Uses micropeptide MIAC

Abstract

This invention discloses the use of micropeptide MIAC, which belongs to the biopharmaceutical technology field. Specifically, it relates to the use of micropeptide MIAC in the preparation of reagents or drugs for the detection, prevention, or treatment of tumors. The tumors include one or more solid tumors and hematological malignancies such as pancreatic cancer, liver cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, melanoma, glioblastoma, neuroblastoma, glioma, osteosarcoma, lymphoma, hematological malignancies, myeloma, cholangiocarcinoma, and prostate cancer. The micropeptide MIAC has the effect of inhibiting the growth, proliferation, and / or migration of many tumor cells, exhibits a broad therapeutic spectrum, and is suitable for the diagnosis, prevention, or treatment of many tumors, particularly malignant tumors.

Description

[Technical Field]

[0001] This application belongs to the field of biopharmaceutical technology and specifically relates to the use of micropeptide MIAC. [Background technology]

[0002] An open reading frame (ORF) is a translatable latent sequence consisting of a series of intraframe sense codons, beginning with a start codon and ending with a stop codon. Translable ORFs are typically noted as coding DNA sequences (CDS) on mRNA, and these sequences are responsible for protein translation.

[0003] Long noncoding RNAs (lncRNAs) are noncoding RNAs longer than 200 nt that are expressed in tissue- and development-specific ways and have been shown to directly regulate multiple functions. LncRNAs also include short open-reading frames (sORFs), which are generally less than 300 nucleotides in length. While these sORFs were previously considered non-coding, recent studies suggest that some sORFs are coding, and their coding products are defined as micropeptides.

[0004] Micropeptides, consisting of fewer than 150 amino acids, are being increasingly found in a wide range of organisms, from bacteria to humans. The functional diversity of these micropeptides, and the emergence of new methods for identifying them, are attracting the attention of the scientific community. All of these studies suggest that micropeptides translated from sORFs play important regulatory roles in many vital physiological processes, including muscle development, amino acid metabolism, immunomodulation, tumor growth, and cell death.

[0005] Maolei Zhang et al. discovered a potentially coding lncRNA in glioma cells through circular RNA sequencing (circRNAseq) and ribosomal peptide chain complex sequencing (RNC-seq) analysis. Experiments revealed that its second exon can form a circular RNA (circRNA) and be translated into an 87-amino acid micropeptide. In vitro and in vitro experiments suggest that this micropeptide can suppress the proliferative capacity of gliomas and inhibit the transcriptional elongation of multiple proto-oncogenes by interacting with the polymerase complex-related factor PAF1. Clinical sample analysis suggests that this micropeptide exhibits significantly lower expression in breast cancer, liver cancer, and gastric cancer compared to cancer-associated tissues, indicating its potential role in the diagnosis and treatment of malignant tumors (Zhang M et al., A peptide encoded by circular form of LINC-PINT suppresses oncogenic transcriptional elongation in glioblastoma. Nat Commun. 2018 Oct 26;9(1):4475).

[0006] Related technologies, such as the Chinese invention patent with publication number CN112442116A, describe the use of micropeptides encoded by lncRNA in the detection and treatment of malignant tumors such as head and neck cancer, thyroid cancer, and kidney cancer.

[0007] However, different tumor tissues exhibit genetic, phenotypic, and immunological heterogeneity, and research on micropeptides, including in the field of tumor therapy, remains insufficient. Therefore, it is necessary to explore the use of micropeptides in different tumors in order to expand the tumor spectrum in which MIAC can play a role. This invention provides a novel solution for tumor therapy by detecting the expression status of MIAC in different tumor cells and their corresponding normal cells, and by evaluating the activity of the micropeptide MIAC in different tumors using in vivo and in vitro experimental systems. [Overview of the project]

[0008] 1. Purpose of the Invention The object of the present invention is to provide the use of the micropeptide MIAC. After a long period of exploration and continuous attempts, the inventors have discovered that the micropeptide MIAC having the amino acid sequence shown in SEQ ID NO: 1 can be used in the preparation of reagents or drugs for the detection, prevention, or treatment of tumors such as pancreatic cancer, liver cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, melanoma, glioblastoma, neuroblastoma, glioma, osteosarcoma, lymphoma, hematological malignancies, myeloma, cholangiocarcinoma, and prostate cancer.

[0009] 2.Technical solution To solve the above problems, the technical solutions adopted in this application are as follows:

[0010] This application provides the use of the micropeptide MIAC in the preparation of reagents or drugs for the detection, prevention, or treatment of tumors. The micropeptide has the amino acid sequence shown in SEQ ID NO: 1, which is MERAGVPGFSPRRSSVEAKMQSTSCSVRKSSTVTAWPAVVLLLSWGQRRGG.

[0011] Furthermore, the above tumors include one or more of the following: pancreatic cancer, liver cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, melanoma, glioblastoma, neuroblastoma, glioma, osteosarcoma, lymphoma, hematological malignancies, myeloma, cholangiocarcinoma, and prostate cancer.

[0012] Furthermore, the reagents or drugs for the prevention or treatment of the above-mentioned tumors include a micropeptide MIAC having at least the amino acid sequence shown in SEQ ID NO: 1, and a pharmaceutically acceptable carrier.

[0013] Furthermore, the prevention or treatment of the above-mentioned tumors includes inhibiting the growth, migration, and / or proliferation of tumor cells.

[0014] Furthermore, the prevention or treatment of the above-mentioned tumors includes inhibiting the proliferation of tumor cells.

[0015] Furthermore, the prevention or treatment of the above-mentioned tumors includes inhibiting the migration of tumor cells.

[0016] Furthermore, the prevention or treatment of the above-mentioned tumors includes inhibiting the growth of tumor cells, particularly their growth within the body.

[0017] Furthermore, the tumor cells involved in the inhibition of tumor cell proliferation described above include the following:

[0018] Pancreatic cancer cells contain one or a combination of MIP-PaCa-2, Aspc-1, and PANC-1. The cancer cells of the liver cancer include one or a combination thereof of HepG2, SMMC-7721, HCCLM3, QGY-7701, and Hep3B. The cancer cells of the colorectal cancer include one or a combination thereof of LOVO, SW480, and HCT-116. The cancer cells of the ovarian cancer include SKOV3 and / or A2780. The cancer cells of the aforementioned cervical cancer include HeLa and / or SIHA. The cancer cells of the bladder cancer include T24 and / or EJ, The melanoma cancer cells include one or a combination thereof of A375, SK-mel-2, and M14. The aforementioned glioblastoma cancer cells contain U87-MG, The aforementioned neuroblastoma cancer cells contain SH-SY5Y, The cancer cells of the glioma contained SHG-44, The osteosarcoma cancer cells include one or a combination thereof of MG-63, Saos-2, and U-2OS. The cancer cells of the lymphoma include one or a combination thereof of U937, Raji, U2932, WSU-DLCL2, OCI-LY10, and JeKo-1. The cancer cells of the blood tumor include any one or a combination of NB4, Reh, K562, Thrp-1, JurKat, and HL60, The cancer cells of the myeloma include any one or a combination of NCI-H929, ARP-1, and RPMI-8226, The cancer cells of the cholangiocarcinoma include QBC939, and / or The cancer cells of the prostate cancer include cell Du145.

[0019] Furthermore, the tumor cells in the inhibition of migration of the above tumor cells include prostate cancer cell Du145, cervical cancer cell HeLa, osteosarcoma cell MG-63, ovarian cancer cell SKOV3, melanoma cell A375, and / or hepatocarcinoma cell Hep3B.

[0020] Furthermore, the tumor cells in the inhibition of proliferation of the above tumor cells include hepatocarcinoma cell Hep3B.

[0021] This application also provides the use of nucleotides encoding the amino acid sequence shown in SEQ ID NO: 1 in the preparation of reagents or drugs for the detection, prevention, or treatment of tumors.

[0022] Furthermore, the above tumors include one or more of pancreatic cancer, liver cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, melanoma, glioblastoma, neuroblastoma, glioma, osteosarcoma, lymphoma, blood tumor, myeloma, cholangiocarcinoma, and prostate cancer.

[0023] Furthermore, the above nucleotides include the nucleotide sequence shown in SEQ ID NO: 2, and the nucleotide sequence shown in SEQ ID NO: 2 is atggagagagcaggggtgcccgggttctctccgcggcgctcatcggtggaggcgaagatgcagagcaccagctgcagtgtcaggaagagctccacagtcaccgcctggccagccgtcgtgctgttgctgagctggggacagagaagaggcggatga.

[0024] This application also provides the use of recombinant vectors in the preparation of tumor reagents or drugs for the detection, prevention, or treatment of tumors. The recombinant vector encodes an amino acid sequence shown in SEQ ID NO: 1 or a nucleotide sequence shown in SEQ ID NO: 2.

[0025] Furthermore, the above tumors include one or more of the following: pancreatic cancer, liver cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, melanoma, glioblastoma, neuroblastoma, glioma, osteosarcoma, lymphoma, hematological malignancies, myeloma, cholangiocarcinoma, and prostate cancer.

[0026] The invention also provides pharmaceutical compositions for treating tumors. These pharmaceutical compositions comprise at least the above-mentioned micropeptide MIAC, the above-mentioned nucleotide, or the above-mentioned recombinant vector, and a pharmaceutically acceptable carrier, wherein the tumor comprises one or more of the following: pancreatic cancer, liver cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, melanoma, glioblastoma, neuroblastoma, glioma, osteosarcoma, lymphoma, hematological malignancy, myeloma, cholangiocarcinoma, and prostate cancer.

[0027] This application also provides a method for detecting, preventing, or treating tumors. The method comprises administering a micropeptide MIAC having the amino acid sequence shown in SEQ ID NO: 1, or a pharmaceutical composition having the micropeptide MIAC, to a subject in need.

[0028] Furthermore, the subjects mentioned above may be humans, mice, or monkeys.

[0029] Furthermore, in the above method, subjects are administered micropeptide MIAC at doses of 0.1-50 mg / kg, 0.1-20 mg / kg, 0.1-10 mg / kg, 0.1-5 mg / kg, 5-20 mg / kg, 10-20 mg / kg, or 5-15 mg / kg.

[0030] 3. Beneficial effects Compared to the prior art, the beneficial effects of this application are as follows:

[0031] (1) The micropeptide MIAC provided in this application is expressed at a significantly lower level in pancreatic cancer, liver cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, melanoma, glioblastoma, neuroblastoma, glioma, osteosarcoma, lymphoma, hematological malignancies, myeloma, cholangiocarcinoma, and prostate cancer cells compared to the corresponding normal cells.

[0032] (2) In using the micropeptide MIAC provided in this application, this micropeptide can significantly suppress the proliferation of tumor cells in pancreatic cancer, liver cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, melanoma, glioblastoma, neuroblastoma, glioma, osteosarcoma, lymphoma, hematological malignancies, myeloma, and cholangiocarcinoma, and can also inhibit the migration of tumor cells in prostate cancer, cervical cancer, osteosarcoma, ovarian cancer, melanoma, and liver cancer.

[0033] (3) In the use of the micropeptide MIAC provided in this application, this micropeptide can significantly suppress the in vivo growth of liver cancer, particularly liver cancer cell Hep3B tumors.

[0034] (4) The micropeptide MIAC provided in this application has the effect of inhibiting the proliferation and migration of various tumor cells, as well as the in vivo tumor growth of liver cancer, exhibiting a broad therapeutic spectrum and being suitable for the diagnosis, prevention, and treatment of many tumors, especially malignant tumors. [Brief explanation of the drawing]

[0035] [Figure 1] This figure shows the results of the expression level analysis of the micropeptide MIAC in different tumor cells and normal cells. [Figure 2A] This figure shows the statistical results of the inhibition of Du145 migration activity in prostate cancer cells by the micropeptide MIAC. [Figure 2B] These are cell staining diagrams of the blank group and each treatment group in a prostate cancer cell Du145 migration activity inhibition study using the micropeptide MIAC. [Figure 3A] This figure shows the statistical results of the inhibition of HeLa migration activity in cervical cancer cells by the micropeptide MIAC. [Figure 3B]These are cell staining diagrams of the blank group and each treatment group in a study inhibiting the HeLa migration activity of cervical cancer cells with the micropeptide MIAC. [Figure 4A] This figure shows the statistical results of the inhibition of osteosarcoma cell MG-63 migration activity by the micropeptide MIAC. [Figure 4B] These are cell staining diagrams for the blank group and each treatment group in a study inhibiting the migration activity of osteosarcoma cells with the micropeptide MIAC (MG-63). [Figure 5A] This figure shows the statistical results of the inhibition of SKOV3 migration activity in ovarian cancer cells by the micropeptide MIAC. [Figure 5B] These are cell staining diagrams for the blank group and each treatment group in a study inhibiting the migration activity of ovarian cancer cells SKOV3 using the micropeptide MIAC. [Figure 6A] This figure shows the statistical results of the inhibition of A375 migration activity in malignant melanoma cells by the micropeptide MIAC. [Figure 6B] These are cell staining diagrams for the blank group and each treatment group in a study inhibiting the migration activity of malignant melanoma cells A375 using the micropeptide MIAC. [Figure 7A] This figure shows the statistical results of the inhibition of Hep3B migration activity in liver cancer cells by the micropeptide MIAC. [Figure 7B] These are cell staining diagrams for the blank group and each treatment group in a study inhibiting the Hep3B migration activity of liver cancer cells with the micropeptide MIAC. [Figure 8] This figure shows the curve indicating that MIAC inhibited the in vivo growth of subcutaneously transplanted liver cancer cells (Hep3B) in nude mice in Example 4. [Figure 9] This is a tumor weight statistics graph taken 22 days after suppression of Hep3B liver cancer cells subcutaneously transplanted into nude mice with MIAC in Example 4. [Modes for carrying out the invention]

[0036] The present application will be further described below, along with specific examples.

[0037] Furthermore, terms such as "up," "down," "left," "right," and "center" used herein are used solely for the purpose of clarifying the explanation and are not intended to limit the scope of implementation. Any changes or adjustments to their relative relationships should be considered within the scope of implementation of this application, provided that they do not substantially alter the technical content.

[0038] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of this application. The term "and / or" as used herein includes any one or more and all combinations of the relevant enumerated items.

[0039] Unless otherwise specified in the examples, the procedures are carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified by the manufacturer, the reagents and equipment used are all standard products available commercially.

[0040] As used herein, the term “about” is used to give flexibility and impreciseness to a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility to a particular variable.

[0041] As used herein, the term "at least one of..." is synonymous with "one or more of...". For example, "at least one of A, B, and C" clearly includes A only, B only, C only, and any combination thereof.

[0042] In this specification, concentrations, quantities, and other numerical data may be presented in range format. Such range formatting is used solely for convenience and brevity, and should be interpreted flexibly to include not only the explicitly stated range limits, but also all individual numbers or subranges within that range (so that each number and subrange is clearly described). For example, a numerical range from approximately 1 to approximately 4.5 should be interpreted to include not only the explicitly stated limit values ​​from 1 to approximately 4.5, but also individual numbers (2, 3, 4, etc.) and subranges (1 to 3, 2 to 4, etc.). The same principle applies to describing only one numerical range, such as "less than approximately 4.5," which should be interpreted to include all the values ​​and ranges described above. Furthermore, such interpretation should apply regardless of the breadth of the described range or feature.

[0043] Micropeptide MIAC (Micropeptide Inhibiting Actin Cytoskeleton) is a human endogenous protein or polypeptide fragment consisting of 51 amino acids, encoded by non-coding RNA (lncRNA). The amino acid sequence of the micropeptide MIAC is MERAGVPGFSPRRSSVEAKMQSTSCSVRKSSTVTAWPAVVLLLSWGQRRGG (Sequence ID 1).

[0044] This application provides for the use of the above-mentioned micropeptide MIAC in the preparation of reagents or drugs for the detection, prevention, or treatment of tumor diseases.

[0045] In some embodiments, the tumor includes a solid tumor or a hematological tumor.

[0046] In some embodiments, the tumors include pancreatic cancer, liver cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, melanoma, glioblastoma, neuroblastoma, glioma, osteosarcoma, lymphoma, hematological malignancies, myeloma, cholangiocarcinoma, and / or prostate cancer.

[0047] The micropeptide MIAC provided in this application has the effect of inhibiting the growth, migration, and / or proliferation of tumor cells in vivo and / or in vivo.

[0048] In some embodiments, the micropeptide MIAC has the effect of inhibiting the proliferation of many tumor cells, including the following:

[0049] Pancreatic cancer cells contain one or a combination of MIP-PaCa-2, Aspc-1, and PANC-1. Cancer cells in liver cancer include one or a combination of HepG2, SMMC-7721, HCCLM3, QGY-7701, and Hep3B. Cancer cells of colorectal cancer contain one or a combination of LOVO, SW480, and HCT-116. Ovarian cancer cells include SKOV3 and / or A2780. Cervical cancer cells include HeLa and / or SIHA. Bladder cancer cells include T24 and / or EJ cells. Melanoma cancer cells contain one or a combination of A375, SK-mel-2, and M14. Glioblastoma cancer cells contain U87-MG, Neuroblastoma cancer cells contain SH-SY5Y, Glioma cancer cells contain SHG-44, Osteosarcoma cancer cells contain one or a combination of MG-63, Saos-2, and U-2OS. Lymphoma cancer cells include one or a combination of U937, Raji, U2932, WSU-DLCL2, OCI-LY10, and JeKo-1. Cancer cells in hematological malignancies contain one or a combination of NB4, Reh, K562, Thrp-1, JurKat, and HL60. Myeloma cancer cells contain one or a combination of NCI-H929, ARP-1, and RPMI-8226. Bile duct cancer cells contain QBC939, Prostate cancer cells contain cell Du145.

[0050] The micropeptide MIAC provided in this application has the effect of inhibiting the proliferation of tumor cells in the ranges of 50-200 μM, 10-200 μM, and 50-100 μM.

[0051] In some embodiments, the micropeptide MIAC can also inhibit the migration of tumor cells, specifically human prostate cancer cells Du145, human cervical cancer cells HeLa, human osteosarcoma cells MG-63, human ovarian cancer cells SKOV3, human malignant melanoma cells A375, and / or human hepatocellular carcinoma cells Hep3B.

[0052] Selectively, the micropeptide MIAC can inhibit tumor cell migration in the ranges of 0.78-50 μM, 0.78-25 μM, 1.56-50 μM, 3.125-50 μM, 6.25-50 μM, 25-50 μM, or 12.5-50 μM.

[0053] The micropeptide MIAC provided in this application can inhibit the in vivo growth of tumor cells. Selectively, MIAC can inhibit the in vivo tumor growth of human hepatocytes. In some embodiments, the micropeptide MIAC inhibits the in vivo tumor growth of hepatocytes Hep3B.

[0054] Therefore, this application also provides a method for detecting, preventing, or treating tumor diseases. The method comprises administering a micropeptide MIAC having or comprising the amino acid sequence shown in SEQ ID NO: 1, or a pharmaceutical composition comprising the micropeptide MIAC, to a subject requiring such intervention.

[0055] The subject may be a human, mouse, monkey, dog, or pig. In some embodiments, the subject is a human, monkey, dog, or mouse.

[0056] When a pharmaceutical composition containing the micropeptide MIAC is administered to a subject, the pharmaceutical composition further comprises at least one pharmaceutically acceptable adjunct.

[0057] The term "pharmaceutically acceptable carrier" refers to an auxiliary material or carrier that does not cause significant irritation to the subject and does not counteract the biological activity and properties of the administered anti-mesothelin antibody or its antigen-binding fragment, and / or any other therapeutic agent in the composition. A pharmaceutically acceptable carrier may enhance or stabilize the composition and may also be used to facilitate the preparation of the composition.

[0058] The pharmaceutical composition comprising the micropeptide MIAC may be administered by an appropriate method depending on the specific applicable form and physicochemical characteristics of the pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may be formulated in the form of a lyophilized or liquid formulation that may contain appropriate pharmaceutical additives in the art. For example, the pharmaceutical composition typically includes one or more pharmaceutical excipients, such as water and a sterile liquid such as oil (including petroleum, animal oil, vegetable oil, or synthetic oil (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.)). When the pharmaceutical composition is administered intravenously, water is a more typical carrier. Saline solution, glucose aqueous solution, and glycerol aqueous solution can also be used as liquid carriers, particularly for injection. Suitable pharmaceutical excipients are known in the art. If necessary, the composition may contain trace amounts of wetting agents, emulsifiers, or pH buffers. The pharmaceutical composition is usually administered by extraintestinal methods such as intradermal, intramuscular, intraperitoneal, intravenous, or subcutaneous injection. For example, it may be administered by intravenous infusion or bolus injection, intracranial, intrathecal, or intranasal (e.g., by inhalation), intradermal, subcutaneous, or transmucosal administration.

[0059] When using micropeptides (MIACs) for tumor detection, prevention, or treatment, the dose varies depending on factors such as the specific condition being treated, the severity of the condition, the individual patient's parameters (including age, health status, size, sex, and weight), the duration of treatment, the nature of any concomitant therapies, the specific route of administration, and the physician's or veterinarian's knowledge, and is gradually increased until the desired effect is achieved. In some embodiments, the dose of the active ingredient may be determined empirically in individuals who have received one or more doses of micropeptides (MIACs).

[0060] In some embodiments, for example, when micropeptide MIAC is administered to a subject at concentrations of 0.1-50 mg / kg, 0.1-20 mg / kg, 0.1-10 mg / kg, 0.1-5 mg / kg, 5-20 mg / kg, 10-20 mg / kg, or 5-15 mg / kg, tumor formation of tumor cells, particularly tumor formation of tumor cells in vivo, can be inhibited.

[0061] In some embodiments, the dose administered to the subject may be converted between species, for example, between humans and mice. Such conversion methods are known in the art and are described, for example, in Xu Shuyun, "Pharmacological Experimental Methodology," 2nd edition [M], People's Medical Press, 1991, the contents of which are incorporated herein by reference.

[0062] The technical solutions of the present invention will be described more clearly and concisely below with reference to examples. It should be understood that these examples are merely illustrative and do not limit the scope of protection of the present invention. The scope of protection of the present invention is limited solely by the claims.

[0063] Example 1 In this study, we detected the expression status of the micropeptide MIAC in different tumor cells and normal cells.

[0064] (1) Cell culture and RNA extraction Human pancreatic ductal epithelial cells HPDE, pancreatic cancer cells PANC-1, Aspc-1, human normal hepatocytes L02, hepatic cancer cells HepG2, QGY-7701, Hep3B, human normal colon cells CCD-18Co, colorectal cancer cells SW480, HCT-116, human normal ovarian epithelial cells IOSE-80, ovarian cancer cells SKOV3, A2780, human cervical epithelial cells HCerEpiC, cervical cancer cells HeLa, SIHA, human normal bladder epithelial cells SV-HUC-1, bladder cancer cells T24, EJ, human epidermal keratinocytes HEKa, melanoma cells SK-mel-2, M14, human brain glial cells T98G, glioma cells SHG Cells -44, neuroblastoma cells SH-SY5Y, human osteoblast cell line hFOB1.19, osteosarcoma cells MG-63, U-2OS, human T lymphocytes H9, lymphoma cells U2932, WSU-DLCL2, JeKo-1, human peripheral blood mononuclear cells PBMC, hematological malignant cells K562, HL60, NCI-H929, RPMI-8226, human intrahepatic cholangiocarcinoma cells HIBEpiC, cholangiocarcinoma cells QBC939, human prostatic epithelial cells HPEpiC, and prostatic cancer cells Du145 were cultured in an incubator at 37°C and 5% CO2 until the density reached 90%. The supernatant was removed, a predetermined volume of Trizol (Life Invitrogen) was added, and total RNA was extracted from different cells according to the instructions.

[0065] (2) Primer design Primers were designed based on the nucleotide sequence information encoding the micropeptide MIAC. The sequence is as follows:

[0066] The upstream primer (SEQ ID NO: 3) is ATGGAGAGAGCAGGGGTGCCCG.

[0067] The downstream primer (SEQ ID NO: 4) is CGCCCTTCTCTGTCCCCAGCTC.

[0068] (3) Detection of the expression status of micropeptide MIAC in different tumor cells and normal cells by real-time quantitative PCR The purity and concentration of the total RNA extracted in the above steps were quantified using a NanoDrop ND-1000 nucleic acid quantifier. Agarose gel quality testing was performed to ensure the integrity of the extracted RNA. (TaKaRa kit (PrimeScript)) TM The extracted total RNA was reverse transcribed into cDNA using the RT reagent kit with gDNA Eraser (Perfect Real Time). (TaKaRa kit (SYBR® Premix Ex Taq)) TM The qPCR reaction was performed using II (Tli RNaseH Plus). The reaction system is shown in Table 1. After homogeneously mixing the above components, the following procedure was performed: pre-denaturation at 95°C for 30s, 40 cycles, 95°C for 5s, and 60°C for 30s.

[0069] [Table 1]

[0070] Based on the melting curve, the specificity of the reaction was determined, and the relative expression level of MIAC was calculated according to Equation 2-ΔΔCt. The results are shown in Figure 1. Since the expression levels of MIAC in human pancreatic cancer, liver cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, melanoma, glioblastoma, neuroblastoma, glioma, osteosarcoma, myeloma, lymphoma, hematological malignancies, and cholangiocarcinoma cells were significantly lower than in corresponding normal cells, the antitumor effect of MIAC in the above tumor cells was investigated.

[0071] Example 2 This example demonstrates the inhibitory effect of micropeptide MIAC on the proliferation of human tumor cells.

[0072] Human pancreatic cancer, liver cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, melanoma, glioblastoma, neuroblastoma, glioma, osteosarcoma, myeloma, lymphoma, hematological malignancies, and cholangiocarcinoma cells were cultured in an incubator at 37°C and 5% CO2 until the cell density reached 90%. The cells were harvested by trypsin digestion, resuspended in culture medium, and counted under a microscope. The cell concentration was approximately 1.0 × 10⁻⁶.5 The cell suspension was adjusted to cells / mL and seeded at a concentration of 100 μL per well in a 96-well plate. The cells were incubated overnight in a 37°C, 5% CO2 incubator. After the cells had fully adhered to the plate walls, 100 μL each of micropeptide MIAC at concentrations of 50 μM, 100 μM, and 200 μM were added to the treatment group, while the culture medium without any drugs served as the negative control group. After a subsequent 72-hour incubation, 100 μL of CCK8 was added to each well of the 96-well plate and dissolved. Using a microplate reader, absorbance (OD) was measured at a measurement wavelength of 450 nm, and the proliferation inhibition rate (PI) was calculated according to the formula PI(%) = 1 - treatment group / negative control group. The experiment was repeated three times independently. Results were expressed as mean ± standard deviation (Mean ± SD), and a statistical t-test was performed. *P<0.05 indicated a statistically significant difference, and **P<0.01 indicated a very significant difference. The results are shown in Tables 2-52.

[0073] [Table 2]

[0074] [Table 3]

[0075] [Table 4]

[0076] [Table 5]

[0077] [Table 6]

[0078] [Table 7]

[0079] Table 8

[0080] Table 9

[0081] Table 10

[0082] Table 11

[0083] Table 12

[0084] Table 13

[0085] Table 14

[0086] Table 15

[0087] Table 16

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[0096] Table 25

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[0098] Table 27

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[0100] Table 29

[0101] Table 30

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[0106] Table 35

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[0114] Table 43

[0115] Table 44

[0116] Table 45

[0117] Table 46

[0118] Table 47

[0119] Table 48

[0120] Table 49

[0121] [Table 50]

[0122] [Table 51]

[0123] [Table 52]

[0124] As can be seen from Tables 2-52, compared to negative controls, micropeptide MIAC, at doses of 50-200 μM, significantly inhibited the proliferation of human pancreatic cancer, liver cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, melanoma, glioma, neuroblastoma, osteosarcoma, myeloma, lymphoma, leukemia, and cholangiocarcinoma cells, to varying degrees, and its proliferation effect was dose-dependent.

[0125] Example 3 This example demonstrates the inhibitory effect of the micropeptide MIAC on the migration of human tumor cells.

[0126] Human prostate cancer, cervical cancer, osteosarcoma, ovarian cancer, melanoma, and hepatocellular carcinoma cells were seeded in Transwell chambers at a rate of 100 μL per well. As a drug group, 100 μL of different doses of the micropeptide MIAC were added to each chamber, while the blank group received no drug. Next, to stimulate cell migration, 0.6 mL of complete medium containing 10% FBS was added to the lower chamber of the Transwell, and the cells were cultured at 37°C and 5% CO2 for 48 hours. The culture medium in the wells was removed, fixed with methanol at room temperature for 30 minutes, stained with 0.1% crystal violet at room temperature for 10 minutes, rinsed with water, and the unmigrating cells in the upper layer were wiped off with a cotton swab. These cells were observed under a microscope, and four fields of view were selected, photographed, and counted. The invasion inhibition rate (MI) was calculated according to the following formula.

[0127]

number

[0128] The results are shown in Tables 53-58 and Figures 2-7.

[0129] [Table 53]

[0130] Figure 2A corresponds to the statistical graphs by group in Table 53, and Figure 2B corresponds to the staining diagrams of cells in each group in Table 53.

[0131] [Table 54]

[0132] Figure 3A corresponds to the statistical graphs by group in Table 54, and Figure 3B corresponds to the staining diagrams of cells in each group in Table 54.

[0133] [Table 55]

[0134] Figure 4A corresponds to the statistical graphs by group in Table 55, and Figure 4B corresponds to the staining diagrams of cells in each group in Table 55.

[0135] [Table 56]

[0136] Figure 5A corresponds to the statistical graphs by group in Table 56, and Figure 5B corresponds to the staining diagrams of cells in each group in Table 56.

[0137] [Table 57]

[0138] Figure 6A corresponds to the statistical graphs by group in Table 57, and Figure 6B corresponds to the staining diagrams of cells in each group in Table 57.

[0139] [Table 58]

[0140] Figure 7A corresponds to the statistical graphs by group in Table 58, and Figure 7B corresponds to the staining diagrams of cells in each group in Table 58.

[0141] As can be seen from Tables 53-58 and Figures 2-7, the micropeptide MIAC significantly inhibited the migration of human prostate cancer cells Du145, human osteosarcoma cells MG-63, human ovarian cancer cells SKOV3, human hepatocellular carcinoma cells Hep3B, human melanoma cells A375, and human cervical cancer cells HeLa within a specified range, compared to a blank control, and the inhibition rate was dose-dependent. This suggests that the micropeptide MIAC can be developed as an antitumor drug by inhibiting the migration of the above-mentioned malignant tumor cells.

[0142] Example 4 In this embodiment, the inhibitory effect of the micropeptide MIAC on the growth of human tumor cells in vivo was provided.

[0143] A large amount of human hepatoma cells Hep3B was cultured and digested with a 0.25% trypsin solution. After digestion, the cell suspension was centrifuged at 1000 rpm for 5 min, and the cells were resuspended in serum-free MEM medium and then counted. The cell concentration was adjusted to 5×10 7 cells / mL. 100 μL of the cell suspension was inoculated into the left axilla of BALB / c nude mice (4 - 6 weeks old, female, body weight 14 - 16 g, acclimated and raised in an SPF-grade animal breeding room for 1 week), and the amount of injected cells was 5×10 6 cells. After inoculation, the growth of tumors at the inoculation site of the nude mice was carefully observed. On the 7th day after inoculation, white nodules appeared at the inoculation site and sometimes moved subcutaneously when touched. As the tumor tissue grew, a hard mass was gradually formed at the inoculation site. The average volume of the tumor tissue reached about 100 mm 3 in about 14 days. The BALB / c nude mice were randomly divided into 7 groups (the physiological saline group was the blank control, the micropeptide MIAC dose of 5 mg / kg was the low dose, the micropeptide MIAC dose of 10 mg / kg was the medium dose, the micropeptide MIAC dose of 15 mg / kg was the medium-high dose, the micropeptide MIAC dose of 20 mg / kg was the high dose, and the positive controls were oxaliplatin 15 mg / kg and sorafenib 50 mg / kg). There were 10 mice in the blank group and 6 mice in each of the remaining groups, and the body weight of the animals at the start of dosing was about 20 g. The volume of the transplanted tumor was measured and recorded every 2 days. The calculation formula for the tumor volume (Tumor volume, TV) was tumor volume = 0.5×a×b 2 where a was the length (mm) of the transplanted tumor and b was the width (mm) of the transplanted tumor.

[0144] The results are shown in Figures 8 and 9. Micropeptide MIAC significantly inhibited the in vivo tumor growth of human hepatocellular carcinoma cells (Hep3B) compared to the blank control group, albeit to varying degrees. At a dose of 20 mg / kg, the therapeutic effect on tumors was comparable to that of the positive drug, suggesting that micropeptide MIAC has excellent in vivo antitumor effects.

Claims

1. Use of micropeptide MIAC in the preparation of reagents or drugs for the detection, prevention, or treatment of tumors, wherein the micropeptide MIAC has the amino acid sequence shown in SEQ ID NO: 1, and the tumor includes one or more of pancreatic cancer, liver cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, melanoma, glioblastoma, neuroblastoma, glioma, osteosarcoma, lymphoma, hematological malignancy, myeloma, cholangiocarcinoma, and prostate cancer.

2. The use of the micropeptide MIAC in the preparation of a reagent or drug for the detection, prevention, or treatment of tumors according to claim 1, characterized in that the reagent or drug for the prevention or treatment of the tumor comprises at least the micropeptide MIAC and a pharmaceutically acceptable carrier.

3. The use of the micropeptide MIAC according to claim 1 or 2 in the preparation of reagents or drugs for the detection, prevention, or treatment of tumors, characterized in that the prevention or treatment of the tumor includes inhibition of the growth, migration, and / or proliferation of tumor cells.

4. The prevention or treatment of the aforementioned tumor includes inhibiting the proliferation of tumor cells. The pancreatic cancer cells include one or a combination thereof of MIP-PaCa-2, Aspc-1, and PANC-1. The cancer cells of the liver cancer include one or a combination thereof of HepG2, SMMC-7721, HCCLM3, QGY-7701, and Hep3B. The cancer cells of the colorectal cancer include one or a combination thereof of LOVO, SW480, and HCT-116. The ovarian cancer cells include SKOV3 and / or A2780. The cancer cells of the aforementioned cervical cancer include HeLa and / or SIHA. The cancer cells of the bladder cancer include T24 and / or EJ, The melanoma cancer cells include one or a combination thereof of A375, SK-mel-2, and M14. The cancer cells of the glioblastoma mentioned above contain U87-MG, The neuroblastoma cancer cells mentioned above contain SH-SY5Y, The cancer cells of the glioma contained SHG-44, The osteosarcoma cancer cells include one or a combination thereof of MG-63, Saos-2, and U-2OS. The lymphoma cancer cells include one or a combination thereof of U937, Raji, U2932, WSU-DLCL2, OCI-LY10, and JeKo-1. The cancer cells of the hematological malignancy include one or a combination thereof of NB4, Reh, K562, Thrp-1, JurKat, and HL60. The cancer cells of the myeloma include one or a combination thereof of NCI-H929, ARP-1, and RPMI-8226. The cancer cells of the aforementioned bile duct cancer include QBC939, and / or The use of the micropeptide MIAC according to claim 3 in the preparation of reagents or drugs for the detection, prevention, or treatment of tumors, characterized in that the cancer cells of the prostate cancer contain cell Du145.

5. The use of the micropeptide MIAC according to claim 3 in the preparation of reagents or drugs for the detection, prevention, or treatment of tumors, characterized in that the prevention or treatment of the tumors comprises inhibiting the migration of prostate cancer cells Du145, cervical cancer cells HeLa, osteosarcoma cells MG-63, ovarian cancer cells SKOV3, melanoma cells A375, and / or hepatoma cells Hep3B.

6. The use of the micropeptide MIAC according to claim 3 in the preparation of reagents or drugs for the detection, prevention, or treatment of tumors, characterized in that the prevention or treatment of the tumor includes inhibition of the growth of hepatocellular carcinoma cells Hep3B.

7. Use of a nucleotide in the preparation of a reagent or drug for the detection, prevention, or treatment of tumors, wherein the nucleotide encodes the amino acid sequence shown in SEQ ID NO: 1, and the tumor includes one or more of the following: pancreatic cancer, liver cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, melanoma, glioblastoma, neuroblastoma, glioma, osteosarcoma, lymphoma, hematological malignancy, myeloma, cholangiocarcinoma, and prostate cancer.

8. The use of the nucleotide according to claim 7 in the preparation of a reagent or drug for the detection, prevention, or treatment of tumors, characterized in that the nucleotide sequence includes the nucleotide sequence shown in SEQ ID NO:

2.

9. Use of a recombinant vector in the preparation of reagents or drugs for the detection, prevention, or treatment of tumors, wherein the recombinant vector encodes an amino acid sequence shown in SEQ ID NO: 1 or a nucleotide sequence shown in SEQ ID NO: 2, and the tumor comprises one or more of the following: pancreatic cancer, liver cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, melanoma, glioblastoma, neuroblastoma, glioma, osteosarcoma, lymphoma, hematological malignancy, myeloma, cholangiocarcinoma, and prostate cancer.

10. A pharmaceutical composition for treating a tumor, wherein the pharmaceutical composition comprises at least the micropeptide MIAC described in claim 1, the nucleotide described in claim 7 or 8, or the recombinant vector described in claim 9, and a pharmaceutically acceptable carrier, wherein the tumor comprises one or more of the following: pancreatic cancer, liver cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, melanoma, glioblastoma, neuroblastoma, glioma, osteosarcoma, lymphoma, hematological malignancy, myeloma, cholangiocarcinoma, and prostate cancer.

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