LACTB gene and its application in diagnosis of prostate cancer

Abstract

The present invention discloses a LACTB gene and its application in diagnosing prostate cancer. The present invention provides an application of a reagent for detecting LACTB in preparing a product for diagnosing prostate cancer / predicting the prognosis of prostate cancer and a product for diagnosing prostate cancer / predicting the prognosis of prostate cancer. The present invention also provides a pharmaceutical composition for treating prostate cancer and a method for screening candidate drugs for treating prostate cancer. The LACTB marker provided by the present invention can diagnose prostate cancer at an early stage, effectively treat it, and predict the prognosis of prostate cancer, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to LACTB gene and its application in diagnosing prostate cancer. Background Art

[0002] Prostate cancer (PCa) is one of the most common malignant tumors of the urinary system in my country and the second leading cause of cancer death in men worldwide, especially in elderly men. A statistical study on global cancer found that the incidence and mortality of this cancer have been rising year by year, accounting for more than 90% of reproductive organ tumor diseases.

[0003] The evolution of prostate cancer shows multi-step and multi-gene complex characteristics. In the early stage of prostate cancer, malignant lesions appear in the prostate capsule, but there are no special clinical manifestations. Even if there is some discomfort, the symptoms are usually confused with frequent urination and urgency caused by common prostate diseases. Generally, when the patient develops obvious symptoms such as swelling and numbness of the lower limbs, bone pain caused by bone metastasis, and renal failure caused by bilateral ureteral obstruction, it is not diagnosed until it has developed to the late stage of prostate cancer. When the tumor is limited to the prostate, it is considered to be curable. Once the tumor breaks through the prostate capsule and spreads and metastasizes, the patient's treatment will be very difficult and the prognosis will be relatively poor. Therefore, the death of prostate cancer patients is generally attributed to the failure to receive timely and effective treatment, resulting in tumor metastasis and death from other diseases.

[0004] At present, traditional prostate puncture causes great pain to patients during the screening process. Prostate specific antigen (PSA), which is mainly found in prostate tissue, is the most widely used indicator for prostate cancer screening, diagnosis and prognosis evaluation worldwide. However, more and more studies have shown that this indicator has many limitations for the diagnosis of prostate cancer, causing both economic and physical harm to most patients. Due to the limitations of existing medical levels, the complexity of clinical detection of prostate cancer, and the hidden clinical manifestations, the early diagnosis and treatment of prostate cancer are not optimistic. This poses a huge threat to human health and social development, and also poses greater challenges to the treatment of prostate cancer. The importance of early screening, treatment and prognosis of prostate cancer in clinical practice is self-evident.

[0005] The current limitations of medical standards, the clinical complexity of prostate cancer, and the unclear pathogenesis have led to low cure and survival rates for prostate cancer patients. Biomarkers can timely predict whether a patient has cancer and evaluate the prognosis in the early stages to assist clinicians in choosing treatment. However, from the current research progress, the number of biomarkers used in clinical early screening of prostate cancer and producing good results is small and has great limitations. Potential prostate cancer biomarkers in the research stage have not passed or have not been clinically verified and tested. Therefore, screening out biomarkers that can be used to guide the diagnosis and prognosis of prostate cancer, so as to achieve effective treatment of prostate cancer patients, is of great significance to improving the quality of life of patients. Summary of the invention

[0006] To remedy the deficiencies of the prior art, the present invention provides a biomarker for diagnosing prostate cancer / predicting the prognosis of prostate cancer, thereby achieving early diagnosis, effective treatment and prognosis prediction of prostate cancer.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides the use of a reagent for detecting LACTB in the preparation of a product for diagnosing prostate cancer / predicting the prognosis of prostate cancer.

[0009] Furthermore, the reagent is selected from an oligonucleotide probe that specifically recognizes the LACTB gene, a primer that specifically amplifies the LACTB gene, or a binding agent that specifically binds to a protein encoded by the LACTB gene.

[0010] A second aspect of the present invention provides a product for diagnosing prostate cancer / predicting the prognosis of prostate cancer, the product comprising a reagent capable of detecting the expression level of LACTB.

[0011] Furthermore, the product includes a chip, a kit or a nucleic acid membrane strip.

[0012] Furthermore, the chip includes a gene chip and a protein chip, the gene chip includes an oligonucleotide probe for the LACTB gene for detecting the transcription level of the LACTB gene, and the protein chip includes a specific binder for the LACTB protein; the kit includes a gene detection kit and a protein detection kit, the gene detection kit includes a reagent or chip for detecting the transcription level of the LACTB gene, and the protein detection kit includes a reagent or chip for detecting the expression level of the LACTB protein.

[0013] Furthermore, the kit includes reagents for detecting the expression level of LACTB gene or protein by RT-PCR method, qRT-PCR method, biochip detection method, Southern blotting method, in situ hybridization method, and immunoblotting method.

[0014] The third aspect of the present invention provides a pharmaceutical composition for treating prostate cancer, comprising an inhibitor of LACTB.

[0015] Further, the inhibitor reduces the expression level of LACTB.

[0016] Furthermore, the inhibitors include nucleic acid inhibitors and protein inhibitors.

[0017] Furthermore, the nucleic acid inhibitor includes shRNA and siRNA.

[0018] Furthermore, the protein inhibitor includes an antibody or a ligand that inhibits the activity of the LACTB protein.

[0019] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0020] The fourth aspect of the present invention provides the use of LACTB in preparing a drug combination for treating prostate cancer.

[0021] Further, the inhibitor reduces the expression level of LACTB.

[0022] Furthermore, the inhibitors include nucleic acid inhibitors and protein inhibitors.

[0023] Furthermore, the nucleic acid inhibitor includes shRNA and siRNA.

[0024] Furthermore, the protein inhibitor includes an antibody or a ligand that inhibits the activity of the LACTB protein.

[0025] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0026] The fifth aspect of the present invention provides the use of LACTB in screening candidate drugs for treating prostate cancer.

[0027] Furthermore, the method for screening candidate drugs for treating prostate cancer is as follows: treating a culture system expressing or containing the LACTB gene or a protein encoded by it with a substance to be screened; and detecting the expression or activity of the LACTB gene or a protein encoded by it in the system; wherein, when the substance to be screened inhibits the expression level or activity of the LACTB gene or a protein encoded by it, the substance to be screened is a candidate drug for treating prostate cancer.

[0028] The sixth aspect of the present invention provides a method for screening candidate drugs for treating prostate cancer, the method comprising treating a culture system expressing or containing the LACTB gene or a protein encoded by it with a substance to be screened; and detecting the expression or activity of the LACTB gene or a protein encoded by it in the system; wherein, when the substance to be screened inhibits the expression level or activity of the LACTB gene or the protein encoded by it, the substance to be screened is a candidate drug for treating prostate cancer.

[0029] A seventh aspect of the present invention provides a system / device for diagnosing prostate cancer / predicting the prognosis of prostate cancer, the system / device comprising:

[0030] 1) Analysis unit: The analysis unit is used to detect the expression level of LACTB in a sample of a subject;

[0031] 2) Evaluation unit: The evaluation unit comprises a stored reference and a data processor, wherein the data processor has implemented an algorithm for comparing the expression level of LACTB detected by the analysis unit with the stored reference, thereby diagnosing prostate cancer / predicting the prognosis of prostate cancer.

[0032] The eighth aspect of the present invention provides a computer-readable storage medium, on which a computer program is stored. When the computer program is executed by a processor, the system / device described in the seventh aspect of the present invention is implemented.

[0033] A ninth aspect of the present invention provides a prostate cancer prognosis model, wherein the detection index of the model includes the expression level of LACTB.

[0034] Furthermore, the detection indicators of the model also include T stage, N stage, age, race, and PSA.

[0035] Furthermore, the model is a nomogram model.

[0036] The tenth aspect of the present invention provides the use of LACTB in constructing a model for diagnosing prostate cancer / predicting the prognosis of prostate cancer.

[0037] Furthermore, the model for predicting the prognosis of prostate cancer is a nomogram model.

[0038] Furthermore, the detection indicators of the nomogram model include T stage, N stage, age, race, and PSA.

[0039] Advantages and beneficial effects of the present invention:

[0040] The present invention discovered for the first time the correlation between LACTB and prostate cancer, which can be used for early diagnosis of prostate cancer, thereby achieving effective treatment for patients, making effective predictions, and improving the quality of life of patients, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the result of immunohistochemical staining test;

[0042] Figure 2 is the LACTB protein expression graph, where 2A is the immunoblot graph and 2B is the LACTB protein expression difference bar graph;

[0043] Figure 3 3A is a graph showing the results of the scratch test and invasion test of prostate cancer by LACTB, wherein 3A is a graph showing the results of the scratch test, 3B is a graph showing the statistical histogram of the scratch test, 3C is a graph showing the results of the invasion test, and 3D is a graph showing the statistical histogram of the invasion test;

[0044] Figure 4 4A is a graph of LACTB expression level bioinformatics analysis, wherein 4A is a graph of LACTB expression level in prostate cancer, and 4B is a graph of LACTB expression level in prostate cancer in paired samples;

[0045] Figure 5 is the ROC curve plot of LACTB prediction in prostate cancer patients;

[0046] Figure 6 is a nomogram for LACTB in prostate cancer patients. DETAILED DESCRIPTION

[0047] The definitions of some terms used in this specification are provided below. Unless otherwise defined, 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 belongs.

[0048] LACTB includes wild type, mutant or fragment thereof. The term encompasses full length, unprocessed LACTB, as well as any form of LACTB derived from processing in cells. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of LACTB. The term encompasses, for example, human LACTB as well as LACTB from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats), gene ID: 114294.

[0049] The terms "diagnosis," "diagnostic," "diagnosing," and variations of these terms refer to the discovery, judgment, or knowledge of an individual's health state or condition based on one or more signs, symptoms, data, or other information associated with the individual. An individual's health state may be diagnosed as healthy / normal (i.e., the absence of a disease or disorder), or may be diagnosed as unhealthy / abnormal (i.e., the presence of a disease or disorder or an assessment of a characteristic). The above terms "diagnosis," "diagnosing," "diagnosing," etc. include early detection of a disease in relation to a specific disease or disorder; the characterization or classification of a disease; the discovery of the progression, cure, or recurrence of a disease; and the discovery of an individual's response to a disease following treatment or therapy.

[0050] The term "prognosis" refers to the expectation regarding medical development (e.g., likelihood of long-term survival, disease-free survival rate, etc.), and includes a positive prognosis or a negative prognosis, wherein the negative prognosis includes disease progression such as recurrence, tumor growth, metastasis, and drug-resistant mortality, and the positive prognosis includes disease remission such as a disease-free state, and disease improvement such as tumor regression or stabilization.

[0051] The term "probe" refers to a molecule that can bind to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, a "probe" generally refers to a polynucleotide probe that can bind to another polynucleotide (often referred to as 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. The probe can be directly or indirectly labeled, including primers. Hybridization methods include, but are not limited to, solution phase, solid phase, mixed phase or in situ hybridization assays.

[0052] As the probe, a labeled probe in which a polynucleotide for cancer detection is labeled with a fluorescent label, a radioactive label, a biotin label, etc. can be used. The labeling method of polynucleotides itself is well known. The presence of the test nucleic acid in the sample can be checked by the following method: fix the test nucleic acid or its amplification product, hybridize with the labeled probe, wash, and measure the label bound to the solid phase. Alternatively, the polynucleotide for cancer detection can also be fixed, the test nucleic acid is hybridized with it, and then the test nucleic acid bound to the solid phase is detected using a labeled probe, etc. In this case, the polynucleotide for cancer detection bound to the solid phase is also referred to as a probe. The method of measuring the test nucleic acid using a polynucleotide probe is also well known in the art. The method can be performed as follows: the polynucleotide probe is brought into contact with the test nucleic acid at Tm or near it (preferably within ±4°C) in a buffer for hybridization, washed, and then the hybridized labeled probe or the template nucleic acid bound to the solid phase probe is measured.

[0053] The size of the polynucleotide used as a probe is preferably 18 or more nucleotides, more preferably 20 or more nucleotides, and the full length of the coding region or less. When used as a primer, the polynucleotide size is preferably 18 or more nucleotides, and 50 or less nucleotides. These probes have a base sequence that is complementary to a specific base sequence of the target gene. Here, the so-called "complementary" only requires hybridization, and may not be completely complementary. These polynucleotides usually have a homology of more than 80%, preferably more than 90%, more preferably more than 95%, and particularly preferably 100% relative to the specific base sequence. These probes can be DNA or RNA, and can also be polynucleotides in which nucleotides in part or all of them are replaced by artificial nucleic acids such as PN, LNA, ENA, GNA, TNA, etc.

[0054] The term "amplification" generally refers to the process of generating multiple copies of a desired sequence.

[0055] The term "primer" means an oligonucleotide, whether naturally present in a purified restriction digest or produced synthetically, which can serve as a starting point for synthesis when placed under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid chain, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer can be single-stranded or double-stranded, and must be long enough to initiate the synthesis of an expected extension product in the presence of an inducing agent. The exact length of the primer depends on many factors, including temperature, primer source, and method of use. For example, for diagnostic applications, depending on the complexity of the target sequence, an oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. The factors involved in determining the appropriate length of the primer are readily known to those skilled in the art.

[0056] Primer or probe of the present invention can use phosphoramidite solid phase support method or other well-known method chemical synthesis.Also can use many means known in the art to modify the nucleotide sequence.Non-limiting examples of these modifications are methylation, capping, displacement and modification between nucleotides with one or more analogs of natural nucleotides, for example, modification of uncharged linker (for example, methyl phosphate, phosphotriester, phosphoramidite, carbamate etc.), or modification of charged linker (for example, phosphorothioate, phosphorodithioate etc.).

[0057] The term "binding agent" includes a receptor for protein LACTB, a lectin that binds to protein LACTB, an antibody to protein LACTB, a peptide antibody to protein LACTB, a bispecific dual binding agent or a bispecific antibody format. Specific binding agents include, but are not limited to, peptides, peptide mimetics, aptamers, spiegelmers, darpins, ankyrin repeat proteins, Kunitz-type domains, antibodies, single domain antibodies and monovalent antibody fragments.

[0058] The term "expression level" or "expressed level" generally refers to the amount of a biomarker in a biological sample. "Expression" generally refers to the process by which information (e.g., genetic coding and / or epigenetic information) is converted into structures that exist and operate in a cell. Therefore, as used herein, "expression" may refer to transcription into polynucleotides, translation into polypeptides, or even polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of polypeptides). Fragments of transcribed polynucleotides, translated polypeptides, or polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of polypeptides) should also be considered expressed, whether they are derived from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of polypeptides (e.g., by proteolysis). "Expressed genes" include genes that are transcribed into polynucleotides (e.g., mRNA) and then translated into polypeptides, as well as genes that are transcribed into RNA but not translated into polypeptides (e.g., transport and ribosomal RNA).

[0059] The term "chip", also referred to as "array", refers to a solid support comprising connected nucleic acid or peptide probes. An array typically comprises a plurality of different nucleic acid or peptide probes attached to a substrate surface at different known locations. These arrays, also referred to as "microarrays", can typically be produced using mechanical synthesis methods or light-guided synthesis methods that incorporate a combination of photolithography and solid phase synthesis methods. The array may comprise a flat surface, or may be a nucleic acid or peptide on a bead, gel, polymer surface, fiber such as an optical fiber, glass, or any other suitable substrate. The array may be packaged in a manner that allows for diagnosis of a fully functional device or other manipulation.

[0060] The term "microarray" is an orderly arrangement of hybridization array elements such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies) on a substrate. The substrate can be a solid substrate, for example, a glass or silica slide, a bead, a fiber optic binder, or a semi-solid substrate, such as a nitrocellulose membrane. The nucleotide sequence can be DNA, RNA, or any arrangement thereof.

[0061] The term "kit" refers to a set of components provided in the context of a system for sequencing nucleotides 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. Such systems may include, for example, systems that allow for storage, confirmation, or delivery of expressed genes from one location to another in one or more cells (e.g., oligonucleotides in appropriate containers, oligonucleotides encoding enzymes, extracellular matrix components) and / or supporting materials (e.g., buffers, culture media, cells, written instructions for performing assays).

[0062] In the present invention, the kit includes reagents for detecting LACTB gene or protein, one or more substances selected from the following group: container, instruction manual, positive control, negative control, buffer, adjuvant or solvent. The components of the kit can be packaged in the form of aqueous medium or in lyophilized form. Suitable containers in the kit usually include at least one vial, test tube, flask, PET bottle, syringe or other container, in which one component can be placed, and preferably, it can be appropriately divided. When there are more than one component in the kit, the kit will usually also include a second, third or other additional container, in which the additional components are placed separately. However, different combinations of components can be contained in a vial. The kit of the present invention will also usually include a container for containing reactants, sealed for commercial sale. Such a container may include an injection molded or blow molded plastic container, in which the required vials can be retained.

[0063] In an embodiment of the present invention, the kit includes but is not limited to a qPCR kit, an ELISA kit, an immunoblotting detection kit, an immunochromatography detection kit, an immunohistochemistry detection kit, a flow cytometry analysis kit, and an electrochemiluminescence detection kit.

[0064] The term "nucleic acid membrane strip" includes a substrate and oligonucleotide probes fixed on the substrate; the substrate can be any substrate suitable for fixing oligonucleotide probes, including but not limited to nylon membrane, nitrocellulose membrane, polypropylene membrane, glass slide, silica gel wafer, micro-magnetic beads.

[0065] The term "pharmaceutical composition" can be prepared using various additives such as buffers, stabilizers, bacteriostats, isotonic agents, chelating agents, pH control agents and surfactants.

[0066] The pharmaceutical composition of the present invention can be administered orally, parenterally, by inhalation spray, topical, rectal, nasal, buccal, vaginal or by implanted drug storage device. The pharmaceutical composition of the present invention can contain any commonly used non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle. In some cases, medicinal acid, alkali or buffer can be used to adjust the pH of the preparation to improve the stability of the compound or its dosage form. The term parenteral used in the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intracorporeal, intralesional, intralesional, and intracranial injection or infusion techniques. As long as the target tissue can be reached, the pharmaceutical composition of the present invention can be given to the receptor by any route.

[0067] The term "inhibitor" refers to any substance that can reduce the activity of LACTB protein, reduce the stability of LACTB gene or protein, down-regulate the expression of LACTB protein, reduce the effective action time of LACTB protein, or inhibit the transcription and translation of LACTB gene. These substances can be used in the present invention as substances useful for down-regulating LACTB, and thus can be used to prevent or treat osteoarthritis.

[0068] In the present invention, the inhibitors include nucleic acid inhibitors, protein inhibitors, proteolytic enzymes, and protein binding molecules. The nucleic acid inhibitors are selected from: interfering molecules that target LACTB or its transcripts and can inhibit LACTB gene expression or gene transcription, including: shRNA (small hairpin RNA), small interfering RNA (siRNA), dsRNA, microRNA, antisense nucleic acid, or constructs that can express or form the shRNA, small interfering RNA, dsRNA, microRNA, and antisense nucleic acid. The protein binding molecules are selected from: substances that specifically bind to the LACTB protein, such as antibodies or ligands that can inhibit the activity of the LACTB protein.

[0069] The term "pharmaceutically acceptable carrier" may include inert ingredients that do not unduly inhibit the biological activity of the compound. A pharmaceutically acceptable carrier should be biocompatible, e.g., non-toxic, non-inflammatory, non-immunogenic, or non-other undesirable reactions or side effects when administered to a subject. Standard pharmaceutical formulation techniques may be used.

[0070] Some examples of substances that can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as tween 80, phosphates, glycine, sorbic acid or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride or zinc salts), colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, methylcellulose, hydroxypropyl methylcellulose, lanolin; sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as Sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol, and phosphate buffered solutions, and other nontoxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and colorants, release agents, coating agents, sweeteners, flavoring agents and perfuming agents, preservatives and antioxidants may also be present in the composition, according to the judgment of the formulator.

[0071] Any oral acceptable dosage form (including but not limited to capsules, tablets, aqueous suspensions or solutions) can be used for oral administration. For tablets for oral administration, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are typically also added. For oral administration in capsule form, available diluents include lactose and dry corn starch. When an aqueous suspension is required for oral administration, the active ingredient is combined with an emulsifier and a suspending agent. If necessary, certain sweeteners, flavorings or coloring agents may also be added.

[0072] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage form may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and aromatics.

[0073] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is mixed with at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and silicic acid, b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic, c) humectants such as glycerol, d) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, e) solution retardants such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glyceryl monostearate, h) absorbents such as kaolin and bentonite, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0074] Solid compositions of similar type may also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols, etc. Solid dosage forms of tablets, lozenges, capsules, pills and granules may be prepared with coatings and shells, such as enteric coatings and other coatings well known in the field of pharmaceutical formulations. They may optionally contain opacifiers and may also have a composition that allows them to release the active ingredient only or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymers and waxes. Solid compositions of similar type may also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols, etc.

[0075] Microencapsulated forms with one or more of the above excipients can also be used in the present invention. Solid dosage forms of tablets, lozenges, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings, controlled release coatings and other coatings well known in the field of pharmaceutical formulation. In such solid dosage forms, the active compound can be mixed with at least one inert diluent such as sucrose, lactose or starch. As is common practice, such dosage forms can also contain additional substances that are not inert diluents, such as tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. For capsules, tablets and pills, the dosage form can also include a buffer. They can optionally contain an opacifier and can also have a composition that allows them to release the active ingredient only or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymers and waxes.

[0076] The term "subject" or "patient" refers to an animal capable of suffering from or having prostate cancer. Examples of subjects include mammals, e.g., humans, non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals.

[0077] In an embodiment of the invention, the subject is a human.

[0078] The term "computer-readable storage medium", such as computer executable code, can take many forms, including but not limited to tangible storage media, carrier media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, such as any storage device in any computer, and volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wires and optical fibers, including wires that constitute the bus within a computer system. Carrier transmission media can take the form of electrical or electromagnetic signals or sound or light waves, such as those generated during radio frequency and infrared data communications. Therefore, common forms of computer-readable media include, for example: floppy disks, floppy disks, hard disks, magnetic tapes, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punched card paper tapes, any other physical storage media with hole patterns, RAM, ROMs, PROMs and EPROMs, FLASH-EPROMs, any other memory chips or boxes, carriers that transmit data or instructions, cables or links that transmit such carriers, or any other media from which a computer can read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0079] The term "nomogram model", also known as the Nomogram model, is a clinical prediction model based on multi-factor regression analysis. It integrates multiple prediction indicators and then uses scaled line segments to draw them on the same plane at a certain ratio, so as to express the relationship between the variables in the prediction model and evaluate the risk of disease. The nomogram can transform complex regression equations into visual graphics, making the results of the prediction model more readable and convenient for evaluating subjects. It is widely used in medical research and clinic.

[0080] In an embodiment of the present invention, the nomogram includes twelve straight lines, which are arranged in sequence from top to bottom and are parallel to each other, and each straight line represents a scale with scale marks;

[0081] The first line is a score scale with a value range of 0-100;

[0082] The second line is the T staging scale. When the staging is T2, the score is 0, and when the staging is T3 or T4, the score is 11;

[0083] The third line is the N-stage scale. When the stage is N0, the score is 0, and when the stage is N1, the score is 26;

[0084] The fourth line is the age scale, when age is ≤60, the score is 0, when age is >60, the score is 17.5;

[0085] The fifth line is the racial scale, where the score is 0 when the race is Asian, 92 when the race is Caucasian, and 100 when the race is Black;

[0086] The sixth line is the PSA scale. When PSA < 4, the score is 0, and when PSA ≥ 4, the score is 32.5;

[0087] The seventh line is the LACTB scale. When LACTB is 1.5, the score is 0; when LACTB is 2.5, the score is 16; when LACTB is 3.5, the score is 32; when LACTB is 4.5, the score is 47.5; when LACTB is 5.5, the score is 63;

[0088] The eighth line is the total score scale, with a value range of 0-240;

[0089] The ninth line is the linear prediction period scale, with a value range of -7-4;

[0090] The tenth line is the 3-year survival rate scale, with a value range of 0.6-0.8;

[0091] The eleventh line is a 5-year survival rate scale, with a numerical range of 0.6-0.8;

[0092] The twelfth line is the 10-year survival rate scale, with a value of 0.5.

[0093] The term "PSA" stands for prostate-specific antigen, a marker primarily used to detect prostate cancer.

[0094] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0095] Example 1 Expression level of LACTB in prostate cancer

[0096] 1.1 Experimental Materials

[0097] Samples: 43 cases of prostate cancer tissues and 43 cases of adjacent cancer tissues, collected from March 1, 2021 to March 1, 2022 at the Second Affiliated Hospital of Dalian Medical University.

[0098] Reagents: The primary antibody LACTB was purchased from Abcam, UK; PBS, peroxidase, secondary antibody, and DAB colorimetric reagent kit were all purchased from Fuzhou Maixin Biotechnology Co., Ltd.

[0099] 1.2 Experimental methods

[0100] 1.2.1 Immunohistochemical staining

[0101] The specimens were fixed with 10% neutral formalin and embedded in paraffin, and then cut into 4 μm sections. The sections were removed by immunohistochemistry film and baked at 60℃ for 1 hour. The Max Vision method was used for immunohistochemistry.

[0102] 1) Dewaxing and hydration: Place the paraffin sections in a 55°C float chamber, bake the sections for 1 hour, dewax them in xylene I and xylene II for 10 minutes each, and hydrate them by soaking them in gradient alcohol (100% I, 100% II, 95%, 85%, 70%) for 2 minutes each;

[0103] 2) Antigen retrieval: rinse gently with water for 10 minutes, add 0.01M sodium citrate micro-liquid and incubate at room temperature for 20 minutes, boil in boiling water for 15 minutes, cool naturally, and soak in PBS for 5 minutes, repeat three times;

[0104] 3) Removal of catalase: add 3% H2O2 and incubate for 10 min in dark to block the activity of endogenous peroxidase, soak in PBS for 5 min, repeat three times;

[0105] 4) Blocking: add 3% BSA blocking solution and block for 30 minutes at room temperature;

[0106] 5) Apply primary antibody: add primary antibody (dilution ratio is 1:100). Put it in a plastic box, cover it, and place it at 4℃ overnight;

[0107] 6) Apply secondary antibody: Soak in PBS for 5 minutes, repeat three times, add secondary antibody (dilution ratio is 1:200) and incubate at 37℃ for 30 minutes;

[0108] 7) DAB color development: Soak in PBS for 5 min, repeat three times, add DAB color development solution (dilution ratio is 1 ml solution I: 1 drop solution II), and develop color in the dark;

[0109] 8) Nuclear staining: rinse slowly with water for 10 minutes, add hematoxylin and incubate for 20 seconds;

[0110] 9) Dehydration: rinse slowly with water for 10 minutes, and soak in gradient alcohol (70%, 85%, 90%, 100% II, 100% I) for 2 minutes each;

[0111] 10) Sealing and observation: Soak in xylene II and xylene I for 2 min each, add neutral gum, cover with a coverslip, leave at room temperature for 24 h, and observe and photograph under an upright microscope.

[0112] Under the microscope, the presence of brown-yellow particles in the tumor cytoplasm was recorded as positive cells. When the ratio of the number of positive cells to the total number of tumor cells was ≥40%, it was considered positive expression, otherwise it was considered negative expression.

[0113] The score of staining intensity was defined as: negative 0, weak 1, moderate 2, strong positive 3; the frequency of positive cells was defined as: when the number of positive cells was less than 5%, the score was 0, when the number of positive cells reached 26%-50%, the score was 2, when the number of positive cells reached 51%-75%, the score was 3, when the number of positive cells was greater than 75%, the score was 4. Finally, a comprehensive score was calculated.

[0114] 1.2.2 BCA protein quantification and preparation

[0115] 1) Prepare the required AB solution (A:B=50:1) according to the amount of sample;

[0116] 2) Take a 96-well plate and add 20, 18, 16, 12, 4 μl of deionized water and 0, 2, 4, 8, 16 μl of 0.5 μg / μl standard protein to the standard protein wells in sequence, and add 19 μl of deionized water and 1 μl of sample protein to the sample wells;

[0117] 3) Add 180 μl of AB solution to each well and mix well. Incubate at 37°C in the dark for 30 min, and measure the absorbance at 589 nm using an ELISA reader.

[0118] 4) Prepare a standard curve based on the measured absorbance value and calculate the corresponding sample protein content;

[0119] 5) Take the sample protein, add 1 / 4 volume of 5× Loading Buffer, mix well, boil in boiling water for 10 minutes, and store at -20℃ for later use.

[0120] 1.2.3 Western Blot Detection

[0121] 1) SDS-PAGE electrophoresis: prepare 10% separation gel and 5% stacking gel, load 30 μg protein solution into each well, add electrophoresis solution, perform electrophoresis at 60V constant voltage for 30 min, and perform electrophoresis at 120V constant voltage until the bottom (about 1 h);

[0122] 2) Transfer: remove the concentrated gel and useless separation gel, cut the PVDF membrane and three-layer filter paper of appropriate size (make sure membrane>gel>filter paper), put the PVDF membrane into methanol for activation for 15 seconds, set the transfer device from anode to cathode in the order of sponge, three-layer filter paper, PVDF membrane, gel, three-layer filter paper, sponge, and transfer the membrane in an ice bath at a constant current of 220mA for 1 hour;

[0123] 3) Blocking: Take out the PVDF membrane and place it in 5% skim milk powder (prepared in TBST) and block it at room temperature for 3 hours;

[0124] 4) Antibody incubation: The PVDF membrane was washed with TBST for 5 min, the PVDF membrane was cut and incubated with rabbit anti-ST6Gal-I (1:500, diluted in TBST) or GAPDH (1:5000, diluted in TBST) primary antibody at 4°C overnight, then the PVDF membrane was washed three times with TBST for 15 min each time, the PVDF membrane was incubated with anti-rabbit secondary antibody (1:8000, diluted in TBST) at room temperature for 1 h, and washed three times with TBST for 15 min each time;

[0125] 5) ECL luminescence: Add the newly prepared ECL luminescence solution (solution A: solution B = 1:1) onto the PVDF membrane, image it using a gel imager, and save it;

[0126] 6) Analysis: The bands were analyzed using Image Lab software.

[0127] 1.3 Experimental Results

[0128] The LACTB content in normal prostate tissue and prostate cancer tissue was detected by immunohistochemical staining. LACTB protein was expressed in the plasma of prostate cancer cells. The expression in cancer tissue was significantly higher than that in adjacent normal tissue ( Figure 1 , P<0.05).

[0129] WB results showed that the expression level of LACTB in prostate cancer tissues was significantly increased compared with that in normal prostate tissues ( Figure 2 ).

[0130] Example 2 Effect of targeted inhibition of LACTB on migration and invasion of prostate cancer cells

[0131] 2.1 Experimental Materials

[0132] Cell line: PC-3 human prostate cancer cell line, cultured in PC-3 complete medium with 10% fetal bovine serum (FBS) at 37°C and 5% CO2.

[0133] 2.2 Experimental methods

[0134] 2.2.1 Construction of LACTB transfection vector

[0135] Specific small interfering RNA (siRNA) targeting LACTB was synthesized and transiently transfected according to the instructions. The silencing level was detected by Western blot 48 hours later, and the stably transfected cells were screened.

[0136] sequence:

[0137] LACTB-homo-520

[0138] Sense5'-3':CCAUGGUUGCUCUUGCCAATT(SEQ ID NO:1)

[0139] Antisense5'-3':UUGGCAAGAGCAACCAUGGTT(SEQ ID NO:2)

[0140] LACTB-homo-1229

[0141] Sense5'-3':CCUUCUGAAAUUUGGGAAUTT(SEQ ID NO:3)

[0142] Antisense5'-3':AUUCCCAAAUUUCAGAAGGTT(SEQ ID NO:4)

[0143] 2.2.2 Targeted inhibition of LACTB regulates the migration ability of PC-3 human prostate cancer cells

[0144] 1) Prepare ruler and marker pen and sterilize them under UV light;

[0145] 2) Seal the 6-well plate with sealing film, place it upside down on the clean bench, and use a marker pen to draw parallel and equidistant lines along the diameter of the 6-well plate for positioning;

[0146] 3) PC-3 cells were routinely cultured in DMEM medium containing 10% FBS. Cells in the logarithmic growth phase were taken, digested, and the cell density was adjusted to 2×10 5 Add the above cell suspension to a 6-well plate, with a volume of 2 mL per well, and place in a 37°C, 5% CO2 incubator for continued culture. Observe the cells until they are in a fusion state, then scratch them;

[0147] 4) Use a 100μL sterile pipette tip to scratch the wells perpendicular to the pre-positioned horizontal line. The pipette tip should be placed perpendicular to the 6-well plate to ensure that the width of the scratch is consistent. After the scratch is completed, slowly aspirate the culture medium with a gun, add an appropriate amount of PBS to wash the cells 3 times to remove cell debris;

[0148] 5) Observe the cell scratch healing at 0 and 24 hours respectively and take photos;

[0149] 2.2.3 Targeted inhibition of LACTB regulates the invasive ability of PC-3 human prostate cancer cells

[0150] 1) Place the Matrigel matrix gel in a 4°C refrigerator overnight to make the Matrigel gel become liquid;

[0151] 2) Dilute Matrigel matrix gel at a ratio of 1:8 using serum-free medium, and then place the Transwell chamber in a 24-well plate;

[0152] 3) Add 100 μl of diluted Matrigel to the Transwell chamber and place it in a cell culture incubator until it solidifies;

[0153] 4) PC-3 cells in the logarithmic growth phase transfected with siNC and siLACTB were digested with trypsin and mixed by pipetting to prepare a single cell suspension;

[0154] 5) Count the cells and adjust the cell density to 5×10 5 cells / ml;

[0155] 6) Place the Transwell chamber into a 24-well plate, add 750 μl of complete culture medium containing 10% FBS into the 24-well plate, add 200 μl of the diluted cell suspension into the Transwell chamber, and culture in a cell culture incubator;

[0156] 7) After 48 h of culture, the chamber was removed, gently washed with PBS, and the cells on the inner membrane of the chamber were wiped off with a cotton swab;

[0157] 8) Add 4% paraformaldehyde to new wells of the 24-well plate and place the chamber in the paraformaldehyde for fixation for 15 minutes;

[0158] 9) Take out the Transwell chamber and wash it with PBS for 5 minutes, then stain it with crystal violet for 10 minutes;

[0159] 10) Remove the staining solution, wash the Transwell chamber with clean water, place it under an inverted microscope to take pictures and select 5 different fields of view to count the number of cells that pass through the chamber membrane, calculate the average value, and draw a bar graph.

[0160] 2.3 Experimental Results

[0161] After transfection with LACTB, the number of PC-3 cells passing through the Transwell chamber was significantly reduced, indicating that targeted inhibition of LACTB can inhibit the migration and invasion of prostate cancer cells ( Figure 3 ).

[0162] Example 3 Application of LACTB in predicting prostate cancer prognosis

[0163] 3.1 Experimental methods

[0164] Bioinformatics analysis of the prostate cancer patient variable LACTB was performed using the TCGA database (https: / / portal.gdc.cancer.gov / ) using R software (version 3.6.3), using the pROC package (version 1.17.0.1), ggplot2 package (version 3.3.3), rms package (version 6.2-0), and survival package (version 3.2-10), and using the statistical methods Mann-Whitney U test, paired sample T test, Log-rank test, and cox regression analysis.

[0165] 3.2 Experimental Results

[0166] The results of Mann-Whitney U test showed that the expression level of LACTB in prostate cancer was higher than that in adjacent tissues and higher than that in Normal tissues. The median difference between the two groups was 0.392 (0.24-0.552), and the difference was statistically significant (P<0.001). The paired sample T test showed that the expression level of LACTB in prostate cancer was higher than that in adjacent tissues. The median difference between the two groups was 0.408 (0.253-0.563), and the difference was statistically significant (t=5.293, P<0.001). Figure 4 ).

[0167] The ROC curve shows that the variable LACTB has a certain degree of accuracy in predicting the outcomes of normal prostate and prostate cancer (AUC = 0.708, CI = 0.641-0.776 ( Figure 5 ).

[0168] A nomogram was established to predict the survival prognosis of prostate cancer patients with high accuracy ( Figure 6 ).

[0169] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of a reagent for detecting LACTB in the preparation of a product for diagnosing prostate cancer.

2. The use according to claim 1, characterized in that: The reagent is selected from an oligonucleotide probe that specifically recognizes the LACTB gene, a primer that specifically amplifies the LACTB gene, or a binding agent that specifically binds to a protein encoded by the LACTB gene.

3. The use according to claim 1, characterized in that: The product includes a chip, a test kit or a nucleic acid membrane strip.

4. The use according to claim 3, characterized in that: The chip includes a gene chip and a protein chip. The gene chip includes an oligonucleotide probe for the LACTB gene for detecting the transcription level of the LACTB gene, and the protein chip includes a specific binding agent for the LACTB protein; the kit includes a gene detection kit and a protein detection kit. The gene detection kit includes a reagent or a chip for detecting the transcription level of the LACTB gene, and the protein detection kit includes a reagent or a chip for detecting the expression level of the LACTB protein.

5. The use according to claim 3, characterized in that: The kit comprises reagents for detecting the expression level of LACTB gene or protein by RT-PCR method, qRT-PCR method, biochip detection method, Southern blotting method, in situ hybridization method and immunoblotting method.

6. Use of LACTB inhibitors in the preparation of pharmaceutical compositions for treating prostate cancer; The inhibitor is siRNA, and the siRNA is selected from: LACTB-homo-520 Sense5'-3': CCAUGGUUGCUCUUGCCAATT Antisense5'-3':UUGGCAAGAGCAACCAUGGTT or LACTB-homo-1229 Sense5'-3':CCUUCUGAAAUUUGGGAAUTT Antisense5'-3':AUUCCCAAAUUUCAGAAGGTT.

7. The use according to claim 6, characterized in that: The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

8. A system / device for diagnosing prostate cancer, characterized in that: The system / device comprises: 1) Analysis unit: The analysis unit is used to detect the expression level of LACTB in the subject sample; 2) Evaluation unit: The evaluation unit comprises a stored reference and a data processor, wherein the data processor has implemented an algorithm for comparing the expression level of LACTB detected by the analysis unit with the stored reference, thereby diagnosing prostate cancer.

9. A computer-readable storage medium, characterized in that: The storage medium stores a computer program, and when the computer program is executed by the processor, the system / device of claim 8 is implemented.

10. A device for a prostate cancer prognostic nomogram model, characterized in that: The detection indexes of the nomogram model include the expression level of LACTB, T stage, N stage, age, race and PSA. Among them, when the stage is T2, the score is 0, and when the stage is T3 or T4, the score is 11; when the stage is N0, the score is 0, and when the stage is N1, the score is 26; when the age is ≤60, the score is 0, and when the age is >60, the score is 17.5; when the race is Asian, the score is 0, when the race is Caucasian, the score is 92, and when the race is black, the score is 100; when PSA <4 ng / ml, the score is 0, when PSA ≥4ng / ml, the score is 32.5; when LACTB is 1.5, the score is 0, when LACTB is 2.5, the score is 16, when LACTB is 3.5, the score is 32, when LACTB is 4.5, the score is 47.5, and when LACTB is 5.5, the score is 63.

11. Application of LACTB in the preparation of a device for diagnosing prostate cancer.

Images