Application of MUC16c_del15Y polypeptide in the preparation of drugs for treating gallbladder cancer

Abstract

The present invention provides a use of a MUC16c_del15Y polypeptide in preparing a drug for treating gallbladder cancer. The MUC16c_del15Y polypeptide competitively inhibits MUC16c in gallbladder cancer cells. The amino acid sequence of the MUC16c_del15Y polypeptide is shown in SEQ ID NO.1, and the amino acid sequence of MUC16c is shown in SEQ ID NO.2. Preparation of the polypeptide comprises: deleting the tyrosine site in wild-type MUC16c, synthesizing the polypeptide, and eluting and purifying. Compared with a control group, the MUC16c_del15Y polypeptide of the present invention inhibits the growth of gallbladder cancer xenografts in nude mice by 46%, demonstrating a significant inhibitory effect. MUC16c is an intracellular fragment of the MUC16 gene product and is similar in sequence to the MUC16c polypeptide. The MUC16c_del15Y polypeptide is rich in a high proportion of hydrophilic amino acids and is highly water-soluble.

Description

Technical Field

[0001] The present invention belongs to the technical field of gallbladder cancer treatment, and specifically relates to the use of a MUC16c_del15Y polypeptide in the preparation of a drug for treating gallbladder cancer. Background Art

[0002] CA-125 is an important tumor marker used in the diagnosis and treatment of gallbladder cancer. It is encoded by the MUC16 gene. The Mucins gene family includes at least 20 members. The mucin products of these Mucins family members are characterized by their large molecular weight and complex O-glycosylation, which has made their study difficult. The MUC16 protein product is a membrane-bound mucin with a molecular weight of 1519 kDa. This large molecular weight has limited its research. Studies have shown that the intracellular C-terminal fragment of the MUC16 protein product (MUC16c) can promote tumorigenesis and metastasis, inducing tumor transformation and invasion. Our previous studies have found that the expression level of MUC16c in pancreatic cancer tissue is positively correlated with serum CA-125 levels. MUC16c also induces Treg enrichment in pancreatic cancer tissue, promoting immune evasion in pancreatic cancer. MUC16c also plays a key role in the growth and metastasis of gallbladder cancer. In gallbladder cancer, MUC16c can bind to ALDOC, causing abnormal glucose sensing in gallbladder cancer cells. This leads to continuous glucose uptake, increased glycolysis, and accelerated cancer growth. Furthermore, MUC16c binds to stathmin1, causing cytoskeletal rearrangements in gallbladder cancer cells and promoting metastasis. It has also been found that the effects of MUC16c may be regulated by Src kinase. Summary of the Invention

[0003] In response to the deficiencies in the prior art, the present invention aims to provide a use of a MUC16c_del15Y polypeptide in the preparation of a drug for treating gallbladder cancer. Therefore, a MUC16c_del15Y polypeptide with a specific deletion site of wild-type MUC16c was prepared, and it was confirmed that the MUC16c_del15Y polypeptide with a functional site deletion can competitively inhibit the action of MUC16c in gallbladder cancer cells.

[0004] To achieve the above object, the solution of the present invention is:

[0005] In a first aspect, the present invention provides a use of a MUC16c_del15Y polypeptide in the preparation of a drug for treating gallbladder cancer.

[0006] Furthermore, the MUC16c_del15Y polypeptide competitively inhibits MUC16c in gallbladder cancer cells.

[0007] Furthermore, the amino acid sequence of the MUC16c_del15Y polypeptide is shown in SEQ ID NO.1.

[0008] Furthermore, the amino acid sequence of MUC16c is shown in SEQ ID NO.2.

[0009] In a second aspect, the present invention provides a medicine or pharmaceutical composition for treating gallbladder cancer, wherein the active ingredient is MUC16c_del15Y polypeptide and further comprises a pharmaceutically acceptable carrier.

[0010] Furthermore, the dosage form of the drug or pharmaceutical composition is selected from injection (intravenous injection, subcutaneous injection) or respiratory administration (nasal spray or tracheal inhalation).

[0011] In a third aspect, the present invention provides a method for preparing the above-mentioned MUC16c_del15Y polypeptide, comprising:

[0012] The tyrosine site in the wild-type MUC16c was deleted, and the peptide was synthesized and purified to obtain the MUC16c_del15Y peptide.

[0013] Furthermore, the amino acid sequence of the MUC16c_del15Y polypeptide is shown in SEQ ID NO.1.

[0014] Furthermore, the amino acid sequence of MUC16c is shown in SEQ ID NO.2.

[0015] Due to the adoption of the above solution, the beneficial effects of the present invention are:

[0016] The MUC16c_del15Y polypeptide of the present invention has a significant inhibitory effect on a gallbladder cancer xenograft model. Compared with the control group, the MUC16c_del15Y polypeptide inhibited the growth of gallbladder cancer xenografts in nude mice by 46%, which is a significant effect. MUC16c is an intracellular fragment of the MUC16 gene product, rich in a high proportion of hydrophilic amino acids and highly water-soluble. Similar to the MUC16c polypeptide sequence, the MUC16c_del15Y polypeptide is rich in a high proportion of hydrophilic amino acids and highly water-soluble. Therefore, the polypeptide is composed of a specific amino acid sequence, is a major component of protein synthesis in the human body, has good water solubility, and has minimal side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a structural diagram of the MUC16 encoding product in Example 1 of the present invention (ED: extracellular domain (ED), TMD: transmembrane domain (TMD), CD: cytoplasmic domain (CD); MUC16c is the CD sequence).

[0018] Figure 2Figure 2 shows the anti-gallbladder cancer growth effect of the MUC16c_del15Y polypeptide in Example 2 of the present invention ((A) flow chart of establishing a gallbladder cancer transplant tumor model and injecting the polypeptide, (B) gallbladder cancer transplant tumor sample, (C) gallbladder cancer transplant tumor sample volume, and (D) gallbladder cancer transplant tumor sample mass).

[0019] Figure 3 This is a diagram showing the immunohistochemical detection of Ki67 in transplanted tumors injected with the MUC16c_del15Y polypeptide in Example 2 of the present invention. DETAILED DESCRIPTION

[0020] The present invention provides an application of a MUC16c_del15Y polypeptide in preparing a medicament for treating gallbladder cancer.

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments and drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] Example 1:

[0023] According to the phosphorylation site analysis, wild-type MUC16c had several potential phosphorylation sites, and these sites were deleted to generate a new fragment MUC16c_del24-25YY.

[0024] The specific sequence of the polypeptide fragment is as follows: Wild type MUC16c: GVLVTTRRRKKEGEYNVQQQCPGYYQSHLDLEDLQ (SEQ ID NO. 2) (such as Figure 1 The wild-type polypeptide fragment (a 35-amino acid fragment at the C-terminus of the MUC16 gene product) has the tyrosine at position 15 deleted (MUC16c_del15Y: GVLVTTRRRKKEGENVQQQCPGYYQSHLDLEDLQ (SEQ ID NO. 1)). These polypeptide fragments are chemically synthesized crude and then purified by HPLC. Purity >90% is considered acceptable for use in experimental testing and analysis. The MUC16c_del15Y polypeptide fragment, which has a functional site deleted, has an amino acid sequence essentially identical to wild-type MUC16c. Through competitive interaction, it can inhibit the action of wild-type MUC16c in cells, thereby achieving an inhibitory therapeutic effect on gallbladder cancer. Further, a mouse xenograft tumor model confirmed that the MUC16c_del15Y polypeptide effectively inhibited gallbladder cancer growth.

[0025] Example 2:

[0026] Gallbladder cancer cells were inoculated subcutaneously into the abdomen of nude mice to establish a xenograft tumor model. Then, MUC16c and MUC16c_del15Y peptide solutions were injected into the tumor site at regular intervals. After the injection, the xenograft tumors were collected and analyzed for volume and mass to evaluate the effect of the peptide fragments on the growth of gallbladder cancer xenograft tumors.

[0027] (a) MUC16c and MUC16c_del15Y peptides were synthesized by Anhui Guoping Pharmaceutical Co., Ltd. with a purity of >90%.

[0028] (b) Twelve Balb / c nude mice (3-4 weeks old) were purchased from Slack. Each nude mouse was subcutaneously inoculated with 2*10 6 After 5 days of growth, SGC-996 cells were randomly divided into MUC16c group (control group) and MUC16c_del5-6TT group, with 6 cells in each group.

[0029] (c) The prepared polypeptide solution was injected into the tumor part once every 3 days for 7 injections. The long diameter and short diameter of the tumor were measured during the growth process.

[0030] (d) The mice were then killed by cervical dislocation, the transplanted tumors were removed, and the tumor volume and mass of each group were measured and analyzed. Figure 2 The results showed that the growth volume of the transplanted tumors in the MUC16c_del15Y group was 46% lower than that in the MUC16c group, and the weight of the transplanted tumors was also reduced by 53%. The results showed that the difference was significant. At the same time, the samples were stored at -80℃ and paraformaldehyde for subsequent testing. Immunohistochemical testing of the transplanted tumor samples confirmed that the Ki67 level in the transplanted tumors in the MUC16c_del15Y group was significantly lower than that in the MUC16c group. Figure 3 ), thus indicating that MUC16c_del15Y has an inhibitory effect on the growth of gallbladder cancer xenografts.

[0031] The above description of the embodiments is intended to facilitate understanding and use of the present invention by those skilled in the art. Those skilled in the art will readily be able to make various modifications to these embodiments and apply the general principles described herein to other embodiments without resorting to creative effort. Therefore, the present invention is not limited to the above-described embodiments. Any improvements or modifications made by those skilled in the art based on the principles of the present invention that do not depart from the scope of the present invention should be considered within the scope of protection of the present invention.

Claims

1. Use of the MUC16c_del15Y polypeptide in the preparation of a drug for treating gallbladder cancer, wherein the amino acid sequence of the MUC16c_del15Y polypeptide is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The MUC16c_del15Y polypeptide competitively inhibits MUC16c in gallbladder cancer cells. The amino acid sequence of MUC16c is shown in SEQ ID NO.

2.

3. A medicine or pharmaceutical composition for treating gallbladder cancer, characterized in that: The active ingredient of the medicine or pharmaceutical composition is the MUC16c_del15Y polypeptide, and further comprises a pharmaceutically acceptable carrier.

4. The drug or pharmaceutical composition according to claim 3, characterized in that: The dosage form of the medicine or pharmaceutical composition is injection.

5. A method for preparing a MUC16c_del15Y polypeptide, characterized in that: It includes: The tyrosine site in wild-type MUC16c was deleted, and the peptide was synthesized and purified to obtain the MUC16c_del15Y peptide; The amino acid sequence of the MUC16c_del15Y polypeptide is shown in SEQ ID NO.1; The amino acid sequence of MUC16c is shown in SEQ ID NO.2.

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