Genes for treating diseases related to AKT / mTOR signaling pathway

Abstract

The application discloses a gene for treating AKT / mTOR signal path related diseases. The application proves through experiments that FAM64A promotes proliferation, invasion, lipid droplet formation and chemotherapy resistance of colorectal cancer through the Akt / mTOR signal path. Up-regulation of FAM64A expression is closely related to occurrence and subsequent development of the colorectal cancer. Abnormal expression of FAM64A can be used as an index of invasive behavior and poor prognosis of the colorectal cancer. The finding provides a new direction for treatment and drug resistance of the Akt / mTOR signal path related diseases, especially treatment and drug resistance of the colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to genes used to treat diseases related to the AKT / mTOR signaling pathway. Background Technology

[0002] The AKT (protein kinase B) / mTOR (mammalian target of rapamycin) signaling pathway plays a crucial role in tumorigenesis and development, chemotherapy resistance, and anti-apoptosis. AKT is a serine / threonine protein kinase that controls cell proliferation, survival, and tissue angiogenesis by activating downstream target molecules. An important downstream target molecule of AKT is mTOR, a key serine / threonine kinase originally discovered and named TOR in yeast. Inhibiting the AKT / mTOR signaling pathway is considered a promising anticancer therapy; therefore, finding methods to inhibit the AKT / mTOR signaling pathway is essential for the treatment of diseases related to this pathway. Summary of the Invention

[0003] To overcome the shortcomings of existing technologies, this invention provides a gene for treating diseases related to the AKT / mTOR signaling pathway.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] The first aspect of the present invention provides the use of a reagent for detecting FAM64A expression levels in the preparation of products for diagnosing colorectal cancer / diagnosing colorectal cancer staging / metastasis / predicting colorectal cancer prognosis.

[0006] Furthermore, the reagents include oligonucleotide probes that specifically recognize the FAM64A gene, primers that specifically amplify the FAM64A gene, or binding agents that specifically bind to the protein encoded by the FAM64A gene.

[0007] Furthermore, the sequences of the primers for specifically amplifying the FAM64A gene are shown in SEQ ID NO:6-7.

[0008] A second aspect of the present invention provides a product for diagnosing colorectal cancer / diagnosing colorectal cancer staging / predicting colorectal cancer prognosis, said product comprising a reagent capable of detecting FAM64A expression levels.

[0009] Furthermore, the reagent also includes a detectable marker.

[0010] Furthermore, the detectable markers include radioactive isotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent components, magnetic particles, and bioluminescent components.

[0011] Furthermore, the products include chips, reagent kits, or nucleic acid membrane strips.

[0012] Furthermore, the kit also includes a buffer or preservative.

[0013] Furthermore, the kit also includes instructions.

[0014] A third aspect of the invention provides the use of an inhibitor of FAM64A in the preparation of a pharmaceutical composition, said pharmaceutical composition being used for any one or more of the following:

[0015] (1) Prevention and / or treatment of diseases related to the AKT / mTOR signaling pathway;

[0016] (2) Inhibit cell migration and / or invasion in diseases related to the AKT / mTOR signaling pathway;

[0017] (3) Promotes apoptosis in cells with diseases related to the AKT / mTOR signaling pathway;

[0018] (4) Increase the sensitivity of diseases related to the AKT / mTOR signaling pathway to chemotherapeutic agents;

[0019] (5) Inhibit lipid droplet formation in diseases related to the AKT / mTOR signaling pathway.

[0020] Furthermore, the inhibitors include nucleic acid inhibitors and protein inhibitors.

[0021] Furthermore, the nucleic acid inhibitors include siRNA, shRNA, and ribozymes.

[0022] Furthermore, the nucleic acid inhibitor is selected from shRNA.

[0023] Furthermore, the sequence of the shRNA is shown in SEQ ID NO:1.

[0024] Furthermore, the chemotherapeutic agents include platinum-based drugs and / or pyrimidine-based drugs.

[0025] Furthermore, the platinum-based drug is selected from cisplatin.

[0026] Furthermore, the pyrimidine drug is selected from uracil analogs.

[0027] Furthermore, the uracil analogue is selected from 5-FU.

[0028] Furthermore, the pharmaceutical composition also includes other pharmaceuticals.

[0029] Furthermore, the other drugs include other drugs for treating AKT / mTOR signaling pathway-related diseases or other drugs that increase the sensitivity of AKT / mTOR signaling pathway-related diseases to chemotherapeutic agents.

[0030] Furthermore, the other drugs for treating diseases related to the AKT / mTOR signaling pathway include antiviral drugs, chemotherapy drugs, targeted therapy drugs, immunotherapy drugs, traditional Chinese medicines, or any combination thereof.

[0031] Furthermore, other drugs that increase the sensitivity of AKT / mTOR signaling pathway-related diseases to chemotherapeutic agents include fatty acid synthesis inhibitors and / or Akt inhibitors.

[0032] Furthermore, the fatty acid synthesis inhibitor includes one or more inhibitors of ACLY, ACC1, and MOGAT2.

[0033] Furthermore, the inhibitors include nucleic acid inhibitors, protein inhibitors, and compounds.

[0034] Furthermore, the nucleic acid inhibitors include siRNA, shRNA, and ribozymes.

[0035] Furthermore, the nucleic acid inhibitor is selected from shRNA.

[0036] Furthermore, the shRNA sequence of ACLY is shown in SEQ ID NO:2.

[0037] Furthermore, the shRNA sequence of ACC1 is shown in SEQ ID NO:3.

[0038] Furthermore, the compound inhibitor for ACLY is selected from SB-204990.

[0039] Furthermore, the compound inhibitor of ACC1 is selected from PF-05175157.

[0040] Furthermore, the Akt inhibitor is selected from AZD5363.

[0041] Furthermore, the diseases related to the AKT / mTOR signaling pathway are selected from cancer.

[0042] Furthermore, the cancer is selected from colorectal cancer.

[0043] A fourth aspect of the present invention provides a pharmaceutical composition comprising an inhibitor of FAM64A.

[0044] Furthermore, the pharmaceutical composition also includes other pharmaceuticals.

[0045] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0046] The fifth aspect of the invention provides the use of FAM64A as a target in screening candidate drugs for the treatment of diseases related to the AKT / mTOR signaling pathway.

[0047] Furthermore, the method for screening candidate drugs for treating AKT / mTOR signaling pathway-related diseases includes: treating a culture system expressing or containing the FAM64A gene or its encoded protein with a substance to be screened; and detecting the expression or activity of the FAM64A gene or its encoded protein in the system; wherein, when the substance to be screened inhibits the expression level or activity of the FAM64A gene or its encoded protein, the substance to be screened is a candidate drug for treating AKT / mTOR signaling pathway-related diseases.

[0048] Furthermore, diseases related to the AKT / mTOR signaling pathway are selected from cancer.

[0049] Furthermore, the cancer is selected from colorectal cancer.

[0050] A sixth aspect of the present invention provides a method for screening candidate drugs for treating diseases related to the AKT / mTOR signaling pathway, the method comprising: treating a culture system expressing or containing the FAM64A gene or its encoded protein with a substance to be screened; and detecting the expression or activity of the FAM64A gene or its encoded protein in the system; wherein, when the substance to be screened inhibits the expression level or activity of the FAM64A gene or its encoded protein, the substance to be screened is a candidate drug for treating diseases related to the AKT / mTOR signaling pathway.

[0051] Furthermore, diseases related to the AKT / mTOR signaling pathway are selected from cancer.

[0052] Furthermore, the cancer is selected from colorectal cancer.

[0053] A seventh aspect of the present invention provides a method for inhibiting cell migration / invasion / promoting apoptosis in AKT / mTOR signaling pathway-related diseases, the method comprising administering an inhibitor of FAM64A.

[0054] Furthermore, diseases related to the AKT / mTOR signaling pathway are selected from cancer.

[0055] Furthermore, the cancer is selected from colorectal cancer.

[0056] Furthermore, the method described is not for therapeutic purposes.

[0057] An eighth aspect of the invention provides a method for enhancing the sensitivity of AKT / mTOR signaling pathway-related diseases to chemotherapeutic agents, the method comprising administering an inhibitor of FAM64A.

[0058] Furthermore, the chemotherapeutic agents include platinum-based drugs and / or pyrimidine-based drugs.

[0059] Furthermore, the platinum-based drug is selected from cisplatin.

[0060] Furthermore, the pyrimidine drug is selected from uracil analogs.

[0061] Furthermore, the uracil analogue is selected from 5-FU.

[0062] Furthermore, the method also includes administering other drugs that increase the sensitivity of AKT / mTOR signaling pathway-related diseases to chemotherapeutic agents.

[0063] Furthermore, diseases related to the AKT / mTOR signaling pathway are selected from cancer.

[0064] Furthermore, the cancer is selected from colorectal cancer.

[0065] Furthermore, the method described is not for therapeutic purposes.

[0066] A ninth aspect of the present invention provides a method for regulating lipid droplet formation in diseases related to the AKT / mTOR signaling pathway, the method comprising administering an inhibitor of FAM64A.

[0067] Furthermore, the method also includes the administration of fatty acid synthesis inhibitors.

[0068] Furthermore, the fatty acid synthesis inhibitor includes one or more inhibitors of ACLY, ACC1, and MOGAT2.

[0069] Furthermore, diseases related to the AKT / mTOR signaling pathway are selected from cancer.

[0070] Furthermore, the cancer is selected from colorectal cancer.

[0071] Furthermore, the method described is not for therapeutic purposes.

[0072] The tenth aspect of the present invention provides a method for regulating the expression of any of the following substances, the method comprising administering an inhibitor of FAM64A, said substance comprising one or more of the following: N-cadherin, MMP9, NF-κB, p53, p-AKT, p-STAT3, p-p38, Caspase7, Caspase1, Gasdemin D, pNrf2, Cyclin D1, p21, p-CDC-25c, and E-cadherin.

[0073] Furthermore, the method described is not for therapeutic purposes.

[0074] Advantages and beneficial effects of the present invention:

[0075] This application experimentally demonstrates that FAM64A promotes the proliferation, invasion, lipid droplet formation, and chemotherapy resistance of colorectal cancer through the Akt / mTOR signaling pathway. Upregulation of FAM64A expression is closely related to the occurrence and subsequent progression of colorectal cancer. Its aberrant expression can serve as an indicator of invasive behavior and poor prognosis in colorectal cancer. This finding provides a new direction for the treatment and drug resistance of diseases related to the Akt / mTOR signaling pathway, especially colorectal cancer. Attached Figure Description

[0076] Figure 1 This is a differential expression plot of FAM64A in the Xiantao database;

[0077] Figure 2 This is a differential expression plot of FAM64A in the UALCAN database;

[0078] Figure 3 This is a differential expression plot of FAM64A in the GEO database;

[0079] Figure 4 This is a graph showing the overall survival and progression-free survival of colorectal cancer patients;

[0080] Figure 5 This is a graph showing the differential expression of FAM64A and survival in colorectal cancer tissue;

[0081] Figure 6 This is a graph showing the effects of overexpression or knockdown of FAM64A on the migration, invasion, and apoptosis of colorectal cancer cells.

[0082] Figure 7 This is a graph showing the effect of FAM64A overexpression or knockdown on the levels of related factors;

[0083] Figure 8 This is a sequence result image;

[0084] Figure 9 This is a drug sensitivity graph for FAM64A overexpression or knockdown;

[0085] Figure 10 This is a graph showing the effect of FAM64A overexpression or knockdown on fatty acid synthesis factor levels.

[0086] Figure 11 This is a diagram showing the effect of FAM64A overexpression or knockdown on lipid droplet formation;

[0087] Figure 12 It is a drug sensitivity graph of overexpression or knockdown of FAM64A, ACC1 or ACLY;

[0088] Figure 13This is a drug sensitivity graph for overexpression or knockdown of FAM64A, Akt agonists, or Akt inhibitors. Detailed Implementation

[0089] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0090] This invention provides the application of reagents for detecting FAM64A expression levels in the preparation of products for diagnosing colorectal cancer, diagnosing colorectal cancer staging / metastasis, and predicting colorectal cancer prognosis.

[0091] In one implementation, FAM64A includes wild-type, mutant, or fragments thereof. The term encompasses full-length, unprocessed FAM64A, as well as naturally occurring variants of FAM64A (e.g., splice variants or allelic variants). The term covers, for example, human FAM64A, as well as FAM64A from any other vertebrate source, including mammalian FAM64A, such as primates and rodents (e.g., mice and rats), gene ID: 54478.

[0092] In one implementation, the staging of colorectal cancer diagnosis refers to TNM staging, specifically stage 1, stage 2, stage 3, and stage 4.

[0093] In one implementation, the diagnosis of colorectal cancer metastasis refers to lymph node involvement (metastasis). When lymph nodes are not involved, it is represented by N0. As the degree and extent of lymph node involvement increase, it is successively represented by N1 to N2. A higher number indicates a greater number of lymph node metastases.

[0094] In one implementation, prognosis refers to expectations regarding medical developments (e.g., the likelihood of long-term survival, disease-free survival, etc.), including positive or negative prognoses. Negative prognoses include disease progression such as relapse, disease growth, metastasis, and drug-resistant mortality, while positive prognoses include disease remission such as a disease-free state, and disease improvement such as regression or stabilization.

[0095] In one implementation, predicting the prognosis of colorectal cancer includes predicting overall survival and progression-free survival.

[0096] The reagents include oligonucleotide probes that specifically recognize the FAM64A gene, primers that specifically amplify the FAM64A gene, or binding agents that specifically bind to the protein encoded by the FAM64A gene.

[0097] In one embodiment, a probe is a molecule capable of binding to a specific sequence, subsequence, or other portion of another molecule. Unless otherwise specified, a probe typically refers to a polynucleotide probe capable of binding to another polynucleotide (often called a target polynucleotide) through complementary base pairing. Depending on the stringency of the hybridization conditions, the probe can bind to a target polynucleotide that lacks complete sequence complementarity with the probe. Probes can be labeled directly or indirectly, including primers. Hybridization methods include, but are not limited to, solution-phase, solid-phase, mixed-phase, or in situ hybridization assays.

[0098] In one implementation, the primer refers to a short nucleic acid sequence, which is a nucleic acid sequence having a short free 3' terminal hydroxyl group (free 3' hydroxyl), and can form a base pair with the complementary template and act as the starting point for the replication template.

[0099] In one embodiment, a binder refers to a naturally occurring or non-naturally occurring molecule that specifically binds to a target. Examples of specific binders include, but are not limited to, proteins, peptides, nucleic acids, carbohydrates, and lipids.

[0100] The reagent also includes a detectable marker.

[0101] In one embodiment, a detectable marker refers to a composition capable of generating a detectable signal indicating the presence of a target polynucleotide in a sample. Suitable markers include, but are not limited to, radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent components, magnetic particles, and bioluminescent components. Therefore, a marker is any composition detectable by a device or method, including but not limited to spectroscopic, photochemical, biochemical, immunochemical, electrochemical, optical, chemical detection devices, or any other suitable device. In some embodiments, the marker can be visually detected without the aid of a device.

[0102] Among them, radioactive isotopes include but are not limited to 3 H, 14 C 35 S, 125 I, 131 I.

[0103] Enzymes include, but are not limited to, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and acetylcholinesterase.

[0104] Fluorescent molecules include, but are not limited to, FITC, rhodamine, and lanthanide phosphors.

[0105] The products include chips, reagent kits, or nucleic acid membrane strips.

[0106] In one implementation, a kit refers to a set of components provided in the context of a system for sequencing and / or isolating nucleotide sequences and / or diagnosing a subject with a disease or infection based on the presence, absence, and / or amount of expressed nucleotide sequences from a sample or cell.

[0107] The kit also includes one or more substances from the following groups: container, positive control, negative control, buffer, preservative, and protein stabilizer.

[0108] The kit may also include an instruction manual, which explains how to use the kit for testing and how to use the test results to assess disease progression and select treatment options.

[0109] The components of the kit can be packaged in an aqueous medium or in a lyophilized form. Suitable containers in the kit typically include at least one vial, test tube, long-necked flask, PET bottle, syringe, or other container for holding one component, and preferably, for appropriate aliquoting. When more than one component is present in the kit, the kit will also typically include a second, third, or other additional container in which the additional components are held separately. However, different combinations of components may be contained in a single vial. The kit of the present invention also typically includes a container for containing the reactants, sealed for commercial sale. Such a container may include injection-molded or blow-molded plastic containers in which the desired vials can be held.

[0110] This invention provides the use of an inhibitor of FAM64A in the preparation of a pharmaceutical composition, said pharmaceutical composition being used for any one or more of the following:

[0111] (1) Prevention and / or treatment of diseases related to the AKT / mTOR signaling pathway;

[0112] (2) Inhibit cell migration and / or invasion in diseases related to the AKT / mTOR signaling pathway;

[0113] (3) Promotes apoptosis in cells with diseases related to the AKT / mTOR signaling pathway;

[0114] (4) Increase the sensitivity of diseases related to the AKT / mTOR signaling pathway to chemotherapeutic agents;

[0115] (5) Inhibit lipid droplet formation in diseases related to the AKT / mTOR signaling pathway.

[0116] In one embodiment, an inhibitor refers to any substance that can reduce the activity of FAM64A protein, decrease the stability of the FAM64A gene or protein, downregulate the expression of FAM64A protein, reduce the effective duration of FAM64A protein, or inhibit the transcription and translation of the FAM64A gene. Such substances can be used in this application as substances useful for downregulating FAM64A, thereby being used for the prevention or treatment of diseases.

[0117] In one embodiment, the inhibitor includes nucleic acid inhibitors and protein inhibitors. The nucleic acid inhibitor is selected from interfering molecules that target FAM64A or its transcripts and are capable of inhibiting FAM64A gene expression or transcription, including but not limited to shRNA, siRNA, ribozymes, antisense oligonucleotides, or constructs capable of expressing or forming said shRNA, siRNA, ribozymes, or antisense oligonucleotides. The protein inhibitor is selected from substances that specifically bind to the FAM64A protein, such as antibodies or ligands capable of inhibiting FAM64A protein activity.

[0118] In a preferred embodiment, the inhibitor is selected from nucleic acid inhibitors.

[0119] The siRNA may include partially purified RNA, substantially pure RNA, synthetic RNA, or recombinant RNA, as well as RNA modified to differ from natural RNA by adding, deleting, substituting, and / or altering one or more nucleotides. These alterations may include adding non-nucleotide substances, such as adding to the end of the siRNA or one or more internal nucleotides; modifications that make the siRNA resistant to nuclease digestion (e.g., using 2'-substituted ribonucleotides or modifying the sugar phosphate backbone); or replacing one or more nucleotides in the siRNA with deoxyribonucleotides.

[0120] shRNA is a non-coding small RNA molecule that can form hairpin structures. shRNA can suppress gene expression through the RNA interference pathway.

[0121] Antisense oligonucleotides (antisense nucleic acid sequences) may include nucleotide sequences that are complementary to sense nucleic acids encoding proteins (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to FAM64AmRNA).

[0122] Ribozymes are a class of RNAs that can be engineered to enzymatically cleave and inactivate other RNA targets in a specific sequence-dependent manner.

[0123] In a specific implementation, the nucleic acid inhibitor is selected from shRNA.

[0124] In one implementation, treatment may refer to therapeutic procedures or preventative measures, wherein the goal is to prevent or slow (mitigate) an undesirable physical condition, impairment, or disease, or to achieve a beneficial or desired clinical outcome. In this application, treatment may refer to both treatment and prevention. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief; reduction of the severity of a condition, impairment, or disease; stabilization (i.e., non-exacerbation) of the state of a condition, impairment, or disease; delaying the onset of a condition, impairment, or disease or slowing its progression; improving the state of a condition, impairment, or disease; and relief (whether partial or complete) (whether detectable or undetectable) or improvement of a condition, impairment, or disease. Treatment may include causing a clinically noticeable response without excessive side effects. Treatment also includes extended survival compared to the expected survival without treatment.

[0125] The chemotherapeutic agents include platinum-based drugs and / or pyrimidine-based drugs.

[0126] In one embodiment, platinum-based drugs include, but are not limited to, cisplatin (DDP), carboplatin, nedaplatin, oxaliplatin, and lobaplatin.

[0127] In a specific implementation, the platinum-based drug is selected from cisplatin.

[0128] In one embodiment, pyrimidine drugs include, but are not limited to, cytosine analogs and uracil analogs.

[0129] In a preferred embodiment, the pyrimidine drug is selected from uracil analogs.

[0130] In one embodiment, uracil analogues include, but are not limited to, 5-fluorouracil (5-FU or f5U), fluorouridine, and capecitabine.

[0131] In a specific implementation, the uracil analogue is selected from 5-fluorouracil (fluorouracil, 5-FU or f5U).

[0132] The pharmaceutical composition also includes other drugs.

[0133] The other drugs mentioned include other drugs for treating diseases related to the AKT / mTOR signaling pathway or other drugs that promote the sensitivity of AKT / mTOR signaling pathway-related diseases to chemotherapeutic agents.

[0134] In one embodiment, when the pharmaceutical composition is used to prevent and / or treat AKT / mTOR signaling pathway-related diseases, the pharmaceutical composition further includes other drugs for treating AKT / mTOR signaling pathway-related diseases; when the pharmaceutical composition is used to increase the sensitivity of AKT / mTOR signaling pathway-related diseases to chemotherapeutic agents, the pharmaceutical composition further includes other drugs that promote the sensitivity of AKT / mTOR signaling pathway-related diseases to chemotherapeutic agents.

[0135] Other drugs for treating diseases related to the AKT / mTOR signaling pathway include antiviral drugs, chemotherapy drugs, targeted therapy drugs, immunotherapy drugs, traditional Chinese medicine, or any combination thereof.

[0136] In one embodiment, the antiviral drug includes entecavir, lamivudine, sofosbuvir, danoprevir, tenofovir disoproxil fumarate, adefovir disoproxil fumarate, oseltamivir, telbivudine, and ritonavir.

[0137] The chemotherapy drugs include fluorouracil, cyclophosphamide, doxorubicin, cisplatin, carboplatin, mitomycin, daunorubicin, epirubicin, gemcitabine, irinotecan, oxaliplatin, and mitoxantrone.

[0138] The targeted therapies include sorafenib, regorafenib, lenvatinib, donafenib, regorafenib, apatinib, and cabozantinib.

[0139] The immunotherapy drugs include atezolizumab, sintilimab, camrelizumab, tislelizumab, bevacizumab, nivolumab, and pembrolizumab.

[0140] The traditional Chinese medicines mentioned include Compound Candida Capsules, Anti-cancer Pills, Huachansu Capsules, Zhenxiang Capsules, and Zhenqi Fuzheng Granules.

[0141] The diseases related to the AKT / mTOR signaling pathway include cancer, organ transplant-related dysregulation (e.g., reduced rejection rate, graft-versus-host disease, etc.), amyotrophic lateral sclerosis, arthritis, allergic encephalomyelitis, immunosuppression-related dysregulation, metabolic disorders (e.g., obesity, diabetes, etc.), intimal thickening after vascular injury, and protein misfolding diseases (e.g., Alzheimer's disease, Gaucher disease, Parkinson's disease, Huntington's disease, cystic fibrosis, macular degeneration, retinitis pigmentosa, diabetic retinopathy, infectious protein diseases, etc.).

[0142] In a preferred embodiment, the AKT / mTOR signaling pathway-related diseases are selected from cancer.

[0143] In one embodiment, cancer includes solid tumors and hematologic malignancies. Solid tumors include, but are not limited to, head and neck cancer, lung cancer, pleural mesothelioma, esophageal cancer, gastric cancer, pancreatic cancer, hepatobiliary cancer, small bowel cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, kidney cancer, urethral cancer, bladder cancer, prostate cancer, penile cancer, testicular cancer, gynecological cancer, ovarian cancer, breast cancer, endocrine system cancer, skin cancer, central nervous system cancer, soft tissue sarcoma, osteosarcoma, and melanoma. Hematologic malignancies include lymphoma, Hodgkin's disease, leukemia, plasma cell tumors, and AIDS-related cancers. Furthermore, all stages of cancer, including primary cancer, metastatic cancer, and recurrent cancer, are included in the scope.

[0144] In a specific implementation, the cancer is selected from colorectal cancer.

[0145] In one embodiment, the dosage form of the pharmaceutical composition includes injectable and oral dosage forms. The oral dosage forms include tablets, capsules, films, and granules.

[0146] The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0147] In one embodiment, pharmaceutically acceptable carriers include, but are not limited to, diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

[0148] The ingredients include diluents such as lactose, sodium chloride, glucose, urea, starch, and water; binders such as starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginate and alginates, xanthan gum, hydroxypropyl cellulose, and hydroxypropyl methylcellulose; surfactants such as polyethylene oxide sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, and hexadecyl alcohol; humectants such as glycerin and starch; adsorbents such as starch, lactose, bentonite, silica gel, kaolin, and soap clay; and lubricants such as zinc stearate and monostearate. The ingredients include: glyceryl fatty acids, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium dodecyl sulfate, etc.; fillers such as mannitol (granular or powdered), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polysaccharides, coupled sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate and sodium bicarbonate, etc.; disintegrants such as crosylvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, crosylcarboxymethyl cellulose sodium, soybean polysaccharides.

[0149] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0150] Example

[0151] 1. Experimental Materials and Methods

[0152] 1) Cell culture and transfection

[0153] Colorectal cancer cell line (SW480) was obtained from the Shanghai Cell Bank, Chinese Academy of Sciences. Cells were stored in RPMI-1640 medium (BD Bioscience) containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO2. FAM64A was overexpressed ectopically using SW480 cells and the plasmid pcDNA3.1-FAM64A-3×Flag, and FAM64A in SW480 cells was knocked down using pGPU6 / GFP-shFAM64A. The shFAM64A targeting sequence is: 5′-TCGCTCAGCTAAGAGTGCTTT-3′ (SEQ ID NO:1). Plasmids pcDNA3.1-ACC1-3×Flag, pcDNA3.1-ATP citrate lyase (ACLY)-3×Flag, shRNA-ACC1 (acetyl-CoA carboxylase 1), and shRNA-ACLY were purchased from HonorGene. The shACLY targeting sequence is 5′-CCTATGACTATGCCAAGACTA-3′ (SEQ ID NO:2), and the shACC1 targeting sequence is 5′-TACAAGGGATACAGGTATTTA-3′ (SEQ ID NO:3). ACC1 (PF-05175157) and the ACLY inhibitor (SB-204990) were purchased from AbMole. All plasmids were transfected with Lipofectamine 3000 (Thermo Fisher Scientific). To investigate cell sensitivity to chemotherapy, they were administered cisplatin (DDP) or 5-fluorouracil (5-FU). Cells were treated with the Akt agonist SC79 (Beyotime) and the Akt inhibitor AZD5363 (Beyotime) to observe changes in cell phenotype and related protein expression.

[0154] 2) Cell proliferation experiment

[0155] Cell Counting Kit-8 (CCK-8) is used to assess the number of viable cells. Initially, 5.0 × 10⁶ cells were counted. 3Cells were seeded per well in a 96-well plate. After cell adhesion, 10 μL of CCK-8 solution was added to each well according to the experimental time points. Cells were incubated at 37°C in a humidified environment with 5% CO2 for 3 hours, and measurements were taken at 450 nm using a microplate reader (Multiskan FC microplate spectrophotometer). The data were carefully analyzed and visualized using Prism 8.

[0156] 3) Flow cytometry detection of apoptosis

[0157] Cell apoptosis experiment collected 1×10 6 Cells were counted. Apoptotic cells were detected using a flow cytometer FC500 (Beckman-Coulter, USA) and stained with Annexin V-phycoerythrin (PE) and 7-aminoactinomycin (7-AAD) (BDPharmingen, USA). The staining method was strictly performed according to the reagent instructions.

[0158] 4) Scratch test

[0159] Cells at 5 × 10 5 Cells were seeded at a density of 100 cells / well in 6-well plates. When cells reached approximately 80%-90% confluence, a monolayer of cells was scraped using a 200 μL pipette tip. Cell debris was then washed three times with PBS. Cells were then cultured in serum-free medium and photographed at 48 h and 72 h. Finally, cell migration area was analyzed using ImageJ software.

[0160] 5) Migration and invasion experiments

[0161] The transfer experiment used 1.0 × 10⁻⁶ 5 Cells were seeded in serum-free RPMI 1640, and 200 μL of cell suspension was cultured in the upper chamber of a Transwell plate (BD Bioscience, USA). The lower chamber contained 600 μL of RPMI 1640 supplemented with 10% FBS. After incubation for 24 hours, non-migrating cells were wiped off and washed with PBS. Migrating cells were fixed with 100% methanol and stained with hematoxylin for 10 minutes. The invasion assay was performed using the same procedure as above, except that the upper chamber of the Transwell plate was coated with the substrate.

[0162] 6) Nile Red Staining

[0163] Cells were cultured overnight in 12-well plates. After removing the culture medium, the cells were washed three times with PBS and fixed with 100% methanol for 20 minutes. Following fixation, the cells were washed three times with PBS and stained with 1 mg / mL Nile Red for 15 minutes. After washing three times with PBS, the cells were stained with 0.5 μg / mL DAPI (Solarbio) for 5 minutes and then imaged under an ECHO fluorescence microscope. Images were acquired and analyzed using ImageJ software (v1.8.0; National Institutes of Health, USA).

[0164] 7) Specimen Information

[0165] Tumor and adjacent normal tissue specimens were preserved at -80℃ at the Affiliated Hospital of Chengde Medical College in 2020-2021. Samples were prepared from paraffin-embedded masses obtained through surgical resection for routine pathological examination. Frozen samples were used for protein and RNA extraction. There were 78 tumor tissue samples and 78 normal tissue samples.

[0166] Inclusion criteria: Pathologically diagnosed with colorectal cancer, and had not received any form of anticancer treatment before surgery.

[0167] Exclusion criteria: Patients with a history of cancer or other malignant tumors; patients who received neoadjuvant chemotherapy or other anti-tumor treatments before surgery.

[0168] Prior to the commencement of the clinical study, patients had not received chemotherapy, radiotherapy, or adjuvant therapy, and all patients provided written consent for the use of tumor tissue in the clinical study. The Ethics Committee of the Affiliated Hospital of Chengde Medical College approved the conduct of this clinical study. All protocols were performed in accordance with relevant guidelines and regulations. Information identifying individual participants was not available during or after data collection.

[0169] 8) Tissue chip

[0170] Pathological specimens were fixed with 4% paraformaldehyde, dehydrated with alcohol, washed with xylene, and then embedded. 4 μm sections were excised from the paraffin blocks and stained with hematoxylin and eosin to guide subsequent histological analysis. Representative areas of adjacent normal tissue and solid tumors were identified under a light microscope. Tissue nuclei were extracted from the paraffin blocks and transferred to recipient pathological blocks. TMA blocks were cut into 4 μm thick sections for immunohistochemical examination.

[0171] 9) Immunohistochemistry

[0172] After baking at 60°C for 2 hours, the slides were dewaxed and rehydrated three times in graded ethanol. Antigen retrieval was performed in a microwave oven for 20 minutes. The slides were then incubated in methanol with 3% hydrogen peroxide for 30 minutes to block endogenous peroxidase activity. Finally, 5% bovine serum albumin was added for 30 minutes to block non-specific binding sites. The slides were then incubated with rabbit anti-FAM64A antibody (ab122605; Abcam) at room temperature for 3 hours. The slides were washed three times with TBST and then incubated with goat anti-rabbit horseradish peroxidase (HRP) conjugated with secondary antibody (1:200; P0399; DAKO, Japan) at 37°C for 2 hours. After washing with TBS, the slides were stained with diaminobenzidine for specific staining, followed by hematoxylin staining. The slides were then dehydrated and mounted. Finally, the slides were observed using a Nikon microscope (Nikon Corporation, Japan).

[0173] 10) RT-PCR

[0174] According to the instructions, total RNA was extracted from colorectal cancer and paired adjacent normal tissues using the Qiagen RNeasy Mini Kit (74104, QIAGEN, Germany), and quantified. Total RNA (2 μg) was reverse transcribed to obtain cDNA. Primers used for real-time PCR were: GAPDH (glyceraldehyde-3-phosphate dehydrogenase), forward 5′-GTCTCCTCTGACTTCAACAGCG-3′ (SEQ ID NO:4) and reverse 5′-ACCACCCTGTTGCTGTAGCCAA-3′ (131 bp) (SEQ ID NO:5); FAM64A, forward 5′-CCATTACGGCGATCAAGG-3′ (SEQ ID NO:6) and reverse 5′-CACAGGCTCTAGGTCACT-3′ (102 bp) (SEQ ID NO:7). A BIO-RAD CFX96™ real-time PCR system was used, employing iTaq™ universal... PCR was performed using green ultramixed buffer (Bio-Rad, Bio-Rad Laboratories, Inc., Singapore). After brief centrifugation of the cDNA microarray plate, 10 μL of quantitative PCR mixture (2×), 1 μL (10 μM) of each primer, and 7 μL of RNase-free ddH2O were added. The experimental procedure was as follows: 95℃ pre-denaturation for 15 min, 95℃ denaturation for 10 s, 60℃ annealing for 20 s, and 72℃ extension for 30 s, for 45 cycles. GAPDH was used as an internal control.

[0175] 11) Protein imprinting assay

[0176] Proteins were extracted from fresh samples and cells using RIPA lysis buffer and quantified using a BCA protein assay kit (NCM; WB6502). Equal volumes of protein were separated by 10% sodium lauryl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF (Millipore) membrane. To reduce nonspecific antigen binding, the membrane was incubated in TBST with 5% skim milk for 1.5 hours at room temperature, followed by overnight incubation with primary antibody at 4°C. After three washes with TBST, the membrane was incubated with anti-mouse or anti-rabbit HRP-conjugated secondary antibody (1:5000; #7074; CST) at room temperature for 2 hours and analyzed using Western Spectrophotometry. TM The ECL chemiluminescence assay kit (K-12045-D50; Advansta, USA) was used to visualize protein bands using a C300 (Azure Biosystems) scanner. Measurements were performed using ImageJ software (v1.8.0) (National Institutes of Health), with GAPDH as an internal control.

[0177] 12) Bioinformatics Analysis

[0178] We analyzed the expression, related genes, and signaling pathways of the FAM64A gene in colorectal cancer using the Xiantao platform (https: / / www.xiantaozi.com / ) and the UALCAN database (http: / / ualcan.path.uab.edu). We assessed the prognostic value of FAM64A in colorectal cancer using Kaplan-Meier (http: / / www.kmplot.com / ). We identified differentially expressed FAM64A genes and related genes in colorectal cancer using the Xiantao database. We constructed a protein-protein interaction (PPI) network using the differentially expressed genes to identify key hub genes and signaling pathways in colorectal cancer. We performed Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) on these related genes in colorectal cancer.

[0179] 13) Statistical analysis

[0180] Data analysis was performed using SPSS 23.0 software. Chi-square tests were used to compare ratios, and t-tests were used to compare means. Kaplan-Meier curves and log-rank tests were used for survival analysis, and Cox proportional hazards regression models were used for multivariate analysis. A p-value less than 0.05 was considered statistically significant.

[0181] 2. Experimental Results

[0182] Compared with normal mucosa, the expression of FAM64AmRNA in colorectal cancer was significantly lower in Xiantao ( Figure 1 ), UALCAN Figure 2 ) and GEO ( Figure 3 The levels were significantly elevated in all databases (p<0.05).

[0183] FAM64AmRNA expression was negatively correlated with overall survival and progression-free survival in colorectal cancer patients. Figure 4 ).

[0184] Western blot and RT-PCR analyses showed that FAM64A expression was significantly increased in colorectal cancer tissues compared to normal tissues. Figure 5 ).

[0185] FAM64A was successfully overexpressed (FAM64A) or knocked down (shFAM64A) in SW480 cells using Western blot. In SW480 cells, FAM64A overexpression increased cell proliferation and reduced apoptosis. Scratch and transwell assays showed that FAM64A-transfected cells exhibited higher migration and invasiveness. Figure 6 Western blot experiments showed that overexpression of FAM64A in SW480 cells was accompanied by upregulation of N-cadherin, MMP9, NF-κB, p53, p-AKT, p-STAT3, p-p38, Caspase7, Caspase1, Gasdemin D, pNrf2, Cyclin D1, p21, and p-CDC-25c, and downregulation of E-cadherin. Knockdown of FAM64A produced the opposite effect. Figure 7 ).

[0186] Sequencing results: Differentially expressed proteins between the two groups were screened based on criteria of (log2|fold-change|≥1.2 and p<0.05) (upregulation) and (log2|fold-change|≤0.83 and p<0.05) (downregulation). Compared with SW480, the results showed that 50 proteins were upregulated and 21 proteins were downregulated in the FAM64A overexpression group, while 59 proteins were upregulated and 55 proteins were downregulated in the shFAM64A group. GO and KEGG pathway enrichment analyses were performed on the differentially expressed proteins. In FAM64A vs sw480, the top three BP categories were transferase activity, transferring phosphorus-containing groups, and glucose transferase activity. CC categories included nucleoplasm part, non-membrane-bounded organelle, and intracellular non-membrane-bounded organelle. The MF class includes regulation of chromosome organization, regulation of chromatin organization, and negative regulation of hemopoiesis. N-Glycan biosynthesis, ribosome biogenesis in eukaryotes, and the insulin signaling pathway are the top three pathways with the most protein enrichment. In shFAM64Avs sw480, the top three pathways in the BP class are transferase activity, transferring phosphorus-containing groups, and protein kinase activity. The CC class includes cellular components, cell and cell part. The MF class includes regulation of chromatin organization, substrate adhesion-dependent cell spreading, and histone phosphorylation. Ribosome biogenesis in eukaryotes, nucleotide metabolism, and the adapelin signaling pathway are the top three pathways with the most protein enrichment. Figure 8 ).

[0187] FAM64A overexpression (ov-FAM64A) led to resistance of SW480 cells to DDP and 5-FU, while its knockdown (sh-FAM64A) increased sensitivity to both drugs. Figure 9 ).

[0188] Western blotting experiments showed that FAM64A overexpression (ovFAM64A) increased the levels of MOGAT2 (monoacylglycerol transferase 2), ACLY, and ACC1, while FAM64A knockdown (sh-FAM64A) had the opposite effect. In the FAM64A knockdown group treated with Akt agonists, the expression of AKT, p-Akt, p-mTor, ACC1, ACLY, and MOGAT2 was reversible, while the opposite results were observed in the FAM64A overexpression group treated with Akt inhibitors. Figure 10 ).

[0189] Nile red staining showed that FAM64A overexpression (ov-FAM64A) promoted SW480 lipid droplet formation, while FAM64A knockdown (sh-FAM64A) inhibited SW480 lipid droplet formation. ACC1 or ACLY overexpression (ovACC1 or ovACLY) or exposure to high glucose (HG) or palmitic acid (PA) restored the effect of FAM64A knockdown on lipid droplet formation, while ACC1 or ACLY knockdown (shACC1 or shACLY) attenuated the effect of FAM64A overexpression on lipid droplet formation. Figure 11 ).

[0190] FAM64A overexpression (FAM64A) promoted chemoresistance to 5-FU and DDP, while FAM64A knockdown (shFAM64A) had the opposite effect. SW480 cells with FAM64A overexpression or knockdown were further aggravated by HG and PA. ACC1 or ACLY overexpression (ovACC1 or ovACLY) restored the effect of FAM64A knockdown on chemoresistance, while ACC1 or ACLY knockdown (shACC1 or shACLY) or its inhibitors (ACC1 inhibitor or ACLY inhibitor) attenuated the effect of FAM64A overexpression on chemoresistance. Figure 12 ).

[0191] Treatment with the Akt agonist SC79 (5 μM) reversed the drug sensitivity of FAM64A knockdown (shFAM64A) SW480 cells to 5-FU and DDP. In contrast, in the FAM64A-overexpression (ovFAM64A) group, treatment with the Akt inhibitor AZD5363 (1 μm) reversed resistance to 5-FU and DDP. Nile red staining showed that SC79 treatment stimulated lipid droplet production in the FAM64A knockdown group, while AZD5363 treatment inhibited lipid droplet formation in the FAM64A-overexpression group. Figure 13 ).

[0192] Fatty acid synthesis largely depends on ACLY, ACC1, and MOGAT2, all of which are associated with chemotactic resistance. FAM64A was observed to enhance the resistance of SW480 cells to 5-FU and DDP by upregulating MOGAT2, ACLY, and ACC1.

[0193] Based on these findings, it is speculated that FAM64A induces drug resistance and lipid droplet formation in colorectal cancer cells through the Akt / mTOR pathway.

[0194] In summary, upregulation of FAM64A expression is closely related to the occurrence and subsequent progression of colorectal cancer. Its abnormal expression can serve as an indicator of invasive behavior and poor prognosis in colorectal cancer. FAM64A promotes the proliferation, invasion, lipid droplet formation, and chemotherapy resistance of colorectal cancer through the Akt / mTOR signaling pathway.

[0195] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Use of a reagent for detecting the expression level of FAM64A gene in the preparation of a product for diagnosing colorectal cancer.

2. Use according to claim 1, characterized in that, The reagent comprises an oligonucleotide probe specifically recognizing the FAM64A gene, a primer specifically amplifying the FAM64A gene, or a binding agent specifically binding to a protein encoded by the FAM64A gene.

3. Use according to claim 2, characterized in that, The sequence of the primer specifically amplifying the FAM64A gene is shown as SEQ ID NO: 6-7.

4. Use according to claim 2, characterized in that, The reagent further comprises a detectable label.

5. Use according to claim 4, characterized in that, The detectable label comprises a radioisotope, an enzyme, a fluorescent molecule, or a magnetic particle.

6. Use according to claim 1, characterized in that, The product comprises a chip, a kit, or a nucleic acid membrane strip.

7. Use according to claim 6, characterized in that, The kit further comprises a buffer or a preservative.

8. Use according to claim 6, characterized in that, The kit further comprises an instruction.

9. Use of an inhibitor of FAM64A in the preparation of a pharmaceutical composition for treating colorectal cancer, wherein the inhibitor is selected from an shRNA, and the sequence of the shRNA is shown as SEQ ID NO:

1.

10. Use according to claim 9, characterized in that, The treatment of colorectal cancer is selected from any one or more of the following: (1) inhibiting the migration and / or invasion of colorectal cancer cells; (2) promoting the apoptosis of colorectal cancer cells; (3) increasing the sensitivity of colorectal cancer to a chemotherapeutic agent; (4) inhibiting the lipid droplet formation of colorectal cancer. The chemotherapeutic agent comprises a platinum drug and / or a pyrimidine drug; the platinum drug is selected from cisplatin; the pyrimidine drug is selected from a uracil analog; and the uracil analog is selected from 5-FU.

11. Use according to claim 9, characterized in that, The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

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