WO2023280001A1

WO2023280001A1 - Application of mir-31-5p in acute myeloid leukemia

Info

Publication number: WO2023280001A1
Application number: PCT/CN2022/101571
Authority: WO
Inventors: 闫道广; 钟文彬
Original assignee: 暨南大学
Priority date: 2021-07-05
Filing date: 2022-06-27
Publication date: 2023-01-12
Also published as: CN113584166B; CN113584166A

Abstract

The present disclosure relates to an application of miR-31-5p in acute myeloid leukemia (AML), particularly, a use of a product for detecting the miR-31-5p in preparation of a reagent, chip, or reagent kit for auxiliary diagnosis of a subject suffering from the AML and/or for prognosis of survival time of the subject, and a use of a miR-31 primary/precursor miRNA (pri/pre-miR-31) and/or a miR-31 mature miRNA (miR-31-5p) in preparation of a drug for treatment of the AML. Introduction of the miR-31-5p into cells by means of gene transduction can induce cell death of bone marrow AML cells and AML leukemic stem cells (AML-LSCs) and enhance the cytotoxicity of cytosine arabinoside as a chemotherapeutic drug, and expression of the miR-31-5p can inhibit the growth of the AML cells and the AML-LSCs, and prolong the lifetime of animals. The present disclosure provides a novel method for AML diagnosis and prognostic assessment, and further provides a gene therapeutic drug for the AML.

Description

Application of miR-31-5p in acute myeloid leukemia

technical field

The disclosure belongs to the field of biomedicine, relates to the diagnosis and treatment of tumors, and specifically relates to the application of miR-31-5p in the diagnosis and treatment of acute myeloid leukemia (AML).

Background technique

Leukemia is a kind of clonal malignant disease originating from hematopoietic stem cells. According to statistics, about 350,000 people worldwide die of leukemia every year. Therefore, leukemia has become the main malignant tumor that threatens human health. Leukemia stem cells (LSCs) are a very small group of cells in leukemia patients, accounting for about 0.1% to 1% of all leukemia cells. LSC was first discovered in acute myeloid leukemia (AML) and has been widely recognized. Whether in long-term cell culture or in animal models, LSCs can cause and maintain leukemia; at the same time, unlike leukemia cells, more than 95% of LSCs are in the "dormant" state of G0 phase, and these cells are not effective for traditional cell cycle chemotherapy. Drug insensitivity, the retained LSC increases the relapse rate of leukemia patients through self-replication (Renewal). Therefore, finding the unique self-protection mechanism of LSC and targeting to kill LSC is an important way to completely cure leukemia.

MicroRNAs (miRNAs) are a class of non-coding small RNA molecules, about 19-25bp in length, including a 6-8nt seed sequence and a 12-17nt supplementary sequence. After the miRNA gene is transcribed, the initial miRNA (pri-miRNA) is produced, which forms a hairpin structure under the processing of Drosha enzyme. According to the order of transcription, it is divided into 5' end arm and 3' end arm, also known as 5p and 3p. The partial sequences of the 5' end arm and the 3' end arm are complementary to form a double-stranded RNA, which becomes a mature miRNA precursor (pre-miRNA) and is transported to the cytoplasm to play a role in post-transcriptional control of gene expression. Once the pre-miRNA enters the cytoplasm from the nucleus, it will be further processed by Dicer enzyme to become a mature miRNA. These miRNAs play an important role in the regulation of gene expression, and are involved in cell differentiation, proliferation, and maintenance of cell homeostasis.

In most solid tumors, miR-31-5p expression was significantly upregulated. Chinese patent CN106636308B discloses a probe combination and kit for detecting skin cancer-related markers, wherein hsa-miR-31-5p is used as a detection of skin cancer-related markers.

However, there is no report on the expression of miR-31-5p in AML cells and AML stem cells. Therefore, a better understanding of biomarkers in acute myeloid leukemia is urgently needed to develop prognostic predictive tools as well as to discover new drugs.

Contents of the invention

In order to solve the deficiencies in the prior art, the purpose of the present disclosure is to screen out acute myeloid leukemia as a molecular diagnostic biomarker, and develop a corresponding diagnostic kit to diagnose and treat acute myeloid leukemia or provide its prognosis.

Specifically, the present disclosure proposes the following technical solutions:

In one aspect, the present disclosure provides a product for detecting miR-31-5p, pri-miR-31 and/or pre-miR-31 in preparation for assisting in diagnosing subjects with acute myeloid leukemia (AML) And/or use in a reagent, chip or kit for prognosing the survival period of a subject.

In one aspect, the present disclosure provides a use of miR-31-5p, pri-miR-31 and/or pre-miR-31 in the preparation of a drug for preventing and/or treating acute myeloid leukemia (AML).

In one aspect, the present disclosure provides a drug for treating acute myeloid leukemia (AML), the drug comprising miR-31 naive miRNA, miR-31 precursor miRNA, and/or mature miR-31-5p.

In one aspect, the present disclosure provides a method of treating acute myeloid leukemia (AML), comprising administering to a subject a therapeutically effective amount of miR-31-5p, pri-miR-31 and/or pre-miR- 31 or a pharmaceutical composition comprising it.

In vitro cell culture experiments have shown that introducing miR-31-5p into cells by gene transduction can induce cell death in AML and AML-LSC. Mouse animal experiments have shown that expression of miR-31-5p can inhibit AML cells and AML-LSC. The growth of cells prolongs the lifespan of mice. The present disclosure provides new methods for diagnosis and prognosis assessment of AML, as well as for the preparation of gene therapy drugs for AML.

Description of drawings:

Figure 1 shows the principle of miRNA tailing reverse transcription.

Figure 2 is the QPCR detection of miR-31-5p expression in bone marrow leukemia stem cells (LSCs) of AML patients and normal human bone marrow hematopoietic stem cells (HSCs).

Figure 3 shows the expression of miR-31-5p detected in AML patient bone marrow cells (AML) and normal human bone marrow cells (BM) by QPCR.

Fig. 4 is a graph showing the relationship between the expression level of miR-31-5p and the prognosis and survival period of AML patients.

Figure 5 shows the effect of miR-31-5p on the ability of AML-LSC colony formation.

Fig. 6 is a graph showing the results of miR-31-5p-induced death of AML-LSC and bone marrow AML cells.

Fig. 7 is a graph showing the results of miR-31-5p enhancing the chemotherapy drug cytarabine (Ara-C) to induce AML-LSC and bone marrow AML cell death.

Figure 8 is a verification of the therapeutic effect of miR-31-5p on AML disease in B-NDG mice.

detailed description

In the present disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation steps used herein are all terms and routine procedures widely used in the corresponding fields. Meanwhile, for a better understanding of the present disclosure, definitions and explanations of related terms are provided below.

As used herein, the term "primary miR-31" or "pri-miR-31" refers to miRNA obtained after transcription of miR-31 gene by RNA polymerase.

As used herein, the term "precursor miR-31" or "pre-miR-31" refers to the original miR-31 (pri-miR-31) under the processing of Drosha enzyme to form a hairpin structure sequence with 71 nucleosides Acid (71nt), its sequence is as follows: 5'-ggagaggaggcaagaugcuggcauagcuguugaacugggaaccugcuaugccaacauauugccaucuuucc-3'(SEQ ID NO3).

As used herein, the term "mature miR-31-5p" or "miR-31-5p" refers to the sequence formed by the processing of the precursor miR-31 (pre-miR-31) by Dicer enzyme, having 21 nucleosides Acid (21nt), its sequence is as follows: 5'-aggcaagaugcuggcauagcu-3' (SEQ ID NO 1).

As used herein, the term "vector" refers to a construct capable of delivering and optionally expressing one or more polynucleotides of interest into a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes , and certain eukaryotic cells, such as producer cells. Vectors can be stable and self-replicating. There is no limitation regarding the types of vectors that can be used. A vector may be a cloning vector, a polynucleotide suitable for propagation and obtaining incorporation with various foreign organisms, a genetic construct or an expression vector. Suitable vectors include prokaryotic expression vectors (such as pUC18, pUC19, Bluescript and their derivatives), mpl8, mpl9, pBR322, pMB9, CoIE1, pCR1, RP4, phage and shuttle vectors (such as pSA3 and pAT28), and viral-based vectors ( eukaryotic expression vectors such as adenovirus, adeno-associated virus, and retroviruses and lentiviruses), and non-viral vectors such as pSilencer 4.1-CMV (LifeTechnologies Corp., Carslbad, CA, USA), pcDNA3, pcDNA3.1/hyg pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1, pML2d and pTDT1.

As used herein, the term "plasmid" refers to a small, circular, double-stranded, self-replicating DNA molecule obtained by genetic engineering techniques capable of transferring genetic material of interest to cells, which results in the production of The product encoded by the genetic material (such as protein polypeptide, peptide or functional RNA) in. In addition, the term "recombinant plasmid" or "plasmid" also refers to a small, circular, double-stranded, self-replicating DNA molecule obtained by genetic engineering techniques used during the preparation of viral vectors as vectors for recombinant vector genomes.

As used herein, the term "viral vector" refers to an agent obtained from a naturally occurring virus by genetic engineering techniques capable of transferring genetic material of interest (such as DNA or RNA) to a cell, resulting in the production of The product encoded by a substance such as a protein polypeptide, peptide or functional RNA.

As used herein, the term "pharmaceutical composition" refers to a formulation of various preparations. Formulations containing therapeutically effective amounts of miR-31-5p, pri-miR-31 and/or pre-miR-31 are in sterile liquid solution, liquid suspension or lyophilized form, optionally comprising stabilizers or excipients .

As used herein, the term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, which is not an active ingredient, and which is nontoxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

As used herein, "treating" an individual suffering from a disease or condition means that the individual's symptoms are partially or fully alleviated, or remain unchanged after treatment. Thus, treatment includes prophylaxis, treatment and/or cure. Prevention refers to preventing an underlying disease and/or preventing worsening of symptoms or development of a disease.

As used herein, "therapeutic effect" means the effect resulting from treatment of an individual that alters, usually ameliorates or ameliorate the symptoms of, or cures a disease or condition.

As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect when administered to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest or partially arrest the symptoms of a disease or disorder.

As used herein, "prophylactically effective amount" or "prophylactically effective dose" refers to the amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the intended prophylactic effect, e.g., prevent or delay a disease or symptom occurrence or recurrence of the disease or symptoms, and to reduce the likelihood of occurrence or recurrence of the disease or symptoms. A full prophylactically effective dose does not have to occur by administering one dose, and can only occur after administering a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.

In one aspect, the present disclosure provides a product for detecting miR-31-5p, pri-miR-31 and/or pre-miR-31 in preparation for assisting in diagnosing subjects with acute myeloid leukemia (AML) And/or the purposes in the reagent, chip or test kit that are used for prognostic experimenter's survival period, wherein, the sequence of miR-31-5p is: 5'-aggcaagaugcuggcauagcu-3'(SEQ ID NO1), pre-miR- The sequence of 31 is: 5'-ggagaggaggcaagaugcuggcauagcuguugaacugggaaccugcuaugccaacauauugccaucuuucc-3' (SEQ ID NO 3).

In some embodiments of the present disclosure, the reagent is capable of detecting the level of miR-31-5p, pri-miR-31 and/or pre-miR-31 in a biological sample.

In some embodiments of the present disclosure, the level of miR-31-5p, pri-miR-31 and/or pre-miR-31 in the biological sample is lower than the corresponding miR-31-5p, pri-miR in the normal control sample A level of -31 and/or pre-miR-31 indicates that the subject has acute myeloid leukemia (AML).

In some embodiments of the present disclosure, the biological sample is selected from one or more of peripheral blood, bone marrow, and tissue suspected of having leukemia cells.

In some embodiments of the present disclosure, the subject can be a human or other mammal.

In some embodiments of the present disclosure, the level of miR-31-5p, pri-miR-31 and/or pre-miR-31 adopts high-throughput sequencing method, miRNA expression profiling chip, quantitative PCR method and/or probe hybridization method for detection.

In some embodiments of the present disclosure, the reagent comprises a forward primer for amplifying miR-31-5p; preferably, the sequence of the forward primer is as follows: 5'-aggcaagatgctggcatagct-3' (SEQ ID NO 2) .

In some embodiments of the present disclosure, miR-31-5p, pri-miR-31 and/or pre-miR-31 in the biological sample are relative to the corresponding miR-31-5p, pri-miR-31 and And/or the transcript level of the pre-miR-31 gene is low, the subjects have a shorter survival period.

In some embodiments of the present disclosure, the chip comprises a solid support and oligonucleotide probes immobilized on the solid support.

The disclosed experiment proves that the increased level of miR-31-5p in cells can directly induce the death of AML and AML-LSC cells, and enhance the cytotoxicity of the chemotherapeutic drug cytarabine. As mentioned above, the miR-31-5p gene is transcribed by RNA polymerase to obtain the initial miR-31 (pri-miR-31), and the initial miR-31 (pri-miR-31) is processed by Drosha enzyme to form a hairpin The structure of the precursor miR-31 (pre-miR-31), the precursor miR-31 (pre-miR-31) is processed by Dicer enzyme to form miR-31-5p. Therefore, the intracellular level of pri-miR-31 and/or pre-miR-31 increases, and pri-miR-31 and/or pre-miR-31 will be processed in the cell to form miR-31-5p, which plays a role in increasing miR-31-5p had the same effect.

In some embodiments of the present disclosure, miR-31-5p, pri-miR-31 and/or pre-miR-31 are natural, artificially synthesized, or can be expressed using miR-31-5p, pri-miR-31 and/or pre-miR-31 DNA fragment expression vector transfected cells, wherein the gene sequence of miR-31-5p is: 5'-aggcaagatgctggcatagct-3'(SEQ ID NO 2); pre-miR- The gene sequence of 31-5p is: 5'-ggagaggaggcaagatgctggcatagctgttgaactgggaacctgctatgccaacatattgccatctttcc-3' (SEQ ID NO 4).

In some embodiments of the present disclosure, the expression vector is selected from vectors selected from plasmids, phages, phagemids, cosmids, viral vectors, virions, prokaryotic expression vectors and eukaryotic expression vectors.

In some embodiments of the present disclosure, the viral vector is selected from the group consisting of retroviral vectors, lentiviruses, adenoviral vectors, adeno-associated viral vectors, herpesvirus vectors, alphavirus vectors, baculoviruses, and vaccinia viruses.

In some embodiments of the present disclosure, the prokaryotic expression vector is selected from an E. coli expression vector and a Bacillus subtilis expression vector.

In some embodiments of the present disclosure, the eukaryotic expression vector is selected from a yeast expression vector, an insect expression vector, or a mammalian expression vector.

In some embodiments of the present disclosure, the drug may be administered alone or in combination with other drugs capable of inhibiting AML.

In some embodiments of the present disclosure, other drugs capable of inhibiting AML are selected from daunorubicin, cytarabine, thioguanine, etoposide, harringtonine, vincristine, prednisone, Mitoxantrone, doxorubicin, cyclophosphamide, carboplatin, decitabine, methotrexate, etoposide, doxorubicin (doxorubicin), cisplatin, dexamethasone, sacola Neo, methylnitrosourea, fluorouracil, 5-fluorouracil, vinblastine, camptothecin, actinomycin-D, mitomycin C, hydrogen peroxide, oxaliplatin, irinotecan, topotecan One or more of Kang, leucovorin, carmustine, streptozocin, paclitaxel, tamoxifen, dacarbazine, imatinib, azacitidine and gemtuzumab.

In some embodiments of the present disclosure, the medicament further comprises a pharmaceutically acceptable carrier.

In some embodiments of the present disclosure, the pharmaceutically acceptable carrier is selected from lactose, dextrose, sucrose, polyvinylpyrrolidone, alginate, gelatin, cellulose, syrup, sorbitol, mannitol, starch, arabic Rubber, Talc, Magnesium Stearate, Calcium Phosphate, Calcium Silicate, Microcrystalline Cellulose, Methyl Cellulose, Methyl Hydroxybenzoate, Propyl Hydroxybenzoate, Mineral Oil, Microcapsules & Microspheres, Nano One or more of particles and liposomes.

In some embodiments of the present disclosure, the dosage form of the drug is solution, injection, oral liquid, suspension, emulsion, extract, powder, granule, suppository, aerosol, granule, tablet or capsule .

In some embodiments of the present disclosure, injections include sterile or sterile solutions, aqueous injections, oil injections, powder injections, etc.; oral liquid dosage forms include solutions, syrups, emulsions, suspensions, etc.; tablets include ordinary Compressed tablets, sugar-coated tablets, effervescent tablets, chewable tablets, multilayer tablets, implanted tablets, sustained-release tablets, controlled-release tablets, etc.

In some embodiments of the present disclosure, the medicament further comprises a dispersant or stabilizer.

In one aspect, the present disclosure provides a reagent, a chip or a kit for assisting in diagnosing that a subject has acute myeloid leukemia (AML) and/or for prognosing the survival of a subject; the reagent, chip or kit comprises Assays for products that detect miR-31-5p and/or its precursors in biological samples.

In one aspect, the present disclosure provides a drug for treating acute myeloid leukemia (AML), the drug comprising miR-31-5p, pri-miR-31 and/or pre-miR-31.

In one aspect, the present disclosure provides a pharmaceutical composition for treating acute myeloid leukemia (AML), the medicine comprising miR-31-5p, pri-miR-31 and/or pre-miR-31, and a pharmaceutically acceptable Carrier.

A medicament or pharmaceutical composition of an embodiment is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal or subcutaneous administration may include the following components: sterile diluents for injection such as water, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; Antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphate, and agents to adjust osmotic pressure, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable pharmaceutically acceptable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and a suitable mixture thereof. Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, the maintenance of the desired particle size in the case of dispersions, and the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases it will be preferable to include isotonic agents, for example, sugars, polyalcohols (such as mannitol, sorbitol), sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent delaying absorption, for example, aluminum monostearate and gelatin.

In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum-drying and freeze-drying to obtain a powder containing the active ingredient plus any additional desired ingredient from a sterile-filtered solution of these ingredients as previously described .

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically separable units suitable as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of a drug calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. miR-31-5p, pri-miR-31 and/or pre-miR-31.

The pharmaceutical composition can be presented in a container, pack, or dispenser together with instructions for administration.

In one embodiment, one or more of miR-31-5p, pri-miR-31 and/or pre-miR-31 may be administered in combination therapy, ie in combination with other drugs capable of inhibiting AML. The term "in conjunction" herein means that the agents are administered substantially simultaneously, simultaneously or sequentially. If administered sequentially, the first of the two compounds is still preferably detectable at the site of treatment at an effective concentration when administration of the second compound is initiated. In one case, "combination" can also include miR-31-5p, pri-miR-31 and/or pre-miR-31 and other therapeutic agents in the kit at the same time.

For example, combination therapy may comprise miR-31-5p, pri-miR-31 and/or pre-miR-31 herein with one or more additional therapeutic agents (e.g., one or more cytokines and growth factor inhibitory agents, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and/or cytotoxic or cytostatic agents, as described in more detail below) are co-formulated and/or co-administered. Such combination therapy may advantageously utilize lower doses of the therapeutic agents administered, thus avoiding possible toxicity or complications associated with various monotherapies.

It should be pointed out that those skilled in the art know that appropriate adjustments and modifications can be made to the sequences of the primers disclosed in the present disclosure according to the sequences of the relevant markers, and these modified primer sequences can still be used to detect the aforementioned markers. The present disclosure also includes these equivalent technical solutions.

Example 1. Collection and cell sorting of clinical samples

1. Collection of AML clinical samples

Randomly select 44 initial bone marrow cases (n=44) from hospitalized acute myeloid leukemia (AML) patients in the Department of Hematology, and collect bone marrow samples from the patients, and number the collected samples (AML-1, AML-2 , ..., AML-44), the patients did not receive any drug treatment; 5 normal human bone marrow samples were collected (n=5). All the above samples were obtained with the approval of the organizational ethics committee.

The processing methods for collecting bone marrow samples from AML patients and normal people are as follows:

Extract 2 mL of bone marrow under sterile conditions and add heparin for anticoagulation. Bone marrow sample collection requirements: heparin anticoagulation, no blood clots, bone marrow volume greater than 2mL, cell sorting within 24 hours after sample collection.

2. Isolation of mononuclear cells

Mononuclear cells were separated from the AML patient bone marrow samples and normal human bone marrow samples collected in step 1 using Dayou's human lymphocyte separation medium (product number 711101X), including the following steps:

A. Dilute the bone marrow sample with an equal volume of pH7.4, 0.01M phosphate buffered saline (PBS). For example, add 1mL of PBS to 1mL of bone marrow sample, and mix evenly by inverting gently.

B. Add a certain volume of separation liquid into a 15mL centrifuge tube, spread the diluted bone marrow sample obtained in step A above the liquid surface of the separation liquid, keep the interface between the two liquid surfaces clear, and obtain a mixture of bone marrow and separation liquid. Ensure that the volume ratio of the lymphocyte separation medium to the diluted bone marrow sample in PBS is 1:2.

C. At room temperature, adjust the centrifugal force of the horizontal rotor of the centrifuge to 700-800g (or 2000-2500rpm/min), and centrifuge the mixture of bone marrow and separation solution obtained in step B for 20-30min.

D. After centrifugation, collect the thin and dense buffy coat layer above the separation liquid, that is, the mononuclear cell (including lymphocyte and monocyte) layer.

E. Dilute the mononuclear cells collected in step D with 5 mL of PBS, and mix upside down to obtain diluted mononuclear cells.

F. At room temperature, adjust the centrifugal force of the horizontal rotor of the centrifuge to 250g (or a rotational speed of 1000rpm/min), and centrifuge the diluted mononuclear cells obtained in step E for 10min.

G. Resuspend the mononuclear cell pellet obtained in step F with PBS or a suitable medium for use.

3. CD34 ⁺ CD38 ^- stem cell sorting

Use Miltenyi's magnetic bead sorting kit to sort the leukemia stem cells (LSC) in the mononuclear cells of AML patients isolated in step 2 or the hematopoietic stem cells (HSC) in the mononuclear cells of normal people, and screen out CD34 ⁺ CD38 ^- stem cells. Specifically include the following steps:

A. Resuspend 2×10 ⁸ mononuclear cells with 600 μL PBS to obtain resuspended mononuclear cells.

B. Add 200 μL FcR blocking solution and 200 μL magnetic bead-coupled anti-CD34 antibody to the resuspended mononuclear cells obtained in step A, and incubate at 4°C for 30 min to obtain blocked and coupled mononuclear cells.

C. Add 50 μL of FITC-labeled anti-CD38 antibody to the blocked and conjugated mononuclear cells obtained in step B, and incubate at 4°C for 10 min to obtain FITC-labeled mononuclear cells.

D. Add the FITC-labeled mononuclear cells obtained in step D to the magnetic bead sorter, let the cells not bound to the CD34 antibody pass through the magnetic field, and retain the cells bound to the CD34 antibody.

E. Wash step D with 500 μL PBS to keep the sorter bound to CD34 antibody cells for 3 times.

F. Remove the magnetic field of the magnetic bead sorter, add 1mL PBS, and quickly push the PBS out of the sorter with the plunger. Collect the cells washed from the sorter, which are CD34 positive cells.

G. Add 20 μL of enzymatic hydrolysis solution to the CD34-positive cells collected in step F, and incubate at 4°C for 10 minutes. Centrifuge at 250g (or 1000rpm/min) for 10min, discard the supernatant, and collect the cell pellet.

H. Suspend the cell pellet collected in step G with 20 μL PBS, add 60 μL enzymatic hydrolysis stop solution, add 100 μL anti-FITC antibody, and incubate at 4°C for 30 min.

I. Add the cells incubated in step H to the magnetic bead sorter, and collect the cells not bound to the FITC antibody, which are CD34 ⁺ CD38 ^- stem cells.

Embodiment 2, detection of miR-31-5p gene expression

1. Total RNA extraction:

A. The mononuclear cells or CD34 ⁺ CD38 ^- stem cells sorted in Example 1 were washed twice with PBS.

B. Add 1mL Trizol lysate, pipette several times to lyse and mix; place at room temperature for 5min to fully separate nucleoprotein.

C. Add 200 μL of chloroform, close the cap tightly, shake vigorously for 15 seconds, and place at room temperature for 5 minutes.

D. Centrifuge at 12,000g at 4°C for 15 minutes, and pipette about 400μL of the upper aqueous phase into another new 1.5mL EP tube. Be careful not to suck up the interfacial layer.

E. Add 500 μL of isopropanol, mix thoroughly, and place at room temperature for 10 minutes.

F. Centrifuge at 12,000g for 10 minutes at 4°C, discard the supernatant, and precipitate the RNA at the bottom of the centrifuge tube.

G. Add 1 mL of 70% ethanol to wash the RNA pellet.

H, 4°C, centrifuge at 7,500g for 5min, discard the supernatant.

I. After the precipitation was air-dried for 3-5 minutes, add 40 μL of DEPC-treated water to dissolve the RNA precipitation, measure the RNA concentration and integrity, and store at -70°C. Measure the ratio of RNA concentration and 260nm/280nm: the purity requirement of total RNA is that the OD260/OD280 value should be between 1.8 and 2.2; the detection of RNA integrity: use 1% agarose gel electrophoresis to detect the integrity of RNA.

2. Synthesize cDNA template by reverse transcription (using Takara Bio's One Step PrimeScript miRNA cDNA synthesis Kit):

miRNA is different from mRNA. The length of mature miRNA is only about 20nt, which is very short. Usually, the forward primer is enough to cover its full length or even more than that, and the reverse primer has nowhere to be placed. In order to achieve qPCR amplification of miRNA, the solution is to try to increase the length of the reverse transcription product during reverse transcription. The most direct way to increase the length of the miRNA reverse transcription product is to increase the length of the miRNA, that is, to add a sequence behind the 3' end of the miRNA, and then perform the reverse transcription reaction. After the miRNA is processed by the tailing method, the length of the obtained cDNA is increased from the original 20nt to more than 80nt, so that the qPCR amplification of the miRNA can be realized.

The tailing method is completed by the joint action of two enzymes, which are PolyA polymerase and reverse transcriptase. PolyA polymerase is responsible for adding PolyA tails to miRNAs, increasing their length. Afterwards, the reverse transcription primer is bound to the PolyA sequence, and the synthesis of the extended version of cDNA is completed by reverse transcriptase. The process is shown in Figure 1.

Prepare the reaction system as follows:

Table 1 reaction system

反应体系reaction system	体积volume
RNA(0.5-8μg)RNA (0.5-8μg)	3.75μL3.75 μL
mRQ缓冲液(2×)mRQ buffer (2×)	5μL5μL
mRQ酶mRQ enzyme	1.25μL1.25 μL
加DEPC水后总体积Total volume after adding DEPC water	10μL10μL

Mix well, and set the following program in the PCR machine:

Table 2 PCR program

步骤step	温度temperature	时间time
11	37℃37°C		60min60min
22	85℃85°C		5s5s
33	4℃4°C	终止termination

After the reaction, the reaction product was stored in a -20°C refrigerator for subsequent experiments.

3. Fluorescence quantitative PCR

With the U6 housekeeping gene as the control, three parallel controls were made for each RNA sample, the amplified target gene was detected in the CFX96 detection system, and the relative quantification of RNA was analyzed by the ΔΔCt method.

Fluorescent quantitative PCR was performed using Takara Bio's kit (SYBR Premix EX Taq Kit).

Table 3 Fluorescent quantitative PCR reaction system

The forward primer is a forward primer for miR-31-5p or internal reference U6, wherein:

The nucleotide sequence of the miR-31-5p forward primer is: 5'-aggcaagatgctggcatagct-3' (SEQ ID NO 2).

The nucleotide sequence of U6 forward primer is: 5'-ggaacgatacagagaagattagc-3' (SEQ ID NO 5).

The reverse primer is the mRQ3' primer provided by the kit, wherein:

The nucleotide sequence of mRQ3' primer is: 5'-ctcaactggtgtcgtgga-3' (SEQ ID NO 6).

Table 4 Fluorescent quantitative PCR reaction conditions

步骤step	温度temperature	时间time	循环数number of cycles
11	95℃95°C	10s10s	--
22	95℃95°C	5s5s	--
33	60℃60℃	30s30s	返回步骤2，重复40个循环Return to step 2 and repeat for 40 cycles

4. Data processing and result analysis

The software configured with fluorescent quantitative PCR was used to process the data of quantitative PCR, and the threshold and baseline of each gene were established, and the melting amplification curve was automatically generated by the software. Obtain the CT value corresponding to each sample response. The relative expression in the samples was calculated using the 2-ΔΔct algorithm, and then the log(2-ΔΔct) of each sample was calculated for plotting.

The results are shown in Figure 2. Compared with the bone marrow hematopoietic stem cells (HSC) of 5 normal people, the expression of miR-31-5p in the bone marrow leukemia stem cells (LSC) of 44 AML patients was significantly down-regulated. Similarly, as shown in Figure 3, the expression of miR-31-5p was also significantly down-regulated in the bone marrow cells (AML) of 44 AML patients compared with the bone marrow cells (BM) of 5 normal people. The above results show that miR-31-5p is a very good diagnostic marker for AML and has a good clinical application prospect.

Example 3. Analysis of the relationship between miR-31-5p gene expression and the prognosis of AML patients from the TCGA database

Download the original miRNA expression of TCGA-LAML in GDC (https://portal.gdc.cancer.gov/) through GDCRNATool (http://bioconductor.org/packages/devel/bioc/html/GDCRNATools.html) R language package data and clinical information. Filter duplicate samples in miRNA raw data and merge miRNA raw count data into a single expression matrix. To perform TMM normalization and Voom transformation, by running the gdcVoomNormalization function, the raw count data will be normalized by the TMM method implemented in edgeR and further transformed by the voom method provided in limma. Use the gdcSurvivalAnalysis toolkit in the R language package GDCRNATools for Kaplan Meier (KM) analysis, import miR-31-5p expression data and clinical information in the TCGA database, and group by miR-31-5p expression, in order to maximize the analysis Sensitivity and to find any potential correlation with survival independent of a preset cutoff (e.g. median), every possible cutoff between the lower and upper quartiles of expression was calculated. Each of these cutoffs was then used for a separate Kaplan Meier (KM) analysis. False discovery rate (FDR) was calculated to correct for multiple hypothesis testing, and results were considered significant only if FDR < 0.05. In the final result, the optimal cut-off value with the lowest p value (in this embodiment, the lowest p value is 0.0284) was used, and it was divided into high expression group N=91, low expression group N=73 (wherein the Cases were considered high miR-31-5p expression, and cases below the cutoff value were considered low miR-31-5p expression), and the gdcKMPlot toolkit was used to draw survival curves. The GDCRNAT tool was used to analyze the relationship between the expression level of miR-31-5p gene and the survival of AML patients. The results in Figure 4 show that the lower the expression level of miR-31-5p, the shorter the survival period of the patient. The above results indicated that the expression level of miR-31-5p was directly related to the prognosis of AML patients.

Example 4, Detection of AML-LSC Clonogenic Ability in Vitro

1. Sample selection and numbering

The leukemia stem cells (AML-LSC) of acute myeloid leukemia were separated according to the method in Example 1. From the 44 AML cases detected in Example 2, the LSCs of 3 different AML patients were selected to carry out the clone formation experiment, and corresponding to Patient sample numbers, the leukemia stem cell (LSC) clone formation experiments of these 3 AML patients were numbered AML-5, AML-8 and AML-9, respectively.

2. AML-LSC culture

AML-LSC cell culture adopts Serum-Free Medium (STEMCELL Technologies Company) medium, in addition, three kinds of stem cell factors need to be added to the medium: rIL3 (final concentration 10 ng/mL, PeproTech Company), rFlt3 (final concentration 10 ng/mL, PeproTech Company) and rSCF (final concentration 25ng/mL, PeproTech Company), the cells were cultured at 37° C. in a 5% CO ₂ incubator, and the medium was replaced or the cells were passaged every 3 days. Note that the cell culture density should be controlled at 10 ³ -10 ⁴ cells/mL to prevent stem cell differentiation.

3. Lentivirus infection

Carry a DNA sequence capable of expressing control RNA (abbreviated as control RNA in the example in Figure 5, and its DNA sequence is 5'-ttctccgaacgtgtcacgt-3') or a DNA sequence capable of expressing miR-31-5p (abbreviated as miR-31- 5p, whose DNA sequence is 5'-aggcaagatgctggcatagct-3') lentiviral particles were purchased from Shanghai Gemma Company (control RNA product batch number G07AZ; miR-31-5p product batch number 170305DZ), control RNA lentivirus and expression miR-31 The lentivirus titer of -5p is greater than 10 ⁹ TU (that is, the number of biologically active virus particles per milliliter) to ensure the infection efficiency.

Lentiviral infection process of AML-LSC:

A. Culture 1×10 ⁴ cells in 0.5 mL medium.

B. Add 5 μg/mL polybrene, and add lentivirus according to the MOI (that is, the number of virus particles infected per cell in a system) as 100. For example, 10 ⁴ cells need to add 10 ⁶ TU of virus.

C, 37°C, 5% CO ₂ incubator for 4 hours.

D. Add 0.5mL culture medium again, and culture in 37°C, 5% CO ₂ incubator for 24 hours.

E. After 24 hours, add 1 mL of culture medium and continue culturing for 24 hours for subsequent experiments.

4. Cloning culture process

A. Take 2000 AML-LSC cells infected with lentivirus for 48 hours and mix them evenly with 2 mL of semi-solid medium Methylcellulose Medium (STEMCELL Technologies).

B. Inoculate the cell/semi-solid medium mixture prepared in step A above in a 6-well plate, and inoculate 3 replicate wells for each AML case cell.

C, 37°C, 5% CO ₂ incubator for 14 days.

5. Data processing and result analysis

Each well was imaged and photographed under a 10× microscope, and the number of cell clones that could be formed in each well was recorded and counted.

Figure 5 shows the effect of lentivirus expressing miR-31-5p on the colony formation ability after infection of AML-LSC. As shown in Figure 5a, three cases of AML-LSC cells infected with lentivirus expressing control RNA could form good cell clones after 14 days of culture. On the contrary, three cases of AML-LSC cells infected with lentivirus expressing miR-31-5p, after 14 days of culture, the number of clones formed was significantly reduced, and the size of clones was also significantly inhibited.

Statistical analysis was performed using GraphPad Prism 8 software. Results show mean ± standard deviation. Statistical methods The t-test was used for comparison between means, *P<0.05, **P<0.01, ***P<0.001 were considered statistically significant. As shown in Figure 5b, the statistical results showed that the clonogenic ability of 3 AML-LSC cells was significantly inhibited by the expression of miR-31-5p. In the control group, about 100 clones could be formed per 2000 AML-LSC cells, while in the miR-31-5p expression group, the number of clones formed per 2000 AML-LSC cells was less than 20.

Example 5, the effect of miR-31-5p on AML-LSC cells and bone marrow AML cells

1. Sample selection and numbering

The leukemia stem cells (AML-LSC) of acute myeloid leukemia are separated according to the method in Example 1, and from the 44 AML cases detected in Example 2, the LSC and bone marrow AML cells of 3 different AML patients are selected for cell death detection Experiments, and corresponding to the patient sample numbers, the cell death detection experiments of these 3 AML patients were numbered AML-5, AML-8 and AML-9, respectively.

2. Culture of AML-LSC and bone marrow AML cells

AML-LSC culture method is the same as embodiment 4.

Bone marrow AML cells were cultured in 1640 medium containing 20% fetal bovine blood at 37°C and 5% CO ₂ .

3. Lentivirus infection

The method of lentivirus infection of AML-LSC is the same as that in Example 4.

The method for infecting bone marrow AML cells with lentivirus was the same as that for infecting AML-LSC in Experimental Example 4.

4. Cell death detection

After the lentivirus-infected AML-LSC and bone marrow AML cells were cultured for 96 hours, the cell death rate was detected (using the LIVE/DEAD cell viability detection kit from THERMO FISHER company):

A. Centrifuge at 1000g for ^5min to collect 1×105 cells;

B. Discard the supernatant, wash the cell pellet once with 1mL PBS, and centrifuge again at 1000g for 5min to collect the cells;

C. Resuspend the cells with 1mL PBS;

D. Add 1 μL of the fluorescent dye provided in the kit and mix well;

E. Incubate at room temperature for 30 minutes in the dark;

F, BD Accuri ^TM C6 (BD) flow cytometry detection.

5. Data processing and result analysis

The results of flow cytometry were analyzed by FlowJo_V10 software, and GraphPad Prism 8 software was used for statistical analysis. Results show mean ± standard deviation. Statistical methods The t-test was used for comparison between means, *P<0.05, **P<0.01, ***P<0.001 were considered statistically significant. Three replicate experiments were performed in parallel for each case.

Taking the fluorescence intensity (Intensity) as the abscissa and the cell number as the ordinate, normal living cells only show a peak with a weaker fluorescence intensity; when the cells die, the fluorescent dye infects more, so a peak with a stronger fluorescence intensity appears. Fig. 6 shows the results of cell death induced by lentivirus expressing miR-31-5p after infection of AML-LSC and bone marrow AML cells. As shown in Figure 6a, the expression of miR-31-5p caused the cells to have a strong fluorescence peak, indicating that the cells had died. The statistical results in Figure 6b show that no matter it is AML-LSC cells or bone marrow AML cells, the cell death rate of three different case control groups is lower than 5%, but the cell death rate of the miR-31-5p expression group is significantly increased, and the AML- 5. The average death rates of AML-LSC and bone marrow AML cells in the three cases of AML-8 and AML-9 were: AML-LSC (41±3, 30.4±3.5 and 39.8±4.8), bone marrow AML cells (43.5 ±5.1, 51.4±4.9 and 43.4±6.6). Therefore, miR-31-5p expression can effectively induce AML-LSC and bone marrow AML cell death.

Example 6. Effects of miR-31-5p combined with chemotherapy drugs on AML-LSC cells and bone marrow AML cells

1. Sample selection and numbering

2. Culture of AML-LSC and bone marrow AML cells

AML-LSC culture method is the same as embodiment 4.

3. Lentivirus infection

The method of lentivirus infection of AML-LSC is the same as that in Example 4.

4. Cell death detection

After the lentivirus-infected AML-LSC and bone marrow AML cells were cultured for 72 hours, they were cultured for 24 hours in the medium containing or not containing 5 μM chemotherapeutic drug cytarabine (Ara-C), and then the cell death rate was detected. The specific detection method is the same as in Example 5.

5. Data processing and result analysis

Figure 7 shows the results of cell death after the lentivirus expressing miR-31-5p infected AML-LSC and bone marrow AML cells treated with cytarabine. As shown in Figures 7a and 7b, whether it was AML-LSC cells or bone marrow AML cells, the cell death rate in the miR-31-5p expression group increased. However, the death rate of AML-LSC cells and bone marrow AML cells was further increased after the chemotherapy drug cytarabine was used in combination. The average death rates of AML-LSC and bone marrow AML cells in the three cases of AML-5, AML-8 and AML-9 were as follows: AML-LSC (36±2.9 increased to 72±6.6, 33.6±4.5 increased to 64.6 ±6.9, and 29.3±3.6 increased to 47.3±4.6), bone marrow AML cells (41.6±3.3 increased to 65.3±6.6, 39.3±6.5 increased to 73.6±4.1, and 46.3±7.1 increased to 69.6±5.7). Therefore, miR-31-5p expression can effectively enhance chemotherapy drug-induced AML-LSC and bone marrow AML cell death.

Example 7. In vivo experiments in animals verify the effect of miR-31-5p expression on AML and AML-LSC

1. Cell culture and virus infection

AML-LSC culture and lentivirus infection methods are the same as in Example 4.

2. Feeding of experimental animals

B-NDG immunodeficient mice aged 4-6 weeks were purchased from Beijing Biocytogen Biotechnology Co., Ltd. After the mice were purchased, they were kept in an SPF-grade animal room, and the experiment was carried out after one week of adaptation to the environment.

3. Treatment of experimental animals before cell transplantation

24 hours before the cell transplantation experiment, B-NDG immunodeficient mice were irradiated by Rad Source RS2000 series X-ray bioirradiation apparatus, and received a radiation dose of 2Gy to destroy the residual immune system.

4. Establishment of B-NDG mouse leukemia model

Within 24 hours, AML-LSC cells infected with lentiviruses expressing control RNA (Control RNA) and miR-31-5p (miR-31-5p) were injected into mice through the tail vein, and the number of injected cells was 1 ×10 ⁶ cells/mouse to establish B-NDG mouse leukemia model. AML-LSC cells of each case were injected into 12 mice.

5. Positive cell detection

After the second week of transplantation, 6 mice in each group were randomly selected for dissection, bone marrow coelomocytes were isolated, stained with anti-human CD45 and CD34 antibodies, and the proportion of positive cells was detected by flow cytometry (CD45 molecules were present on all leukocytes). There is expression, and the anti-human CD45 antibody can specifically recognize the human leukocytes transplanted in mice, so the ratio of CD45 ⁺ cells reflects the ratio of human AML cells transplanted into mice, and the CD45 ⁺ CD34 ⁺ cells reflect the transplanted human leukocytes AML-LSC). The specific method is as follows:

A. Take the mouse bone marrow, remove the red blood cells with the red blood cell lysate, and obtain the bone marrow cells.

B. Wash the cells once with PBS, and collect the cells by centrifugation at 1000 rpm for 5 minutes.

C. Cell staining buffer (Biolegend Company) was used to resuspend the cells, and the cell density was adjusted to 10 ⁶ cells/mL.

D. Take 200 μL of cell suspension and add 20 μL of FcR blocking reagent.

E. Add 5 μL FITC-labeled anti-human CD45 antibody and 5 μL APC-labeled anti-human CD34 antibody.

F. At room temperature, incubate in the dark for 30 minutes, and mix by inverting every 10 minutes during this period.

G, BD Accuri ^TM C6 (BD) flow cytometry detection.

6. Drawing of mouse survival curve

The remaining 6 mice continued to be fed, the time of death was recorded, and the survival curve was drawn.

7. Data processing and result analysis

The results of flow cytometry were analyzed by FlowJo_V10 software, and GraphPad Prism 8 software was used for statistical analysis. Results show mean ± standard deviation. Statistical methods The t-test was used for comparison between means, *P<0.05, **P<0.01, ***P<0.001 were considered statistically significant. Survival curves were drawn using the Survival function of GraphPad Prism 8 software.

Figure 8 shows the verification of the therapeutic effect of miR-31-5p expression on AML disease in B-NDG mice. in:

The results in Figure 8a show that, after 2 weeks of transplantation, AML cells (hCD45 ⁺ cells) were successfully implanted in the bone marrow cavity of the mice treated by the control group, and the three cases of AML-5, AML-8 and AML-9 The implantation rates of AML cells were: 40.5±6.9, 32.5±6.2 and 32.3±7.3; on the contrary, the cell implantation rates in the miR-31-5p expression group were significantly decreased, respectively: 4±3, 6.3±4.5 and 7.7 ±4.4.

The results in Figure 8b show that, 2 weeks after transplantation, AML-LSC cells (hCD45 ⁺ CD34 ⁺ cells) were successfully implanted in the bone marrow cavity of mice treated with the control group, and AML-5, AML-8 and AML-9 The engraftment rates of AML-LSC cells in the three cases were: 9.6±3.9, 11.2±4.6, and 8.1±3.8; on the contrary, the cell engraftment rates in the miR-31-5p expression group were significantly decreased, respectively: 1.5±1.1 , 1.1±0.7 and 2.4±1.5.

The results in Figure 8c show that mice treated with the control group died from the 15th day after transplantation, and all mice died within 30 days. The median survival periods were 19 days, 20 days and 19 days, respectively; in contrast, only a few mice in the miR-31-5p expression group died, and most of the mice survived for more than 40 days.

These results indicate that the expression of miR-31-5p in the B-NDG mouse model can inhibit the growth of AML and AML-LSC cells in the mouse bone marrow and significantly prolong the lifespan of the mice.

The above-mentioned embodiment is a preferred implementation mode of the present disclosure, but the implementation mode of the present disclosure is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, All simplifications should be equivalent replacement methods, and all are included in the protection scope of the present disclosure.

Claims

Products for detecting miR-31-5p, pri-miR-31 and/or pre-miR-31 are used in the preparation of auxiliary diagnosis of subjects suffering from acute myeloid leukemia (AML) and/or for prognosing the survival of subjects Use in reagents, chips or kits that are out of date;

Preferably, the reagent is capable of detecting the level of miR-31-5p, pri-miR-31 and/or pre-miR-31 in a biological sample;

Preferably, the level of miR-31-5p, pri-miR-31 and/or pre-miR-31 in the biological sample is lower than the corresponding miR-31-5p, pri-miR-31 and/or in the normal control sample or the level of pre-miR-31 indicates that the subject has acute myeloid leukemia (AML);

Preferably, the biological sample is selected from one or more of peripheral blood, bone marrow and tissues suspected of having leukemia cells;

Preferably, said subject may be a human or other mammal.
The use according to claim 1, wherein, the level of the miR-31-5p, pri-miR-31 and/or pre-miR-31 adopts high-throughput sequencing method, miRNA expression profiling chip, quantitative PCR method and / or probe hybridization method for detection;

Preferably, the sequence of the miR-31-5p is shown in SEQ ID NO 1; preferably, the sequence of the pre-miR-31 is shown in SEQ ID NO 3;

Preferably, the reagent comprises a forward primer for amplifying miR-31-5p; preferably, the sequence of the forward primer is shown in SEQ ID NO 2;

Preferably, miR-31-5p, pri-miR-31 and/or pre-miR-31 in the biological sample are compared to the corresponding miR-31-5p, pri-miR-31 and/or pre-miR-31 in the normal control sample. If the transcript level of the miR-31 gene is low, the subject has a shorter survival period.
The use according to claim 1 or 2, wherein the chip comprises a solid phase carrier and oligonucleotide probes immobilized on the solid phase carrier.
Use of miR-31-5p, pri-miR-31 and/or pre-miR-31 in the preparation of drugs for the prevention and/or treatment of acute myeloid leukemia (AML), wherein the sequence of the miR-31-5p is as follows Shown in SEQ ID NO 1; The sequence of the pre-miR-31 is shown in SEQ ID NO 3.
The use according to claim 4, wherein miR-31-5p, pri-miR-31 and/or pre-miR-31 are natural, artificially synthesized or can be expressed by using miR-31-5p, pri-miR -31 and/or pre-miR-31 DNA fragment expression vector transfected cells obtained, wherein, the gene sequence of the miR-31-5p is shown in SEQ ID NO 2; the pre-miR-31 The gene sequence is shown in SEQ ID NO 4.
The use according to claim 5, wherein the expression vector is selected from the group consisting of plasmids, phages, phagemids, cosmids, viral vectors, virus particles, prokaryotic expression vectors and eukaryotic expression vectors;

Preferably, the viral vector is selected from retroviral vectors, lentiviruses, adenoviral vectors, adeno-associated viral vectors, herpesvirus vectors, alphavirus vectors, baculoviruses and vaccinia viruses;

Preferably, the prokaryotic expression vector is selected from an E. coli expression vector and a Bacillus subtilis expression vector;

Preferably, the eukaryotic expression vector is selected from yeast expression vectors, insect expression vectors or mammalian expression vectors.
The use according to any one of claims 4-6, wherein the drug can be administered alone or in combination with other drugs capable of inhibiting AML;

Preferably, the other drugs capable of inhibiting AML are selected from daunorubicin, cytarabine, thioguanine, etoposide, harringtonine, vincristine, prednisone, mitoxantrone , doxorubicin, cyclophosphamide, carboplatin, decitabine, methotrexate, etoposide, doxorubicin (doxorubicin), cisplatin, dexamethasone, saccomesine, methyl Nitrosourea, fluorouracil, 5-fluorouracil, vinblastine, camptothecin, actinomycin-D, mitomycin C, hydrogen peroxide, oxaliplatin, irinotecan, topotecan, leucovorin One or more of , carmustine, streptozocin, paclitaxel, tamoxifen, dacarbazine, imatinib, azacitidine and gemtuzumab.
The use according to any one of claims 4-7, wherein, preferably, the medicament further comprises a pharmaceutically acceptable carrier;

Preferably, the pharmaceutically acceptable carrier is selected from lactose, dextrose, sucrose, polyvinylpyrrolidone, alginate, gelatin, cellulose, syrup, sorbitol, mannitol, starch, acacia, talc, Magnesium Stearate, Calcium Phosphate, Calcium Silicate, Microcrystalline Cellulose, Methyl Cellulose, Methyl Hydroxybenzoate, Propyl Hydroxybenzoate, Mineral Oil, Microcapsules & Microspheres, Nanoparticles, Lipid one or more of the body.
The use according to any one of claims 4-8, wherein the dosage form of the drug is solution, injection, oral liquid, suspension, emulsion, extract, powder, granule, suppository, aerosol granules, tablets or capsules;

Preferably, injections include sterile or sterilized solutions, water injections, oil injections, powder injections, etc.; oral liquid dosage forms include solutions, syrups, emulsions, suspensions, etc.; tablets include ordinary compressed tablets, sugar-coated tablets, Effervescent tablets, chewable tablets, multi-layer tablets, implant tablets, sustained-release tablets, controlled-release tablets, etc.;

Preferably, the medicament further contains a dispersant or a stabilizer.
A method of treating acute myeloid leukemia (AML), comprising administering to said subject a therapeutically effective amount of miR-31-5p, pri-miR-31 and/or pre-miR-31 or a medicament comprising the same combination.