CN111549130A

CN111549130A - Application of LncRNA MAGI2-AS3 in acute myelogenous leukemia product and detection kit thereof

Info

Publication number: CN111549130A
Application number: CN202010294487.4A
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: 2020-08-18

Abstract

The invention discloses a long-chain non-coding RNA for treating acute myeloid leukemia, and particularly relates to LncRNAMAGI2-AS 3. The invention discloses application of LncRNAMAGI2-AS3 in preparing a product for treating acute myeloid leukemia. The invention also discloses a pharmaceutical composition for treating acute myeloid leukemia and a kit for evaluating LncRNAMAGI2-AS 3. 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. The kit can be one of evaluation means of expression level of LncRNAMAGI2-AS3, and has better measuring and guiding effects on the LncRNAMAGI2-AS3 when being used AS a pharmaceutical composition.

Description

Application of LncRNA MAGI2-AS3 in acute myelogenous leukemia product and detection kit thereof

Technical Field

The invention relates to a long-chain non-coding RNA related to acute myeloid leukemia and application thereof in acute myeloid leukemia, wherein the long-chain non-coding RNA is LncRNAMAGII 2-AS 3. 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. According to the National Institutes of Health (NIH), 2.4 out of 10 million people per year are diagnosed with acute myeloid leukemia, with a mortality rate of about 1.2% of cancer deaths. 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 a major contributor to acute myeloid leukemia relapse and chemotherapy 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, several novel immunotherapeutic strategies, including immune checkpoint inhibitors, chimeric antigen receptor T cells, and bispecific antibody therapies, etc., are also being gradually adopted in current acute myeloid leukemia therapy. Although several new approaches to the treatment of acute myeloid leukemia have been proposed recently, in addition to acute promyelocytic leukemia, half of them are so far incurable, which still leads to death in more than half of young patients and 90% of elderly patients. 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. It has been reported in the literature that expression of long non-coding RNAs (longnoncodingnrnas, LncRNAs) may suggest recurrent mutations, clinical features and prognosis in acute myeloid leukemia. In addition, the long-chain non-coding RNA can be used as a biomarker for prognosis of acute myelogenous leukemia. Based on the expression of long non-coding RNAs, it is possible to protect these patients from other toxic side effects. This suggests that a portion of these long non-coding RNAs may play an important role in leukemia development. The length of the Long non-coding RNA is generally between 200 and 100000 nucleotides, and can be divided into five types of antisense Long non-coding RNA (antisense LncRNA), intron non-coding RNA (intron tran), Long endogenous non-coding RNA (lincRNA), Promoter-associated LncRNA (Promoter-associated LncRNA), and untranslated region LncRNA (UTlassococcated LncRNA) according to the source. The long-chain non-coding RNA has a structure similar to mRNA, has a ployA tail and a promoter structure through splicing, and has dynamic expression and different splicing modes in the differentiation process to form different LncRNA; LncRNA can regulate and control the expression of genes on epigenetic, transcriptional and post-transcriptional levels, and can also participate in a plurality of important regulation and control processes such as X chromosome silencing, genome imprinting, chromatin modification, transcriptional activation, transcriptional interference, intranuclear transportation and the like, and the occurrence, development and prevention of human diseases are closely related to the expression. Overall, these results provide a new perspective for studying the existence of acute myeloid leukemia and its associated potential long-chain non-coding RNA, and also provide a new research idea for the diagnosis and/or treatment of acute myeloid leukemia.

As a newly discovered long non-coding RNA, lncrnaagi 2-AS3 can inhibit breast cancer cell growth by targeting the Fas/FasL signaling pathway. In addition, the long-chain non-coding RNAMAGI2-AS3 was finally determined to be one of the down-regulated genes by analysis of 3,005 differentially expressed genes between normal human hematopoietic stem cells and LSCs from patients with acute myelogenous leukemia in the GSE17054 database. In addition to long non-coding RNAs, studies have shown that epigenetic and genetic heterogeneity (e.g., DNA methylation) may underlie various biological subtypes in acute myeloid leukemia. For example, TET2 (Ten-ElevenTranslocation-2) is a DNA demethylase that converts 5-methyl-cytosine (5-methyl-cytosine) to 5-hydroxymethyl-cytosine (5-hydroxymethyl-cytosine). In a previous study, 53% of patients with chronic myelogenous leukemia (chronic myelogenous leukemia) have a TET2 missense or nonsense mutation, and more importantly, a TET2 mutation increases the overall methylation of chronic myelogenous leukemia. Similar to TET2, leucine-rich repeats and immunoglobulin-like domain-1 (Leucine-rich repeats and immunoglobulin-like domains-1, LRIG1) also play a role in hematopoiesis and leukemia formation. LRIG1 has been developed to be significantly downregulated in chronic lymphocytic leukemia (chronic lymphocytic leukemia). More interestingly, LRIG1 has been shown to maintain stem cell quiescence and its expression is down-regulated in acute myeloid leukemia. Therefore, it is speculated that the long-chain non-coding RNA MAGI2-AS3 may play a promoting or inhibiting role in acute myeloid leukemia by relating to TET2/LRIG1 or other ways, so that the long-chain non-coding RNA MAGI2-AS3 may be a promising biomarker for diagnosing and/or treating the acute myeloid leukemia in the 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 purposes of the invention is to provide the application of a long-chain non-coding RNA marker in screening candidate drugs for treating acute myeloid leukemia; the invention also aims to provide a kit for evaluating the expression level of the long-chain non-coding RNA.

In order to study the role of long-chain non-coding RNA associated with acute myeloid leukemia in acute myeloid leukemia, appropriate long-chain non-coding RNA was selected. LncRNAMAGI2-AS3 which can be used AS an acute myeloid leukemia marker is found from AML tissues through bioinformatics technology and modern molecular biology technology.

Therefore, in one aspect of the invention, the invention provides the use of LncRNAMAGI2-AS3 in the preparation of a product for treating acute myeloid leukemia according to the use of LncRNAMAGI2-AS3 gene.

Preferably, LncRNAMAGI2-AS3 is shown in SEQ ID NO. 1.

Preferably, the target gene of lncrnaagi 2-AS3 of the present invention is MMP 9.

Preferably, lncrnamaigi 2-AS3 of the invention recruits demethylases to mediate demethylation of the MMP9 promoter.

Preferably, the methylase of the invention is TET 2.

In still another aspect, the present invention provides a pharmaceutical composition for treating acute myeloid leukemia, comprising lncrnaagi 2-AS 3.

Preferably, the pharmaceutical composition of the present invention is a plasmid containing lncrnaagi 2-AS 3.

In still another aspect, the present invention provides a kit comprising a reagent for detecting the expression level of lncrnaagi 2-AS 3.

Preferably, the reagent is a primer that specifically amplifies LncRNAMAGI2-AS 3.

Preferably, the primer sequence for specifically amplifying LncRNA MAGI2-AS3 is shown AS SEQ ID NO.2 and SEQ ID NO. 3.

The invention screens a marker LncRNAMAGI2-AS3 for treating acute myeloid leukemia from AML tissues by the technical means of bioinformatics and existing molecular biology (LncRNAMAGI2-AS3 is low expressed in AML tissues). On the basis, the invention provides the application of LncRNAMAGI2-AS3 in preparing a product for treating acute myeloid leukemia; in still another aspect, the present invention also provides a pharmaceutical composition for treating acute myeloid leukemia based on lncrnaagi 2-AS 3; in still another aspect, the present invention also provides a kit for detecting the expression level of lncrnaagi 2-AS 3. The LncRNAMAGI2-AS3 and the pharmaceutical composition thereof have good inhibition effect on acute myeloid leukemia, and have important reference and practical significance in the treatment of acute myeloid leukemia. In addition, when the kit for detecting LncRNAMAGI2-AS3 provided by the invention is applied to the treatment of acute myeloid leukemia AS a pharmaceutical composition, LncRNAMAGI2-AS3 provides good help for evaluating the duration of action of the drug, more accurate administration time and the like, and has better measuring and guiding effects when LncRNAMAGI2-AS3 is applied AS a pharmaceutical composition.

Drawings

Wherein denotes P <0.05 with significant differences.

Wherein AMLSC represents an abbreviation of Acute myeloloid leukemia stem cells, translated into Acute myelogenous leukemia stem cells; MAGI2-AS3 is an abbreviation for LncRNAMAGI2-AS 3; TET2 is an abbreviation for Translocation-2, MMP9 is an abbreviation for matrix metalloprotein 9; abbreviation of 5-Aza-dC (5-Aza-2' -deoxycytidine ).

FIG. 1LncRNAMAGI2-AS3 is low expressed in AMLSC.

FIG. 1A: expression of MAGI2-AS3 in AML chip GSE17054, data expressed AS mean ± SD, unpaired Student's ttest, n ═ 13; FIG. 1B: flow cytometry sorting AMLSC and normal bone marrow hematopoietic stem cells; FIG. 1C: qRT-PCR detection of LncRNA MAGI2-AS3 expression levels in AMLSC and Normal bone marrow hematopoietic stem cells (Normal group n ═ 20, AMLSC group n ═ 51)

FIG. 2LncRNAMAGI2-AS3 inhibits AMLSC self-renewal.

FIG. 2A: qRT-PCR detects the expression level of MAGI2-AS3 after the MAGI2-AS3 is over-expressed in AMLSC; FIG. 2B: cell balling experiments detect the formation capacity (x 200) of AMLSC cells after over-expressing MAGI2-AS 3; FIG. 2C: soft agar colony formation assay the ability of AMLSC clones to form was tested after overexpression of MAGI2-AS 3.

FIG. 3LncRNAMAGI2-AS3 inhibits AMLSC self-renewal by MMP 9.

FIG. 3A: analyzing the correlation between the expression of MAGI2-AS3 and MMP9 in the data of the GSE17054 chip; FIG. 3B: detecting MMP9 expression in AMLSC and normal bone marrow hematopoietic stem cells by qRT-PCR; FIG. 3C: detecting MMP9 expression conditions in AMLSC and normal bone marrow hematopoietic stem cells by Western blot; FIG. 3D: qRT-PCR detects the MMP9 expression condition after over-expressing MAGI2-AS3 in AMLSC; FIG. 3E: westernblot detects the expression condition of MMP9 after over-expressing MAGI2-AS3 in AMLSC; FIG. 3F: westernblot detects the expression condition of MMP9 after overexpression of MMP9 in AMLSC; FIG. 3G: cell balling experiments were performed to detect the formation capacity (x 200) of each group of AMLSC cells; FIG. 3H: detecting the cloning forming capability of AMLSC of each group by a soft agar colony forming experiment; FIG. 3I: detecting MMP9 expression in each group of AMLSCs by qRT-PCR; FIG. 3J: westernblot detects the MMP9 expression in each group of AMLSCs; FIG. 3K: cell balling experiments were performed to detect the formation capacity (x 200) of each group of AMLSC cells; FIG. 3L: the soft agar colony formation assay measures the ability of each group of AMLSC clones to form.

FIG. 4 the MMP9 promoter region is capable of methylation.

FIG. 4A: CPG island enrichment analysis of MMP9 promoter region; FIG. 4B: MS-PCR detects the methylation level of MMP9 promoter in the Normal group and AMLSC group; FIG. 4C: MS-PCR detection of MMP9 promoter methylation level after treatment with 5-Aza-dC; FIG. 4D: qRT-PCR detection of MMP9 expression after treatment with 5-Aza-dC; FIG. 4E: and (3) detecting the MMP9 expression condition after the Western blot is treated by 5-Aza-dC.

FIG. 5LncRNAMAGI2-AS3 mediates MMP9 demethylation by recruiting the demethylase TET 2.

FIG. 5A: the lnctatlas online website analyzes the cell location of MAGI2-AS 3; FIG. 5B-FISH detects the localization of MAGI2-AS3 in cells; FIG. 5C: nuclear-cytoplasmic fractionation experiments were performed to detect the cellular localization of MAGI2-AS 3; FIG. 5D: prediction of binding capacity between the MAGI2-AS3 sequence and the TET2 protein sequence, wherein the fact that both the RF value and the SVM value are more than 0.5 indicates that the protein possibly has the binding capacity; FIG. 5E: the LongTarget website predicts the binding site of MAGI2-AS3 to the MMP9 promoter region; FIG. 5F: RNA-pull-down experiment, Westernblot to detect pulled down protein; FIG. 5G: RIP experiment, qRT-PCR detection of MAGI2-AS3 under immunoprecipitation; FIG. 5H: ChIP assay, qPCR detects TET2 promoter.

FIG. 6LncRNAMAGI2-AS3 inhibits AMLSC self-renewal by MMP 9.

FIG. 6A: westernblot measures the expression level of TET2 in each group; FIG. 6B: detecting MMP9 expression level in each group by qRT-PCR; FIG. 6C: westernblot detects the MMP9 expression level in each group; FIG. 6D: cell balling experiments were performed to detect the formation capacity (x 200) of each group of AMLSC cells; FIG. 6E: the soft agar colony formation assay measures the ability of each group of AMLSC clones to form.

FIG. 7 in vivo results.

FIG. 7A: quantitatively detecting the AMLSC content in peripheral blood by flow cytometry; FIG. 7B: and (5) counting the survival rate of the leukemia mouse model.

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 long-chain non-coding RNA in AML tissue through a bioinformatics technology and a molecular biology technology after extensive and intensive research, finds long-chain non-coding RNA segments with obvious expression difference, and discusses the relationship between the long-chain non-coding RNA segments and the occurrence of acute myeloid leukemia, thereby finding better ways and methods for the targeted therapy of the acute myeloid leukemia. Experiments prove that LncRNA MAGI2-AS3 in AML tissues is remarkably reduced, and further experiments prove that the growth of AMLSC can be influenced by changing the expression level of LncRNA MAGI2-AS3, which suggests that LncRNA MAGI2-AS3 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.

Based on the findings of the inventors, the present invention provides the use of LncRNA MAGI2-AS3 in the preparation of a product for treating acute myeloid leukemia, and a pharmaceutical composition prepared therefrom and the use of the pharmaceutical composition in the treatment of acute myeloid leukemia, the properties of which are not important to the present invention AS long AS it contains the functionality of LncRNA MAGI2-AS3 gene, for example, the pharmaceutical composition of the present invention may be a recombinant plasmid constructed with the synthetic sequence of LncRNA MAGI2-AS3 gene, which can inhibit self-renewal of AMLSC (detailed in the preferred embodiments of the present invention). On the basis, the invention designs an agent or other components aiming at improving the gene expression of LncRNA MAGI2-AS3 according to the nucleotide sequence of LncRNA MAGI2-AS3 and the action mechanism of LncRNA MAGI2-AS3 in acute myeloid leukemia (MAGI2-AS3 promotes the expression of MMP9 by demethylating DNA mediated by demethylase TET2 so AS to inhibit the self-renewal of stem cells of the acute myeloid leukemia), and also designs an agent or other components directly improving the gene expression of MMP9, wherein the agents or components can play a role in treating the acute myeloid leukemia and all belong to the protection scope of the invention. For example, the present invention provides a plasmid capable of up-regulating LncRNA MAGI2-AS3 or up-regulating MMP 9. Of course, artificially synthesized LncRNA MAGI2-AS3 or MMP9 directly fall within the scope of this patent.

The LncRNA MAGI2-AS3 or MMP9 of the present invention can be chemically synthesized or prepared by transcribing an expression cassette in a recombinant nucleic acid construct into single stranded RNA. Can be delivered into the cell by using an appropriate transfection reagent, or can also be delivered into the cell using a variety of techniques known in the art.

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.

In the examples of the present invention, the nucleotide sequence of a representative human LncRNA MAGI2-AS3 gene is shown in SEQ ID NO. 1. The full-length LncRNA MAGI2-AS3 nucleotide sequence or a fragment thereof of the present invention can be obtained by PCR amplification, recombination, or artificial synthesis.

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 LncRNA MAGI2-AS 3.

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 LncRNA MAGI2-AS3, 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 LncRNA MAGI2-AS 3. When the LncRNA MAGI2-AS3 is used AS a pharmaceutical composition for treating acute myeloid leukemia, the kit is used for evaluating the expression level of the LncRNA MAGI2-AS3, so that the kit provides help for evaluating the duration of action of the drug, more accurate administration time and the like, and has better measuring and guiding effects when the LncRNA MAGI2-AS3 is used AS the pharmaceutical 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: clinical specimen collection

51 patients with confirmed diagnosis of AML from 2016 to 2018 and 12 months of age in the department of hematology of our hospital were collected, 32 men, 19 women, 18-72 years of age, and the median age was 45 years of age. Diagnostic typing according to FAB criteria, wherein M₁Example 3, M₂27 example, M₄Example 15, M₅Example 6. With 20 healthy donor bone marrow as a normal control, the study was informed consent of the patients and signed an informed consent form, approved by the ethical committee of my hospital and strictly adhered to the declaration of helsinki.

The method 2 comprises the following steps: mononuclear cell isolation

4ml of bone marrow of AML patients and healthy donors was aseptically extracted, 4ml of Ficoll lymph (model: 17-1440-03, Shanghai Hua Yasi Chuang Biotech Co., Ltd., Shanghai, China) was added to a 15ml centrifuge tube, bone marrow was slowly added, and centrifuged at 2000 Xg for 10min, and the liquid was divided into 3 layers. And sucking the film between the 1 st layer and the 2 nd layer, putting the film into another clean 15ml centrifugal tube, cleaning the film twice by using Hank liquid, and adding the frozen stock solution for storage for later use.

The method 3 comprises the following steps: flow cytometry sorting of bone marrow stem cells

The frozen mononuclear cells were thawed to prepare a cell suspension, the cell suspension was transferred into a sterile 1.5mL EP tube at a cell concentration of 1X 107 cells/mL, anti-CD34-FITC (1:50, ab8536, Abcam, Cambridge, UK) and anti-CD123-PE (1:500, ab21562, Abcam, Cambridge, UK) were added to the tube, CD34+ CD123+ cells and CD34+ CD 123-cells were sorted and collected on a flow cytometer (Becton Dickinson), and the purity of the sorted stem cells was identified. The CD34+ CD123+ cells finally screened were Acute Myeloid Leukemia Stem Cells (AMLSC), and the CD34+ CD 123-cells were Normal bone marrow stem cells (Normal).

The method 4 comprises the following steps: cell transfection and grouping

AMLSC are divided into 8 groups: oe-NC group (transfection pCMV6-AC-GFP-NC plasmid), oe-MAGI2-AS3 group (transfection pCMV6-AC-GFP-MAGI2-AS3 plasmid), oe-MMP9 group (transfection pCMV 9-AC-GFP-MMP 9 plasmid), oe-MAGI 9-AS 9 + sh-NC group (co-transfection pCMV 9-AC-GFP-MAGI 9-AS 9 plasmid and pGPU 9/Neo-NC), oe-MAGI 9-AS 9 + sh-MMP9 group (co-transfection pCMV 9-AC-MAGI 9-AS 9 plasmid and pGPU 9/Neo-MMP 9 plasmid), oe-MAGI 9-AS 9 + sh-TET 9 group (co-transfection pCMV 9-AC-MAGI 9-AS 9 plasmid and pGPU 9/Neo-MMP 9 plasmid), oe-MAGI 9-AS 9 + sh-TET 9 group (co-GFP-MTC-ELISA group (co-MTC-ELISA 5 group) and (co-MTA-MTC-ELISA) group (co-MTC-MTA-MTC-ELISA) were treated with pCMV- pCMV6-AC-GFP and pGPU6/Neo were transfected using lipofectamin 2000(Invitrogen, Calsbad, CA, USA) kit (cat # 11668019, purchased from Thermo fisher), purchased from Wuhan vast Ling Biotech Ltd (Wuhan, China) and Jima gene (Shanghai, China), respectively. Mu.g of the desired plasmid and 10. mu.L of LLIPOFECTAMINE 2000 were diluted in 250. mu.L of serum-free Opti-MEM (Gibco) medium and gently mixed. Standing at room temperature for 5min, mixing the two solutions uniformly, standing for 20min, adding the mixed solution into a culture hole, placing at 37 ℃, culturing in a 5% CO2 incubator for 6h, replacing with a complete culture medium, continuing culturing for 48h, collecting cells, detecting the transfection effect, and using the cells for subsequent experiments.

The method 5 comprises the following steps: qRT-PCR

Tissue and fine tissue were extracted using TRIzol (Invitrogen, Calsbad, Calif., USA)Total RNA in cells, nanodrop2000 microultraviolet spectrophotometer (1011U, nanodrop, USA) measures the concentration and purity of total RNA extracted. The primers of LncRNA MAGI2-AS3 were designed by reverse transcription of RNA to cDNA according to the instruction of PrimeScriptRTreagent Kit (RR047A, Takara, Japan) and synthesized by TaKaRa (Table 1). Real-time fluorescent quantitative PCR detection is carried out by using an ABI7500 quantitative PCR instrument (7500, ABI, USA) under the reaction conditions of pre-denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 20s, extension at 72 ℃ for 34s and 40 cycles. Relative quantification method using GAPDH as internal reference primer (2)^-△△CTMethod) for calculating relative transcription level of target gene, △△ Ct (△ Ct experimental group) - △ Ct control group, △ Ct (target gene) -Ct (internal reference), and mRNA (mRNA) of target gene (2)^-△△Ct. Each experiment was repeated three times.

TABLE 1 qRT-PCR reaction primer sequences

The method 6 comprises the following steps: western bolt

Extracting total protein from cell with RIPA lysate (P0013C, Biyunyan, Shanghai, China) containing PMSF, incubating on ice for 30min, centrifuging at 4 deg.C and 8000g for 10min, and collecting supernatant. Total protein concentration was measured with BCA kit. After boiling 50. mu.g of protein in 2 XSDS loading buffer for 5min at 100 ℃, each of the above samples was subjected to SDS-PAGE gel electrophoresis, the proteins were transferred to PVDF membrane, 5% skim milk powder was blocked for 1H at room temperature, and then PVDF membrane was incubated with diluted primary anti-rabbit anti-CD34 (1:10000, ab81289, Abcam, Cambridge, UK), CD123(1:1500, ab200687, Abcam, Cambridge, UK) and MMP9(1:1000, #12752, Cell Signaling Technology, USA), TET2 (1:2000, ab94580, Abcam, Cambridge, UK), GAPDH (ab9485, 1:2500, Abcam, Cambridge, UK) as internal references, washed 3 times at TBST, 10min each time, the membrane was incubated with HRP-labeled secondary antibody goat anti-rabbit H & IgG & 051H & 2000 (Abcam, Abcam & K), and washed overnight with glass (Abch & S & ltd) (Abch & ltd & gtH & ltd, Abch & ltd & gth & ltd & gth & ltd & gtwash, and wash (. Equal amounts of solution A and solution B were mixed in an ECL fluorescence detection kit (cat # BB-3501, Ameshame, UK), and applied onto a membrane by dripping, and the membrane was photographed by a Bio-Rad image analysis system (BIO-RAD, USA) and analyzed by quantitative One v4.6.2 software, and the relative protein content was expressed as the gray value of the corresponding protein band/the gray value of the GAPDH protein band. The experiment was repeated three times and the mean value was taken.

The method 7 comprises the following steps: cell balling experiment

Preparing single cell suspension from AMLSC of each transfection group, and adjusting cell density to 1 × 10⁴After each ml, the suspension cell pellet is inoculated into a low-adsorption 24-well plate containing serum-free medium, the suspension cell pellet is cultured, half-quantitative liquid change is carried out every 2 days, after 10 days of continuous culture, CKX4l is observed and photographed under an inverted optical microscope (OLYMPUS, Japan), the number of newly formed suspension cell pellets in each well is counted and averaged, and the experiment is repeated for 3 times.

The method 8 comprises the following steps: soft agar colony formation assay

Paving bottom agar: the vessels used were autoclaved, 0.7% low melting point agarose was prepared in fresh DMEM medium and stored at 4 ℃ for future use. When the agar is laid, 0.7 percent of agarose is heated and melted, 2mL of the agarose is sucked into a culture dish with the diameter of 100mm, the culture dish is gently shaken, the bottom-layer agar is uniformly laid, and the agar is cooled and solidified for later use; cell inoculation, culture and colony identification: when cells were inoculated, 1mL of the cell suspension and an equal volume of 0.7% agarose solution were diluted to 0.35% agarose cell mixture per 100cm²Inoculation of about 1 × 10⁴For each cell, 3 replicates were set for each group of samples. After the upper agar is solidified, dripping 2-3 mL of culture solution on the surface of the upper agar to avoid crushing, and carrying out 5% CO treatment at 37 DEG C₂Culturing, replacing the cell with liquid every 2-3 days, and terminating the culture after 1 month; taking out the culture dish, placing the culture dish under an inverted microscope for counting, judging that only the mass of more than or equal to 50 cells is 1 cell colony, and photographing for preservation.

The method 9: nuclear mass separation experiment

In accordance with PARIS^TMKit Protein and RNA Isolation System(Life TechnoloCollecting AMLSC, washing with PBS, digesting with pancreatin, centrifuging at 4 deg.C for 5min with 500g PBS, discarding the supernatant, adding 500 μ L Cell fractionation Buffer, gently blowing, standing on ice for 5-10 min, centrifuging at 4 deg.C for 5min with 500g, transferring the supernatant (cytoplasm) into a new 2mL sterile enzyme-free tube, centrifuging at 4 deg.C for 5min, adding 500 μ L Cell fractionation Buffer into the precipitate (Cell nucleus), gently mixing with a bomb, adding 500 μ L room temperature 2 × lysine/binding solution, gently blowing, mixing, standing on ice, discarding the supernatant, adding precooled 500 μ L Cell fractionation Buffer, mixing, immediately vortexing, adding 500 μ L absolute ethanol, gently blowing, mixing, placing the adsorption column into a collection tube, adding 700 μ L reaction solution, adding 30 g electrophoresis solution, discarding 30 μ L Cell fractionation Buffer, centrifuging at 12030 μ L, adding 12030 μ L rRNA, centrifuging at 12030 μ L, collecting RNA, adding 12030 μ L rRNA, centrifuging at 12030 μ L, eluting with 30 μ L centrifugation liquid, adding 12035 μ L RNA, centrifuging at 12035 μ L, adding RNA, centrifuging at 12035 μ L, collecting RNA, centrifuging at 12035 μ L, adding RNA, centrifuging at 12035 μ L, centrifuging at 30 μ L, adding RNA, centrifuging at room temperature, and collecting RNA.

TABLE 2 Nuclear plasmid isolation internal reference qRT-PCR primer sequences

The method 10 comprises the following steps: Methylation-Specific PCR (MS-PCR)

MS-PCR detects the methylation condition of the MMP9 promoter. DNA extraction: extracting genome DNA by using a Beijing Tiangen Biotechnology Co Ltd genome DNA extraction kit according to the kit specification, extracting the genome DNA, measuring the DNA concentration and purity by using an ultraviolet spectrophotometry, and storing in a refrigerator at-80 ℃ for later use. MS-PCR: adopting EZ DNAmethylation Kit (ZymoResearch, USA) to treat DNA with sodium sulfite, utilizing a reaction column to desulfurize and purify, using the purified DNA for subsequent PCR reaction, and designing methylation and non-methylation primers aiming at CPG island enrichment region of MMP9 gene promoter. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 45s, denaturation at 58 ℃ (methylated)/denaturation at 57 ℃ (unmethylated) for 45s, annealing at 72 ℃ for 45s, and 35 cycles; finally, extension is carried out for 10min at 72 ℃. The reaction product is subjected to agarose gel electrophoresis, gel electrophoresis imaging and image analysis by an image analysis system. If the CpG island in the promoter region of the MMP9 gene is completely methylated, only a methylation primer can amplify a target band; if the target band is completely unmethylated, only the unmethylated primer can amplify the target band; if the methylation is partial, the target band can be amplified by both pairs of primers. Partial methylation is classified as a methylation category. Each experiment was repeated 3 times.

TABLE 3 primer sequences

The method 10 comprises the following steps: RNA-pull-down

The fragment was transcribed in vitro using T7 RNA polymerase (Ambion, USA) to LncRNAMAGI2-AS3, then treated with RNeasy Plus Mini Kit (Qiagen, Germany), DNase I (Qiagen, Germany), and purified with RNeasy Mini Kit. The 3' end of the purified RNA was biotinylated with a biotin RNA labeling cocktail (Ambion, USA). Mu.g of labeled RNA was added to the RNA structure buffer (10mmol/L Tris pH7, 0.1mol/LKCl, 10mmol/L MgCl)₂) AMLSC 3 mu g is added into cell lysate (Sigma, USA) for 1h at 4 ℃, the lysate is centrifuged for 10min at 12000 × g 4 ℃, the supernatant is collected and transferred into an RNase-free centrifuge tube, 400ng biotinylated RNA is added into 500 mu LRIPbuffer and mixed with cell lysate for incubation for 1h at room temperature, streptavidin magnetic beads are added into each binding reaction and incubated for 1h at room temperature, finally, RIPbuffer is used for washing for 5 times, 5 × sample buffer is added, and Westernblot detection of eluted TET2 protein is carried out after incubation for 5min at 95 ℃, and IgG (ab172730, 1:100, Abcam, UK) is used as a negative control in the experiment.

The method 11 comprises the following steps: RNA co-immunoprecipitation assay (RIP, RNA-Binding Protein Immunoprecipitation)

The binding of LncRNAMAGI2-AS3 to TET2 protein was detected using RIP kit (millipore, USA). The test cells were washed with pre-cooled PBS and the supernatant was discarded. Cells were lysed with an equal volume of RIPA lysate (P0013B, picui, china) in ice for 5min, and centrifuged at 12000 × g at 4 ℃ for 10min to obtain the supernatant. Incubating and coprecipitating the cell extract and the antibody, and specifically comprising the following steps: 50 μ L of magnetic beads were washed and resuspended in 100 μ L of RIPWash Buffer for each coprecipitation reaction, and 5 μ g of antibody was added for incubation according to experimental groups for binding. After washing, the magnetic bead-antibody complex is resuspended in 900. mu.L RIP Wash Buffer, and 100. mu.L of cell extract is added and incubated overnight at 4 ℃. The sample is placed on a magnetic seat to collect the magnetic bead-protein complex. And digesting the sample by proteinase K, and extracting RNA for subsequent PCR detection. The antibodies used in RIP were: rabbit anti-TET 2 (1:100, ab94580, Abcam, UK) was mixed well for 30min at room temperature with IgG (1:100, ab172730, Abcam, UK) as negative control.

The method 12 comprises the following steps: chromatin immunoprecipitation experiment (ChIP)

Taking AMLSC, adding 1% formaldehyde when the cell fusion degree reaches 70-80%, fixing at room temperature for 10min to make the DNA and Protein in the cell be fixed and crosslinked, after crosslinking, carrying out ultrasonic treatment and random fragmentation, each time carrying out ultrasonic treatment for 10s, spacing for 10s, circulating for 15 times to break the fragments into fragments with proper size, centrifuging at 12000 Xg at 4 ℃, collecting supernatant and dividing into two tubes, respectively adding rabbit anti-IgG (1:100, ab172730, Abcam, UK) as a negative control antibody and rabbit anti-TET 2 (1:100, ab230358, Abcam, UK) as a target Protein specific antibody at 4 ℃ for overnight incubation, utilizing Protein agar/Sepharose to precipitate endogenous DNA-Protein complex, after short centrifugation, sucking and discarding supernatant, washing non-specific complex, carrying out crosslinking release at 65 ℃ overnight, extracting phenol/chloroform, purifying and recovering DNA fragments, MMP9 promoter fragment enrichment in combination with TET2 was tested with MMP9 gene promoter fragment specific primers.

Method 13: in vivo experiments

24 NOD/SCID mice are 18-22 g in body mass and 6-8 weeks old. Limited laboratory animals from Schlekschada, HunanThe company randomly divides NOD/SCID mice into two groups, namely an oe-NC group and an oe-MAGI2-AS3 group, wherein 20 mice are in each group, the two groups are subjected to whole body X-ray irradiation for 1.8Gy, and the oe-MAGI2-AS3 group, and after 24 hours, AMLSC tail vein injection of stable transfection MAGI2-AS3 in logarithmic growth phase is carried out for 5 × 10⁶One/only. oe-NC group: AS a control for the oe-MAGI2-AS3 group. Taking 4 surviving mice every 2 weeks, taking tail vein blood, detecting the AMLSC content in peripheral blood, observing and detecting the survival rates of two groups of mice. All animal experiments in this study were in accordance with local laboratory animal care and approval by the medical ethics committee of our hospital.

Example 1: LncRNAMAGI2-AS3 is low expressed in Acute Myelogenous Leukemia Stem Cells (AMLSC)

By differential analysis of the GSE17054 chip, it was found that long-chain non-coding RNA MAGI2-AS3 was significantly down-regulated in AML stem cells compared to normal bone marrow hematopoietic stem cells (fig. 1A).

To further confirm the expression of MAGI2-AS3 in AMLSC, we collected acute myeloid leukemia patients and normal human bone marrow, isolated mononuclear cells, and successfully sorted by flow cytometry to collect CD34⁺CD123⁺Cells (acute myeloid leukemia stem cells: AMLSC) and CD34⁺CD123^-Cells (Normal bone marrow hematopoietic stem cells: Normal) (FIG. 1B). The qRT-PCR detection result shows that: compared with normal bone marrow hematopoietic stem cells, the expression level of MAGI2-AS3 in AMLSC was significantly reduced (FIG. 1C).

The research results show that: LncRNAMAGI2-AS3 was low expressed in AMLSC.

Example 2: LncRNAMAGI2-AS3 inhibits AMLSC self-renewal

To investigate the effect of lncrnaagi 2-AS3 on AMLSC, we used cell spheronization and soft agar colony formation experiments to examine the effect of MAGI2-AS3 on the self-renewal capacity of AMLSC after overexpression of MAGI2-AS3 in AMLSC. First, the expression level of MAGI2-AS3 was significantly increased after qRT-PCR detection of MAGI2-AS3 over-expressed in AMLSC (FIG. 2A). The results of the cell balling experiment and the soft agar colony forming experiment show that: the balling and clonogenic capacity of AMLSC was significantly reduced after overexpression of MAGI2-AS3 compared to the control group (fig. 2B-C).

The above experimental results show that: LncRNAMAGI2-AS3 inhibits AMLSC self-renewal.

Example 3: LncRNAMAGI2-AS3 inhibits AMLSC self-renewal by MMP9

MMP9 has been reported to be down-regulated in leukemia (PMID: 30747385). We speculate that MAGI2-AS3 may influence AML progression by modulating MMP9 expression. qRT-PCR and Western blot are used for detecting the expression condition of MMP9 in AMLSC and normal bone marrow hematopoietic stem cells, and the result shows that: the expression level of MMP9 was significantly reduced in AMLSC compared to normal bone marrow hematopoietic stem cells (fig. 3B-C). Upon overexpression of MAGI2-AS3 in AMLSC, it was found that: MMP9 expression was significantly increased compared to the control group (fig. 3D-E). Shows that: MMP9 was low expressed in AMLSC, and MAGI2-AS3 promoted expression of MMP 9. To further investigate the effect of MMP9 on the self-renewal capacity of AMLSCs. We over-expressed MMP9 in AMLSC and the Westernblot results show that: after overexpression of MMP9, the expression level of MMP9 protein is obviously increased (FIG. 3F). The results of the cell balling experiment and the soft agar colony forming experiment are shown as follows: upon overexpression of MMP9, the balling and clonogenic capacity of AMLSC was significantly reduced compared to the control group (fig. 3G-H). Shows that: MMP9 inhibits AMLSC self-renewal. To study the effect of MAGI2-AS3 on the self-renewal capacity of AMLSCs by modulating MMP 9. We divided the cells into two groups: oe-MAGI2-AS3+ sh-NC group and oe-MAGI2-AS3+ sh-MMP9 group. And (3) displaying a Western blot detection result: compared with the oe-MAGI2-AS3+ sh-NC group, the expression level of MMP9 in the oe-MAGI2-AS3+ sh-MMP9 group is obviously reduced (FIGS. 3I-J). The results of the cell balling experiment and the soft agar colony forming experiment are shown as follows: compared with the oe-MAGI2-AS3+ sh-NC group, the oe-MAGI2-AS3+ sh-MMP9 group AMLSC has significantly improved balling-up and clonogenic abilities (FIGS. 3K-L).

The research results show that: lncrnamaigi 2-AS3 inhibits amlc self-renewal by promoting the expression of MMP 9.

Example 4: the MMP9 promoter region in AML can be methylated

The current study indicates that TET2 can be linked to demethylation of the promoter region of MMP9 and further that methylation-specific PCR (MS-PCR) is used to detect whether the promoter of MMP9 is methylated in the Normal group and AMLSC group, and the results show that: the MMP9 promoter region was unmethylated in the Normal group, while the MMP9 promoter region was methylated in the AMLSC group (FIG. 4B). After treatment of AMLSC with the methyltransferase inhibitor 5-Aza-dC, it was found that: the methylation level of the promoter region of MMP9 was significantly reduced in the 5-Aza-dC group compared to the control group (FIG. 4C). The qRT-PCR and Western blot detection results show that: MMP9 expression was significantly increased in the 5-Aza-dC group compared to the control group (FIGS. 4D-E). The above studies show that: the MMP9 promoter region can be methylated, thereby inhibiting the expression of MMP 9.

Example 5: LncRNAMAGI2-AS3 mediates MMP9 demethylation by recruiting the demethylase TET2

To study the promoter methylation regulation mechanism of LncRNAMAGI2-AS3 on MMP9, we found that MAGI2-AS3 was mainly localized to the nucleus by lncALAS online website (http:// lncALAS. crg. eu /) analysis (FIG. 5A), and that FISH detected the localization of MAGI2-AS3 in cells found that: MAGI2-AS3 was mainly distributed in the nucleus (FIG. 5B). Further validation using nuclear-cytoplasmic separation experiments: MAGI2-AS3 was mainly localized to the nucleus (FIG. 5C), suggesting that its regulatory function may occur at the transcriptional level. TET2 is a DNA demethylase, analysis of RPIseq database (http:// pridb. gdcb. iastate. edu/RPISeq /) revealed that MAGI2-AS3 may have a binding relationship with TET2 (FIG. 5D), and it was speculated that MAGI2-AS3 may promote DNA demethylation of MMP9 by recruiting TET2 to MMP9 promoter. To confirm this hypothesis, we performed RNA-pull-down and RIP experiments to test the recruitment of MAGI2-AS3 to TET2 after overexpression of MAGI2-AS3 in AMLSC. The results of the RNA-pull-down experiment show that: MAGI2-AS3 pulls down TET2, indicating: MAGI2-AS3 can bind to TET2 (FIG. 5F). The RIP experiment results show that: the amount of MAGI2-AS3 immunoprecipitated by TET2 was significantly increased after overexpression of MAGI2-AS3, AS compared to the control group, indicating that: mage 2-AS3 recruited TET2 and, after overexpression of mage 2-AS3, recruited more TET2 (fig. 5G). Next, we tested the enrichment of TET2 in the MMP9 promoter region by ChIP experiments, and the experimental results showed that: the enrichment of TET2 in the MMP9 promoter region was significantly increased after overexpression of MAGI2-AS3 compared to the control group (fig. 5H).

The research results show that: MAGI2-AS3 can promote DNA demethylation by MMP9 by recruiting TET2 to the MMP9 promoter.

Example 6: LncRNA MAGI2-AS3 mediates MMP9 demethylation through TET2, and inhibits AMLSC self-renewal

To investigate the effect of MAGI2-AS3 on the self-renewal capacity of AMLSCs to mediate MMP9 demethylation via TET2, we divided AMLSCs into two groups: the Western blot detection results show that the Western blot detection results of the oe-MAGI2-AS3+ sh-NC group and the oe-MAGI2-AS3+ sh-TET2 group: compared with the oe-MAGI2-AS3+ sh-NC group, the expression level of TET2 in the oe-MAGI2-AS3+ sh-TET2 group is remarkably reduced (FIG. 6A). The qRT-PCR and Western blot detection results show that: compared with the oe-MAGI2-AS3+ sh-NC group, the expression level of MMP9 in the oe-MAGI2-AS3+ sh-TET2 group is obviously reduced (FIG. 6B-C). The LncRNA MAGI2-AS3 can mediate the demethylation of MMP9 through TET2 and promote the expression of MMP 9. Cell balling experiments and soft agar colony formation experiments tested each group of self-renewal capacity of AMLSC and the results showed: the balling and clonogenic capacities of AMLSCs in the oe-MAGI2-AS3+ sh-TET2 group were significantly increased compared to the oe-MAGI2-AS3+ sh-NC group (FIGS. 6D-E).

The research results show that: lncrnaagi 2-AS3 mediates MMP9 demethylation via TET2, inhibiting AMLSC self-renewal.

Example 7: in vivo experiments

To further investigate the effect of lncrnaagi 2-AS3 on leukemia in vivo, we constructed a leukemia model by tail vein injection of AMLSC in NOD/SCID mice. Taking peripheral blood every 2 weeks, quantitatively detecting the content of AMLSC in the peripheral blood by using flow cytometry, and finding that: the content of AMLSC was significantly reduced in the oe-MAGI2-AS3 group compared to the oe-NC group (FIG. 7A). Through statistics on the survival rate of a leukemia mouse model, the following results are found: overall survival was significantly increased in mice of the oe-MAGI2-AS3 group compared to the oe-NC group (fig. 7B).

The research results show that: in leukemia mice, MAGI2-AS3 can inhibit AMLSC self-renewal and improve the survival rate of leukemia mice.

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> LncRNA MAGI2-AS3 in preparation of product for treating acute myeloid leukemia and detection kit thereof

<160>5

<170>PatentIn version 3.5

<210>1

<211>918

<212>DNA

<213>LncRNA MAGI2-AS3

<400>1

attgccctgc gccccgagcg gtcccaccgc ccggcaaaac actgagcaag ccgctgctct 60

caccttgctt gacacacttg agccggaggg ggtccttgca gtgtttgatc acggccagca 120

cgtccctgat ggtgagcccc gccacggggg tctcgttcac ctccagcagc agctcctccg 180

acaccaattt gctgccgctc tcataggcca ccttgccggg cttcacctcc cccagtaatg 240

tcttacaata acagaatgca ggagagcaca tatcaatgaa gaaaagacaa aatgtactta 300

tccatgaagc tgaggttaag aaaccagtgt cttcaagagc cagggacagc actgggtctg 360

tgcagagttg agagatggtg atgacatgtg acagagctcc ttgctgctca aattttatgc 420

cagataaaat attcaccttc tcggactccc tggcaccagg catggccatg taatcctaca 480

gcacagctga tgaaagagaa gacatcttgc tgctatttga caagcaagaa gataaaagct 540

ttcctataaa gatgatggag caaaaaaata gaaggggcct agactcccta ccttcacact 600

tcctgctata aaagcattgc tattataagg ttaatctgca gaaacagact tctatgtcat 660

atgcttaaat gcatcaaaat tcatacaata tccattaaat tggtaaaagt atccatttta 720

aaggattaat ctgagggacc cacagacact taacacaatc aaatctcaga aaaaaaatca 780

gttatttcat tgtgattttg aggctgatct tttatgatct tttagttatc aaactgttaa 840

atacatttta actttcaaat tttatgaata aaggagatga atcctgttat tttctcagtg 900

aaaaaaaaaa aaaaaaaa 918

<210>2

<211>20

<212>DNA

<213> Artificial Sequence (Aruifisial Sequence)

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caccttgctt gacaacttga 20

<210>3

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<212>DNA

<213> Artificial Sequence (Artificial Sequence)

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cattacagct cggctactgc 20

Claims

1. The long-chain non-coding RNA is LncRNAMAGI2-AS3, and is characterized by application of LncRNAMAGI2-AS3 in preparation of a product for treating acute myeloid leukemia.

2. The use according to claim 1, wherein lncrnaagi 2-AS3 is represented by SEQ ID No. 1.

3. The use according to claim 1, wherein the target gene of lncrnaagi 2-AS3 is MMP 9.

4. The use of claim 1, wherein lncrnaagi 2-AS3 recruits a demethylase enzyme that mediates demethylation of the MMP9 promoter.

5. The use of claim 4, wherein the methylase is TET 2.

6. A pharmaceutical composition for treating acute myeloid leukemia, comprising lncrnaagi 2-AS 3.

7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition is a plasmid comprising lncrnaagi 2-AS 3.

8. A kit comprising reagents for detecting the expression level of lncrnaagi 2-AS 3.

9. The kit of claim 8, wherein the reagent is a primer that specifically amplifies LncRNA MAGI2-AS 3.

10. The kit of claim 9, wherein the primer sequence for specific amplification lncrnaagi 2-AS3 is shown AS SEQ ID No.2 and SEQ ID No. 3.