation and invasion ability of rectal cancer cells.

[0043] An embodiment of the present invention further provides a drug for reducing liver metastasis of rectal cancer, comprising a MUC12 gene inhibitor, a fructose metabolism pathway inhibitor, and / or a DNMT1 gene overexpression preparation.

[0044] In some specific embodiments, the drug further comprises a pharmaceutically acceptable excipient.

[0045] In some specific embodiments, the dosage form of the drug is a liquid dosage form, a solid dosage form or a paste dosage form.

[0046] This invention aims to address the shortcomings of existing technologies and provide a novel strategy for inhibiting colorectal cancer liver metastasis. It specifically reveals and targets the DNMT1-MUC12-fructose metabolic pathway to effectively inhibit the liver metastasis ability of colorectal cancer cells. The technical problem addressed by this invention is to elucidate the molecular regulatory mechanism of fructose metabolic reprogramming during colorectal cancer liver metastasis and, based on this, to provide novel methods and uses for intervening in tumor metastasis.

[0047] To achieve the above-mentioned purpose, the present invention provides the following technical solutions:

[0048] The present study found that the transmembrane mucin MUC12 plays a key regulatory role in the process of colorectal cancer liver metastasis. Specifically, high expression of MUC12 can directly upregulate the gene transcription levels of key enzymes in the fructose metabolism pathway - ketohexokinase (KHK) and aldolase B (ALDOB), significantly enhancing the ability of tumor cells to metabolize and utilize fructose, thereby promoting the colonization and proliferation of colorectal cancer cells in the liver, and ultimately accelerating the occurrence and development of liver metastasis. Conversely, by reducing the expression of MUC12, the expression levels of KHK and ALDOB in tumor cells can be significantly reduced, weakening the metabolic utilization of fructose by tumor cells, thereby effectively inhibiting the proliferation and invasion of tumor cells. The present invention clearly reveals for the first time the important mechanism of the MUC12-KHK / ALDOB axis in colorectal cancer liver metastasis, namely, MUC12 activates the gene expression of key enzymes of fructose metabolism through transcription, allowing tumor cells to obtain metabolic growth advantages in the fructose-rich liver microenvironment, thereby promoting the process of tumor metastasis.

[0049] The present invention further discovered that the expression of MUC12 is finely regulated by epigenetic mechanisms. Specifically, DNA methyltransferase 1 (DNMT1) can regulate the expression level of the MUC12 gene by modifying the DNA methylation of the MUC12 gene promoter region. The present invention's research shows that in colorectal cancer cells and liver metastasis tissues with high liver metastasis ability, the degree of methylation of the MUC12 gene promoter region is significantly reduced, which is highly correlated with the high expression of MUC12; conversely, by increasing DNMT1 activity, the DNA methylation level of the MUC12 promoter region can be significantly enhanced, thereby inhibiting the expression of MUC12. In other words, DNMT1 negatively regulates MUC12 through an epigenetic silencing mechanism. When DNMT1 activity is absent or reduced, the MUC12 gene promoter region is relieved of methylation inhibition, resulting in high expression of MUC12, and thereby triggering enhanced activity of the downstream fructose metabolism pathway. This study systematically elucidates the DNMT1-MUC12-KHK / ALDOB molecular pathway for the first time: DNMT1, as an upstream switch of epigenetic regulation, determines the expression status of MUC12; while MUC12, as an important effector molecule, directly enhances the transcription of KHK and ALDOB, key enzymes in fructose metabolism, thereby driving the occurrence and progression of liver metastasis.

[0050] Based on the above research findings, the present invention provides a method and strategy for effectively inhibiting colorectal cancer liver metastasis, namely, targeted intervention of the aforementioned DNMT1-MUC12-fructose metabolic pathway. Specific intervention measures include, but are not limited to: blocking excessive fructose metabolism by tumor cells by increasing DNMT1 activity or reducing MUC12 expression or function; or directly inhibiting the activity of key enzymes KHK and ALDOB in the fructose metabolic pathway to block the energy supply of tumor cells downstream. For example, MUC12 expression or function can be inhibited through gene silencing, RNA interference, or neutralizing antibodies, restoring KHK and ALDOB expression to normal levels and significantly reducing the ability of tumor cells to form liver metastases in vivo. The technical solutions of the present invention also include the use of the above intervention measures to prepare pharmaceutical compositions for preventing and treating colorectal cancer liver metastasis, or using MUC12 expression or its methylation status for liver metastasis risk assessment or early diagnosis.

[0051] Example 1

[0052] 1 Establishment and identification of a highly invasive liver metastasis cell model

[0053] 1.1 First, human colorectal cancer cell lines with different liver metastasis abilities were constructed to study the relationship between MUC12 and the fructose metabolism pathway. Human colorectal cancer cells (such as HCT116 or SW series cells) were injected into BALB / c nude mice via the spleen to induce liver metastasis (luciferase signal expressed by tumor cells was detected by in vivo bioluminescence imaging technology, and ≥1×10 5 The number of photons / s was used as a criterion for determining tumorigenesis / metastasis formation. Subsequently, cells were isolated from the liver metastases and expanded in vitro. The above in vivo transfer-isolation process was repeated three times to obtain a cell line with high liver metastasis ability (denoted as F3 cells). The original parental cell line was designated as F0 cells.

[0054] Nude mice were randomly divided into two groups, with 6 mice in each group, and were treated as follows:

[0055] Experimental group (F3 cell group): 1×10 6 F3 cells; control group (F0 cell group): 1×10 6 F0 cells.

[0056] 1.2 After cell injection, the mice were continuously housed and observed. Approximately 8 weeks later, the mice were dissected to observe and record the number and size of liver metastases. Further samples were collected for cellular and molecular biology tests, including transcriptome sequencing, real-time quantitative PCR (RT-qPCR), and Western blot analysis of MUC12 expression levels to clarify the relationship between MUC12 and liver metastasis potential.

[0057] Results from mouse experiments showed that after splenic injection, F3 cells formed significantly higher numbers and volumes of metastatic nodules in the mouse liver than F0 cells, clearly demonstrating the significantly enhanced liver metastatic potential of F3 cells. Transcriptome sequencing analysis revealed significant enrichment and upregulation of genes involved in the fructose metabolism pathway in F3 cells. Furthermore, RT-qPCR and Western blot analysis revealed significantly higher expression of MUC12 in F3 cells compared to F0 cells, suggesting that MUC12 may be involved in liver metastasis.

[0058] Effects of 2MUC12 on fructose metabolism and tumor cell invasion ability (in vitro experiment)

[0059] 2.1 To verify the role of MUC12 in fructose metabolism, MUC12 was overexpressed in F0 cells and knocked down in F3 cells, and then analyzed by culturing in high-fructose or high-glucose medium.

[0060] Using lentiviral vector-mediated gene overexpression technology, a cDNA fragment encoding the full-length human MUC12 gene sequence (MUC12 standard transcript NM_001164462.2 on NCBI RefSeq, corresponding to the encoded protein NP_001157934.1) was first cloned into a lentiviral vector (such as pLVX-Puro or pCDH-CMV-MCS-EF1) containing a fluorescent protein (such as GFP or mCherry) reporter gene to construct a MUC12 overexpression lentiviral plasmid. The constructed plasmid was then co-transfected with a packaging plasmid (such as psPAX2 or pMD2.G) into 293T cells to produce lentiviral particles carrying the MUC12 overexpression vector. The viral supernatant was collected and concentrated, and then infected into human colorectal cancer F0 cells. After selection with puromycin, an F0 cell line with stable and high expression of MUC12 (denoted as F0-MUC12 overexpressing cells) was obtained. Cells infected with an empty vector were used as a control (denoted as F0-Control cells).

[0061] Using shRNA-mediated gene silencing technology, a short hairpin RNA (shRNA) oligonucleotide sequence specifically targeting the human MUC12 gene was designed and synthesized and ligated into a lentiviral RNA interference vector (e.g., pLKO.1) to construct a MUC12-shRNA lentiviral plasmid. This plasmid was then co-transfected with a packaging plasmid into 293T cells for lentiviral packaging. The lentiviral supernatant was collected and used to infect F3 cells, which were selected and amplified with puromycin to obtain an F3 cell line with stably silenced MUC12 gene expression (denoted as F3-shMUC12 cells). Cells infected with an empty vector served as a control (denoted as F3-shControl cells).

[0062] 2.2 RT-qPCR and Western blot experiments showed that MUC12 overexpression significantly upregulated the expression of fructose metabolism-related genes, including ALDOB, KHK, and GLUT5, in F0 cells. Conversely, MUC12 knockdown significantly reduced the expression of these genes in F3 cells. This effect was particularly pronounced under fructose culture conditions. Chromatin immunoprecipitation (ChIP) experiments further demonstrated that MUC12 could directly enrich in the promoter regions of the KHK and ALDOB genes and enhance their transcriptional activity.

[0063] Functional experiments, including CCK-8 cell proliferation assays and Transwell migration and invasion assays, demonstrated that MUC12 overexpression significantly promoted cell proliferation, migration, and invasion, while MUC12 knockdown significantly inhibited these abilities. These results suggest that MUC12 enhances the invasive and metastatic potential of tumor cells in a fructose-rich environment by promoting fructose metabolism.

[0064] Regulatory effects of 3DNMT1 on MUC12 and fructose metabolism

[0065] To further explore the role of DNMT1 in this mechanism, researchers used the DNA methylation inhibitor 5-Aza to reduce DNMT1 activity in F0 cells. The results showed increased demethylation of the MUC12 promoter, significantly increased expression of MUC12 and its downstream fructose metabolism enzymes, and enhanced cell proliferation and migration under high-fructose conditions.

[0066] Conversely, overexpression of DNMT1 in F3 cells increased MUC12 promoter methylation, decreased expression of MUC12 and its downstream enzymes, and significantly weakened cell proliferation and invasion. Seahorse energy metabolism analysis further confirmed that DNMT1 significantly alters the ratio of glycolysis to oxidative metabolism in cells by regulating MUC12. These experiments demonstrate that DNMT1-regulated MUC12 expression is a key mechanism determining enhanced fructose metabolism and liver metastasis.

[0067] 4 Animal models verify the role of MUC12-mediated fructose metabolism in liver metastasis

[0068] Using a nude mouse model of liver metastasis via splenic injection, researchers found that stable knockdown of MUC12 in tumor cells significantly reduced the number and volume of liver metastases formed. Conversely, cells overexpressing MUC12 significantly increased the liver metastasis burden in mice. These in vivo experiments demonstrate that MUC12 expression levels determine the metastatic potential of tumor cells in the liver microenvironment, further supporting the role of MUC12 in promoting liver metastasis.

[0069] 4.1 To evaluate the effect of changes in MUC12 expression on the liver metastasis ability of colorectal cancer cells, a nude mouse spleen injection liver metastasis model was established. The specific experimental steps are as follows:

[0070] (1) Animal preparation and cell pretreatment:

[0071] Animal selection: BALB / c-nu female nude mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.), 6 weeks old, weighing 18-20 g. The nude mice were housed in an SPF-grade barrier environment with free access to food and water.

[0072] Cell preparation: Human colorectal cancer cells (such as F3 or MUC12 stable knockdown F3 cells) in the logarithmic growth phase of in vitro culture were obtained, digested with trypsin, and resuspended in PBS to form a single cell suspension. The cell concentration was adjusted to 2.5×10 6 Cells / 50 μL PBS were set aside and placed on ice until use.

[0073] (2) Nude mouse anesthesia and surgical procedures:

[0074] Anesthesia method: nude mice were placed in an anesthesia induction box and inhaled 2% isoflurane gas until they entered an anesthesia state. After anesthesia, they were transferred to a 37°C constant temperature operating table.

[0075] Spleen exposure: After disinfecting the abdomen of the nude mouse with iodine, use sterile surgical scissors to make an incision of about 1 cm along the midline of the abdomen to expose the abdominal cavity. Use a sterile cotton swab to gently push away the tissue around the spleen and gently pull out the spleen.

[0076] Cell injection: Use a 1mL insulin syringe (equipped with a 29G needle) to slowly and evenly inject the prepared cell suspension into the tail end of the spleen. Insert the needle to a depth of approximately 1 / 3-1 / 2 the length of the spleen. Slowly push the cell suspension in, ensuring that the spleen is slightly bulging after the injection. Then, slowly withdraw the needle after approximately 30 seconds to prevent backflow of the fluid.

[0077] (3) Postoperative treatment:

[0078] Hemostasis and suturing: Use a sterile cotton swab to gently press the spleen injection site for about 30 seconds to stop bleeding. After confirming that there is no bleeding, gently return the spleen to the abdominal cavity. Then, use 4-0 absorbable sutures to suture the abdominal wall and skin layer by layer.

[0079] Recovery and Observation: After surgery, nude mice were kept warm on a 37°C thermostatic blanket until they fully recovered. Their weight, diet, and activity were closely monitored for 3 days. Appropriate analgesics (e.g., ibuprofen syrup, 100 mg / kg, administered orally once daily for 3 consecutive days) were administered.

[0080] Dynamic observation of tumor growth:

[0081] Starting from the 7th day after surgery, the liver tumor metastasis was observed weekly using the small animal in vivo imaging system (IVIS), and the body weight and mental state of the nude mice were recorded regularly.

[0082] The experiment was terminated at 8 weeks after surgery, and the nude mice were killed for sample collection.

[0083] (4) Sampling and pathological analysis:

[0084] 4.1 Liver tissue collection: After killing nude mice, the liver was quickly dissected and removed. After washing with PBS, the metastatic nodules on the liver surface were photographed, the number of metastases was counted, and the major and minor diameters of the metastases were measured to calculate the total tumor volume (volume = major diameter × minor diameter 2 × 0.5).

[0085] Tissue fixation: Part of the liver tissue was fixed in 4% paraformaldehyde for 24 hours and then embedded in paraffin.

[0086] HE staining pathological analysis steps:

[0087] Paraffin-embedded tissues were sliced into 5 μm thick sections.

[0088] The sections were dewaxed with xylene and then hydrated with graded ethanol.

[0089] Hematoxylin staining was performed for about 5 minutes, followed by differentiation with 1% hydrochloric acid ethanol for 3-5 seconds, and blueing with tap water for 15 minutes.

[0090] The sections were stained with eosin for 2 minutes, dehydrated with graded ethanol, and transparentized with xylene. The sections were then sealed with neutral resin and the morphology and infiltration of the metastatic tumors were observed under a microscope.

[0091] 4.2 Immunohistochemical analysis (such as Ki67):

[0092] After the sections were dewaxed and hydrated, antigens were retrieved at high temperature (EDTA buffer, pH 8.0).

[0093] 3% H2O2 blocks endogenous peroxidase.

[0094] Block with 10% goat serum at room temperature for 20 minutes.

[0095] Ki67 primary antibody (diluted 1:400) was added and incubated at 4°C overnight.

[0096] The next day, the sections were washed with PBS and incubated with secondary antibodies at 37°C for 30 minutes.

[0097] After DAB color development and hematoxylin counterstaining, the sections were dehydrated with graded ethanol, transparentized with xylene, and sealed with neutral resin. The percentage of positive cells was observed under a microscope.

[0098] 5. The promoting effect of high fructose diet on liver metastasis and the mediating mechanism of MUC12

[0099] In nude mice, a high-fructose diet significantly increased liver metastasis burden and tumor cell proliferation in the nude mouse model. However, this effect was abolished in MUC12-knockdown cells, indicating that the high-fructose diet's promotion of liver metastasis is dependent on MUC12. Biochemical analysis revealed increased glycogen and lipid deposition in liver metastases in the high-fructose group, suggesting that tumor cells utilize excess fructose to support their growth and invasion.

[0100] 6. Applicability of the validation mechanism in human patients using clinical tumor sample analysis

[0101] The present invention also detects primary tumors and liver metastasis tissues of clinical colorectal cancer patients.

[0102] The clinical tissue specimens used in this example were all obtained from the colorectal cancer tissue bank established by the Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. The collection of relevant tissue specimens and the conduct of this study strictly adhered to ethical guidelines and were approved by the Medical Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (Ethics Approval No.: [TJ-IRB2023-XXX]). All patients or their legal guardians signed written informed consent.

[0103] This example included a total of 50 patients with colorectal cancer liver metastases who underwent surgical treatment at Tongji Hospital, affiliated to Tongji Medical College of Huazhong University of Science and Technology, between January 2020 and June 2023. All patients were diagnosed with colorectal adenocarcinoma and confirmed to have liver metastases by preoperative imaging examinations (including but not limited to CT, MRI, or PET-CT) and postoperative pathological examinations.

[0104] The specific inclusion criteria are as follows:

[0105] Age range: 18-75 years, male or female; clinical and postoperative pathological diagnosis of primary colorectal adenocarcinoma with liver metastasis; no prior anti-tumor treatment, including chemotherapy, radiotherapy, targeted therapy, or immunotherapy; good general condition (ECOG score 0-1), with no obvious surgical contraindications; complete clinical pathological data and follow-up data; normal function of important organs such as the heart, liver, and kidneys before surgery; and quality and quantity of tissue samples removed after surgery meeting research requirements.

[0106] The specific exclusion criteria are as follows:

[0107] Patients with a history of familial adenomatous polyposis (FAP), hereditary non-polyposis colorectal cancer (HNPCC), or inflammatory bowel disease (such as Crohn's disease or ulcerative colitis); patients with concurrent or previous diagnosis of other malignant tumors (excluding colorectal liver metastasis); patients with severe infection or systemic metabolic disorders before surgery; patients with distant metastases to other sites besides colorectal cancer (such as lung metastasis, brain metastasis, bone metastasis, etc.); patients with severe immune system defects or autoimmune diseases; pregnant or breastfeeding women; and patients who are unable to cooperate with follow-up or research procedures.

[0108] Sample collection and processing methods:

[0109] During the tumor surgery, the following samples were collected: (1) primary colorectal cancer lesion tissue; (2) corresponding adjacent normal intestinal tissue (normal mucosa at least 5 cm from the tumor edge); and (3) liver metastasis tissue. Each tissue sample was quickly frozen within 30 minutes after ex vivo. The tissue specimen processing steps were as follows: after ex vivo, the tissue was quickly rinsed with normal saline, necrotic tissue and fat tissue were removed, and the tissue was immediately cut into small pieces of approximately 0.5 cm × 0.5 cm × 0.5 cm, placed in clearly labeled cryotubes, and quickly frozen in liquid nitrogen. The pieces were then transferred to a -80°C freezer for long-term storage. In addition, peripheral blood (5-10 ml) was collected from all patients before surgery and collected in EDTA anticoagulant tubes. After centrifugation at 3000 rpm for 10 minutes, the plasma was aliquoted into cryotubes and stored at -80°C for subsequent testing and analysis.

[0110] The entire process of collecting, processing, and preserving the above clinical specimens and patient information was strictly carried out in accordance with the research plan approved by the Ethics Committee of Tongji Hospital to ensure the authenticity, scientific nature, and ethical compliance of the research.

[0111] Results showed that MUC12, KHK, and ALDOB expression levels were significantly higher in liver metastases than in primary lesions, and MUC12 expression was positively correlated with fructose metabolism enzymes. Methylation sequencing further confirmed decreased methylation in the MUC12 promoter region in liver metastases, consistent with elevated MUC12 expression. These clinical data further support the important role of the DNMT1–MUC12–fructose metabolism pathway discovered in this study in human colorectal cancer liver metastasis and suggest that this pathway could be used for patient prognosis assessment and as a target for therapeutic intervention.

[0112] Figure 1 The paper demonstrates the epigenetic regulatory mechanism of DNA methyltransferase 1 (DNMT1) on the expression of the transmembrane mucin MUC12 gene, as well as the transcriptional activation of key fructose metabolism enzymes KHK and ALDOB by MUC12.

[0113] Under normal conditions, DNMT1 epigenetically inhibits MUC12 gene expression by catalyzing DNA methylation in the MUC12 gene promoter region (indicated by the "solid T-shaped arrow" in the figure). When DNMT1 expression or function is reduced, the methylation inhibition of the MUC12 gene promoter region is relieved (indicated by the "dashed T-shaped arrow" in the figure), leading to a significant increase in MUC12 protein expression. Highly expressed MUC12, a transmembrane protein, is not only localized to the cell membrane, but its intracellular domain also enters the nucleus and specifically binds to the promoter regions of the genes for KHK and ALDOB, key enzymes in fructose metabolism, activating their transcriptional expression (indicated by the "solid arrow" in the figure). Increased KHK and ALDOB expression significantly enhances tumor cells' ability to uptake and degrade fructose, generating sufficient energy and metabolic intermediates. This promotes tumor cell proliferation, invasion, and survival in the liver microenvironment and accelerates the development and progression of liver metastases (indicated by the "thick solid arrow" in the figure). The technical intervention strategy of the present invention is to block the key links of the above-mentioned DNMT1-MUC12-KHK / ALDOB signaling pathway (the intervention target is indicated by a "red prohibition symbol" in the schematic diagram), for example, by restoring DNMT1 activity, inhibiting MUC12 expression or function, or directly inhibiting the key enzymes of fructose metabolism, KHK and ALDOB, thereby inhibiting the excessive metabolic utilization of fructose by tumor cells and achieving the purpose of inhibiting the development of liver metastasis.

[0114] Figure 2 We systematically investigated the association between MUC12 expression and colorectal cancer liver metastasis (CRLM). Clinically, immunohistochemistry (IHC) staining was performed in primary tumor tissues from colorectal cancer patients and their paired liver metastases to compare MUC12 expression levels. Univariate and multivariate regression analyses were used to assess its association with CRLM progression. Furthermore, receiver operating characteristic (ROC) curves were used to analyze the potential value of MUC12 as a diagnostic biomarker for CRLM. At the cellular level, Western blot and real-time quantitative PCR (RT-qPCR) were used to examine MUC12 expression in primary CRC cell lines and CRLM metastatic cell lines. The results showed that MUC12 expression was significantly elevated in liver metastases, and its expression level was closely associated with the development and progression of liver metastases, suggesting that MUC12 may serve as a potential molecular marker and targeted intervention candidate for CRLM.

[0115] Figure 3The mechanism of action of MUC12 in driving the progression of colorectal cancer liver metastasis (CRLM) in vitro and in vivo through metabolic reprogramming was comprehensively elucidated. In vitro, the function of MUC12 was analyzed in three dimensions: (1) CCK-8, Transwell, and EMT marker assays were used to evaluate its effects on the proliferation, migration, and epithelial-mesenchymal transition of colorectal cancer cells; (2) ECAR and OCR changes were detected using a Seahorse metabolic analyzer to clarify the regulatory ability of MUC12 on glycolysis and oxidative phosphorylation levels; (3) a high-fructose metabolic environment was simulated to investigate the effects of different fructose concentrations on the malignant phenotype of CRLM cells. In vivo, a mouse CRLM model was constructed to evaluate the regulation of MUC12 on liver metastasis potential, including tumor growth (body weight / liver ratio), tumor proliferation ability (Ki67 immunohistochemical staining), liver tissue glycogen accumulation (PAS-D staining), and lipid deposition (Oil Red O staining). At the same time, combined with high-fructose diet intervention, it was further verified whether MUC12 enhances liver metastasis ability through fructose metabolic reprogramming. The results of the study showed that MUC12 significantly enhances the glucose metabolism ability of CRLM cells by upregulating the expression of key fructose metabolism enzymes KHK and ALDOB, thereby driving their malignant behavior and is a key metabolic regulatory factor mediating the progression of CRLM.

[0116] Figure 4A systematic study revealed the key regulatory role of the DNMT1–MUC12–KHK / ALDOB axis in fructose metabolism reprogramming and liver metastasis in colorectal cancer. First, IHC and WB / RT-qPCR assays revealed a positive correlation between the expression levels of DNMT1, MUC12, KHK, and ALDOB in primary tumors, metastatic tissues, and cell models from CRC patients. Further experiments using MSP-PCR and 5-Aza treatment confirmed that DNMT1 inhibits MUC12 transcription by regulating the DNA methylation status of the MUC12 promoter region. Subsequently, ChIP-qPCR, DNA pull-down, and luciferase reporter assays confirmed that MUC12 directly binds to the KHK and ALDOB promoters, activating their transcriptional activity. Functional restoration experiments systematically evaluated MUC12's regulatory capacity on KHK / ALDOB, fructose metabolism activity (ECAR, OCR), tumor cell proliferation and invasion (CCK-8, Transwell), and expression of EMT markers by constructing multiple expression / interference models. Results showed that downregulation of DNMT1 leads to demethylation of the MUC12 promoter and increased expression. MUC12 enhances fructose metabolism by activating KHK and ALDOB transcription, thereby promoting tumor cell invasion and metastasis, leading to CRLM (colorectal cancer liver metastasis). This study proposes for the first time a closed-loop mechanism of "epigenetic regulation-glycometabolism reprogramming-promoting metastatic phenotype," providing a potential diagnostic marker and epigenetic intervention target for colorectal cancer liver metastasis.

[0117] Construction of a highly metastatic colorectal cancer cell line and its bioluminescence imaging analysis Figure 5. Among them, (A) Schematic diagram shows the construction process of the highly invasive colorectal cancer cell line F3. By performing three rounds of liver metastasis selection on primary Caco-2 cells (F0) in the spleen of mice, the F3 cell line with enhanced liver metastasis ability was successfully established. (B) BLI in mice showed that the metastatic ability of the F3 cell line in the liver was significantly higher than that of the F0 cell line, and the bioluminescence intensity was significantly enhanced. (C) The number and volume of liver metastases in mice in the F3 cell line group were significantly increased by naked eye. (D) Representative H&E staining shows sections of liver tissue and liver metastases in the two groups. The bar graph on the right shows the quantitative data, and the metastatic area is marked with a black line. The scale bars are 500μm and 100μm, respectively. (E) The LW / BW ratio of mice in the F3 group was significantly higher than that in the F0 group. (F) IHC analysis showed that the proportion of Ki67-positive cells in liver metastases in the F3 group was significantly increased, suggesting its stronger proliferation ability. (G) Kaplan-Meier survival curves showed that mice injected with F3 cells had significantly shorter survival than mice injected with F0 cells (Log-rank test, P < 0.01). (H) IHC analysis showed significantly decreased E-cadherin expression in liver metastases in the F3 group, suggesting enhanced EMT activity. Data are expressed as mean ± standard error of the mean (SEM), **P < 0.01, ***P < 0.001.

[0118] Reprogramming of fructose metabolism and analysis of its metabolic characteristics in colorectal cancer liver metastasis cell lines Figure 6 . Among them, (A) KEGG pathway enrichment analysis revealed that fructose and mannose metabolic pathways were significantly enriched in the differentially expressed genes between the highly invasive F3 cell line and the low-invasive F0 cell line, suggesting a potential key role of fructose metabolism in colorectal cancer liver metastasis. (B, C) Seahorse energy metabolism analysis showed that the ECAR of F3 cells increased significantly after treatment with 10mM fructose, indicating enhanced glycolytic capacity, while F0 cells showed no significant changes under the same conditions. (D, E) The OCR of F3 cells also showed certain changes after fructose treatment, further indicating that their mitochondrial respiratory activity was relatively stable and the metabolic reprogramming was more significant. Data are expressed as mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001.

[0119] The high expression of MUC12 in colorectal cancer tissues is significantly correlated with the prognosis of patients. Figure 7(A) A heatmap of the top 10 differentially expressed genes in transcriptome sequencing between F0 and F3 cells shows that MUC12 expression is significantly elevated in F3 cells, which have a strong liver metastatic potential (marked by the yellow box). (B, C) RT-qPCR and Western blot experiments further confirmed that MUC12 expression is significantly higher in F3 cells than in F0 cells. (D) Analysis of TCGA pan-cancer data revealed that MUC12 expression is significantly elevated in various cancers, particularly in digestive system tumors (e.g., gastric adenocarcinoma (STAD), bile duct carcinoma (CHOL), and pancreatic adenocarcinoma (PAAD), marked in red). MUC12 expression is particularly prominent in colorectal cancer (COAD and READ, marked by the yellow box). (E) Further bioinformatics analysis confirmed that MUC12 expression is significantly elevated in colorectal cancer (CRC) tissues (n = 620) compared with normal tissues (n = 789) (Wilcox test, ****P = 8.2e-45). (F) Representative IHC staining images of TMA (upper image, scale bar 200 μm; lower image, scale bar 50 μm) and semiquantitative statistical analysis showed that MUC12 expression levels were significantly higher in colorectal cancer tissues than in adjacent normal tissues (n = 90, ****P < 0.0001). (G) Kaplan-Meier survival analysis indicated that patients with high MUC12 expression had significantly lower overall survival than those with low MUC12 expression (Log-rank test, P < 0.05). (H) Chi-square test further confirmed that the proportion of patients with high MUC12 expression in colorectal cancer tissues was significantly higher than that in adjacent normal tissues (P < 0.001). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0120] In vivo experiments verify the role of MUC12 in promoting liver metastasis of colorectal cancer. Figure 8Among them, (A) After the spleen injection model was established in nude mice, changes in liver tumor burden were dynamically monitored by BLI. The results showed that the luminescence signal intensity in the liver area of mice in the MUC12 knockdown group was significantly lower than that in the control group (the right side shows the quantitative statistical results of the luminescence signal). (B) Macroscopic observation of the liver of nude mice at the end of the experiment showed that a large number of obvious tumor metastatic nodules were distributed on the liver surface of the control group (indicated by red arrows), while only a small number of isolated and smaller nodules were observed in the MUC12 knockdown group. (C) H&E staining of liver tissue showed that the area of metastatic foci formed in the MUC12 knockdown group was significantly smaller than that in the control group (yellow circles mark the metastatic foci area, and the bar graph on the right shows the relative quantitative results of metastatic area; scale bar: 500μm). (D) LW / BW quantification results further confirmed that MUC12 knockdown significantly reduced the liver metastatic burden in mice. (E) Kaplan-Meier survival curve analysis showed that the median survival time of mice in the MUC12 knockdown group was significantly prolonged (Log-rank test, *P<0.05). The above data are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

[0121] MUC12 enhances fructose metabolism by regulating the transcriptional activity of fructose metabolizing enzymes KHK and ALDOB, and promotes the proliferation, migration and invasion of colorectal cancer cells. Figure 9 (A, B) Cell proliferation assays showed that MUC12 overexpression significantly promoted the proliferation of F0 cells in fructose medium, particularly under fructose conditions, while the effect was weaker in glucose medium. Conversely, MUC12 knockdown significantly inhibited the proliferation of F3 cells in fructose medium. (C) Transwell migration and invasion assays showed that MUC12 overexpression significantly enhanced the migration and invasion abilities of F0 cells, particularly under fructose conditions. Conversely, MUC12 knockdown significantly impaired the migration and invasion abilities of F3 cells. Quantitative results are shown in the bar graphs on the right. (D) RT-qPCR results showed that MUC12 overexpression significantly upregulated the expression of ALDOB, KHK, and GLUT5 in F0 cells, particularly under fructose conditions. In F3 cells, the expression of these genes was significantly decreased after MUC12 knockdown, particularly under fructose conditions. (E) ChIP-qPCR analysis showed that MUC12 directly binds to the KHK and ALDOB promoter regions in fructose-treated F3 cells and significantly enhances their transcriptional activity. The enrichment effect is particularly pronounced under fructose culture conditions. Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

[0122] In summary, the clinical research data in this example are highly consistent with the results of the aforementioned cell and animal experiments, further supporting the important role of the DNMT1–MUC12–fructose metabolic pathway discovered by the present invention in human colorectal cancer liver metastasis, and confirming the clinical application potential of this pathway as a target for prognosis assessment and therapeutic intervention in colorectal cancer patients.

[0123] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Application of MUC12 gene in the preparation of drugs for reducing liver metastasis of rectal cancer.

2. Application of MUC12 gene inhibitors in the preparation of drugs for reducing rectal cancer liver metastasis.

3. The use according to claim 2, characterized in that The MUC12 gene inhibitor includes at least one of the following: siRNA or shRNA targeting MUC12; antisense nucleic acid targeting the MUC12 gene; Neutralizing antibodies or antigen-binding fragments thereof that can specifically bind to the MUC12 protein and block its function.

4. Application of fructose metabolism pathway inhibitors in the preparation of drugs for reducing liver metastasis of rectal cancer.

5. The use according to claim 4, characterized in that The fructose metabolism pathway inhibitor is selected from KHK enzyme inhibitors and / or ALDOB enzyme inhibitors.

6. Use of a DNMT1 gene overexpression preparation in the preparation of a drug for reducing rectal cancer liver metastasis, characterized in that: Overexpression of DNMT1 reduces the expression of the MUC12 gene and reduces the proliferation and invasion ability of rectal cancer cells.

7. A drug for reducing liver metastasis of rectal cancer, characterized in that: These include MUC12 gene inhibitors, fructose metabolism pathway inhibitors, and / or DNMT1 gene overexpression preparations.

8. The drug according to claim 7, characterized in that The drug also includes pharmaceutically acceptable excipients.

9. The drug according to claim 8, characterized in that The dosage form of the medicine is a liquid dosage form, a solid dosage form or a paste dosage form.

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