CN113584166A - Application of miR-31-5p in acute myeloid leukemia - Google Patents

Abstract

Translated fromChinese

本公开涉及miR‑31‑5p在急性髓系白血病中的应用，其中包括检测miR‑31‑5p的产品在制备用于辅助诊断受试者患有急性髓系白血病(AML)和/或用于预后受试者生存期的试剂、芯片或试剂盒中的用途，以及miR‑31初始/前体miRNA(pri/pre‑miR‑31)和/或者miR‑31成熟miRNA(miR‑31‑5p)在制备治疗AML药物中的用途。通过基因转导的方法将miR‑31‑5p导入细胞能诱导骨髓AML细胞和白血病干细胞(AML‑LSC)细胞死亡，并增强化疗药物阿糖胞苷的细胞毒性；表达miR‑31‑5p能抑制AML细胞和AML‑LSC细胞的生长，延长动物寿命。本公开提供了用于AML诊断和预后评估的新方法，同时还提供了用于制备AML的基因治疗药物。The present disclosure relates to the application of miR-31-5p in acute myeloid leukemia, including the preparation of products for detecting miR-31-5p for auxiliary diagnosis of subjects suffering from acute myeloid leukemia (AML) and/or for use in Use in reagents, chips or kits for prognosing survival of a subject, and miR-31 naive/precursor miRNA (pri/pre-miR-31) and/or miR-31 mature miRNA (miR-31-5p) Use in the preparation of medicines for the treatment of AML. The introduction of miR-31-5p into cells by gene transduction can induce cell death of bone marrow AML cells and leukemia stem cells (AML-LSC) and enhance the cytotoxicity of the chemotherapeutic drug cytarabine; expression of miR-31-5p can inhibit the Growth of AML cells and AML‑LSC cells to prolong animal lifespan. The present disclosure provides new methods for AML diagnosis and prognostic assessment, as well as gene therapy drugs for the preparation of AML.

Description

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

Technical Field

The disclosure belongs to the field of biological medicine, relates to diagnosis and treatment of tumors, and particularly relates to application of miR-31-5p in diagnosis and treatment of Acute Myeloid Leukemia (AML).

Background

Leukemia is a type of clonal malignant disease derived from hematopoietic stem cells, and it is statistical that about 35 million people die of leukemia worldwide each year. Thus, leukemia has become a major malignancy that currently threatens human health. Leukemia Stem Cells (LSCs) are a very small population of cells present in leukemia patients, accounting for approximately 0.1% to 1% of all leukemia cells. LSCs were first discovered and widely recognized in Acute Myeloid Leukemia (AML). LSCs can cause and maintain leukemia, both in long-term cell culture and in animal models; meanwhile, unlike leukemia cells, more than 95% of LSCs are in a "dormant" state at stage G0, and these cells are insensitive to traditional cell-cycling chemotherapeutic drugs, and the remaining LSCs increase the recurrence rate of leukemia patients through self-replication (Renewal). Therefore, the unique self-protection mechanism of LSC is found, and the targeted killing of LSC is an important way to cure leukemia completely.

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules approximately 19-25bp in length, including 6-8nt seed sequences and 12-17nt supplemental sequences. The miRNA gene is transcribed to produce a primary miRNA (pri-miRNA), which forms a hairpin structure under processing by Drosha enzyme, and is divided into 5 'and 3' end arms, also referred to as 5p and 3p, depending on the order of transcription. The sequences of the 5 'end arm and the 3' end arm are complemented to form double-stranded RNA, which becomes a mature miRNA precursor (pre-miRNA), and the mature miRNA precursor is transported to cytoplasm and plays a role in controlling gene expression after transcription. The pre-miRNA, once it enters the cytoplasm from the nucleus, is immediately further processed by Dicer enzymes to become a mature miRNA. These mirnas play an important role in regulating gene expression, and participate in cell differentiation, proliferation, and maintenance of cell homeostasis.

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

However, there is no report on the expression of miR-31-5p in AML cells and AML stem cells. Therefore, there is an urgent need for better understanding of biomarkers in acute myeloid leukemia to develop prognostic prediction tools and to discover new drugs.

Disclosure of Invention

In order to solve the defects of the prior art, the purpose of the disclosure is to screen out the acute myeloid leukemia as a molecular diagnosis biomarker and develop a corresponding diagnosis kit for diagnosing, treating or providing prognosis of the acute myeloid leukemia.

Specifically, the present disclosure proposes the following technical solutions:

in one aspect, the disclosure provides a use of a product for detecting miR-31-5p, pri-miR-31 and/or pre-miR-31 in preparation of a reagent, a chip or a kit for assisting in diagnosing Acute Myeloid Leukemia (AML) in a subject and/or for prognosing survival of the subject.

In one aspect, the disclosure provides a use of miR-31-5p, pri-miR-31 and/or pre-miR-31 in preparation of a medicament for preventing and/or treating Acute Myeloid Leukemia (AML).

In one aspect, the present disclosure provides a medicament comprising a miR-31 primary miRNA, a miR-31 precursor miRNA, and/or a mature miR-31-5p for treating Acute Myeloid Leukemia (AML).

In one aspect, the disclosure provides a method of treating Acute Myeloid Leukemia (AML) comprising administering to the subject a therapeutically effective amount of miR-31-5p, pri-miR-31, and/or pre-miR-31, or a pharmaceutical composition comprising the same.

In vitro cell culture experiments show that miR-31-5p is introduced into cells by a gene transduction method to induce death of AML and AML-LSC cells, and mouse animal experiments show that miR-31-5p is expressed to inhibit growth of AML cells and AML-LSC cells and prolong the life of mice. The present disclosure provides novel methods for diagnosis and prognosis evaluation of AML, and also for preparing gene therapy drugs for AML.

Description of the drawings:

figure 1 shows the principle of miRNA tailing reverse transcription.

FIG. 2 shows QPCR assays of miR-31-5p expression in myeloid Leukemia Stem Cells (LSCs) and normal human myeloid Hematopoietic Stem Cells (HSCs) of AML patients.

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

FIG. 4 is a graph of the relationship between the expression level of miR-31-5p and the prognostic survival of AML patients.

FIG. 5 is a graph showing the effect of miR-31-5p on the clonality of AML-LSC.

FIG. 6 is a graph showing the results of miR-31-5p inducing AML-LSC and myeloid AML cell death.

FIG. 7 is a graph showing the results of miR-31-5 p-potentiating the AML-LSC and myeloid AML cell death induced by cytarabine (Ara-C), a chemotherapeutic drug.

FIG. 8 shows that miR-31-5p has therapeutic effects on AML diseases in B-NDG mice.

Detailed Description

In the present disclosure, unless defined otherwise, scientific and technical terms used herein have the meanings that are commonly understood by those of skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures used herein are all terms and conventional procedures used extensively in the relevant art. Meanwhile, for 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 a miRNA that results from transcription of a miR-31 gene by RNA polymerase.

As used herein, the term "precursor miR-31" or "pre-miR-31" means that the initial miR-31(pri-miR-31) forms a hairpin sequence under processing by Drosha enzyme, having 71 nucleotides (71nt), and its sequence is as follows: 5'-ggagaggaggcaagaugcuggcauagcuguugaacugggaaccugcuaugccaacauauugccaucuuucc-3' (SEQ ID NO 3).

As used herein, the term "mature miR-31-5 p" or "miR-31-5 p" refers to the sequence of precursor miR-31(pre-miR-31) formed under the processing of Dicer enzyme, having 21 nucleotides (21nt), and having the following sequence: 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. The vector may be stable and may be self-replicating. There is no limitation as to the type of vector that may be used. The vector may be a cloning vector, suitable for propagation and obtainment of polynucleotides, gene constructs or expression vectors which may be combined with various foreign organisms. Suitable vectors include prokaryotic expression vectors (e.g., pUC18, pUC19, Bluescript and derivatives thereof), mpl8, mpl9, pBR322, pMB9, CoIE1, pCR1, RP4, phage and shuttle vectors (e.g., pSA3 and pAT28), and eukaryotic expression vectors based on viral vectors (e.g., adenovirus, adeno-associated virus, and retrovirus and lentivirus), as well as non-viral vectors, such as pSilencer 4.1-CMV (Life technologies Corp., Carslbad, CA, USA), pcDNA3, pcDNA3.1/hyg pHGS/Zeo, pCR3.1, Fl/His, pIND/HCMV 2, pSV40/Zeo 38, pTRACER-HCMV,pUB 6/pSV 5-His, pVAXl,pVSV 2,pTSV 596l 2,pBVL 2 andpBVpVL 2.

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 into a cell, which results in the production of a product encoded by the genetic material (e.g., a protein polypeptide, peptide, or functional RNA) in the target cell. Furthermore, 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 (e.g., DNA or RNA) to a cell, which results in the production of a product encoded by the genetic material (e.g., a protein polypeptide, peptide or functional RNA) in the target cell.

As used herein, the term "pharmaceutical composition" refers to a preparation of various preparations. Formulations containing a therapeutically effective amount of miR-31-5p, pri-miR-31 and/or pre-miR-31 are sterile liquid solutions, liquid suspensions, or lyophilized forms, optionally including stabilizers or excipients.

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

As used herein, "treating" an individual having 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 prevention of the underlying disease and/or prevention of worsening of symptoms or disease progression.

As used herein, "therapeutic effect" means an effect resulting from treatment of an individual that alters, typically ameliorates or improves a symptom of a disease or disease condition, or cures the disease or disease 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 upon administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, block, or partially block the symptoms of the disease or disorder.

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

In one aspect, the present disclosure provides a use of a product for detecting miR-31-5p, pri-miR-31 and/or pre-miR-31 in preparation of a reagent, a chip or a kit for assisting in diagnosing Acute Myeloid Leukemia (AML) in a subject and/or for prognosing survival of a subject, wherein the sequence of miR-31-5p is as follows: 5'-aggcaagaugcuggcauagcu-3' (SEQ ID NO 1), wherein the sequence of the pre-miR-31 is: 5'-ggagaggaggcaagaugcuggcauagcuguugaacugggaaccugcuau gccaacauauugccaucuuucc-3' (SEQ ID NO 3).

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

In some embodiments of the disclosure, a level of miR-31-5p, pri-miR-31 and/or pre-miR-31 in the biological sample that is lower than the level of a corresponding miR-31-5p, pri-miR-31 and/or pre-miR-31 in a normal control sample 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 disclosure, the subject may be a human or other mammal.

In some embodiments of the disclosure, the level of miR-31-5p, pri-miR-31, and/or pre-miR-31 is detected using high throughput sequencing methods, miRNA expression profiling chips, quantitative PCR methods, and/or probe hybridization methods.

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

In some embodiments of the disclosure, a subject has shorter survival if the transcriptional level of miR-31-5p, pri-miR-31 and/or pre-miR-31 in the biological sample is low relative to the corresponding miR-31-5p, pri-miR-31 and/or pre-miR-31 gene in a normal control sample.

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

In one aspect, the disclosure provides a use of miR-31-5p, pri-miR-31 and/or pre-miR-31 in preparation of a medicament for preventing and/or treating Acute Myeloid Leukemia (AML).

The disclosed experiment proves that the increase of the intracellular level of miR-31-5p can directly induce the death of AML and AML-LSC cells and enhance the cytotoxicity of chemotherapeutic drug cytarabine. As described above, the miR-31-5p gene is transcribed by RNA polymerase to obtain initial miR-31(pri-miR-31), the initial miR-31(pri-miR-31) forms precursor miR-31(pre-miR-31) with a hairpin structure under the processing of Drosha enzyme, and the precursor miR-31(pre-miR-31) forms miR-31-5p under the processing of Dicer enzyme. Therefore, the pri-miR-31 and/or pre-miR-31 is increased in intracellular level, and the pri-miR-31 and/or pre-miR-31 is processed in cells to form miR-31-5p, so that the same effect of increasing miR-31-5p is achieved.

In some embodiments of the disclosure, miR-31-5p, pri-miR-31 and/or pre-miR-31 is natural, artificially synthesized or obtained by transfecting cells with an expression vector that can express a DNA fragment of miR-31-5p, pri-miR-31 and/or pre-miR-31, wherein the gene sequence of miR-31-5p is: 5'-aggcaagatgctggcatagct-3' (SEQ ID NO 2); the gene sequence of the pre-miR-31-5p is as follows: 5'-ggagaggaggcaagatgctggcatagctgttgaactgggaacctgctat gccaacatattgccatctttcc-3' (SEQ ID NO 4).

In some embodiments of the present disclosure, the expression vector is selected from the group consisting of a plasmid, a phage, a phagemid, a cosmid, a viral vector, a viral particle, a prokaryotic expression vector, and a eukaryotic expression vector.

In some embodiments of the present disclosure, the viral vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a herpesvirus vector, an alphavirus vector, a baculovirus, and a vaccinia virus.

In some embodiments of the present disclosure, the prokaryotic expression vector is selected from the group consisting of an escherichia coli expression vector, 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, the other drug capable of inhibiting AML is selected from one or more of daunorubicin, cytarabine, thioguanine, etoposide, cephalotaxine, vincristine, prednisone, mitoxantrone, doxorubicin, cyclophosphamide, carboplatin, decitabine, methotrexate, etoposide, doxorubicin (adriamycin), cisplatin, dexamethasone, sabcomeline, methylnitrosourea, fluorouracil, 5-fluorouracil, vinblastine, camptothecin, actinomycin-D, mitomycin C, hydrogen peroxide, oxaliplatin, irinotecan, topotecan, 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 one or more of lactose, dextrose, sucrose, polyvinylpyrrolidone, alginate, gel, cellulose, syrup, sorbitol, mannitol, starch, gum arabic, talc, magnesium stearate, calcium phosphate, calcium silicate, microcrystalline cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, mineral oil, microcapsules and microspheres, nanoparticles, liposomes.

In some embodiments of the present disclosure, the pharmaceutical formulation is a 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, water injections, oil injections, powder injections, and the like; oral liquid dosage forms include solutions, syrups, emulsions, suspensions and the like; the tablet includes common compressed tablet, sugar coated tablet, effervescent tablet, chewable tablet, multilayer tablet, implant tablet, sustained release tablet, controlled release tablet, etc.

In some embodiments of the present disclosure, the medicament further comprises a dispersing agent or a stabilizing agent.

In one aspect, the present disclosure provides a reagent, chip or kit for aiding in the diagnosis of Acute Myeloid Leukemia (AML) in a subject and/or for prognosing survival in a subject; the reagent, chip or kit comprises a product for detecting miR-31-5p and/or a precursor thereof in the biological sample.

In one aspect, the present disclosure provides a medicament comprising miR-31-5p, pri-miR-31 and/or pre-miR-31 for treating Acute Myeloid Leukemia (AML).

In one aspect, the present disclosure provides a pharmaceutical composition for treating Acute Myeloid Leukemia (AML), the medicament comprising miR-31-5p, pri-miR-31 and/or pre-miR-31, and a pharmaceutically-acceptable carrier.

In one aspect, the disclosure provides a method of treating Acute Myeloid Leukemia (AML) comprising administering to the subject a therapeutically effective amount of miR-31-5p, pri-miR-31, and/or pre-miR-31, or a pharmaceutical composition comprising the same.

The medicaments or pharmaceutical compositions of the embodiments are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., 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 solutions, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for adjusting the 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 formulations may 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 (herein water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable pharmaceutically acceptable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, n.j.) or Phosphate Buffered Saline (PBS). In all cases, the compositions 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 resistant to the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved 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 which delays 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 such ingredient as previously described.

It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unit doses for the subject to be treated; each unit containing a predetermined amount of miR-31-5p, pri-miR-31 and/or pre-miR-31 calculated to produce the desired therapeutic effect in combination with the desired pharmaceutical carrier.

The pharmaceutical composition may be placed in a container, package, or dispenser with instructions for administration.

In one embodiment, one or more of said miR-31-5p, pri-miR-31 and/or pre-miR-31 can be administered in a combination therapy, i.e., in combination with other drugs capable of inhibiting AML. The term "in combination" as used herein means that the agents are administered substantially simultaneously, simultaneously or sequentially. If given sequentially, the first of the two compounds is still preferably detected at an effective concentration at the treatment site at the beginning of administration of the second compound. In one instance, "combination" can also be a kit comprising miR-31-5p, pri-miR-31 and/or pre-miR-31 and other therapeutic agents together.

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

It should be noted that the skilled artisan will recognize that appropriate modifications and adaptations can be made to the primer sequences of the present disclosure based on the sequence of the relevant marker, and that such modified primer sequences can still be used to detect the marker. The present disclosure also includes such equivalent techniques.

Example 1 clinical sample Collection and cell sorting

1. Collection of AML clinical specimens

Randomly collecting 44 initial bone marrow cases (n-44) from hematology hospitalized Acute Myelogenous Leukemia (AML) patients, collecting bone marrow samples of the patients, and numbering the collected samples (AML-1, AML-2, … …, AML-44), the patients did not receive any drug treatment; normal human bone marrow samples were collected in 5 cases (n-5). All the above samples were obtained with the consent of the tissue ethics committee.

The processing method for collecting AML patient bone marrow samples and normal human bone marrow samples is as follows:

2mL of bone marrow was extracted under aseptic conditions, and heparin was added for anticoagulation. Collecting requirements of bone marrow specimens: heparin is anticoagulated, no blood clots exist, the bone marrow amount is more than 2mL, and cell sorting is carried out within 24 hours after the specimen is collected.

2. Isolation of mononuclear cells

Mononuclear cells were sorted from the AML patient bone marrow sample and the normal human bone marrow sample collected in step 1 using a dauyu human lymphocyte isolate (cat # 711101X) comprising the steps of:

A. bone marrow samples were diluted with an equal volume of 0.01M Phosphate Buffered Saline (PBS) at pH 7.4. For example, 1mL of bone marrow sample is added to 1mL of PBS and mixed well by gentle inversion.

B. And D, adding a certain volume of separation liquid into a 15mL centrifuge tube, flatly paving the diluted bone marrow sample obtained in the step A above the liquid level of the separation liquid, and keeping the interface of the two liquid levels clear to obtain a mixture of the bone marrow and the separation liquid. Ensuring that the volume ratio of the lymphocyte separating medium to the bone marrow sample after PBS dilution is 1: 2.

C. And C, under the condition of room temperature, regulating the centrifugal force of a horizontal rotor of the centrifuge to 700-800 g (or the rotating speed of 2000-2500 rpm/min), and centrifuging the mixture of the bone marrow and the separation liquid obtained in the step B for 20-30 min.

D. After the centrifugation is finished, collecting a thin and compact white film layer on the separation liquid, namely: a layer of mononuclear cells (including lymphocytes and monocytes).

E. The mononuclear cells collected in step D were diluted with 5mL of PBS and mixed by inverting the top and bottom to obtain diluted mononuclear cells.

F. The diluted mononuclear cells obtained in step E were centrifuged for 10min at room temperature with the horizontal rotor of the centrifuge adjusted to a centrifugal force of 250g (or 1000 rpm/min).

G. Resuspending the mononuclear cell pellet obtained in step F in PBS or suitable medium.

3、CD34⁺CD38^-Stem cell sorting

Sorting Leukemia Stem Cells (LSC) in the AML patient mononuclear cells separated in thestep 2 or Hematopoietic Stem Cells (HSC) in normal human mononuclear cells by adopting a magnetic bead sorting kit of Meitian whirlpool company, and screening out CD34⁺CD38^-A stem cell. The method specifically comprises the following steps:

A. resuspend with 600. mu.L PBS2×10⁸And (4) obtaining the resuspended mononuclear cells.

B. And (3) adding 200 mu L of FcR blocking solution and 200 mu L of anti-CD 34 antibody coupled with magnetic beads to the resuspended mononuclear cells obtained in the step A, and incubating for 30min at 4 ℃ to obtain the mononuclear cells subjected to blocking and coupling treatment.

C. To the blocked and conjugated mononuclear cells obtained in step B, 50. mu.L of FITC-labeled anti-CD 38 antibody was added, and incubated at 4 ℃ for 10min to obtain FITC-labeled mononuclear cells.

D. The FITC-labeled treated mononuclear cells obtained in step D were added to a magnetic bead sorter, and the cells not bound to the CD34 antibody were passed through a magnetic field, leaving the cells bound to the CD34 antibody.

E. Wash step D with 500 μ Ι _ PBS retains the sorter for 3 times that bound CD34 antibody cells.

F. The magnetic field of the magnetic bead sorter was removed, 1mL of PBS was added, and the PBS was quickly pushed out of the sorter by the plunger. The cells washed from the sorter were collected as CD34 positive cells.

G. And F, adding 20 mu L of enzymolysis liquid into the CD34 positive cells collected in the step F, and incubating for 10min at 4 ℃. Centrifuging at 250g (or 1000rpm/min) for 10min, discarding the supernatant, and collecting the cell precipitate.

H. And (3) suspending the cell precipitate collected in the step G by using 20 mu L of PBS, adding 60 mu L of enzymolysis stop solution, adding 100 mu L of anti-FITC antibody, and incubating for 30min at 4 ℃.

I. Adding the cells incubated in the step H to a magnetic bead sorter, and collecting the cells which are not combined with the FITC antibody, namely CD34⁺CD38^-A stem cell.

Example 2 detection of miR-31-5p Gene expression

1. Total RNA extraction:

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

B. Adding 1mL of Trizol lysate, blowing and beating the Trizol lysate for several times by using a pipettor, and uniformly mixing the Trizol lysate and the pipette lysate; the mixture was left at room temperature for 5min to separate the nucleoprotein thoroughly.

C. Add 200. mu.L chloroform, cover the tube tightly, shake vigorously for 15s, and let stand at room temperature for 5 min.

D. Centrifuge at 12,000g for 15min at 4 ℃ and aspirate approximately 400. mu.L of the upper aqueous phase into another new 1.5mL EP tube. Care was taken not to draw in the intermediate interface layer.

E. Adding 500 μ L isopropanol, mixing well, and standing at room temperature for 10 min.

F. Centrifugation was carried out at 12,000g for 10min at 4 ℃ and the supernatant was discarded, and RNA was precipitated at the bottom of the tube.

G. The RNA pellet was washed by adding 1mL of 70% ethanol.

H. Centrifuge at 7,500g for 5min at 4 ℃ and discard the supernatant.

I. And (3) air-drying the precipitate for 3-5 min, adding 40 mu L of DEPC treated water to dissolve the RNA precipitate, measuring the concentration and integrity of the RNA, and storing at-70 ℃. Determination of RNA concentration and 260nm/280nm ratio: the purity requirement of total RNA is that the OD260/OD280 value should be between 1.8 and 2.2; detection of RNA integrity: the integrity of the RNA was checked by electrophoresis on a 1% agarose gel.

2. cDNA templates were synthesized by reverse transcription (using One Step PrimeScript miRNA cDNA Synthesis Kit from Takara Bio):

miRNA differs from mRNA in that the length of mature miRNA is only about 20nt, which is very short, and usually the forward primer is enough to cover the full length and even the remaining one, and the reverse primer is installed everywhere. To achieve qPCR amplification of mirnas, the solution is to try to increase the reverse transcription product length at the time of reverse transcription. The most direct method for increasing the length of the reverse transcription product of miRNA is to increase the length of miRNA, i.e. adding a sequence behind the 3-terminal of miRNA, and then carrying out reverse transcription reaction. After miRNA is processed by a tailing method, the length of the obtained cDNA is increased from the original 20nt to more than 80nt, so that qPCR amplification of miRNA can be realized.

The tailing method is performed by the combined action of two enzymes, which are PolyA polymerase and reverse transcriptase, respectively. PolyA polymerase is responsible for adding PolyA tails to mirnas, increasing their length. The reverse transcription primer was then bound to the PolyA sequences and the synthesis of the extended cDNA version was completed by reverse transcriptase. The process is shown in figure 1.

The reaction system was prepared as follows:

TABLE 1 reaction System

Reaction system	Volume of
		RNA(0.5-8μg)	3.75μL
mRQ buffer (2X)	5μL
		mRQ enzyme	1.25μL
Total volume of DEPC water added	10μL

Mix well and set up the following program on the PCR instrument:

TABLE 2PCR procedure

Step (ii) of	Temperature of	Time
			1	37℃	60min
2	85℃	5s
			3	4℃	Terminate

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

3. Fluorescent quantitative PCR

Using U6 housekeeping gene as control, 3 parallel controls are made for each RNA sample, the amplified target gene is detected in CFX96 detection system, and the relative quantification of RNA is analyzed by delta Ct method.

Fluorescent quantitative PCR was performed using a Kit of Takara Bio (SYBR Premix EX Taq Kit).

TABLE 3 fluorescent quantitative PCR reaction System

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

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

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

The reverse primer is mRQ 3' primer provided by the kit, wherein:

mRQ 3' primer has the nucleotide sequence: 5'-ctcaactggtgtcgtgga-3' (SEQ ID NO 6).

Fluorescent quantitative PCR reaction condition

Step (ii) of	Temperature of	Time	Number of cycles
				1	95℃	10s
2	95℃	5s
				3	60℃	30s	Returning to thestep 2, repeating 40 cycles

4. Data processing and result analysis

And processing the data of the quantitative PCR by using software configured with the fluorescent quantitative PCR, establishing a threshold value and a base line of each gene, and automatically generating a lysis amplification curve by the software. And obtaining the corresponding CT value of each sample reaction. The relative expression levels in the samples were plotted by calculating the log (2- Δ Δ ct) for each sample using the 2- Δ Δ ct algorithm.

As a result, as shown in FIG. 2, the expression of miR-31-5p was significantly reduced in myeloid Leukemia Stem Cells (LSCs) of 44 AML patients, as compared to that of 5 normal human myeloid Hematopoietic Stem Cells (HSCs). Similarly, as shown in fig. 3, the expression of miR-31-5p was also significantly down-regulated in bone marrow cells (AML) of 44 AML patients compared to bone marrow cells (BM) of 5 normal persons. The results show that the miR-31-5p is a very good AML diagnosis marker and has a very good clinical application prospect.

Example 3 analysis of miR-31-5p Gene expression from TCGA database in relation to prognosis of AML patients

The original miRNA expression data and clinical information of TCGA-LAML in GDC (https:// portal. GDC. cancer. gov /) were downloaded via GDCRNATool (http:// bioconductor. org/packages/level/bioco/html/GDCRNATools. html) R language package. Duplicate samples in the miRNA raw data were filtered, and the raw count data for mirnas were merged into a single expression matrix. TMM normalization and Voom conversion are performed, and by running the gdcVoomNormalization function, the raw count data will be normalized by the TMM method implemented in edgeR and further converted by the Voom method provided in limma. Kaplan Meier (KM) analysis is carried out by using a gdcSurvival analysis toolkit in R language package GDCRNATools, miR-31-5p expression data and clinical information imported into a TCGA database are grouped through miR-31-5p expression quantity, and each possible cut-off value between the lower quartile and the upper quartile of expression is calculated in order to improve the sensitivity of analysis to the maximum extent and find any potential relevance to survival regardless of a preset cut-off value (such as a median). Each of these cut-off values was then used for a separate Kaplan Meier (KM) analysis. The False Discovery Rate (FDR) was calculated to correct for multiple hypothesis testing, and results were considered significant only if FDR < 0.05. The optimal cut-off with the lowest p-value (in this example the lowest p-value is 0.0284) was used in the final results, divided into high expression group N91, low expression group N73 (where cases above the cut-off were considered high for miR-31-5p expression and cases below the cut-off were considered low for miR-31-5p expression), and the gdcpmplot toolkit was used to plot survival curves. And analyzing the survival relationship between the miR-31-5p gene expression level and the AML patient by using a GDCRNAT tool. FIG. 4 shows that the lower the expression level of miR-31-5p, the shorter the survival time of the patient. The above results indicate that the expression level of miR-31-5p is directly related to the prognosis of AML patients.

Example 4 detection of the ability of AML-LSC to clone in vitro

1. Sample selection and numbering

Leukemia stem cells of acute myeloid leukemia (AML-LSC) were isolated according to the method of example 1, and from 44 AML cases examined in example 2, LSCs of 3 different AML patients were selected for clonogenic experiments, and the Leukemia Stem Cell (LSC) clonogenic experiments of the 3 AML patients were numbered AML-5, AML-8 and AML-9, respectively, corresponding to the patient sample numbers.

2. AML-LSC culture

AML-LSC cell culture adopts Serum-Free Medium (STEMCELL Technologies), and 3 stem cell factors are added into the Medium: rIL3 (final concentration 10ng/mL, PeproTech Co.), rFlt3 (final concentration 10ng/mL, PeproTech Co.) and rSCF (final concentration 25ng/mL, PeproTech Co.), cell culture at 37 deg.C and 5% CO₂In the incubator, medium replacement or cell passaging was performed every 3 days. Note that the cell culture density should be controlled at 10³～10⁴one/mL to prevent stem cell differentiation.

3. Lentivirus infection

Lentiviral particles carrying a DNA sequence capable of expressing control RNA (abbreviated as control RNA in the example of the figure, the DNA sequence of which is 5'-ttctccgaacgtgtcacgt-3') or a DNA sequence capable of expressing miR-31-5p (abbreviated as miR-31-5p in the example of the figure, the DNA sequence of which is 5'-aggcaagatgctggcatagct-3') were purchased from Shanghai Gilmar (control RNA product batch No. G07 AZ; miR-31-5p product batch No. 170305DZ), and the titer of the control RNA lentivirus and the lentivirus expressing miR-31-5p was greater than 10⁹TU (i.e. the number of biologically active virus particles per ml) to ensure infection efficiency.

Lentiviral infection process of AML-LSC:

A. take1X 10⁴Cells were cultured in 0.5mL of medium.

B. 5. mu.g/mL polybrene was added and lentivirus was added at a MOI (i.e.the number of virus particles infected per cell in a system) of 100. E.g. 10⁴The amount of virus added to each cell is 10⁶TU。

C、37℃，5％CO₂The culture was carried out in an incubator for 4 hours.

D. Then, 0.5mL of medium was added thereto, and 5% CO was added thereto at 37 ℃₂The culture was carried out in an incubator for 24 hours.

E. After 24 hours, 1mL of the medium was added and the culture was continued for 24 hours for subsequent experiments.

4. Cloning and culturing process

A. 2000 AML-LSC cells infected with lentivirus for 48 hours were mixed well with 2mL of semisolid Medium Methelculose Medium (STEMCELL Technologies, Inc.).

B. The cell/semi-solid medium mixture prepared in step a above was seeded in 6-well plates, 3 replicate wells per AML case cell.

C、37℃，5％CO₂The culture was carried out in an incubator for 14 days.

5. Data processing and result analysis

Images of each well were taken under a 10 × microscope, and the number of cell clones that could be formed in each well was recorded and counted.

FIG. 5 shows the effect of a lentivirus expressing miR-31-5p on clonality following infection of AML-LSC. As shown in FIG. 5a, 3 cases of AML-LSC cells infected with a lentivirus expressing control RNA all formed good cell clones after 14 days of culture. In contrast, the number of clones formed by 3 AML-LSC cells infected with the miR-31-5p lentivirus after 14 days of culture is obviously reduced, and the clone size is also obviously inhibited.

Statistical analysis was performed usingGraphPad Prism 8 software. Results show mean ± standard deviation. Statistical methods comparisons between the mean values were made using a t-test, where P <0.05, P <0.01, and P <0.001 were assigned as statistical significance. As shown in FIG. 5b, statistical results showed that the clonogenic capacity of 3 AML-LSC cells was significantly inhibited by the expression of miR-31-5 p. In the control group, about 100 clones were formed per 2000 AML-LSC cells, while in the miR-31-5 p-expressing group, the number of clones formed per 2000 AML-LSC cells was less than 20.

Example 5 Effect of miR-31-5p on AML-LSC cells and myeloid AML cells

1. Sample selection and numbering

Leukemia stem cells of acute myeloid leukemia (AML-LSC) were isolated according to the method of example 1, and from 44 AML cases examined in example 2, LSC and myeloid AML cells of 3 different AML patients were selected for cell death detection experiments, and the cell death detection experiments of the 3 AML patients were numbered AML-5, AML-8 and AML-9, respectively, corresponding to the patient sample numbers.

2. Culture of AML-LSC and myeloid AML cells

AML-LSC was cultured in the same manner as in example 4.

Culturing AML cell in 1640 culture medium containing 20% fetal calf blood at 37 deg.C under 5% CO₂。

3. Lentivirus infection

The AML-LSC lentivirus infection method is the same as in example 4.

The method for infecting myeloid AML cells with lentivirus was the same as that used in Experimental example 4 for infecting AML-LSC.

4. Cell death detection

Cell death assays were performed 96 hours after incubation of lentivirus-infected AML-LSC and myeloid AML cells (using the LIVE/DEAD cell viability assay kit from THERMO FIHER):

A. centrifuging at 1000g for 5min to collect 1 × 10⁵(ii) individual cells;

B. discarding the supernatant, washing the cell precipitate with 1mL PBS for 1 time, centrifuging 1000g again for 5min, and collecting the cells;

C. resuspend cells with 1mL PBS;

D. adding 1 mul of fluorescent dye provided by the kit, and mixing uniformly;

E. incubating for 30 minutes at room temperature in a dark place;

F、BD Accuri^TMc6(BD) flow cytometer detection.

5. Data processing and result analysis

The flow cytometry detection result is subjected to data analysis through FlowJo _ V10 software, and statistical analysis is performed throughGraphPad Prism 8 software. Results show mean ± standard deviation. Statistical methods comparisons between the mean values were made using a t-test, where P <0.05, P <0.01, and P <0.001 were assigned as statistical significance. Each case was run in parallel in 3 replicates.

Taking the fluorescence Intensity (Intensity) as the abscissa and the cell number as the ordinate, normal living cells only show a peak with weaker fluorescence Intensity; when the cell is dead, the fluorescent dye is more infected, so that a peak with stronger fluorescence intensity appears. FIG. 6 shows a graph of the results of cell death induction following infection of AML-LSC and myeloid AML cells with a lentivirus expressing miR-31-5 p. As shown in FIG. 6a, the expression of miR-31-5p causes the cells to have stronger fluorescence peaks, which indicates that the cells die. FIG. 6b shows the statistics that the mortality of the cells in the control group of 3 different cases, whether AML-LSC cells or bone marrow AML cells, is less than 5%, but the cell mortality of the miR-31-5p expression group is significantly increased, and the average mortality of AML-LSC and bone marrow AML cells of 3 cases, AML-5, AML-8 and AML-9, respectively, is: AML-LSC (41 + -3, 30.4 + -3.5 and 39.8 + -4.8), and myeloid 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 myeloid AML cell death.

Example 6 Effect of miR-31-5p in combination with chemotherapeutic drugs on AML-LSC cells and myeloid AML cells

1. Sample selection and numbering

Leukemia stem cells of acute myeloid leukemia (AML-LSC) were isolated according to the method of example 1, and from 44 AML cases examined in example 2, LSC and myeloid AML cells of 3 different AML patients were selected for cell death detection experiments, and the cell death detection experiments of the 3 AML patients were numbered AML-5, AML-8 and AML-9, respectively, corresponding to the patient sample numbers.

2. Culture of AML-LSC and myeloid AML cells

AML-LSC was cultured in the same manner as in example 4.

Culturing AML cell in 1640 culture medium containing 20% fetal calf blood at 37 deg.C under 5% CO₂。

3. Lentivirus infection

The AML-LSC lentivirus infection method is the same as in example 4.

The method for infecting myeloid AML cells with lentivirus was the same as that used in Experimental example 4 for infecting AML-LSC.

4. Cell death detection

After 72 hours of culture of lentivirus-infected AML-LSC and myeloid AML cells, culture was continued for 24 hours in medium with or without 5. mu.M of the chemotherapeutic drug cytarabine (Ara-C), respectively, and then cell death rate assay was performed. The specific detection method was the same as in example 5.

5. Data processing and result analysis

The flow cytometry detection result is subjected to data analysis through FlowJo _ V10 software, and statistical analysis is performed throughGraphPad Prism 8 software. Results show mean ± standard deviation. Statistical methods comparisons between the mean values were made using a t-test, where P <0.05, P <0.01, and P <0.001 were assigned as statistical significance. Each case was run in parallel in 3 replicates.

FIG. 7 is a graph showing the results of cell death following cytarabine treatment after infection of AML-LSC and myeloid AML cells with a lentivirus expressing miR-31-5 p. As shown in FIGS. 7a and 7b, cell mortality was increased in the miR-31-5 p-expressing group regardless of AML-LSC cells or myeloid AML cells. However, the combined use of the chemotherapeutic agent cytarabine further increased the mortality of AML-LSC cells and myeloid AML cells. The mean change in mortality of AML-LSC and myeloid AML cells in 3 cases AML-5, AML-8 and AML-9, respectively, was: AML-LSC (36 + -2.9 to 72 + -6.6, 33.6 + -4.5 to 64.6 + -6.9, and 29.3 + -3.6 to 47.3 + -4.6), myeloid AML cells (41.6 + -3.3 to 65.3 + -6.6, 39.3 + -6.5 to 73.6 + -4.1, and 46.3 + -7.1 to 69.6 + -5.7). Therefore, miR-31-5p expression can effectively enhance the induction of AML-LSC and myeloid AML cell death by chemotherapeutic drugs.

Example 7 in vivo animal experiments to verify the Effect of miR-31-5p expression on AML and AML-LSC

1. Cell culture and viral infection

AML-LSC culture and lentivirus infection were performed as described in example 4.

2. Raising of laboratory animals

The B-NDG immunodeficient mice of 4-6 weeks old were purchased from Beijing Baiosaoxi chart Biotechnology Ltd. The mice were purchased and then bred in SPF animal houses, adapted to the environment for 1 week and then subjected to the experiment.

3. Treatment of laboratory animals prior to cell transplantation

24 hours before the cell transplantation experiment, the B-NDG immunodeficient mouse is irradiated by a Rad Source RS2000 series X-ray biological irradiator, the irradiation dose is 2Gy, and the residual immune system is damaged.

4. Establishment of B-NDG mouse leukemia model

Injecting AML-LSC cells infected with expression control RNA (control RNA) and miR-31-5p (miR-31-5p) lentivirus into mice through tail vein within 24 hours, wherein the number of the injected cells is 1 × 10⁶And (4) establishing a B-NDG mouse leukemia model for each cell/mouse. AML-LSC cells from each case were injected into 12 mice separately.

5. Positive cell detection

At2 weeks after transplantation, 6 mice from each group were randomly dissected, marrow cavity cells were isolated, stained with anti-human CD45 and CD34 antibodies, and the proportion of positive cells was determined by flow cytometry (CD45 molecules are expressed on all leukocytes, and anti-human CD45 antibody specifically recognizes human leukocytes transplanted in mice, so the proportion of CD45+ cells reflects the proportion of human AML cells transplanted in mice, and CD45⁺CD34⁺Cells reflect transplanted human AML-LSC). The specific method comprises the following steps:

A. taking mouse bone marrow, removing red blood cells by using red blood cell lysate to obtain bone marrow cells.

B. The cells were washed 1 time with PBS and harvested by centrifugation at 1000rpm for 5 min.

C. Cell staining buffer (Biolegend) resuspend cells and adjust cell density to 10⁶Individual cells/mL.

D. mu.L of cell suspension was taken and 20. mu.L of FcR blocking reagent was added.

E. mu.L of FITC-labeled anti-human CD45 antibody and 5. mu.L of APC-labeled anti-human CD34 antibody were added.

F. Incubate in the dark for 30min at room temperature, during which the mixture is inverted every 10 min.

G、BD Accuri^TMC6(BD) flow cytometer detection.

6. Drawing of mouse survival curve

The remaining 6 mice were kept on stock, the time to death was recorded and a survival curve was plotted.

7. Data processing and result analysis

The flow cytometry detection result is subjected to data analysis through FlowJo _ V10 software, and statistical analysis is performed throughGraphPad Prism 8 software. Results show mean ± standard deviation. Statistical methods comparisons between the mean values were made using a t-test, where P <0.05, P <0.01, and P <0.001 were assigned as statistical significance. Survival curves were plotted using survivval function ofGraphPad Prism 8 software.

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

FIG. 8a shows the results of control treated mice, AML cells (hCD 45) 2 weeks after transplantation⁺Cells) were successfully implanted in the mouse's bone marrow cavity, and the implantation rates of AML cells in 3 cases, AML-5, AML-8 and AML-9, were: 40.5 +/-6.9, 32.5 +/-6.2 and 32.3 +/-7.3; in contrast, the cell implantation rate of the miR-31-5p group is obviously reduced, and the cell implantation rate is respectively as follows: 4 + -3, 6.3 + -4.5 and 7.7 + -4.4.

FIG. 8b shows the results of control treated mice, AML-LSC cells (hCD 45) 2 weeks after transplantation⁺CD34⁺Cells) were successfully implanted in the mouse bone marrow cavity, and the implantation rates of AML-LSC cells in 3 cases, AML-5, AML-8 and AML-9, were: 9.6 +/-3.9, 11.2 +/-4.6 and 8.1 +/-3.8; in contrast, the cell implantation rate of the miR-31-5p group is obviously reduced, and the cell implantation rate is respectively as follows: 1.5 + -1.1, 1.1 + -0.7 and 2.4 + -1.5.

FIG. 8c shows the results that control treated mice died from day 15 after transplantation, all mice died within 30 days, and median survival times were 19 days, 20 days and 19 days for AML-5, AML-8 and AML-9 control mice implanted with cells of 3 cases, respectively; in contrast, the mice in the miR-31-5p expression group only die in a few cases, and the survival period of most of the mice is more than 40 days.

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

The above embodiments are preferred embodiments of the present disclosure, but the embodiments of the present disclosure are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present disclosure should be regarded as equivalent replacements within the scope of the present disclosure.

Sequence listing

<110> river-south university

Application of <120> miR-31-5p in acute myelogenous leukemia

<130> MTI21012

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> RNA

<213> Homo sapiens

<400> 1

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<210> 2

<211> 21

<212> DNA

<213> Homo sapiens

<400> 2

aggcaagatg ctggcatagc t 21

<210> 3

<211> 71

<212> RNA

<213> Homo sapiens

<400> 3

ggagaggagg caagaugcug gcauagcugu ugaacuggga accugcuaug ccaacauauu 60

gccaucuuuc c 71

<210> 4

<211> 71

<212> DNA

<213> Homo sapiens

<400> 4

ggagaggagg caagatgctg gcatagctgt tgaactggga acctgctatg ccaacatatt 60

gccatctttc c 71

<210> 5

<211> 23

<212> DNA

<213> Homo sapiens

<400> 5

ggaacgatac agagaagatt agc 23

<210> 6

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<212> DNA

<213> Homo sapiens

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Claims (10)

1. Use of a product for detecting miR-31-5p, pri-miR-31 and/or pre-miR-31 in preparation of a reagent, a chip or a kit for assisting diagnosis of Acute Myeloid Leukemia (AML) in a subject and/or for prognosis of survival of the subject;

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

preferably, a level of miR-31-5p, pri-miR-31 and/or pre-miR-31 in the biological sample that is lower than the level of the corresponding miR-31-5p, pri-miR-31 and/or pre-miR-31 in a normal control sample 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 tissue suspected of having leukemic cells;

preferably, the subject may be a human or other mammal.

2. The use according to claim 1, wherein the level of miR-31-5p, pri-miR-31 and/or pre-miR-31 is detected using a high throughput sequencing method, a miRNA expression profiling chip, a quantitative PCR method and/or a probe hybridization method;

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

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

preferably, the transcriptional level of miR-31-5p, pri-miR-31 and/or pre-miR-31 in the biological sample is low relative to the corresponding miR-31-5p, pri-miR-31 and/or pre-miR-31 gene in a normal control sample, then the subject has a shorter survival.

3. The use according to claim 1 or 2, wherein the chip comprises a solid support and oligonucleotide probes immobilized on the solid support.

Use of miR-31-5p, pri-miR-31 and/or pre-miR-31 in preparation of a medicament for preventing and/or treating Acute Myeloid Leukemia (AML), wherein the sequence of miR-31-5p is shown in SEQ ID NO 1; the sequence of the pre-miR-31 is shown as SEQ ID NO 3.

5. The use of claim 4, wherein miR-31-5p, pri-miR-31 and/or pre-miR-31 is natural, artificially synthesized or obtained by transfecting cells with an expression vector capable of expressing a DNA fragment of miR-31-5p, pri-miR-31 and/or pre-miR-31, wherein the gene sequence of miR-31-5p is shown in SEQ ID NO 2; the gene sequence of the pre-miR-31 is shown in SEQ ID NO 4.

6. The use according to claim 5, wherein the expression vector is selected from the group consisting of a plasmid, a phage, a phagemid, a cosmid, a viral vector, a viral particle, a prokaryotic expression vector, and a eukaryotic expression vector;

preferably, the viral vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a herpesvirus vector, an alphavirus vector, a baculovirus and a vaccinia virus;

preferably, the prokaryotic expression vector is selected from an escherichia coli expression vector and a bacillus subtilis expression vector.

Preferably, the eukaryotic expression vector is selected from a yeast expression vector, an insect expression vector or a mammalian expression vector.

7. The use according to any one of claims 4-6, wherein the medicament can be administered alone or in combination with other drugs capable of inhibiting AML;

preferably, the other drug capable of inhibiting AML is selected from one or more of daunorubicin, cytarabine, thioguanine, etoposide, cephalotaxine, vincristine, prednisone, mitoxantrone, doxorubicin, cyclophosphamide, carboplatin, decitabine, methotrexate, etoposide, doxorubicin (adriamycin), cisplatin, dexamethasone, safrole, methylnitrosourea, fluorouracil, 5-fluorouracil, vinblastine, camptothecin, actinomycin-D, mitomycin C, hydrogen peroxide, oxaliplatin, irinotecan, topotecan, folinic acid, carmustine, streptozocin, paclitaxel, tamoxifen, dacarbazine, imatinib, azacitidine, and gemtuzumab ozogale.

8. 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 one or more of lactose, dextrose, sucrose, polyvinylpyrrolidone, alginates, gelatin, cellulose, syrup, sorbitol, mannitol, starch, gum arabic, talc, magnesium stearate, calcium phosphate, calcium silicate, microcrystalline cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, mineral oil, microcapsules and microspheres, nanoparticles, and liposomes.

9. The use according to any one of claims 4 to 8, wherein the medicament is in the form of a solution, injection, oral liquid, suspension, emulsion, extract, powder, granule, suppository, aerosol, granule, tablet or capsule;

preferably, the injection comprises sterile or sterilized solution, water injection, oil injection, powder injection and the like; oral liquid dosage forms include solutions, syrups, emulsions, suspensions and the like; the tablet includes common compressed tablet, sugar coated tablet, effervescent tablet, chewable tablet, multilayer tablet, implant tablet, sustained release tablet, controlled release tablet, etc.

Preferably, the medicament further comprises a dispersing agent or a stabilizing agent.

10. A method of treating Acute Myeloid Leukemia (AML), comprising administering to the subject a therapeutically effective amount of miR-31-5p, pri-miR-31, and/or pre-miR-31, or a pharmaceutical composition comprising same.

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