CN117144006A

CN117144006A - LACTB gene and its application in diagnosing prostate cancer

Info

Publication number: CN117144006A
Application number: CN202311044862.XA
Authority: CN
Inventors: 范博; 刘志宇; 王黎; 马爽; 李潇; 腾奇亮; 王禹潼; 陈思杉
Original assignee: Second Hospital of Dalian Medical University
Current assignee: Second Hospital of Dalian Medical University
Priority date: 2023-08-18
Filing date: 2023-08-18
Publication date: 2023-12-01
Anticipated expiration: 2043-08-18
Also published as: CN117144006B

Abstract

The invention discloses a LACTB gene and application thereof in diagnosing prostate cancer. The invention provides application of a reagent for detecting LACTB in preparing a product for diagnosing/predicting prognosis of prostate cancer and a product for diagnosing/predicting prognosis of prostate cancer, and 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 invention can be used for early diagnosis and effective treatment of the prostate cancer, and can be used for prognosis prediction of the prostate cancer, so that the LACTB marker has a wide application prospect.

Description

LACTB gene and application thereof in diagnosing prostate cancer

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a LACTB gene and application thereof in diagnosing prostate cancer.

Background

Prostate cancer (PCa) is one of the most common urinary system malignant tumors in our country at present, and is the second leading cause of cancer in men worldwide, especially in elderly men. In a statistical study on global cancers, the incidence and mortality rate of the cancers are found to rise year by year, accounting for more than 90% of the neoplastic diseases of the reproductive organs.

The characteristics of multi-step and multi-gene recombination are shown in the evolution process of the prostate cancer. In the early stage of prostate cancer, malignant lesions appear in the prostate capsule, but no special clinical manifestation exists, and even if the symptoms are uncomfortable, the symptoms are often confused with frequent urination, urgent urination and the like caused by the conventional diseases of the prostate. In general, the patients are diagnosed until the patients have obvious symptoms such as edema and numbness of lower limbs, bone pain caused by bone metastasis, renal failure caused by bilateral ureter obstruction and the like, namely, the patients develop advanced stages of prostate cancer. When the tumor is confined to the prostate, it is considered possible to cure, and once the tumor breaks through the prostate capsule and spreads and metastasizes, the patient's treatment can be very tricky and the prognosis can be relatively poor. Therefore, mortality in prostate cancer patients is generally due to unobtainable and timely effective treatment, resulting in tumor metastasis leading to mortality from other diseases.

At present, the traditional prostate puncture brings great pain to patients in the screening process. Prostate specific antigen (Prostate specific antigen, PSA), which is predominantly present in prostate tissue, is the most widely used index for prostate cancer screening, diagnosis and prognosis worldwide. However, more and more researches show that the index has more limitations on prostate cancer diagnosis, and the index brings economic and physical double injuries to most patients. Early diagnosis and therapeutic effects of prostate cancer are not optimistic due to limitations in existing medical levels, complexity of clinical detection of prostate cancer, and blindness of clinical manifestations. This presents a great threat to human health and social development, as well as a greater challenge in the treatment of prostate cancer. The clinical early screening, treatment and prognosis of prostate cancer is of self-evident importance.

The current restrictions on medical level, clinical complexity of prostate cancer, unknown pathogenesis of prostate cancer, etc., result in lower cure rate and survival rate for prostate cancer patients. Biomarkers can then timely predict whether a patient is cancerous at an early stage and prognosis to assist the clinician in the selection of treatments. However, from the current research progress, the number of biomarkers clinically applied to early screening of prostate cancer and producing good effects is small and limited. None of the potential prostate cancer biomarkers at the study stage passed or were clinically validated and detected. Therefore, biomarkers which can be used for guiding the diagnosis and prognosis of the prostate cancer are screened, so that the prostate cancer patients can be effectively treated, and the method has important significance for improving the survival quality of the patients.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention provides a biomarker for diagnosing/predicting the prognosis of the prostate cancer, and realizes early diagnosis, effective treatment and prognosis prediction of the prostate cancer.

In order to achieve the above purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides the use of an agent for detecting LACTB in the manufacture of a product for diagnosing/prognosticating prostate cancer.

Further, the agent is selected from the group consisting of 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.

In a second aspect the invention provides a product for diagnosing/prognosticating prostate cancer, said product comprising an agent capable of detecting the level of expression of LACTB.

Further, the product comprises a chip, a kit or a nucleic acid membrane strip.

Further, the chip comprises a gene chip comprising an oligonucleotide probe for the LACTB gene for detecting the transcription level of the LACTB gene, a protein chip comprising a specific binding agent for the LACTB protein; the kit comprises a gene detection kit and a protein detection kit, wherein the gene detection kit comprises a reagent or a chip for detecting the transcription level of the LACTB gene, and the protein detection kit comprises a reagent or a chip for detecting the expression level of the LACTB protein.

Further, the kit comprises reagents for detecting the expression level of the LACTB gene or protein by an RT-PCR method, a qRT-PCR method, a biochip detection method, a southern blotting method, an in situ hybridization method and an immunoblotting method.

In a third aspect the invention provides a pharmaceutical composition for use in the treatment of prostate cancer, the pharmaceutical composition comprising an inhibitor of LACTB.

Further, the inhibitor reduces the expression level of LACTB.

Further, the inhibitors include nucleic acid inhibitors, protein inhibitors.

Further, the nucleic acid inhibitor comprises shRNA, siRNA.

Further, the protein inhibitors include antibodies or ligands that inhibit the activity of the LACTB protein.

Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In a fourth aspect, the invention provides the use of LACTB in the manufacture of a pharmaceutical composition for the treatment of prostate cancer.

Further, the inhibitor reduces the expression level of LACTB.

Further, the inhibitors include nucleic acid inhibitors, protein inhibitors.

Further, the nucleic acid inhibitor comprises shRNA, siRNA.

In a fifth aspect, the invention provides the use of LACTB in the screening of candidate agents for the treatment of prostate cancer.

Further, 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 the LACTB gene with a substance to be screened; and detecting the expression or activity of the LACTB gene or protein encoded thereby in the system; wherein the substance to be screened is a candidate drug for treating prostate cancer when the substance to be screened inhibits the expression level or activity of the LACTB gene or a protein encoded thereby.

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

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

1) Analysis unit: the analysis unit is used for detecting the expression level of the LACTB in the sample of the subject;

2) An evaluation unit: the evaluation unit contains a stored reference and a data processor that has implemented an algorithm for comparing the expression level of the LACTB detected by the analysis unit with the stored reference, thereby diagnosing/predicting the prognosis of prostate cancer.

An eighth aspect of the present invention provides a computer readable storage medium having stored thereon a computer program which when executed by a processor implements the system/apparatus of the seventh aspect of the present invention.

In a ninth aspect, the invention provides a prognostic model of prostate cancer, the detection indicator of which includes the level of expression of LACTB.

Further, the detection indexes of the model also comprise T stage, N stage, age, race and PSA.

Further, the model is a nomogram model.

In a tenth aspect the invention provides the use of LACTB in the construction of a model for diagnosing/predicting the prognosis of prostate cancer.

Further, the model for predicting the prognosis of prostate cancer is a nomographic model.

Further, the detection indexes of the alignment chart model comprise T stage, N stage, age, race and PSA.

The invention has the advantages and beneficial effects that:

the invention discovers the correlation between the LACTB and the prostate cancer for the first time, can be used for early diagnosis of the prostate cancer, further realizes effective treatment of patients, effectively predicts the patients, improves the survival quality of the patients, and has wide application prospect.

Drawings

FIG. 1 is a graph of immunohistochemical staining assay results;

FIG. 2 is a graph showing the expression of the LACTB protein, wherein 2A is an immunoblot graph and 2B is a histogram showing the difference in the expression of the LACTB protein;

fig. 3 is a graph of the results of a scoring experiment and an invasion experiment of the prostate cancer of LACTB, wherein 3A is a graph of the results of the scoring experiment, 3B is a statistical histogram of the scores, 3C is a graph of the results of the invasion experiment, and 3D is a statistical histogram of the invasion;

FIG. 4 is a graph of a LACTB expression level biological analysis, wherein 4A is a graph of a prostate cancer LACTB expression level and 4B is a graph of a prostate cancer LACTB expression level in a paired sample;

FIG. 5 is a graph of the predicted ROC of LACTB in a prostate cancer patient;

FIG. 6 is a nomogram in a patient with LACTB prostate cancer.

Detailed Description

The following provides definitions of some of the terms used in this specification. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

LACTB includes wild-type, mutant or fragments thereof. The term encompasses full length, unprocessed LACTB, as well as any form of LACTB derived from processing in a cell. 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.

the terms "diagnosis," "diagnostic," "diagnosing," and variations of these terms refer to the discovery, judgment, or cognition of an individual's state of health or condition based on one or more signs, symptoms, data, or other information associated with the individual. The health status of an individual may be diagnosed as healthy/normal (i.e., no disease or condition present) or may be diagnosed as unhealthy/abnormal (i.e., there is an assessment of disease or condition or characteristic). The terms "diagnosis", "diagnostic", "diagnosing" and the like include early detection of a disease in relation to a particular disease or condition; characteristics or classification of disease; discovery of progression, cure, or recurrence of disease; following treatment or therapy of an individual, a response to the disease is found.

The term "prognosis" refers to an expectation of medical development (e.g., likelihood of long-term survival, disease-free survival, etc.), including positive prognosis including disease progression such as recurrence, tumor growth, metastasis, and drug-resistant mortality, or negative prognosis including disease remission such as disease-free status, disease improvement such as tumor regression or stabilization.

The term "probe" refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a target polynucleotide) by complementary base pairing. Depending on the stringency of the hybridization conditions, the probe is able to bind to a target polynucleotide that lacks complete sequence complementarity with the probe. Probes may be labeled directly or indirectly, and include primers. Hybridization means include, but are not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

As the probe, a labeled probe in which a polynucleotide for detecting cancer is labeled with a fluorescent label, a radiolabel, a biotin label, or the like can be used. Methods for labeling polynucleotides are known per se. The test sample can be checked for the presence of the test nucleic acid by: immobilization of the test nucleic acid or its amplificates, hybridization with labeled probes, washing, and determination of the label bound to the solid phase. Alternatively, a polynucleotide for detecting cancer may be immobilized, a test nucleic acid is hybridized thereto, and then the test nucleic acid bound to the solid phase is detected using a labeled probe or the like. In this case, the polynucleotide for detecting cancer bound to the solid phase is also referred to as a probe. Methods for assaying test nucleic acids using polynucleotide probes are also well known in the art. The method can be performed as follows: the polynucleotide probe is contacted with the test nucleic acid at or near Tm (preferably within + 4 ℃) in a buffer for hybridization, washed, and then assayed for hybridized labeled probe or template nucleic acid bound to a solid phase probe.

The size of the polynucleotide used as a probe is preferably 18 nucleotides or more, more preferably 20 nucleotides or more, and the entire length of the coding region or less. When used as primers, the polynucleotide is preferably 18 nucleotides or more in size, and 50 nucleotides or less in size. These probes have a base sequence complementary to a specific base sequence of the target gene. The term "complementary" as used herein is not limited to being completely complementary as long as it is hybridized. These polynucleotides generally have homology of 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 100% or more with respect to the specific base sequence. These probes may be DNA or RNA, or may be polynucleotides in which a part or all of the nucleotides are replaced with artificial nucleic acids such as PN, LNA, ENA, GNA, TNA.

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

The term "primer" means an oligonucleotide, whether naturally occurring in a purified restriction digest or synthetically produced, which is capable of acting as a point of origin of synthesis when placed under conditions that induce synthesis of a primer extension product complementary to a nucleic acid strand, i.e., in the presence of a nucleotide and an inducer, such as a DNA polymerase, and at a suitable temperature and pH. The primer may be single-stranded or double-stranded and must be long enough to prime the synthesis of the desired extension product in the presence of the inducer. The exact length of the primer depends on many factors, including temperature, primer source, and method of use. For example, for diagnostic applications, an oligonucleotide primer will typically contain 15-25 or more nucleotides, although it may contain fewer nucleotides, depending on the complexity of the target sequence. Factors involved in determining the appropriate length of the primer will be readily apparent to those skilled in the art.

Primers or probes of the invention may be chemically synthesized using a phosphoimide solid support method or other well known methods. The nucleic acid sequence may also be modified using a number of means known in the art. Non-limiting examples of such modifications are methylation, capping, substitution with one or more analogs of the natural nucleotide, and modification between nucleotides, for example, modification of uncharged linkers (e.g., methyl phosphate, phosphotriester, phosphoimide, carbamate, etc.), or modification of charged linkers (e.g., phosphorothioate, phosphorodithioate, etc.).

The term "binding agent" includes receptors for protein LACTB, lectins binding to protein LACTB, antibodies against protein LACTB, peptide antibodies (peptide bodies) against protein LACTB, bispecific dual binding agents or bispecific antibody forms. Specific binding agents include, but are not limited to, peptides, peptidomimetics, aptamer, spiegelmer, darpin, ankyrin repeat proteins, kunitz-type domains, antibodies, single domain antibodies, and monovalent antibody fragments.

The term "expression level" or "level of expression" generally refers to the amount of a biomarker in a biological sample. "expression" generally refers to the process by which information (e.g., genetic code and/or epigenetic information) is transformed into structures that are present and run in a cell. Thus, as used herein, "expression" may refer to transcription into a polynucleotide, translation into a polypeptide, or even modification of a polynucleotide and/or polypeptide (e.g., post-translational modification of a polypeptide). Fragments of a transcribed polynucleotide, of a translated polypeptide, or of a polynucleotide and/or polypeptide modification (e.g., post-translational modification of a polypeptide) are also considered to be expressed, whether they originate from transcripts produced by alternative splicing or from degraded transcripts, or from post-translational processing of a polypeptide (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).

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

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

The term "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. Such systems may include, for example, systems that allow for storage, validation, or delivery of expressed genes from one location to another in one or more cells (e.g., oligonucleotides in a suitable container, oligonucleotides encoding enzymes, extracellular matrix components) and/or support substances (e.g., buffers, media, cells, written instructions for performing an assay).

In the present invention, the kit comprises reagents for detecting the LACTB gene or protein, one or more selected from the group consisting of: a container, instructions for use, positive control, negative control, buffer, adjuvant, or solvent. The components of the kit may be packaged in aqueous medium or in lyophilized form. Suitable containers in the kit typically include at least one vial, test tube, flask, baud bottle, syringe, or other container in which one component may be placed, and preferably, an appropriate aliquot may be performed. Where more than one component is present in the kit, the kit will also typically contain a second, third or other additional container in which the additional components are placed separately. However, different combinations of components may be contained in one vial. The kits of the invention will also typically include a container for holding the reagents, sealed for commercial sale. Such containers may include injection molded or blow molded plastic containers in which the desired vials may be retained.

In embodiments of the invention, the kits include, but are not limited to, qPCR kits, ELISA kits, immunoblot detection kits, immunochromatographic detection kits, immunohistochemical detection kits, flow cytometry analysis kits, electrochemiluminescence detection kits.

The term "nucleic acid membrane strip" includes a substrate and oligonucleotide probes immobilized on the substrate; the substrate may be any substrate suitable for immobilization of oligonucleotide probes including, but not limited to, nylon membranes, nitrocellulose membranes, polypropylene membranes, glass sheets, silica gel wafers, micro magnetic beads.

The term "pharmaceutical composition" may be prepared using different additives such as buffers, stabilizers, bacteriostats, isotonic agents, chelating agents, pH controlling agents and surfactants.

The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or by an implanted reservoir. The pharmaceutical compositions of the present invention may contain any of the usual non-toxic pharmaceutically acceptable carriers, adjuvants or excipients. In some cases, a pharmaceutically acceptable acid, base or buffer may be used to adjust the pH of the formulation to improve the stability of the formulated compound or dosage form thereof. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrajugular, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may be administered to the subject by any route so long as the target tissue is achieved.

The term "inhibitor" refers to any substance that decreases the activity of a LACTB protein, decreases the stability of a LACTB gene or protein, down-regulates the expression of a LACTB protein, decreases the effective duration of action of a LACTB protein, or inhibits the transcription and translation of a LACTB gene, which can be used in the present invention as a substance useful for down-regulating LACTB and thus can be used for the prevention or treatment of osteoarthritis.

In the present invention, the inhibitors include nucleic acid inhibitors, protein inhibitors, proteolytic enzymes, protein binding molecules. Wherein the nucleic acid inhibitor is selected from the group consisting of: an interfering molecule targeting LACTB or a transcript thereof and capable of inhibiting LACTB gene expression or gene transcription, comprising: shRNA (small hairpin RNA), small interfering RNA (siRNA), dsRNA, microrna, antisense nucleic acid, or constructs capable of expressing or forming the shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid. The protein binding molecule is selected from the group consisting of: substances which bind specifically to the LACTB protein, such as antibodies or ligands which inhibit the activity of the LACTB protein.

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

Some examples of substances that may 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 tween80, phosphate, 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 silica, magnesium trisilicate, polyvinylpyrrolidone, 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 carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc powder; 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; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants, such as sodium lauryl sulfate and magnesium stearate, and colorants, mold release agents, coating agents, sweeteners, flavoring agents, and fragrances, preservatives, and antioxidants can also be present in the composition at the discretion of the formulator.

The composition may be in any orally acceptable dosage form (including, but not limited to, capsules, tablets, aqueous suspensions or solutions) for oral administration. For tablets for oral administration, common carriers include lactose and corn starch. Typically, a lubricant such as magnesium stearate is also added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When an aqueous suspension is required for oral administration, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweeteners, flavoring agents or coloring agents may also be added.

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 forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed 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 starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic, c) humectants such as glycerin, d) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents 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 forms may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar, high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also have a composition such that they 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 a similar type may also be employed as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar, high molecular weight polyethylene glycols and the like.

Microencapsulated forms with one or more of the above excipients may also be used in the present invention. Solid dosage forms of tablets, dragees, 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 pharmaceutical formulating art. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also contain, as is common practice, additional substances other than inert diluents, such as tabletting lubricants and other tabletting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and may also have a composition such that they 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.

The term "subject" or "patient" refers to an animal that is capable of suffering from or suffering from 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.

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

The term "computer-readable storage medium" such as computer-executable code may take many forms, including but not limited to, tangible storage media, carrier wave media, or physical transmission media. Nonvolatile storage media includes, for example, optical or magnetic disks, such as any storage devices in any computer, volatile storage media including dynamic memory, main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier wave transmission media can take the form of electrical or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency and infrared data communications. Thus, common forms of computer-readable media include, for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, RAM, ROM, PROM and EPROM, FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, a cable or link transporting such a carrier wave, or any other medium 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.

The term Nomogram model is also called as a Nomogram model, is a clinical prediction model, is established on the basis of multi-factor regression analysis, integrates a plurality of prediction indexes, and adopts line segments with scales to draw on the same plane according to a certain proportion, so that the model is used for expressing the interrelationship among various variables in the prediction model and evaluating the disease risk. The alignment chart can convert a complex regression equation into a visualized graph, so that the result of the prediction model is more readable, the evaluation of a subject is facilitated, and the method is widely used in medical research and clinic.

In an embodiment of the present invention, the alignment chart includes twelve straight lines which are sequentially arranged from top to bottom and are parallel to each other, each straight line represents a scale, and scales are carved;

wherein the first row is a fractional scale, and the numerical range is 0-100;

a second behavior T stage scale, wherein the score is 0 when the stage is T2, and the score is 11 when the stage is T3 or T4;

the third behavior N stage scale, when stage is N0, the score is 0, when stage is N1, the score is 26;

a fourth behavior age scale, wherein the score is 0 when the age is less than or equal to 60, and is 17.5 when the age is more than 60;

The fifth element is a staff gauge, the score is 0 when the artificial species is yellow, 92 when the artificial species is white, and 100 when the artificial species is black;

a sixth behavior of the PSA scale, wherein when PSA is less than 4, the score is 0, and when PSA is more than or equal to 4, the score is 32.5;

a seventh behavior, the scale of LACTB, with a score of 0 when LACTB is 1.5, a score of 16 when LACTB is 2.5, a score of 32 when LACTB is 3.5, a score of 47.5 when LACTB is 4.5, and a score of 63 when LACTB is 5.5;

an eighth behavior total score scale, the value range is 0-240;

a ninth behavior linear prediction period scale with a numerical range of-7-4;

the survival rate scale of the tenth action in 3 years is in the range of 0.6-0.8;

the eleventh action is a 5-year survival rate scale, and the numerical range is 0.6-0.8;

twelfth, 10 year survival scale, value 0.5.

The term "PSA" is a prostate specific antigen, primarily a marker used to examine prostate cancer.

The invention is further illustrated below in connection with specific embodiments. It should be understood that the particular embodiments described herein are presented by way of example and not limitation. The principal features of the invention may be used in various embodiments without departing from the scope of the invention.

EXAMPLE 1LACTB expression level in prostate cancer

1.1 Experimental materials

Sample: the 43 cases of prostate cancer tissue and the 43 cases of paracancerous tissue were collected at the second hospital unit affiliated with university of Daian university at 2021, 3-1.3-2022.

Reagent: an anti-LACTB available from Abcam corporation, england; PBS, peroxidase, secondary antibodies, DAB chromogenic reagent kits were all purchased from Fuzhou Michaelis technology Co., ltd.

1.2 Experimental methods

1.2.1 immunohistochemical staining detection

The specimens were fixed with 10% neutral formalin and then embedded in conventional paraffin, serial sections were performed at 4 μm, the specimens were fished out with special film for immunohistochemistry, and baked at 60℃for 1h. The Max Vision method was used for immunohistochemistry.

1) Dewaxing and hydration: placing paraffin slice into 55 deg.C buoyancy tank, baking slice for 1 hr, soaking in xylene I and xylene II for 10min for dewaxing, soaking in gradient alcohol (100% I, 100% II, 95%, 85%, 70%) for 2min for hydration;

2) Antigen retrieval: slightly washing with water for 10min, adding 0.01M sodium citrate micro-solution, incubating at normal temperature for 20min, boiling with boiling water for 15min, naturally cooling, soaking in PBS for 5min, and repeating for three times;

3) Removing catalase: dropwise adding 3%H ₂ O ₂ Incubation for 10min in dark, blocking the activity of endogenous peroxidase, soaking in PBS for 5min, and repeating for three times;

4) Closing: dropwise adding 3% BSA blocking solution, and blocking for 30min at room temperature;

5) Applying an antigen: first antibody (dilution ratio 1:100) was added dropwise. Placing into a plastic box, covering with a cover, and placing at 4deg.C overnight;

6) And (3) secondary antibody: soaking in PBS for 5min, repeating for three times, dripping secondary antibody (dilution ratio is 1:200), and incubating at 37deg.C for 30min;

7) DAB color development: soaking in PBS for 5min, repeating for three times, dripping DAB color development liquid (dilution ratio is 1ml solution I:1 drop solution II), and developing in dark place;

8) Nuclear dyeing: slowly washing with water for 10min, and dropwise adding hematoxylin for incubation for 20s;

9) Dehydrating: slowly washing with water flow for 10min, and soaking with gradient alcohol (70%, 85%, 90%, 100% II, 100% I) for 2min;

10 Sealing and observing: and respectively soaking xylene II and xylene I for 2min, dripping neutral gum, covering a cover glass, standing at room temperature for 24h, and observing and photographing under a normal microscope.

Observing the occurrence of brown yellow particles of the tumor cell slurry under a microscope, marking the brown yellow particles as positive cells, and marking positive expression when the ratio of the number of the positive cells to the total number of the tumor cells is more than or equal to 40%, or else marking negative expression.

The score of the staining intensity was defined as: negative score 0, weak score 1, medium score 2, strong positive score 3; the frequency of positive cells is defined as: the score was 0 when the number of positive cells was less than 5%, 2 when the number of positive cells was 26% -50%, 3 when the number of positive cells was 51% -75%, and 4 when the number of positive cells was greater than 75%. And finally, comprehensively scoring.

1.2.2BCA protein quantification and preparation

1) The desired AB solution was formulated according to the amount of sample (a: b=50:1);

2) Taking a 96-well plate, sequentially adding deionized water 20, 18, 16, 12, 4 mu l and 0.5 mu g/mu l of standard protein 0, 2, 4, 8 and 16 mu l into the standard protein well, and adding 19 mu l of deionized water and 1 mu l of sample protein into the sample well;

3) 180 μl of AB solution is added into each hole, the mixture is uniformly mixed, and after incubation for 30min at 37 ℃ in dark, the absorbance value of 589nm wavelength is measured by an enzyme-labeled instrument;

4) A standard curve is manufactured according to the measured light absorption value, and the protein content of a corresponding sample is calculated;

5) Sampling protein, adding 1/4 volume of 5×Loanding Buffer, mixing, boiling in boiling water for 10min, and preserving at-20deg.C.

1.2.3Western Blot detection

1) SDS-PAGE electrophoresis: preparing 10% of separation gel and 5% of concentrated gel, loading 30 mug of protein solution into each hole, adding electrophoresis liquid, performing 60V constant voltage electrophoresis for 30min, and performing 120V constant voltage electrophoresis to the bottom (about 1 h);

2) Transferring: cutting off concentrated gel and useless separating gel, cutting PVDF film and three layers of filter paper (ensuring film > gel > filter paper) with proper size, placing the PVDF film into methanol for activating for 15s, arranging a rotary die device from an anode to a cathode according to the sequence of sponge, three layers of filter paper, PVDF film, gel, three layers of filter paper and sponge, and rotating the film for 1h under a 220mA constant flow ice bath;

3) Closing: taking out the PVDF film, putting the PVDF film into 5% skimmed milk powder (prepared by TBST), and sealing the PVDF film for 3 hours at room temperature;

4) Antibody incubation: washing PVDF membrane with TBST for 5min, shearing PVDF membrane and incubating rabbit anti-ST 6Gal-I (1:500, TBST dilution) or GAPDH (1:5000, TBST dilution) primary antibody, overnight at 4deg.C, then washing PVDF membrane with TBST three times each for 15min, incubating anti-rabbit secondary antibody (1:8000, TBST dilution) at room temperature for 1h, and washing BST three times each for 15min;

5) ECL luminescence: dripping newly configured ECL luminous liquid (A liquid: B liquid=1:1) onto a PVDF film, imaging by using a gel imager, and storing;

6) Analysis: the strips were analyzed using Image Lab software.

1.3 experimental results

The content of LACTB in normal prostate tissue and prostate cancer tissue was detected by immunohistochemical staining and LACTB protein was expressed in the plasma of prostate cancer cells. Expression in cancer tissues was significantly higher than in paracancerous normal tissues (fig. 1, p < 0.05).

WB results showed that the expression level of LACTB was significantly increased in prostate cancer tissue compared to normal prostate tissue (fig. 2).

Example 2 Targeted inhibition of the Effect of LACTB on prostate cancer cell migration invasion

2.1 Experimental materials

Cell lines: PC-3 human prostate cancer cell line, 10% Fetal Bovine Serum (FBS) was added to PC-3 complete medium at 37℃and 5% CO ₂ Is cultured.

2.2 Experimental methods

2.2.1 construction of the LACTB transfection vector

Specific small interfering RNAs (siRNAs) targeting the LACTB are synthesized, transient transfection is carried out according to the specification, the silencing level is detected by Western blot after 48 hours, and stably transfected cells are selected.

Sequence:

LACTB-homo-520

Sense5'-3'：CCAUGGUUGCUCUUGCCAATT

Antisense5'-3'：UUGGCAAGAGCAACCAUGGTT

LACTB-homo-1229

Sense5'-3'：CCUUCUGAAAUUUGGGAAUTT

Antisense5'-3'：AUUCCCAAAUUUCAGAAGGTT

2.2.2 targeted inhibition of the modulation of the ability of LACTB to migrate to PC-3 human prostate cancer cells

1) Preparing a ruler and a Marker pen for ultraviolet sterilization;

2) Sealing the 6-hole plate by using a sealing film, pouring the 6-hole plate on an ultra-clean bench, and marking lines in parallel and equidistantly along the diameter of the 6-hole plate by using a Marker pen for positioning;

3) PC-3 cells were routinely cultured in DMEM medium containing 10% FBS. Taking logarithmic growth phase cells, digesting the cells, and adjusting the cell density to 2×10 ⁵ And (3) one/mL for later use. Adding the above cell suspension into 6-well plate, adding 2mL of the cell suspension per well volume, and placing at 37deg.C, 5% CO ₂ Culturing in incubator, observing the cell to be fused, and scoring;

4) According to the direction perpendicular to the transverse line which is positioned in advance, a 100 mu L sterile gun head is used for scribing, the gun head is placed to be perpendicular to the 6-hole plate, and the width of the scribing is ensured to be consistent. After the scratch is finished, a gun is used for slowly sucking out the culture solution, a proper amount of PBS is added to wash the cells for 3 times, and cell fragments are removed;

5) Observing the healing condition of the cell scratch at 0 h and 24h respectively, and photographing;

2.2.3 Targeted inhibition of the modulation of the ability of LACTB to invade PC-3 human prostate cancer cells

1) Placing Matrigel matrix glue in a refrigerator at 4 ℃ overnight to change Matrigel glue into liquid state;

2) Matrigel was diluted 1:8 with serum-free medium, after which the Transwell chamber was placed in 24 plates;

3) Adding 100 mu l of diluted Matrigel into a Transwell chamber, and placing the Matrigel into a cell incubator to be solidified;

4) PC-3 cells in logarithmic growth phase of transfected siNC and siLACTB are digested by pancreatin, and are uniformly mixed by blowing to prepare single cell suspension;

5) Cell counting was performed to adjust the cell density to 5X 10 ⁵ Cells/ml;

6) The Transwell chamber was placed in a 24-well plate, 750. Mu.l of complete medium containing 10% FBS was added to the 24-well plate, 200. Mu.l of diluted cell suspension was added to the Transwell chamber, and the mixture was placed in a cell incubator for culturing;

7) After 48h of culture, taking out the cell, slightly cleaning the cell by using PBS, and wiping off cells of the inner membrane of the cell by using a cotton swab;

8) Adding 4% paraformaldehyde into a new hole of a 24-hole plate, and placing a small chamber into the paraformaldehyde for fixing for 15min;

9) Taking out the Transwell chamber, cleaning the chamber for 5min by using PBS, and then dyeing for 10min by using crystal violet;

10 Cleaning a Transwell cell by using clear water, photographing under an inverted microscope, selecting 5 different fields of view, counting the number of cells passing through the cell membrane, calculating an average value, and drawing a histogram.

2.3 experimental results

After transfection of LACTB, the number of PC-3 cells passing through the Transwell chamber was significantly reduced, demonstrating that targeted inhibition of LACTB was able to inhibit migration and invasiveness of prostate cancer cells (fig. 3).

EXAMPLE 3 use of LACTB in predicting prognosis of prostate cancer

3.1 Experimental methods

The prostate cancer patient variables LACTB were analyzed bioinformatically by TCGA database (https:// portal.gdc.cancer.gov /), using R software (version 3.6.3), using pROC package (version 1.17.0.1), gglot 2 package (version 3.3.3), rms package (version 6.2-0) and survivinal package (version 3.2-10), and using statistical methods Mann-Whitney U test, paired sample T test, log-rank test and cox regression analysis.

3.2 experimental results

Mann-Whitney U test results show that the expression level of LACTB in prostate cancer is higher than paracancerous and than Normal, the median of the difference between the two groups is 0.392 (0.24-0.552), and the difference has statistical significance (P < 0.001); paired sample T-test showed that the expression level of LACTB in prostate cancer was higher than paracancerous, the difference between the two groups was 0.408 (0.253-0.563), the difference was statistically significant (t=5.293, p < 0.001) (fig. 4).

As can be seen from the ROC curve, the predictive power of the variable LACTB has a certain accuracy in predicting normal prostate and prostate cancer outcome auc=0.708, ci=0.641-0.776 (fig. 5).

The nomogram was established to predict survival prognosis of prostate cancer patients with higher accuracy (figure 6).

The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims

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

preferably, the agent is selected from the group consisting of 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.

2. A product for diagnosing/prognosticating prostate cancer, comprising an agent capable of detecting the expression level of LACTB;

preferably, the product comprises a chip, a kit or a nucleic acid membrane strip;

Preferably, the chip comprises a gene chip comprising oligonucleotide probes for the LACTB gene for detecting the transcription level of the LACTB gene, a protein chip comprising a specific binding agent for the LACTB protein; the kit comprises a gene detection kit and a protein detection kit, wherein the gene detection kit comprises a reagent or a chip for detecting the transcription level of the LACTB gene, and the protein detection kit comprises a reagent or a chip for detecting the expression level of the LACTB protein;

preferably, the kit comprises reagents for detecting the level of expression of the LACTB gene or protein by RT-PCR, qRT-PCR, biochip assay, southern blotting, in situ hybridization, immunoblotting.

3. A pharmaceutical composition for treating prostate cancer, wherein the pharmaceutical composition comprises an inhibitor of LACTB;

preferably, the inhibitor reduces the expression level of LACTB;

preferably, the inhibitor comprises a nucleic acid inhibitor, a protein inhibitor;

preferably, the nucleic acid inhibitor comprises shRNA, siRNA;

preferably, the protein inhibitor comprises an antibody or ligand that inhibits the activity of the LACTB protein;

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

Use of lactb in the preparation of a pharmaceutical composition for the treatment of prostate cancer;

preferably, the inhibitor reduces the expression level of LACTB;

preferably, the nucleic acid inhibitor comprises shRNA, siRNA;

Use of lactb for screening candidate drugs for the treatment of prostate cancer;

preferably, the method of 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 the LACTB gene with a substance to be screened; and detecting the expression or activity of the LACTB gene or protein encoded thereby in the system; wherein the substance to be screened is a candidate drug for treating prostate cancer when the substance to be screened inhibits the expression level or activity of the LACTB gene or a protein encoded thereby.

6. A method for screening a candidate drug for the treatment of prostate cancer, which comprises treating a culture system expressing or containing a LACTB gene or a protein encoded thereby with a substance to be screened; and detecting the expression or activity of the LACTB gene or protein encoded thereby in the system; wherein the substance to be screened is a candidate drug for treating prostate cancer when the substance to be screened inhibits the expression level or activity of the LACTB gene or a protein encoded thereby.

7. A system/device for diagnosing/prognosticating prostate cancer, said system/device comprising:

8. A computer readable storage medium, characterized in that the storage medium has stored thereon a computer program which, when executed by a processor, implements the system/apparatus of claim 7.

9. A prognostic model for prostate cancer, wherein the detection indicators of the model include the expression level of LACTB;

preferably, the detection index of the model further comprises T stage, N stage, age, race and PSA;

preferably, the model is a nomogram model.

Use of lactb in constructing a model for diagnosing/predicting prognosis of prostate cancer;

preferably, the model for predicting the prognosis of prostate cancer is a nomographic model;

Preferably, the detection index of the alignment chart model comprises T stage, N stage, age, race and PSA.