Application of LDLR (Low-Density Lipoprotein Receptor) in screening or preparing medicine for treating acute myelogenous leukemia

Abstract

The invention discloses an application of LDLR in screening or preparing a medicine for treating acute myelogenous leukemia, the expression level of the LDLR in acute myelogenous leukemia cells is detected for the first time so as to characterize the conditions of proliferation, apoptosis, cycle and the like of the acute myelogenous leukemia cells, and the LDLR is taken as a target spot; by knocking down the expression quantity of the LDLR, the proliferation of acute myelogenous leukemia cells can be inhibited, the apoptosis of the acute myelogenous leukemia cells can be promoted, the cycle of the acute myelogenous leukemia cells can be retarded in the S phase, the in-vitro proliferation of the acute myelogenous leukemia cells can be accurately and effectively inhibited, and the application prospect is wide. A new thought and a new direction are provided for screening or preparing the medicine for diagnosing and treating the acute myelogenous leukemia.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of LDLR in screening or preparation of a drug for treating acute myeloid leukemia. BACKGROUND

[0002] Acute myeloid leukemia (AML) is a malignant clonal disease with high invasiveness in the field of blood system, which seriously threatens the life and health of patients. The core pathological feature is that myeloid progenitor cells show uncontrolled proliferation, accompanied by expansion and blocked differentiation process. This series of abnormal changes lead to ineffective state of regular hematopoiesis, and further cause life-threatening anemia and transfusion dependence. The clinical manifestations of AML are usually acute, and symptoms attributable to bone marrow failure appear rapidly. If effective treatment is not given in time, the condition will deteriorate rapidly within weeks or months.

[0003] In recent years, with the continuous deepening of the research on the pathophysiology of the disease, people's understanding of AML has made significant progress, and a large number of new drugs are in the clinical trial stage. However, the current treatment of AML is still not optimistic. In addition, the existing standard chemotherapy regimen not only brings heavy economic burden to patients, but also causes serious side effects in the treatment process. At present, the research on the molecular mechanism of AML is not thorough, and there is still a lack of specific target for effectively treating AML, which undoubtedly becomes the key bottleneck restricting the improvement of AML treatment effect.

[0004] Low-density lipoprotein receptor (LDLR) is a cell surface glycoprotein, which mainly functions to bind and internalize circulating cholesterolic lipoprotein particles, plays a key role in cholesterol metabolism, is a key receptor for maintaining cholesterol homeostasis, and is widely expressed in mammals. It has been found in current research that LDLR is abnormally expressed in various cancers, such as colon cancer, prostate cancer, lung cancer, breast cancer and liver cancer. However, there is no relevant report on the specific role and molecular mechanism of LDLR in acute myeloid leukemia. SUMMARY

[0005] The present application provides the application of LDLR in screening or preparation of a drug for treating acute myeloid leukemia.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] The application of LDLR in screening or preparation of a drug for treating acute myeloid leukemia.

[0008] In the present application, the gene name of the low-density lipoprotein receptor (Low-Density Lipoprotein Receptor, LDLR) is low density lipoprotein receptor, the gene ID on NCBI is 3949, and the sequence number is NM_000527.5.

[0009] The present application also aims to protect the use of the LDLR inhibitor in screening or preparing a drug for treating acute myeloid leukemia, characterized in that the LDLR inhibitor inhibits the proliferation of acute myeloid leukemia cells, promotes the apoptosis of acute myeloid leukemia cells, and blocks the cell cycle of acute myeloid leukemia cells by knocking down the expression level of LDLR.

[0010] In the present application, by detecting the expression level of LDLR in acute myeloid leukemia cells, the proliferation ability, apoptosis degree, cell cycle progression, etc. of acute myeloid leukemia cells are characterized, and it is found that high expression of LDLR promotes the proliferation of acute myeloid leukemia cells, reduces the apoptosis of acute myeloid leukemia cells, and promotes the cell cycle of acute myeloid leukemia cells. Based on this, the present application takes LDLR as a target, and by knocking down the expression level of LDLR, the proliferation of acute myeloid leukemia cells is inhibited, the apoptosis of acute myeloid leukemia cells is promoted, and the cell cycle of acute myeloid leukemia cells is blocked in S phase, which can precisely and effectively inhibit the in vitro proliferation of acute myeloid leukemia cells, and provide a new idea and direction for the development of screening or preparing diagnostic and therapeutic drugs for acute myeloid leukemia.

[0011] Further, the drug is a drug for regulating the MAPK signal pathway.

[0012] The MAPK (mitogen-activated protein kinases) signal pathway is an important intracellular signal transduction pathway, which is a key signal pathway for cell growth, proliferation, differentiation, apoptosis, and stress response under normal and pathological conditions. In the present application, after the LDLR inhibitor knocks down the expression level of LDLR, it is found that the related proteins of the MAPK signal pathway change significantly, the MAPK signal pathway is inhibited, thereby the proliferation of acute myeloid leukemia cells is inhibited, the apoptosis of acute myeloid leukemia cells is promoted, and the cell cycle of acute myeloid leukemia cells is blocked in S phase, so as to treat acute myeloid leukemia.

[0013] Further, the LDLR inhibitor is used to inhibit the transcription level of the LDLR gene, the expression level of the LDLR protein, or the activity of the LDLR protein.

[0014] Further, the LDLR inhibitor is an LDLR gene inhibitor for inhibiting the transcription level of the LDLR gene.

[0015] In the present application, the LDLR gene inhibitor is a lentivirus vector prepared by taking the LDLR gene as a target, and has an inhibiting effect on the transcription level of the LDLR gene.

[0016] Further, the LDLR gene inhibitor includes any one or more of double-stranded RNA, short hairpin RNA, antisense oligonucleotide and small interfering RNA.

[0017] In the present application, the double-stranded RNA (dsRNA), short hairpin RNA (shRNA), antisense oligonucleotide (ASO) and small interfering RNA (siRNA) can all play a regulatory role on the transcription level of the LDLR gene.

[0018] Further, the LDLR gene inhibitor is short hairpin RNA, and the short hairpin RNA contains sequences as shown in SEQ ID NO: 5 and SEQ ID NO: 6.

[0019] The present application also aims to protect the use of LDLR in the preparation of a kit for diagnosing acute myeloid leukemia.

[0020] Further, the kit is used for detecting the expression level of the LDLR gene.

[0021] Further, the kit includes a forward primer with a sequence as shown in SEQ ID NO: 1 and a reverse primer with a sequence as shown in SEQ ID NO: 2.

[0022] The present application also aims to protect the use of LDLR as a drug target in the screening or preparation of a drug for treating acute myeloid leukemia.

[0023] The present application has the following beneficial effects:

[0024] The present application first detects the expression level of LDLR in acute myeloid leukemia cells, and characterizes the proliferation ability, apoptosis degree, cell cycle progression, etc. of the acute myeloid leukemia cells by detecting the expression level of LDLR in the acute myeloid leukemia cells, and finds that the high expression of LDLR promotes the proliferation of the acute myeloid leukemia cells, reduces the apoptosis of the acute myeloid leukemia cells, and promotes the cell cycle of the acute myeloid leukemia cells. Based on this, the present application takes LDLR as a target, and can inhibit the proliferation of the acute myeloid leukemia cells, promote the apoptosis of the acute myeloid leukemia cells, and make the cell cycle of the acute myeloid leukemia cells arrest in S phase by knocking down the expression amount of LDLR, so that the in-vitro proliferation of the acute myeloid leukemia cells can be precisely and effectively inhibited, and a new idea and direction are provided for the development of screening or preparing a drug for diagnosing and treating acute myeloid leukemia. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 A result graph of qRT-RCR method for detecting the expression of LDLR gene in leukemia cell lines and healthy human peripheral blood mononuclear cells in example 1 of the present application;

[0026] Figure 2 A result graph of Western blotting method for detecting the expression of LDLR protein in leukemia cell lines and healthy human peripheral blood mononuclear cells in example 1 of the present application;

[0027] Figure 3 A result graph of qRT-RCR method for detecting the knockdown efficiency of LDLR in leukemia cell lines in example 2 of the present application;

[0028] Figure 4 A result graph of Western blotting method for detecting the knockdown efficiency of LDLR in leukemia cell lines in example 2 of the present application;

[0029] Figure 5 A result graph of CCK-8 for detecting the proliferation of leukemia cells in example 2 of the present application;

[0030] Figure 6 A result graph of FCM for detecting the apoptosis rate of THP-1 cells after knocking down LDLR in example 3 of the present application;

[0031] Figure 7 A result graph of FCM for detecting the apoptosis rate of NB4 cells after knocking down LDLR in example 3 of the present application;

[0032] Figure 8 A result graph of Western blotting method for detecting the expression level of apoptosis-related proteins of THP-1 and NB4 cells after knocking down LDLR in example 3 of the present application;

[0033] Figure 9Figure for distribution ratio of cell cycle of THP-1 after knocking down LDLR by FCM in Example 3 of the present application;

[0034] Figure 10 Figure for distribution ratio of cell cycle of NB4 after knocking down LDLR by FCM in Example 3 of the present application;

[0035] Figure 11 Figure for expression level of related proteins in S phase of THP-1 and NB4 after knocking down LDLR by Western blotting in Example 3 of the present application;

[0036] Figure 12 Figure for expression level of MAPK signaling pathway related proteins of THP-1 and NB4 after knocking down LDLR by Western blotting in Example 4 of the present application. DETAILED DESCRIPTION

[0037] In order to better understand the technical solutions of the present application, achieve the purposes and beneficial effects, the present application is further specifically described by the following examples, but it should not be understood as limiting the present application. Some non-essential improvements and adjustments made by those skilled in the art according to the above content of the application are also regarded as falling within the protection scope of the present application. Unless otherwise specified, the experimental methods in the examples are usually carried out according to the conventional conditions or the conditions suggested by the manufacturers of reagents. Unless otherwise specified, the reagents and equipment used in the present application are conventional reagents and equipment in the technical field.

[0038] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0039] Example 1: Expression of LDLR in acute myeloid leukemia cells

[0040] 1. Experimental materials

[0041] Acute myeloid leukemia cell lines: KG1a, THP-1, NB4, HL-60 and U937 cells were purchased from American Type Culture Collection (ATCC).

[0042] 2. Experimental methods

[0043] 2.1 Extraction of peripheral blood mononuclear cells of healthy people

[0044] The peripheral blood sample of healthy people was added to the surface of density gradient separation liquid (i.e. Ficoll separation liquid), then centrifuged at 650g for 30min to form four layers, and the cells in the chylomicron layer were collected, washed twice with phosphate buffered saline (PBS), and centrifuged to obtain peripheral blood mononuclear cells (PBWC) for standby.

[0045] 2.2 Cultivation of acute myeloid leukemia cells

[0046] KG1a, THP-1, NB4, HL-60 and U937 cells were taken and cultivated in RPMI-1640 medium containing 10% fetal bovine serum, 1% penicillin-streptomycin solution (100 U / mL) in a 37°C incubator containing 5% CO2, and subcultured every 2 days on average to maintain logarithmic growth of the cells.

[0047] 2.3 Detection of expression levels of genes by real-time fluorescent quantitative PCR (qRT-PCR)

[0048] RNA extraction: logarithmically growing cells and normal human peripheral blood mononuclear cells in step 2.2 were collected, washed twice with PBS, centrifuged at 4000 rpm for 3 min, and the supernatant was discarded. 1 mL of RNA extraction reagent TRIzol was added to the cell precipitate, mixed thoroughly, and allowed to stand on ice for 10 min. Then, an equal volume of chloroform was added to the TRIzol, mixed, allowed to stand on ice, and then centrifuged at 12000 g for 15 min at 4°C. The supernatant was transferred to a clean EP tube free of enzymes, an equal volume of isopropanol was added, mixed, allowed to stand on ice, and then centrifuged at 12000 g for 10 min at 4°C. The supernatant was discarded, 1 ml of freshly prepared 75% ethanol solution was added, the precipitate was washed, and then centrifuged at 13000 g for 15 min at 4°C. The supernatant was discarded, and after the ethanol completely evaporated, the precipitate was dissolved in an appropriate amount of RNase free double-distilled water. Finally, the concentration and purity of each extracted RNA were determined.

[0049] Reverse transcription: each extracted RNA was subjected to reverse transcription according to Table 1. Reaction conditions: 37°C for 15 min, 85°C for 5 s, 4°C cooling, and short-term storage of the cDNA at -20°C.

[0050] Table 1: Reverse transcription reaction system (20 μL system)

[0051] Reagent Amount 5x PrimeScript RT Master Mix 4.0 μL total RNA 1.0 μg RNase Free ddH2O added up to 20.0 μL

[0052] qRT-PCR: The real-time fluorescent quantitative PCR reaction system is shown in Table 2. Reaction conditions: first, 95°C pre-denaturation for 30 s; second, 95°C denaturation for 5 s, 58°C annealing for 30 s, 72°C extension for 20 s, fluorescence collection, and 39 cycles; melting curve conditions: (65°C-95°C) fluorescence collection every 0.5°C increase. β-actin was used as an internal reference, and the relative quantitative value results were calculated using 2-ΔΔCt. The primers of each gene were synthesized by Shanghai Generay Biotech Co., Ltd. and provided, and the sequences are shown in Table 3.

[0053] Table 2: qRT-PCR reaction system (10 μL system)

[0054] Reagent Amount (μL) cDNA 1.0 TB Green Premix Ex Taq II 5.0 forward primer 0.4 reverse primer 0.4 ddH2O 3.2

[0055] Table 3: Primer sequences for qRT-PCR

[0056]

[0057]

[0058] 2.4 Western blotting to detect protein expression levels

[0059] Protein extraction: Collect each cell and normal human peripheral blood mononuclear cells in the logarithmic growth phase in step 2.2, wash twice with PBS at 5000 rpm x 4 min; add 2 to 3 times the volume of RIPA lysis buffer containing protease inhibitors to the precipitate, mix thoroughly; lyse the cells on ice for 30 min, vortex once every 10 min, repeat 3 times, centrifuge at 13000 rpm x 30 min at 4°C; aspirate the supernatant into a pre-cooled new EP tube, detect the protein concentration by the BCA method, obtain each protein sample, add the remaining protein sample to 1 / 4 volume of 5x loading buffer, denature after boiling, and store at -40°C;

[0060] SDS-PAGE gel electrophoresis: prepare 10% separating gel and slowly pour the gel, slowly add an appropriate amount of anhydrous ethanol to the surface layer of the gel, and stand at 37°C for 30 min; discard the anhydrous ethanol, pour the prepared 5% concentrated gel on the top layer of the completely solidified separating gel, and insert the comb, and stand at 37°C for 20 min; remove the comb, add 1x SDS electrophoresis buffer; add 50 μg of protein sample to the loading well, and add protein marker to both sides of the sample well as a molecular weight reference; after filling the glass plate with electrophoresis buffer, separate the proteins by two-step electrophoresis: first step, voltage 80V, current 150mA, 30 min; second step, voltage 120V, current 200mA, 60-70 min; wherein, the formula of 10% separating gel and 5% concentrated gel is shown in Table 4;

[0061] Table 4: Formula of 10% separating gel and 5% concentrated gel

[0062]

[0063]

[0064] Transfer: according to the molecular weight of the protein to be detected, cut the appropriate size of polyvinylidene fluoride (PVDF) membrane and soak it in methanol for 30 s, wash it with distilled water for 2 min, then soak the PVDF membrane and filter paper in pre-cooled wet transfer liquid; cut the appropriate gel and soak it in pre-cooled wet transfer liquid, and finally place the sponge, filter paper, PVDF membrane and gel in the positive electrode to negative electrode direction, with a constant current of 220 mA for electrophoretic transfer;

[0065] Blocking: after the transfer is completed, the PVDF membrane is placed in 5% protein blocking solution and blocked at room temperature for 2 h;

[0066] Antibody incubation: wash the residual blocking solution on the membrane with TBST, and after the residual liquid on the membrane is absorbed with qualitative filter paper, place the membrane on a wax plate, add the primary antibody working solution, evenly cover the PVDF membrane, and incubate at 4°C overnight; after recovering the primary antibody working solution, place the PVDF membrane in TBST and wash it 3 times, 10 min each time; finally, add horseradish peroxidase-labeled secondary antibody to the PVDF membrane, incubate at room temperature for 1.5 h, wash with TBST 3 times, 10 min each time;

[0067] Color development and imaging: in the dark room, place the PVDF membrane on a wax plate, then add the prepared chemiluminescence reagent (mix A and B in equal proportions) to cover the PVDF membrane, and display the image in the imaging system.

[0068] 3、Experimental results

[0069] The expression of LDLR gene in leukemia cell lines and healthy human peripheral blood mononuclear cells was detected by qRT-RCR method, and the results are shown in Figure 1 Compared with healthy people, the expression level of LDLR in RNA of the leukemia group was significantly increased;

[0070] The expression of LDLR protein in leukemia cell lines and healthy human peripheral blood mononuclear cells was detected by Western blotting, and the results are shown in Figure 2 Compared with healthy people, the expression level of LDLR in protein of the leukemia group was significantly increased.

[0071] Example 2: Effect of LDLR knockdown on acute myeloid leukemia cell proliferation

[0072] 1、Experimental materials

[0073] Acute myeloid leukemia cell lines: THP-1, NB4 cells were purchased from American Type Culture Collection (ATCC).

[0074] 2、Experimental methods

[0075] 2.1 Construction of leukemia cell lines stably expressing shLDLR (shLDLR gene is a short hairpin RNA (shRNA) sequence designed for low-density lipoprotein receptor (LDLR) to inhibit the expression of LDLR gene by RNA interference technology)

[0076] Lentivirus packaging, sequencing identification and titer determination: lentivirus packaging is obtained by co-transfecting 293T cells with the target gene vector and packaging plasmid, collecting the supernatant to obtain virus particles; sequencing identification requires extracting viral RNA and reverse transcribing it into cDNA, and verifying the correctness of the gene by PCR amplification and sequencing; titer determination uses qPCR to quantify viral genome copy number (GC / mL) or calculates infectious units (IU / mL) by target cell infection experiment; shRNA lentivirus vector targeting LDLR gene (shLDLR) is synthesized by Shanghai Jimabio Pharmaceutical Technology Co., Ltd.

[0077] The shRNA sequence is:

[0078] sh LDLR#1: 5'-GATGAAGTTGGCTGCGTTAAT-3' (SEQ ID NO. 5);

[0079] sh LDLR#2: 5'-CAGAGGATGAGGTCCACATTT-3' (SEQ ID NO. 6);

[0080] Lentivirus infection of cells and screening of stable cell lines: THP-1 and NB4 leukemia cells with high expression of LDLR were used as experimental objects; first, the above cells were collected and seeded in a new 24-well plate to ensure a density of 1 x 10 5 cells per well; then, 15-25 μL of virus solution with a titer of 1 x 10 8 TU / mL and 20 μL of HitransG P were added to each well, and the cell suspension in each well was adjusted to 500 μL with cell culture medium and mixed thoroughly; after incubation at 37°C in a 5% CO2 incubator for 48-72 h, the cell state and fluorescence intensity were observed, and the culture dish and medium were changed; finally, when the infection efficiency reached about 80%, 2 μg / mL puromycin was added for 7-14 d to obtain stable cell lines, which were further expanded and stored for use.

[0081] 2.2 Detection of LDLR knockdown efficiency by qRT-PCR

[0082] The stable cell lines THP-1 and NB4 in step 2.1 were used for the experiment, and the experimental method was the same as that in step 2.3 of Example 1.

[0083] 2.3 Detection of LDLR knockdown efficiency by Western blotting

[0084] Take the stably transfected cell strain THP-1 and NB4 in step 2.1 for experiment, and the experimental method is the same as step 2.4 in example 1.

[0085] 2.4 Detection of leukemia cell proliferation by cell counting kit-8 (CCK-8)

[0086] Take the stably transfected cell strain THP-1 and NB4 in step 2.1, and inoculate 1.0 x 10 4 cells per well in a 96-well plate, set 5 replicate wells for each group, and add 10 μL CCK-8 reagent per well when cultured to the corresponding time, mix well and continue to culture for 3 h; detect the absorbance (OD) value at 450 nm wavelength.

[0087] 3. Experimental results

[0088] 3.1 Detection of LDLR knockdown efficiency in acute myeloid leukemia cells

[0089] The knockdown efficiency of LDLR in leukemia cell lines was detected by qRT-RCR method, and the results are shown in Table 1. In THP-1 cells, shLDLR#1 has higher knockdown efficiency, and in NB4 cells, shLDLR#2 has higher knockdown efficiency. Figure 3

[0090] The knockdown efficiency of LDLR in leukemia cell lines was detected by Western blotting method, and the results are shown in Table 2. In THP-1 cells, shLDLR#1 has higher knockdown efficiency, and in NB4 cells, shLDLR#2 has higher knockdown efficiency. Figure 4

[0091] 3.2 Effect of LDLR knockdown on proliferation of acute myeloid leukemia cells

[0092] Based on the results in 3.1, shLDLR#1 with higher knockdown efficiency in THP-1 cells and shLDLR#2 with higher knockdown efficiency in NB4 cells were used for subsequent experiments.

[0093] The proliferation ability of THP-1 and NB4 cells was detected by CCK-8 experiment, and the results are shown in Table 3. After knockdown of LDLR, the proliferation of acute myeloid leukemia cells was inhibited. Figure 5

[0094] Example 3: Effect of LDLR knockdown on apoptosis and cell cycle of acute myeloid leukemia cells

[0095] 1. Experimental materials

[0096] ​​​Acute myeloid leukemia cell lines: THP-1, NB4 cells were purchased from American Type Culture Collection (ATCC).

[0097] 2. Experimental method

[0098] 2.1 Flow cytometry (FCM) method for detecting acute myeloid leukemia cell apoptosis

[0099] Take the stable cell strain THP-1 and NB4 in step 2.1 of Example 2, wash with pre-cooled PBS 3 times, 1500 rpm x 5 min; take 5 x 10 5 PBS resuspended cells, 300g x 5min, discard the supernatant, add 500ul of diluted 1x Annexin V Binding Buffer working solution to resuspend the cells; add 5ul of Annexin V-APC and 5ul of DAPI staining solution to the cell suspension, mix gently, incubate at room temperature for 15min, and then detect immediately.

[0100] 2.2 Flow cytometry (FCM) method for detecting acute myeloid leukemia cell cycle

[0101] Take the stable cell strain THP-1 and NB4 in step 2.1 of Example 2, collect 1 x 10 6 PBS, 1500 rpm x 5 min; discard the supernatant, resuspend the cells in 0.1 ml PBS buffer, slowly add 500ul of pre-cooled 75% ethanol, shake while adding, slowly add, fix overnight for detection.

[0102] 2.3 Western blotting method for detecting acute myeloid leukemia cell apoptosis related protein expression level

[0103] Take the stable cell strain THP-1 and NB4 in step 2.1 of Example 2 for experiment, and the experimental method is the same as step 2.4 in Example 1.

[0104] 2.4 Western blotting method for detecting acute myeloid leukemia cell cycle related protein expression level

[0105] Take the stable cell strain THP-1 and NB4 in step 2.1 of Example 2 for experiment, and the experimental method is the same as step 2.4 in Example 1.

[0106] 3. Experimental results

[0107] 3.1 Effect of LDLR knockdown on acute myeloid leukemia cell apoptosis

[0108] The apoptosis rate of acute myeloid leukemia cells was detected by flow cytometry (FCM), and the results are shown in Figure 6 and Figure 7 As shown, after knocking down LDLR, the apoptosis rates of THP-1 and NB4 cells were significantly increased compared with the blank control group;

[0109] The expression levels of apoptosis-related proteins of acute myeloid leukemia cells were detected by Western blotting, and the results are shown in Figure 8 As shown, after knocking down LDLR, the apoptosis of THP-1 and NB4 cells was promoted, specifically, the protein levels of Bax and Cle-PARP were increased, and the protein level of Bcl-2 was decreased.

[0110] 3.2 Effect of knocking down LDLR on the cell cycle of acute myeloid leukemia cells

[0111] The cycle distribution ratio of acute myeloid leukemia cells was detected by flow cytometry (FCM), and the results are shown in Figure 9 and Figure 10 As shown, after knocking down LDLR, the S phase ratio of THP-1 and NB4 cells was increased, which proved that the cell cycle of acute myeloid leukemia cells was arrested in the S phase;

[0112] The expression levels of S phase-related proteins of acute myeloid leukemia cells were detected by Western blotting, and the results are shown in Figure 11 As shown, after knocking down LDLR, the protein expression of Cyclin E1 and p27 was decreased, and the protein expression of Cyclin A2 and Cyclin D1 was increased.

[0113] Example 4: Effect of knocking down LDLR on the MAPK signaling pathway of acute myeloid leukemia cells

[0114] 1. Experimental materials

[0115] Acute myeloid leukemia cell lines: THP-1 and NB4 cells were purchased from American Type Culture Collection (ATCC).

[0116] 2. Experimental methods

[0117] 2.1 Western blotting detection of the expression levels of MAPK signaling pathway-related proteins of acute myeloid leukemia cells

[0118] Stably transfected cell strains THP-1 and NB4 in step 2.1 of Example 2 were taken for experiments, and the experimental method was the same as step 2.4 in Example 1.

[0119] 3. Experimental results

[0120] The expression levels of MAPK signaling pathway related proteins were detected by Western blotting, and the results are shown in Table 1. Figure 12 As shown in Table 1, after knocking down LDLR, the related proteins of MAPK pathway changed significantly, the expressions of phosphorylated JNK, phosphorylated p38MAPK and phosphorylated ERK were inhibited, which proved that knocking down LDLR inhibited the MAPK signaling pathway, thereby inhibiting the progression of AML.

[0121] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. Application of LDLR in screening or preparing drugs for treating acute myeloid leukemia.

2. Use of an LDLR inhibitor in screening or preparing a drug for treating acute myeloid leukemia, characterized in that: The LDLR inhibitor inhibits the proliferation of acute myeloid leukemia cells, promotes the apoptosis of acute myeloid leukemia cells, and blocks the cell cycle of acute myeloid leukemia cells by knocking down the expression level of LDLR.

3. The use according to claim 1 or 2, characterized in that The drug is a drug that regulates the MAPK signaling pathway.

4. The use according to claim 2, characterized in that The LDLR inhibitor is used to inhibit the transcription level of the LDLR gene, the expression level of the LDLR protein or the activity of the LDLR protein.

5. The use according to claim 4, characterized in that The LDLR inhibitor is an LDLR gene inhibitor, which is used to inhibit the transcription level of the LDLR gene.

6. The use according to claim 5, characterized in that The LDLR gene inhibitor includes any one or more of double-stranded RNA, short hairpin RNA, antisense oligonucleotide and small interfering RNA.

7. The use according to claim 6, characterized in that The LDLR gene inhibitor is a short hairpin RNA; the short hairpin RNA comprises the sequences shown in SEQ ID NO: 5 and SEQ ID NO:

6.

8. Application of LDLR in the preparation of a kit for diagnosing acute myeloid leukemia.

9. The use according to claim 8, characterized in that The kit is used to detect the expression level of the LDLR gene.

10. The use according to claim 8, characterized in that The kit includes a forward primer with a sequence as shown in SEQ ID NO: 1 and a reverse primer with a sequence as shown in SEQ ID NO: 2.

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