CN111518900A

CN111518900A - Application of miR-1246 as marker for diagnosing and treating acute myeloid leukemia

Info

Publication number: CN111518900A
Application number: CN202010294501.0A
Authority: CN
Inventors: 李丽丽; 刘佳利; 黄娅
Original assignee: Hunan Keyu Biomedical Technology Co ltd
Current assignee: Hunan Keyu Biomedical Technology Co ltd
Priority date: 2020-04-15
Filing date: 2020-04-15
Publication date: 2020-08-11

Abstract

The invention discloses a miRNA marker for diagnosing and treating acute myelogenous leukemia, and particularly relates to miRNA miR-1246. The invention discloses application of miR-1246in preparation of a product for diagnosing acute myeloid leukemia and a pharmaceutical composition for treating acute myeloid leukemia. The invention also discloses a kit for diagnosing acute myeloid leukemia and a pharmaceutical composition for treating acute myeloid leukemia. The kit of the present invention can be used as one of the means for diagnosing acute myeloid leukemia. The pharmaceutical composition has a good inhibition effect on acute myeloid leukemia, and has important reference and practical significance in the treatment of acute myeloid leukemia.

Description

Application of miR-1246 as marker for diagnosing and treating acute myeloid leukemia

Technical Field

The invention relates to miRNA related to acute myeloid leukemia and application thereof in acute myeloid leukemia, wherein the miRNA is miR-1246. Belongs to the field of biological medicine, in particular to the technical field of molecular biology.

Background

Acute Myeloid Leukemia (AML) is a malignancy caused by immature hematopoietic cells of the myeloid lineage in the bone marrow. This is a highly heterogeneous disease, with patients mostly elderly and with a high mortality rate. Acute myeloid leukemia is also an aggressive hematological tumor with multiple genotypes, phenotypes, epigenetic characteristics and net response to intervention against leukemia, with resistance to cytotoxic therapy. It is characterized by high recurrence rate, which is the leading reason for the failure of acute myeloid leukemia treatment. Despite its heterogeneity, acute myeloid leukemia exhibits common features of avoiding programmed cell death and resistance to cytotoxic stimuli. Regenerative and quiescent Leukemic Stem Cells (LSCs) are a rare population of acute myeloid leukemia cells and are also the primary cause of acute myeloid leukemia cell virus resistance. Allogeneic hematopoietic cell transplantation has been developed for decades to eradicate LSCs to control acute myeloid leukemia and has been successfully used in the treatment of acute myeloid leukemia. In addition, new immunotherapeutic approaches, including immune checkpoint inhibitors, chimeric antigen receptor T-cells and bispecific antibody therapies, are also increasingly used in current acute myeloid leukemia therapies. Although several new approaches to the treatment of acute myeloid leukemia have been proposed recently, half of them are incurable to date except acute promyelocytic leukemia. At present, the detailed and precise mechanisms associated with the development and progression of acute myeloid leukemia are still unclear, which prevents scientists from establishing better strategies to eliminate LSCs and cure acute myeloid leukemia.

Adult bone marrow is the main part of hematopoiesis, and provides a research direction for the occurrence and development of malignant hematopoietic diseases such as acute myeloid leukemia and the like. Under physiological conditions, the interaction between the bone marrow microenvironment and hematopoietic stem cells maintains a delicate balance of stem cell compartment proliferation, differentiation and homeostasis. In malignant cases, however, acute myeloid leukemia cells infiltrate the bone marrow, interfering with normal hematopoietic stem cell-microenvironment homeostasis. Recent findings suggest that the delivery of acute myeloid leukemia exosome-derived micrornas (mirnas) into benign hematopoietic stem and progenitor cells may lead to loss of cellular hematopoietic function. miRNAs are small non-coding RNAs, about 20 nucleotides in length, and are closely related to various biological and pathological processes such as cell proliferation, differentiation, apoptosis, canceration and the like; miRNAs participate in various stages of tumor development, and show that the abnormal expression of miRNA plays an important regulatory role in the expression of known oncogenes or cancer suppressor genes in the process of tumor development. In addition, research shows that malignant hematopoietic stem cells shed exosomes, form intracellular senescence-promoting signals and enhance drug resistance. Collectively, these results provide a new perspective for studying the presence of potential miRNAs associated with acute myeloid leukemia and secreted exosomes thereof, and also provide a new research approach for the diagnosis and/or treatment of acute myeloid leukemia.

Exosomes (exosomes) are a group of small particles encapsulated by lipid bilayers, 30-140nm in length, and secrete exosomes from endogenous sources, and various somatic cells in the human body. When an exosome is secreted by a host cell into a recipient cell, the biological activity of the recipient cell may be modulated by the small molecule substance carried by the exosome. Exosomes mediate cell-cell communication mainly by: firstly, through combination with target cell membrane protein, the exosome membrane protein can activate a signal channel in a target cell; secondly, in the extracellular matrix, the exosome membrane protein which is cut by the protease can be used as a ligand to be combined with a receptor on a cell membrane, so as to activate a signal path in the cell; thirdly, directly fusing with target cell membrane, non-selectively releasing messenger RNA (mRNA), protein and macromolecular RNA carried by the target cell membrane, and transferring the molecules among cells:

c1. exogenous RNA activators and the therapeutic potential of exosomes. Extert Opin Biol The.2012Jun; 12Suppl 1: S189-97.). Regulated non-coding RNAs (e.g., miRNAs, long non-coding RNAs, circular RNAs, etc.) play an important role in gene translation and transcription, as well as in physiological and pathological processes such as inflammation, ontogenesis and angiogenesis, and the proliferation, differentiation and apoptosis of leukemia cells are also affected by them, one of whichThese may be used as biomarkers for predicting prognosis. miRNAs play an important role in almost all aspects of acute myeloid leukemia cell proliferation, differentiation and survival. For example, miR-126 with high biological activity promotes leukemia formation of LSCs and chemotherapy resistance; research also shows that miR-34c-5p can accelerate the aging of LSCs, thereby providing a new idea for treating acute myeloid leukemia. Therefore, screening and further studying exosomes containing specific miRNAs may be one research and development direction for the diagnosis and/or treatment of acute myeloid leukemia.

miR-1246 secreted by exosome is used as one of miRNA, and can enhance the invasiveness of liver cancer stem cells in the aspects of self-renewal, drug resistance, tumor occurrence and metastasis. After the expression of miR-1246 is down-regulated, the migration and invasion of liver cancer cells can be inhibited. In addition, the over-expression of miR-1246in prostate cancer cells obviously inhibits the growth of transplanted tumors in vivo, increases apoptosis, and reduces the in vitro proliferation, invasion and migration capacity of prostate cancer cells; miR-1246 secreted by the exosome is a promising biomarker for prostatic cancer, and has a diagnosis potential for predicting disease invasiveness. Therefore, we speculate that miR-1246 secreted by exosome in acute myeloid leukemia is also a promising biomarker for diagnosing and/or treating acute myeloid leukemia. The invention proves the conjecture from the aspects of molecular mechanism and application through in vivo and in vitro experiments and provides a diagnostic kit and a therapeutic drug combination aiming at the acute myeloid leukemia based on the conjecture.

Disclosure of Invention

In order to make up the defects of the prior art, one of the objectives of the present invention is to provide a miRNA marker related to the occurrence and development of acute myeloid leukemia, which can be used as a specific diagnostic marker for acute myeloid leukemia and applied to the discovery of acute myeloid leukemia; the invention also aims to provide application of the miRNA marker in screening candidate drugs for treating acute myeloid leukemia.

In order to study the role of mirnas associated with acute myeloid leukemia in acute myeloid leukemia, suitable mirnas were screened. miR-1246 which can be used as an acute myeloid leukemia marker is found from AML cell exosomes by a bioinformatics technology and a modern molecular biology technology.

Therefore, the invention provides the application of the miRNA and the reagent for detecting the expression level of the miRNA in the preparation of products for diagnosing acute myeloid leukemia, wherein the miRNA is miR-1246.

Preferably, the reagent provided by the invention is a primer for specifically amplifying miR-1246.

On the other hand, on the basis of the application of the miR-1246, the invention provides a kit for diagnosing acute myeloid leukemia, and the kit comprises a reagent for detecting the expression level of the miR-1246.

Preferably, the primer sequence of the specific amplification miR-1246 disclosed by the invention is shown in SEQ ID NO.1 and SEQ ID NO. 2.

In another aspect, the invention provides a pharmaceutical composition for treating acute myeloid leukemia according to the application of miR-1246 gene.

Preferably, the pharmaceutical composition comprises an inhibitor of miR-1246 gene.

In another aspect, the invention also provides a miR-1246 gene-based pharmaceutical composition, which is an inhibitor of the miR-1246 gene, and the inhibitor is si-miR-1246 of miR-1246.

Preferably, the miR-1246 gene inhibitor is an exosome obtained after plasmid containing si-miR-1246 is transfected into AML cells.

Preferably, plasmids containing si-miR-1246 according to the invention are used at a concentration of 75nM when transfecting AML cells.

Preferably, the sequence of si-miR-1246 provided by the invention is shown in SEQ ID NO. 3.

The invention screens a marker miR-1246(miR-1246 is highly expressed in AML exosomes) for diagnosing and treating acute myeloid leukemia from AML extracellular exosomes by the technical means of bioinformatics and existing molecular biology. On the basis, the invention provides application of a reagent for detecting miR-1246in preparation of a product for diagnosing acute myeloid leukemia. The application comprises a kit for diagnosing acute myeloid leukemia and a pharmaceutical composition for treating acute myeloid leukemia. The kit of the present invention can be used as one of the means for diagnosing acute myeloid leukemia. The pharmaceutical composition has a good inhibition effect on acute myeloid leukemia, and has important reference and practical significance in the treatment of acute myeloid leukemia.

Drawings

Wherein denotes P <0.05 with significant differences.

FIG. 1miR-1246 is highly expressed in AML cell-derived exosomes.

Wherein figure 1a. differential gene expression heatmap of chip GSE 55025; FIG. 1B is a differential gene volcano plot of chip GSE 55025;

FIG. 1C is a plot of miR-1246 expression cassettes in GSE55025 on a chip, with the left cassette for normal cell expression and the right cassette for exosome expression;

FIG. 1D, labeling of AML cell lines with CD34-PE and CD38-FITC antibodies, flow cytometry analysis of CD34+ CD38- (i.e., LSCs) cell number; FIG. 1E. qRT-PCR detection of miR-1246 expression in AML cell lines (KG1a and Molm-14) and LSCs; FIG. 1F Transmission Electron Microscopy (TEM) observation of the morphology of AML cell-derived exosomes; FIG. 1G nanoparticle tracking analysis to detect exosome concentration and particle size; western blotting detection of exosome-specific marker proteins CD63 and TSG101 expression; qRT-PCR detection of miR-1246 expression in AML cell lines (KG1a and Molm-14) and their corresponding exosomes is shown in FIG. 1I.

Figure 2 targeting of AML exosomes to LSCs promotes their survival.

Wherein FIG. 2A. LSCs are observed under fluorescent microscope for uptake of CFSE-labeled AML exosomes (CFSE: green, DAPI: blue,. times.400); FIG. 2B.MTS assay detects changes in cell viability of LSCs after treatment with AML exosomes; figure 2c. colony formation assay test the effect of AML exosomes on LSCs colony formation number; figure 2d. flow cytometry to examine the effect of AML exosomes on apoptosis of LSCs; figure 2e differentiation assay detects the change in differentiation capacity of LSCs after treatment with AML exosomes.

FIG. 3AML extracellular exosomes miR-1246 are able to target Leukemic Stem Cells (LSCs) to promote their survival.

FIG. 3A shows that exosomes are extracted from AML cells after miR-1246 is silenced or overexpressed, exosomes and LSCs are cultured together, and expression conditions of miR-1246in each group of LSCs are detected by qRT-PCR; FIG. 3B. MTS assay for proliferative Activity of groups of LSCs;

FIG. 3℃ colony formation assay the cell colony forming ability of LSCs was tested; FIG. 3D. flow cytometry detection of apoptosis of LSCs; figure 3e differentiation assay detects the differentiation capacity of LSCs.

FIG. 4 silencing miR-1246 inhibits AML stem cell transplantation tumor growth.

Wherein, in the graph of fig. 4A and fig. 4b, a nude mouse subcutaneous transplantation tumor model is established by injecting KG1a stem cells (KG1a-SCs) subcutaneously, and tumor forming volume and weight of each group of mice are observed in real time after fixed-point subcutaneous injection of a solvent control and Exo-miR-1246 inhibitor; and FIG. 4C, extracting RNA of each group of mouse tumor tissues, and detecting the expression condition of miR-1246in each group of tumor tissues by qRT-PCR.

Detailed Description

The invention is further illustrated below with reference to specific examples. The various starting materials mentioned in the following examples are all commercially available unless otherwise specified.

The invention detects the expression level of miRNA in AML cell exosome through extensive and intensive research and bioinformatics technology and molecular biology technology, finds miRNA segments with obvious expression difference, and discusses the relationship between the miRNA segments and the occurrence of acute myeloid leukemia, thereby finding better ways and methods for the detection and targeted therapy of acute myeloid leukemia. Through screening, the miR-1246in the AML extracellular exosome is found to be significantly up-regulated, and a large number of experiments prove that the miRNA has higher positive detection rate; further cell experiments prove that the change of the expression level of AML extracellular exosomes can influence the growth of LSCs, and the miR-1246 can be used as a drug target for the precise treatment of acute myeloid leukemia.

"biomarker" and "marker" are used interchangeably to refer to a molecular indicator of a specific biological property, biochemical characteristic or aspect, which can be used to determine the presence or absence and/or severity of a particular disease or condition. In the present invention, "marker" refers to a parameter associated with one or more biomolecules (i.e., "biomarker"), such as naturally or synthetically produced nucleic acids (i.e., individual genes, as well as coding and non-coding DNA and RNA). "marker" in the context of the present invention also includes reference to a single parameter which may be calculated or otherwise obtained by taking into account expression data from two or more different markers. In the present invention, the term "biomarker" refers to a gene, a fragment or a variant of a gene associated with acute myeloid leukemia.

In the embodiment of the invention, the nucleotide sequence SEQ ID NO.4 of a representative human miR-1246 gene. The miR-1246 nucleotide full-length sequence or the fragment thereof can be obtained by a PCR amplification method, a recombination method or an artificial synthesis method.

In the present invention, gene expression can be determined using any method known in the art. It will be appreciated by those skilled in the art that the means by which gene expression is determined is not an important aspect of the present invention. The expression level of the biomarker can be detected at the transcriptional level. The mirnas of the present invention are detected using a variety of nucleic acid techniques known to those of ordinary skill in the art, including but not limited to: nucleic acid sequencing, nucleic acid hybridization, and nucleic acid amplification techniques. Illustrative, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because it is less stable in cells and more susceptible to nuclease attack in experiments.

Another illustrative, non-limiting example of a nucleic acid sequencing technique includes next generation sequencing (deep sequencing/high throughput sequencing), which is a unimolecular cluster-based sequencing-by-synthesis technique based on proprietary reversible termination chemical reaction principles. Random fragments of genome DNA are attached to an optically transparent glass surface during sequencing, hundreds of millions of clusters are formed on the glass surface after the DNA fragments are extended and subjected to bridge amplification, each cluster is a monomolecular cluster with thousands of identical templates, and then four kinds of special deoxyribonucleotides with fluorescent groups are utilized to sequence the template DNA to be detected by a reversible edge-to-edge synthesis sequencing technology.

The nucleic acid amplification technique of the invention is selected from the group consisting of Polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), Transcription Mediated Amplification (TMA), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA) and Nucleic Acid Sequence Based Amplification (NASBA). Among them, PCR requires reverse transcription of RNA into DNA before amplification (RT-PCR), TMA and NASBA to directly amplify RNA.

The polymerase chain reaction, commonly referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of a target nucleic acid sequence; transcription-mediated amplification of TMA autocatalytically synthesizes multiple copies of a target nucleic acid sequence under conditions of substantially constant temperature, ionic strength, and pH, wherein the multiple RNA copies of the target sequence autocatalytically generate additional copies; the ligase chain reaction of LCR uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of a target nucleic acid; other amplification methods include, for example: nucleic acid sequence-based amplification commonly known as NASBA; amplification of the probe molecule itself using an RNA replicase (commonly referred to as Q β replicase); a transcription-based amplification method; and self-sustained sequence amplification.

Nucleic acid hybridization techniques of the invention include, but are not limited to, In Situ Hybridization (ISH), microarrays, and Southern or Northern blots. In Situ Hybridization (ISH) is a hybridization of specific DNA or RNA sequences in a tissue section or section using a labeled complementary DNA or RNA strand as a probe (in situ) or in the entire tissue if the tissue is small enough (whole tissue embedded ISH). DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and locate mRNA and other transcripts (e.g., ncRNA) within tissue sections or whole tissue embedding. Sample cells and tissues are typically treated to fix the target transcript in situ and to increase probe access. The probe is hybridized to the target sequence at high temperature, and then excess probe is washed away. The localization and quantification of base-labeled probes in tissues labeled with radiation, fluorescence or antigens is performed using autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes labeled with radioactive or other non-radioactive labels to detect two or more transcripts simultaneously.

The invention provides a kit which can be used for detecting the expression of miR-1246.

In certain embodiments, the kit comprises one or more probes that specifically bind to mRNA of one or more biomarkers. In certain embodiments, the kit further comprises a wash solution. In certain embodiments, the kit further comprises reagents for performing hybridization assays, mRNA isolation or purification means, detection means, and positive and negative controls. In certain embodiments, the kit further comprises instructions for using the kit. The kit may be customized for home use, clinical use, or research use. For example, the kit provided by the invention is based on qRT-PCR experimental sources, the invention not only provides a primer for detecting miR-1246, but also provides a specific detection method, and on the basis, the invention can refine the qRT-PCR detection kit for detecting the expression level of miR-1246.

Such a kit may employ, for example, a test strip, membrane, chip, tray, test strip, filter, microsphere, slide, multiwell plate, or optical fiber. The solid support of the kit can be, for example, a plastic, a silicon wafer, a metal, a resin, a glass, a membrane, a particle, a precipitate, a gel, a polymer, a sheet, a sphere, a polysaccharide, a capillary, a film, a plate, or a slide. The biological sample may be, for example, a cell culture, cell line, tissue, oral tissue, gastrointestinal tissue, organ, organelle, biological fluid, blood sample, urine sample, or skin inhibitor and drug (composition).

Based on the discovery of the inventor, the invention provides an inhibitor of miR-1246, the property of which is not important to the invention, as long as the inhibitor inhibits the functional expression of miR-1246 gene, for example, the inhibitor of the invention can be an interfering molecule which takes miR-1246 gene as a target sequence and can inhibit miR-1246 gene, and comprises the following components: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA microrna, antisense nucleic acid. The inhibitors are useful as substances for down-regulating miR-1246 and can be used for treating acute myeloid leukemia.

As a preferable mode of the invention, the inhibitor of miR-1246 is siRNA specific to miR-1246. As used herein, the inhibitor is an exosome obtained after transfection of AML cells with a plasmid containing the siRNA, thereby obtaining a stable pharmaceutical composition capable of targeting LSCs. In the embodiment of the invention, the si-miR-1246 can specifically inhibit the expression of miR-1246in AML cells, so that AML cell exosomes with low expression or no expression of miR-1246 are obtained, and a stable pharmaceutical composition capable of targeting LSCs is obtained, so that the growth of the LSCs is inhibited, and the generation and development of acute myeloid leukemia are inhibited.

The nucleic acid inhibitor of the present invention, such as siRNA, can be chemically synthesized or can be prepared by transcribing an expression cassette in a recombinant nucleic acid construct into single-stranded RNA. Nucleic acid inhibitors, such as siRNA, can be delivered into cells by using appropriate transfection reagents, or can also be delivered into cells using a variety of techniques known in the art.

The pharmaceutical composition comprises an inhibitor of miR-1246 and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers include, but are not limited to, diluents, binders, surfactants, humectants, adsorbent carriers, lubricants, fillers, disintegrants. Preferably, the present invention uses AML cell exosomes as the final use form of the pharmaceutical composition (the exosomes are obtained after AML cells are transfected by plasmids containing si-miR 1246), and in the embodiments of the present invention, the pharmaceutical composition of the present invention targets the growth of LSCs, thereby achieving the inhibition of the occurrence and development of acute myeloid leukemia. In addition, in another embodiment of the present invention, the pharmaceutical composition of the present invention has a good effect of inhibiting the occurrence and development of acute myeloid leukemia in a nude mouse tumorigenic model.

Of course, the pharmaceutical composition of the invention may also be used in combination with other drugs for the treatment of acute myeloid leukemia, and other therapeutic compounds may be administered simultaneously with the main active ingredient, even in the same composition.

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.

Experimental methods

The method comprises the following steps: cell culture and sorting

Human AML cell lines KG1a and Molm-14(ATCC, Manassas, VA) were cultured in suspension in DMEM medium containing 10% Fetal Bovine Serum (FBS) and 100U/mL penicillin and streptomycin. The Leukemia Stem Cells (LSCs) are CD34⁺CD38^-KG1a cells (K), all KG1a cells were CD34⁺LSCs were therefore enriched by indirect labeling with CD38-FITC antibody and anti-FITC magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, the cell suspension was centrifuged at 125g for 10min and washed with PBS. The cell pellet was then suspended in isolation buffer (PBS containing 0.5% BSA) and mixed with CD38^-FITC antibody was incubated for 30 minutes. After PBS washing, the cell pellet was incubated for 15 minutes in separation buffer containing anti-FITC microspheres. After washing, the filtrate (CD 34)⁺CD38^-Cells) were collected using LD column of the mitimacs separator system. Throughout the sorting process, cells were placed at 4 ℃ and analyzed by flow cytometry. The remaining cells after depletion of LSCs were defined as AML cells and used for co-culture studies.

The method 2 comprises the following steps: exosome isolation, identification and labeling

By heating at 4 ℃ at 1 × 10⁶g ultracentrifugation for 16 h (Beckman CoulterA)After incubation of AML cell lines (KG1a and Molm-14) for 48-72 hours, culture medium was harvested and exosomes were isolated by ultracentrifugation briefly, cell culture medium was centrifuged sequentially at 300g for 10 minutes, 2,000g for 15 minutes and 12,000g for 30 minutes to remove floating cells and cell debris, then through a 0.22 μm filter the supernatant was centrifuged at 4 ℃ at 1 × 10⁶g further ultracentrifugation for 2 hours, washing in Phosphate Buffered Saline (PBS), and a second ultracentrifugation under the same conditions. Finally, about 100ml of PBS was used to resuspend the pellet and stored at-80 ℃ for use or immediate use.

The morphology of exosomes was observed using Transmission Electron Microscopy (TEM). The exosome concentration and size were determined using nanoparticle tracking analysis (Nanoparticletracking analysis). Isolated exosomes were diluted 1:10 and observed on a NanosightNS300 nanoparticle detector (Malvern, Westborough, MA, USA). For exosome protein quantification, exosome particles were dissolved in RIPA buffer and quantified using BCA protein assay kit (Thermo FisherScientific, Rockford, IL, USA). Exosome western blot analysis antibodies were used as follows: TSG101(ab125011,1:1000), CD63 (ab134045,1:1000), Histone (ab1791,1: 1000).

Exosomes were labeled with CFSE (10 μ M; Thermo Fisher Scientific) for 30min at 37 ℃. The labeled exosomes were then washed with PBS and centrifuged for 1h at 100,000g to remove excess dye. For exosome in vitro tracking analysis, CFSE labeled exosomes were co-cultured with LSCs in culture medium for 4 h. Finally, the sample was observed with an Olympus BX41 microscope (MagnaFire, Olympus) equipped with a CCD.

The method 3 comprises the following steps: grouping and transfection of cells

Cultured AML cells were divided into the following groups: the plasmid vector comprises i.an inhibitor-NC (negative control group, transfection of an unloaded plasmid), ii.miR-1246 inhibitor (transfection of a si-miR-1246 plasmid group, inhibition of expression of miR-1246), iii.a micic-NC (negative control group, transfection of an unloaded plasmid), and iv.a miR-1246 micc (transfection of a miR-1246 group, promotion of expression of miR-1246). The cells were inoculated 24h before transfectionAnd (3) inoculating the cells into a 12-well plate of a DMEM medium, re-suspending the cells in a serum-free DMEM medium when the confluence degree of the cells reaches about 70%, inoculating the cells into the 12-well plate, and performing transfection according to the grouping. After transfection, the temperature was further increased to 37 ℃ with 5% CO₂And (4) incubating for 6-8 h under the condition, continuously culturing for 24-48 h after the complete culture medium is replaced, and carrying out subsequent experiments. The successful transfected AML cells of the above groups were used for exosome isolation. 75nM miR-1246inhibitor or negative control, 100nM miR-1246 imic or negative control, were purchased from Ruibo Biotechnology, Inc., Guangzhou.

The method 4 comprises the following steps: cell viability assay (Cell viability assay)

Logarithmic growth phase LSCs were inoculated into 96-well plates (100. mu.L/well) and cultured to the logarithmic growth phase. Cells were transfected 24h later, 3 replicates per group. The proliferation activity of LSCs cells is detected by using an MTS cell proliferation and toxicity detection kit (BB-4203-3, BestBio), namely 10 mu of LMTS solution is added into each well after 48 hours of transfection, and the cells are incubated for 1-4 hours at 37 ℃. And detecting the light absorption value of each hole at 490nm by using a multifunctional microplate reader. Blank wells (media and MTS solution, no cells) and control wells (cells only) were set simultaneously, with 3-5 duplicate wells per group. Cell viability (%) × (experimental cell OD-blank cell OD)/(control cell OD-blank cell OD) × 100%. The experiment was repeated 3 times.

The method 5 comprises the following steps: cell Differentiation analysis (Differentiation assay)

In differentiation experiments, LSCs were cultured in IMDM medium containing 10% FBS and added with 100ng/mL rhSCF (Recombinant human stem cell factor), 100ng/mL rhFlt3 (Recombinant human soluble Flt3 ligand), and 100ng/mL rhTPO (Recombinant human platelet factor) at 37 deg.C with 5% CO₂Culturing for 24h under the condition. Followed by treatment with 1. mu.g/mL AML exosomes for 7 d. LSCs were collected and labeled with CD19-PE, CD33-APC, CD3-APC, and CD235a-FITC for 30min, and then detected and analyzed by flow cytometry (FACS Calibur (BD)) (Cell Quest software (BD)).

The method 6 comprises the following steps: apoptosis assay (Apoptosis analysis)

Apoptosis analysis was performed using Annexin V and Propidium iodide (Thermo Fischer Scientific). Briefly, LSCs were collected and washed with PBS and binding buffer. Then incubated with 5. mu. Lannexin V for 15min in the dark. After washing with PBS and binding buffer 5 μ L propidium iodide was added. The cell mixture was then incubated at 2-8 ℃ and analyzed by flow cytometry using a FACSAria II Special sequence System (BD Biosciences).

The method 7 comprises the following steps: qRT-PCR analysis

Total RNA was extracted from cells or exosomes using miRNeasy or RNeasy kits (Qiagen, Germantown, MD, USA). Reverse transcription into cDNA was performed by SuperScript III First-Strand Synthesis kit (Invitrogen) with oligo (dT) Priming according to the instructions. Then, qRT-PCR reactions were performed using SYBR Green PCR kit (Applied Biosystems) in 96-well plates and ABI PRISM 7500 real-time PCR system (Bio-tek). The PCR conditions were 95 ℃ for 10min, 50cycles at 95 ℃ for 30s,60 ℃ for 30s, and 72 ℃ for 1 min. Primer sequences are shown in table 1, all primer sequences were synthesized by the department of biotechnology, sharp Bo, Guangzhou. GAPDH was used as an internal control. By using 2^-ΔΔCTThe relative expression amount is calculated. For miRNA quantification, reverse transcription and qRT-PCR were performed using TaqMan assay kits (Applied Biosystems) with U6 snRNA as an endogenous control.

TABLE 1 qRT-PCR primers

The method 8 comprises the following steps: western blotting

Extracting total protein of tissue or cell by using high-efficiency RIPA lysate (R0010, Solarbio) strictly according to the instruction. After 15min of lysis at 4 ℃ and centrifugation at 15000rpm/min for 15min, the supernatant was extracted and the protein concentration of each sample was determined using the BCA kit (20201ES76, assist in san Biotech, Inc., China). And (3) quantifying according to different concentrations, transferring the protein to a PVDF membrane by a wet transfer method after separating the protein by polyacrylamide gel electrophoresis, and blocking for 1h at room temperature by 5% BSA. Drop-wise dilution of primary antibody (rabbit antibody, both from Cell Signaling Technology): GAPDH (#5174,1: 1000). Shaking table incubation was performed overnight at 4 ℃. TBST membrane washing 5min × 3 times, adding HRP-labeled goat anti-rabbit IgG (#7074,1:2000) diluent at room temperature for 1h. Washing membrane with TBST for 5min × 3 times, adding developer, and developing. ImageJ 1.48u software (National institutes of health) was used for protein quantification, and the ratio of the grey scale value of each protein to the grey scale value of internal reference GAPDH was used for protein quantification. The experiment was repeated 3 times.

The method 9: nude mouse transplantation tumor experiment

NOD/SCID/IL2r gamma (null) mice (NSG) (6-9week-old) (Hunan Slek. Jingda.) all mice were injected subcutaneously with a corresponding concentration of 0.5 × 10⁵The experimental groups were i.control (blank control group, no AML cell exosomes were injected), ii. Exo-inhibitor-NC (negative control group, normal AML cell exosomes were injected, normal expression of miR-1246 thereof), iii. Exo-miR-1246inhibitor (experimental group, AML cell exosomes injected with si-miR-1246 plasmid, miR-1246 expression thereof was inhibited), real-time measurement and recording of nude mouse transplanted tumor volume change, V a × B, and targeted subcutaneous injection into mice²/2(mm³) The equation calculates the tumor volume, where a is the maximum diameter and B is the perpendicular diameter. When the tumor volume reaches 1,000mm³When the miR-1246 expression is detected, mice are sacrificed, tumors are excised and weighed, total RNA and protein of tumor tissues are extracted, and the miR-1246 expression condition in each group of tumor tissues is detected. The study conforms to the principles of animal protection and use and is approved by the ethical committee of experimental animals.

Example 1: high expression of miR-1246in AML cell-derived exosome

The miRNA with expression difference in AML cell exosomes are screened out by analyzing a GSE55025 chip, wherein 53 high-expression miRNAs and 45 low-expression miRNAs are included, the difference significance of miR-1246in the different miRNAs is the highest, and an expression heat map and a volcano map are drawn by using an R language (shown in figures 1A-B). From the boxplots, it can be seen that miR-1246 has significantly high expression in AML cell exosomes (fig. 1C). It is speculated that miR-1246 may be associated with leukemia progression. Therefore, we chose miR-1246 as the subject of our study.

Leukemic Stem Cells (LSCs) begin in the progression of AMLPlays an important role in mediating relapse, drug resistance and the like of AML. However, the study of the action of the AML exosomes on LSCs is rare, and in order to study whether the AML exosomes can act on LSCs and affect their biological functions, firstly, we first separated LSCs from AML cell lines (KG1a and Molm-14) by magnetic bead sorting. The successful sorting of LSCs was determined by measuring the proportion of LSCs in AML cell lines (KG1a and Molm-14), and LSCs isolated from AML cell lines and sorted magnetic beads (KG1a-SCs and Molm-14-SCs) were stained with antibodies to CD34-PE and CD38-FITC and flow analyzed. As shown in FIG. 1D, more than 95% of the cells were CD34 after microsphere screening⁺CD38^-. The expression conditions of miR-1246in AML cell lines (KG1a and Molm-14) and LSCs (KG1a-SCs and Molm-14-SCs) are detected by utilizing qRT-PCR, and as a result, the expression of miR-1246in the LSCs is remarkably reduced compared with the AML cell lines (FIG. 1E). Immediately afterwards, the expression of miR-1246in the AML secretory exosomes is further verified, the AML cell line secretory exosomes are separated and purified, and then are analyzed and identified by TEM, Nanoparticle tracking analysis and Westernblotting, and the AML cell line secretory exosomes are observed to be between 50 and 150nm in size and express an exosome specific marker protein (FIGS. 1F-H). The expression condition of miR-1246in exosome is detected by utilizing qRT-PCR, and the result shows that the expression of miR-1246in the source exosome of AML cell line is obviously higher than that of the AML cell line (figure 1I), and is consistent with the chip result.

The results show that miR-1246 is highly expressed in the AML cell-derived exosomes and is lowly expressed in LSCs.

Example 2: targeting of AML cell-derived exosomes to Leukemia Stem Cells (LSCs) to promote survival thereof

From the above results, we guess whether the exosomes derived from AML cells can enter LSCs to affect the in vitro biological function and hematopoietic differentiation of LSCs, and finally affect the development of leukemia. After CFSE-labeled AML cell-derived exosomes and LSCs are cultured for 4h, the exosomes are observed to be endocytosed by the LSCs under a fluorescence microscope, and the exosomes are found to be internalized by the LSCs (FIG. 2A), which indicates that the AML cell exosomes can target the LSCs.

To further study the effect of AML exosomes on LSCs biological phenomena in vitro and cell differentiation, we next examined LSCs cell viability after AML exosomes treatment by MTS method, and examined the effect on LSCs colony formation number by colony formation experiment. The results showed that proliferation activity and cell colony number of LSCs were positively correlated to AML cell exosome dose compared to PBS group, where Exo is exosome (exosome) abbreviation (fig. 2B-C). Flow cytometry is used for detecting the influence of the AML extracellular exosomes on the apoptosis condition of the LSCs, and as a result, the AML extracellular exosomes can obviously inhibit the apoptosis of the LSCs (fig. 2D). Differentiation experiments tested the change of differentiation capacity of LSCs after treatment of AML exosomes, and the results showed that the differentiation capacity of LSCs was negatively correlated with exosome dose, i.e., CD19, CD3, and CD235a positive cells were significantly decreased, and CD33 positive cells were significantly increased (fig. 2E).

The results show that the AML extracellular bodies promote the cell activity and the clone formation of the LSCs, and inhibit the apoptosis and the hematopoietic differentiation of the LSCs.

Example 3: AML cell exosome miR-1246 targeting Leukemia Stem Cells (LSCs) to promote survival of the LSCs

Wherein Exo-mix-NC, Exo-miR-1246 mix, Exo-inhibitor-NC and Exo-miR-1246inhibitor represent groups corresponding to co-culture of AML cell exosomes and LSCs, wherein the AML cell transfection inhibitors-NC (negative control group, transfection no-load plasmid), miR-1246inhibitor (transfection si-miR-1246 plasmid group, inhibition of expression of miR-1246), mix-NC (negative control group, transfection no-load plasmid) and miR-1246 mix (transfection miR-1246 group, promotion of expression of miR-1246) are obtained after AML cell transfection. Specific methods for producing exosomes are described in detail in method 3 of the present invention.

To verify whether AML extracellular secretion affects LSCs biological properties and hematopoietic differentiation in vitro by delivering miR-1246. After AML cells silence or miR-1246 is over-expressed, exosomes are separated, after the separated exosomes are co-cultured with LSCs, expression conditions of miR-1246in all groups of LSCs are detected by qRT-PCR (quantitative reverse transcription-polymerase chain reaction), and miR-1246 expression in the LSCs in the Exo-miR-1246 micic co-cultured group is remarkably increased compared with that in the Exo-mimic-NC co-cultured group; compared with the Exo-inhibitor-NC co-culture group, miR-1246 expression in the Exo-miR-1246 co-culture group is remarkably reduced (FIG. 3A). In addition, the proliferation activity, apoptosis, colony formation number, and differentiation ability of each group of LSCs were examined. The MTS method is used for detecting cell proliferation activity, colony formation experiment is used for detecting the number of cell colonies, flow cytometry is used for detecting the apoptosis condition, and differentiation experiment is used for detecting the cell differentiation capacity, and the results show that compared with an Exo-mimic-NC group, the cell proliferation activity and the number of colonies of LSCs in the Exo-miR-1246mimic group are obviously improved, and the apoptosis and differentiation capacities are obviously reduced; compared with the Exo-inhibitor-NC group, the cell proliferation activity and colony count of the LSCs in the Exo-miR-1246inhibitor group are remarkably reduced, and the apoptosis and differentiation capacities are remarkably increased (FIGS. 3B-E).

The results show that miR-1246in AML exosomes can target LSCs so as to promote survival of the LSCs.

Example 4: silencing miR-1246 for inhibiting growth of AML stem cell transplantation tumor

To verify whether miR-1246 can inhibit in vivo tumorigenesis of AML stem cells, KG1a-SCs is subcutaneously injected into a nude mouse to establish a subcutaneous tumor transplantation model, and mouse tumor formation is observed and recorded in real time by site-specific subcutaneous injection of a physiological saline control, Exo-inhibitor-NC and Exo-miR-1246 inhibitor. The results showed that both tumor volume and weight were significantly increased in the Exo-inhibitor-NC group compared to the saline control group, while both tumor volume and weight were significantly decreased in the Exo-miR-1246inhibitor group mice compared to the Exo-inhibitor-NC group (FIGS. 4A-B). RNA and protein of tumor tissues of all groups of mice are extracted, expression conditions of miR-1246in the tumor tissues are detected by utilizing qRT-PCR, and the miR-1246in the Exo-inhibitor-NC group is found to be remarkably increased compared with a normal saline control group, while the miR-1246in the Exo-miR-1246inhibitor group is remarkably reduced compared with the miR-1246in the Exo-inhibitor-NC group (figure 4C).

The results show that miR-1246 can inhibit in-vivo tumor formation of AML stem cells.

The above description is not intended to limit the invention, nor is the invention limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the spirit of the invention.

<110> Hunan province, scientific and scientific Co., Ltd

Application of <120> miR-1246 as marker for diagnosing and treating acute myeloid leukemia

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<170>PatentIn version 3.5

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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<213> si-miR-1246 Sequence [ Artificial Sequence ]

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<213> nucleotide sequence of miR-1246 gene

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Claims

1. An miRNA, wherein the miRNA is miR-1246, and is characterized in that the application of a reagent for detecting the expression level of miR-1246in the preparation of a product for diagnosing acute myeloid leukemia.

2. The use according to claim 1, wherein the reagent is a primer that specifically amplifies miR-1246.

3. A kit for diagnosing acute myeloid leukemia is characterized by comprising a reagent for detecting miR-1246 expression level.

4. The kit according to claim 3, wherein the reagent is a primer for specific amplification of miR-1246.

5. The kit according to claim 4, wherein the primer sequence of the specific amplification miR-1246 is shown in SEQ ID No.1 and SEQ ID No. 2.

6. An application of miR-1246 gene, which is characterized in that the miR-1246 gene is used for preparing a pharmaceutical composition for treating acute myeloid leukemia.

7. The use according to claim 6, wherein the pharmaceutical composition comprises an inhibitor of the miR-1246 gene.

8. A pharmaceutical composition for treating acute myeloid leukemia, which comprises an inhibitor of miR-1246 gene.

9. The pharmaceutical composition of claim 8, wherein the inhibitor of the miR-1246 gene is si-miR-1246.

10. The composition according to claim 8, wherein the inhibitor of miR-1246 gene is an exosome obtained after transfection of AML cells with plasmid containing si-miR-1246.

11. The composition according to claim 10, wherein the plasmid containing si-miR-1246 is used at a concentration of 75nM when transfecting AML cells.

12. The composition of claim 10, wherein the sequence of si-miR-1246 is shown in SEQ id No. 3.