Use of an inhibitor of the CCDC154 gene in the preparation of a medicament for treating colorectal cancer or improving prognosis

Abstract

The present invention belongs to the field of biopharmaceutical technology, and particularly relates to the application of an inhibitor of the CCDC154 gene in the preparation of a drug for treating colorectal cancer or improving the prognosis of colorectal cancer. The present invention discovers in the puncture or surgical resection specimens of human colorectal cancer patients that the overall survival period of patients with low CCDC154 expression is significantly longer than that of patients with high CCDC154 expression, indicating that CCDC154 can be used as a biomarker for predicting the prognosis of colorectal cancer patients. A kit including an inhibitor of the CCDC154 gene can be prepared for detecting colorectal cancer or monitoring the prognosis of colorectal cancer, providing a new method and tool for the individualized evaluation of the prognosis of colorectal cancer and the formulation of treatment strategies.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to the use of an inhibitor of the CCDC154 gene in the preparation of a drug for treating colorectal cancer or improving the prognosis of colorectal cancer. Background Art

[0002] Colorectal cancer (CRC) is one of the most common malignant gastrointestinal tumors and remains the second leading cause of cancer-related deaths globally, with an estimated 1.4 million new cases and 693,900 related deaths in 2020. The combined treatment method of surgical resection and adjuvant chemotherapy has become the standard treatment method and has achieved significant improvement in the overall prognosis of colorectal cancer. However, a series of epigenetic and metabolic changes endow colorectal cancer cells with high migratory and invasive abilities, resulting in a 5-year survival rate of approximately 12% for metastatic colorectal cancer. For decades, the American Joint Committee on Cancer / International Union Against Cancer Tumor-Node-Metastasis (TNM) staging system has been the gold standard for assessing the prognosis of colorectal cancer. However, many patients with the same stage have different clinical prognoses, and even the prognosis of stage IIb tumors is often worse than that of stage IIIa tumors, indicating that the TNM staging system has limitations in predicting the prognosis of colorectal cancer. Therefore, there is an urgent need for a method that combines the TNM staging system and other prognostic stratification parameters to guide decision-making and reveal individualized treatment strategies. In the past few years, more and more studies have shown that colorectal cancer prognosis prediction based on molecular markers is a promising field. By analyzing a large number of clinical samples, researchers have discovered many biomarkers related to the prognosis of colorectal cancer, and these biomarkers can be used as auxiliary tools for prognosis assessment. These biomarkers cover multiple aspects, such as gene expression, protein expression, DNA methylation, etc. Among them, certain biomarkers have been proven to be closely related to important prognostic indicators such as the survival rate, recurrence risk, and treatment response of patients.

[0003] However, in current studies, no single biomarker has been found that can comprehensively and accurately predict the prognosis of colorectal cancer. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide the use of an inhibitor of the CCDC154 gene in the preparation of a drug for treating colorectal cancer or improving the prognosis of colorectal cancer, so as to solve the limitations of existing means for treating colorectal cancer and predicting the prognosis of colorectal cancer.

[0005] To achieve the above purpose, the solutions adopted by the present invention are as follows:

[0006] In the first aspect, the present invention provides the use of an inhibitor of the CCDC154 gene in the preparation of a drug for treating colorectal cancer or improving the prognosis of colorectal cancer.

[0007] Preferably, the inhibitors of the CCDC154 gene include:

[0008] (1) The mRNA and recombinant protein of CCDC154, and / or

[0009] (2) Any one of siRNA, miRNA, IncRNA, gRNA and antisense nucleotides that can specifically inhibit the expression of the CCDC154 gene;

[0010] And in vivo or in vitro delivery vectors for delivering the above (1) and / or (2).

[0011] Preferably, the inhibitors of the CCDC154 gene can inhibit the proliferation of tumor cells in the HCT116 colorectal cancer cell line.

[0012] Preferably, the inhibitors of the CCDC154 gene can inhibit the cloning of tumor cells in the HCT116 colorectal cancer cell line.

[0013] Preferably, the inhibitors of the CCDC154 gene can inhibit the tumorigenicity of tumor cells in nude mouse colorectal cancer.

[0014] In a second aspect, the present invention also provides a kit for detecting colorectal cancer or monitoring the prognosis of colorectal cancer, comprising the inhibitors of the CCDC154 gene as described above.

[0015] In a third aspect, the present invention also provides a pharmaceutical composition for treating colorectal cancer or improving the prognosis of colorectal cancer, the pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of an active ingredient, the active ingredient comprising the inhibitors of the CCDC154 gene as described above.

[0016] Preferably, the dosage form of the pharmaceutical composition is selected from any one of tablets, powders, injections, capsules, suspensions, pastes, gels, coatings, medicated films, sustained-release agents and microspheres.

[0017] Preferably, the inhibitors of the CCDC154 gene include:

[0018] (1) The mRNA and recombinant protein of CCDC154, and / or

[0019] (2) Any one of siRNA, miRNA, IncRNA, gRNA and antisense nucleotides that can specifically inhibit the expression of the CCDC154 gene;

[0020] And in vivo or in vitro delivery vectors for delivering the above (1) and / or (2).

[0021] The present invention provides a new prognostic prediction marker CCDC154 for colorectal cancer and its related kit. CCDC154 is a protein containing a coiled-coil domain (CCDC154, Coiled-Coil Domain Containing 154), which is widely expressed in human and mouse tissues and is mainly localized in endosomes at the subcellular level. Currently, it has been reported that CCDC154 is related to the pathogenesis of osteopetrosis and is a novel osteopetrosis-related gene involved in cell cycle regulation and cell growth inhibition. Overexpression of CCDC154 inhibits the proliferation activity and soft agar colony formation ability of HEK293 cells by inducing G2 / M cell cycle arrest, without affecting the apoptosis ability. CCDC154 has also been reported to be used as a target for treating liver cancer. For example, in Chinese Patent Document CN110863047A, it was found that after downregulating the expression of human CCDC154 gene by RNAi method, the proliferation of liver cancer cells can be effectively inhibited and cell apoptosis can be promoted, and the growth process of liver cancer can be effectively controlled.

[0022] In the present invention, through careful screening and analysis of a large number of clinical samples, it is found that CCDC154 is related to the prognosis of colorectal cancer patients. The examples of the present invention show that by detecting the expression level of CCDC154, the prognostic risk and disease progression of patients can be more accurately evaluated. The background technology part of the present invention summarizes colorectal cancer as one of the global causes of death, the limitations of the TNM staging system, and the forefront research on the prognostic prediction of colorectal cancer based on molecular markers. The present invention provides a new marker CCDC154 and its related kit, providing a new method and tool for the individualized assessment of colorectal cancer prognosis and the formulation of treatment strategies. Brief Description of the Drawings

[0023] Figure 1 In the examples of the present invention, using the TCGA-COADREAD dataset, it was screened and found that CCDC154 was significantly highly expressed in colorectal cancer (CRC); in the figure, A is the volcano plot of differential genes between tumor tissues and normal tissues, B is the Venn diagram of the intersection of differential genes and CCDC family members, and C is the high expression of CCDC154 in colorectal cancer tissues.

[0024] Figure 2 In the examples of the present invention, after transfecting to inhibit CCDC154 in the HCT116 colorectal cancer cell line, the cell viability of colorectal cancer cells was detected by MTT method at 24, 48, 72, 96, and 120 hours after transfection, and it was found that knocking out CCDC154 inhibited the proliferation of tumor cells.

[0025] Figure 3In the embodiment of the present invention, after knocking out CCDC154 in the HCT116 cell line, the colony formation ability of the tumor cell line was detected using a colony formation assay, and it was found that knocking out CCDC154 reduced the colonies of tumor cells.

[0026] Figure 4 This is the nude mouse tumorigenesis experiment involved in the embodiment of the present invention. After subcutaneous transplantation of tumors in nude mice, it was found that both the tumor volume and body weight of the mice decreased significantly after knocking out CCDC154; in the figure, A is the result of photographing subcutaneous transplanted tumors, B is the statistical result of the tumor volume of the mice, and C is the statistical result of the body weight of the mice.

[0027] Figure 5 This is the immunohistochemical staining result of CCDC154 in adjacent cancerous tissues and colorectal cancer tissues involved in the embodiment of the present invention.

[0028] Figure 6 This is the survival curve of the high-expression group and low-expression group of CCDC154 involved in the embodiment of the present invention.

[0029] Figure 7 This is the receiver operating characteristic curve of CCDC154 based on overall survival involved in the embodiment of the present invention.

[0030] Figure 8 This is the multivariate COX regression forest plot of CCDC154 and clinicopathological parameters involved in the embodiment of the present invention. Detailed implementation manners

[0031] The present invention provides an application of an inhibitor of the CCDC154 gene in the preparation of a drug for treating colorectal cancer or improving the prognosis of colorectal cancer.

[0032] The embodiment of the present invention found that in the puncture or surgical resection specimens of human colorectal cancer patients, the overall survival of patients with high expression of CCDC154 was significantly shorter than that of patients with low expression of CCDC154, indicating that CCDC154 can be used as a biomarker for predicting the prognosis of colorectal cancer patients, and a kit including an inhibitor of the CCDC154 gene can be prepared for detecting colorectal cancer or monitoring the prognosis of colorectal cancer.

[0033] In the present invention, the inhibitor of the CCDC154 gene includes:

[0034] (1) mRNA and recombinant protein of CCDC154, and / or

[0035] (2) Any one of siRNA, miRNA, IncRNA, gRNA, and antisense nucleotides that can specifically inhibit the expression of the CCDC154 gene;

[0036] and an in vivo or in vitro delivery vehicle for delivering the above (1) and / or (2).

[0037] As a specific embodiment of the present invention, the in vivo or in vitro delivery vehicle of the present invention can be lipid nanoparticles. The "lipid nanoparticles" referred to in the present invention refer to particles having at least one nanoscale dimension (such as 1-1000 nm). Lipid nanoparticles can be included in a pharmaceutical composition for delivering an active agent or therapeutic agent (which is an inhibitor of the CCDC154 gene in the present invention) to a target site (such as cells, tissues (such as diseased tissues like tumor tissues), organs). In some embodiments, the lipid nanoparticles of the present invention contain nucleic acids. Such lipid nanoparticles generally contain one or more helper lipid molecules, one or more cholesterol or cholesterol derivatives, and / or one or more polymer-conjugated lipid molecules. The helper lipid molecules can be one or more neutral lipid molecules. The active agent or therapeutic agent (which is an inhibitor of the CCDC154 gene in the present invention) is encapsulated in the lipid portion of the lipid nanoparticles or in an aqueous space enclosed by some or all of the lipid portions of the lipid nanoparticles, thereby protecting it from enzymatic degradation or not causing other undesirable effects induced by the mechanisms of the host organism or cells, such as adverse immune responses.

[0038] As is well known in the art, the average diameter of lipid nanoparticles can be about 30 nm to about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm or about 150 nm, and the lipid nanoparticles are substantially non-toxic.

[0039] The "gene" referred to in the present invention refers to a nucleic acid (such as DNA or RNA) sequence that contains a partial length or the entire length of the coding sequence necessary to produce a polypeptide or precursor polypeptide.

[0040] In addition, the inhibitor of the CCDC154 gene of the present invention can also be used as a component of an active ingredient for preparing a pharmaceutical composition for treating colorectal cancer or predicting the prognosis of colorectal cancer.

[0041] Generally speaking, the pharmaceutical composition of the present invention can be administered in an effective amount by any acceptable administration method for other similar uses. For example, the pharmaceutical composition of the present invention can be administered orally, parenterally, transdermally, topically, rectally or intranasally.

[0042] When used as a medicine, the present invention is usually administered in the form of a pharmaceutical composition. These compositions can be prepared by methods well-known in the pharmaceutical field. These compositions contain at least one active compound, which in the present invention is an inhibitor of the CCDC154 gene. When formulating the compositions provided by the present invention, the active ingredient is usually mixed with a pharmaceutically acceptable excipient or carrier, diluted by a pharmaceutically acceptable excipient or carrier, or encapsulated in a capsule, sachet, paper or other form of container. When the pharmaceutically acceptable excipient or carrier is used as a diluent, it can be a solid, semi-solid or liquid substance, which can be a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, capsules, elixirs, suspensions, emulsions, solutions, syrups, sprays (as solids or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injection solutions, and sterile packaged powders.

[0043] Some typical pharmaceutically acceptable excipients or carriers include lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup and methylcellulose. Additionally, lubricants (such as talc, magnesium stearate and mineral oil), wetting agents, emulsifying and suspending agents, preservatives (such as methyl paraben and propyl paraben), sweeteners and flavoring agents can also be included. The pharmaceutical composition of the present invention can achieve rapid, sustained or delayed release of the drug active ingredient after administration to a patient through specific dosage forms, which is also a widely used method in this field.

[0044] The amount of the active ingredient, that is, the inhibitor of the CCDC154 gene in the present invention, in the pharmaceutical composition and unit dosage form can be changed or greatly adjusted according to the specific application situation, the activity of the specific compound and the expected concentration.

[0045] "Treatment" means any treatment of a disease in a mammal, including: (1) preventing the disease, that is, preventing the development of the symptoms of the clinical disease; (2) inhibiting the disease, that is, preventing the development of the clinical symptoms; (3) alleviating the disease, that is, causing the regression of the clinical symptoms.

[0046] In the embodiments of the present invention, it is found that in the puncture or surgical resection specimens of human colorectal cancer patients, the overall survival of patients with high expression of CCDC154 is significantly shorter than that of patients with low expression of CCDC154, indicating that CCDC154 can be used as a biomarker for predicting the prognosis of colorectal cancer patients.

[0047] The following will further describe the technical solutions of the present invention in conjunction with specific embodiments, but the protection scope of the present invention is not limited to these embodiments. Any change or equivalent replacement that does not deviate from the concept of the present invention is included in the protection scope of the present invention.

[0048] Embodiment:

[0049] I. Screening of prognostic markers for colorectal cancer:

[0050] As Figure 1 shown, the TCGA-COADREAD dataset was used to screen prognostic markers for colorectal cancer, and it was found that CCDC154 was significantly highly expressed in colorectal cancer (CRC).

[0051] II. Tumor cell proliferation experiment, cloning experiment and tumorigenicity experiment of colorectal cancer cell lines:

[0052] As Figure 2 shown, after transfection to inhibit CCDC154 in the HCT116 colorectal cancer cell line, the cell viability of colorectal cancer cells was detected by the MTT method at 24, 48, 72, 96 and 120 hours after transfection, and it was found that the proliferation of tumor cells was inhibited after knocking out CCDC154.

[0053] As Figure 3 shown, after knocking out CCDC154 in the HCT116 cell line, the colony formation ability of the tumor cell line was detected by the colony formation assay, and it was found that knocking out CCDC154 reduced the colonies of tumor cells.

[0054] Figure 4 This is the xenograft tumor experiment in nude mice involved in the embodiments of the present invention. After subcutaneous transplantation of tumors to nude mice, it was found that both the tumor volume and body weight of the mice decreased significantly after knocking out CCDC154.

[0055] III. Sensitivity and specificity tests of CCDC154 gene as a biomarker for predicting the prognosis of colorectal cancer patients:

[0056] (1) In the puncture or surgical resection specimens of patients initially diagnosed with colorectal cancer (the samples were selected from Zhongshan Hospital, Fudan University, a total of 82 people), the expression level of CCDC154 in the colorectal cancer tissues of the patients was analyzed by immunohistochemical staining (a 1:50 dilution, Cat. ab184841, Abcam) (as Figure 5 shown).

[0057] (2) Eighty-two patients with colorectal cancer were divided into a high-expression group and a low-expression group of CCDC154 according to the median expression level of CCDC154. According to the expression level of CCDC154, the overall survival curve was plotted using the Kaplan-Meier method, and statistical analysis was performed using the log-rank test.

[0058] (3) As Figure 6 shown, according to the analysis results, the OS (Overall Survival) of patients with low expression of CCDC154 (n = 41) was significantly longer than that of patients with high expression of CCDC154 (n = 41), indicating that CCDC154 can specifically serve as a biomarker for predicting the prognosis of colorectal cancer patients.

[0059] (4) The ROC receiver operating characteristic curve analysis was performed on 82 patients with colorectal cancer according to the immunohistochemical quantitative results of CCDC154 and the overall survival (OS). It was found that the area under the curve (AUC) value was 0.751 (as Figure 7 shown). This indicates that CCDC154 has a good effect in predicting the prognosis of colorectal cancer.

[0060] (5) Multivariate Cox regression analysis was performed on 82 patients with colorectal cancer according to the immunohistochemical quantitative results of CCDC154 and the corresponding clinicopathological parameters (including age, gender, KRAS mutation status, BRAF mutation status, tumor size, and AJCC stage) (as Figure 8 shown). It was found that the p-value of the expression level of CCDC154 was 0.022, and the HR (hazard ratio) was 2.135, indicating that CCDC154 can be used as an independent prognostic biomarker for colorectal cancer patients.

[0061] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of a reagent for detecting the CCDC154 gene in the preparation of a kit for detecting colorectal cancer or monitoring the prognosis of colorectal cancer.

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