A nucleic acid drug combination for inducing apoptosis of myeloid leukemia cells and its application

Abstract

The present invention discloses a nucleic acid drug combination for inducing apoptosis of myeloid leukemia cells and its application. The pharmaceutical composition is two anti-leukemia nucleic acid drug compositions, mainly composed of miR-5581-5P mimics and TRIM22 siRNA. This product uses a nucleic acid drug composition to induce an apoptosis model of myeloid leukemia cells in vitro for the first time, avoiding the disadvantage of traditional chemical drugs accompanied by cytotoxic effects, and is a safer and more effective apoptosis induction method and potential therapeutic drug. The pharmaceutical composition provides a basis for the next step of animal experiments and drug clinical trials for the treatment of leukemia tumors, and also provides a basis for the study of the molecular mechanism of leukemia cell apoptosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell biomedicine, and in particular relates to a nucleic acid drug combination for inducing apoptosis of myeloid leukemia cells and an application thereof. Background Art

[0002] Acute myeloid leukemia is a type of malignant clonal disease of myeloid hematopoietic stem cells. Due to the enhanced self-renewal ability of leukemia cells, uncontrolled proliferation, differentiation disorders, and blocked apoptosis, leukemia cells stagnate at different stages of cell development. In the bone marrow and other hematopoietic organs, leukemia cells proliferate in large numbers, inhibiting normal hematopoiesis and infiltrating other organs and tissues. The main drugs currently used for clinical treatment are chemotherapy drugs, targeted antibodies, and small molecule compounds. However, the former has large toxic side effects, the latter has large individual differences, and there are relapses, drug resistance, and off-target effects. Therefore, it is necessary to actively explore other effective leukemia treatment drugs or drug combinations.

[0003] MicroRNA (miRNA) is an endogenous small RNA with a length of 18-24 nucleotides. It often works in two ways. One is to bind to the mRNA paired with its own nucleotide sequence and inhibit its translation. The other is to prevent gene expression through mRNA degradation, playing an important role in the post-transcriptional regulation of target genes. miRNA plays an important role in cell proliferation, differentiation, and apoptosis. miRNAs have also been reported to play an important role in the progression of various cancers by directly targeting oncogenes or tumor suppressor genes. MiRNAs similar to tumor suppressor genes include a group of non-coding RNAs, the loss of which promotes the development of cancer. This type of miRNA acts similarly to protein-encoded tumor suppressor genes. Oncogenic miRNAs are a class of miRNAs that are upregulated to accelerate tumor formation and development. Oncogenic miRNAs affect the occurrence of tumors by targeting mRNA encoded by tumor suppressor genes. Therefore, if miRNA interference drugs can be used, they can play an anti-leukemia role. For example, current studies have found that miRNAs are found in many cancers such as lung cancer, gastric cancer, breast cancer, ovarian cancer, and cervical cancer. Some specific miRNAs have been shown to play a key role in hematopoietic differentiation and the formation and maintenance of leukemia. For these reasons, miRNAs may become interesting targets for the development of new therapeutic strategies in tumor treatment. For example, some studies have found that miR-192 plays a pro-oncogenic role in APL, and downregulating the expression of miR-192 promotes ATRA-induced APL cell differentiation. Other studies have found that miRNA-15b plays a role as a tumor suppressor in APL. The expression level of miRNA-15b increases after APL is treated with ATRA. After overexpression of miRNA-15b, ATRA-induced AML cells are promoted to differentiate into myeloid cells, and the proliferation of leukemia cell lines can be inhibited.

[0004] Previous studies have also confirmed that certain key regulatory transcriptional proteins are associated with differentiation, apoptosis, and proliferation of leukemia. For example, the gene for the three-motif protein TRIM22 is located on chromosome 11p15 and encodes proteins involved in gene regulation, ubiquitination, DNA recombination, and DNA repair. The TRIM family is involved in different biological processes, such as antiviral activity, tumorigenesis, cell proliferation, and differentiation. TRIM22 is mainly expressed in peripheral blood leukocytes and lymphoid tissues, such as the spleen and thymus. The expression level of TRIM22 in monocytes is higher than that in CD4 + and CD8 + It is 2 times higher in T lymphocytes and nearly 1.5 times higher than in B lymphocytes. Current studies have shown that TRIM22 may have a tumor suppressor effect because TRIM22 is a target gene of p53, which is a well-known regulator of cell growth and death and acts as a tumor suppressor gene. For example, some studies have shown that TRIM22 is downregulated in breast cancer tissues and cell lines and is associated with p53-mediated induced deletion. Clinical studies have also suggested the tumor suppressor effect of TRIM22. Compared with Wilms patients who have not relapsed, the expression of TRIM22 gene in relapsed patients is 10 times lower. Some TRIM family members play an important role in leukemia. For example, TRIM19 plays an important role in inhibiting the growth and tumor formation of acute promyelocytic leukemia cells. Overexpression of TRIM22 interferes with the clonal growth of monocytic U937 cells. Overexpression of TRIM22 promotes apoptosis of THP-1 cells, and TRIM22 mediates apoptosis through a caspase-9-dependent pathway. The above studies all show that TRIM22 is lowly expressed in tumors and acts as a tumor suppressor. However, some studies have also confirmed that TRIM22 plays a pro-cancer role. TRIM22 is highly expressed in cancer and is found in both solid and non-solid tumors. In non-small cell lung cancer, TRIM22 expression is upregulated, and A549 tumor cell proliferation and invasion are increased, suggesting that TRIM22 is closely related to tumor progression and metastasis. In hematological tumors, downregulation of the TRIM22 gene inhibits the proliferation and invasion of CML cells, slows down the cell cycle, and promotes cell apoptosis. TRIM22 loss can induce cell cycle arrest and apoptosis by inhibiting the activation of the PI3K / Akt / mTOR pathway. Our previous studies have shown that TRIM22 expression increases during the differentiation of leukemia into granulocyte cells, so it is speculated that it has the effect of promoting differentiation and inhibiting apoptosis.

[0005] The miRNAs related to TRIM22 changes were further screened out through gene chips, and the binding sites were screened and predicted using TargetScan software. Then, after transfecting cells with miRNAmimic or inhibitor to detect the effects of miRNA changes on TRIM22 and the biological functions of leukemia cells, it was preliminarily found that miRNA-5581-5P had targeted binding and negative regulatory effects on TRIM22.

[0006] However, there is no more in-depth research and disclosure on the application effect of the combination of the two in the prior art. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides a drug combination for inducing apoptosis of leukemia cells and its application. The present invention has found through experimental studies that the combination of miRNA-5581-5P mimics and TRIM22 siRNA has the effect of inducing apoptosis of myeloid leukemia cells. The product is suitable for in vitro apoptosis induction experiments of leukemia cells, provides a cell apoptosis model for studying the anti-leukemia mechanism of nucleic acids, and provides an experimental basis for zoological and clinical research.

[0008] In order to achieve the above technical objectives, the technical solution implemented by the present invention is:

[0009] On the one hand, the present invention provides a nucleic acid drug combination for inducing apoptosis of myeloid leukemia cells, comprising miR-5581-5P mimics and TRIM22 siRNA, the sequence of miR-5581-5P mimics being shown in the sequence listing SEQ ID NO.1 and SEQ ID NO.2, and the sequence of TRIM22 siRNA being shown in the sequence listing SEQ ID NO.3 and SEQ ID NO.4.

[0010] Preferably, the molar ratio of miR-5581-5P mimics to TRIM22 siRNA is 1.5-2:1.

[0011] Furthermore, the molar ratio of miR-5581-5P mimics and TRIM22 siRNA was 2:1.

[0012] Preferably, in the pharmaceutical composition, the concentration range of miR-5581-5P mimics is 50 to 300 nM; the concentration range of TRIM22 siRNA is 25 to 200 nM.

[0013] Furthermore, in the pharmaceutical composition, the concentration range of miR-5581-5P mimics is 200 nM; the concentration range of TRIM22 siRNA is 100 nM.

[0014] Preferably, the pharmaceutical composition comprises 10 μL Hiperfect transfection reagent, 10 μL miR-5581-5Pmimics and 10 μL TRIM22 siRNA, and 170 μL RPMI1640 culture medium.

[0015] On the other hand, the present invention also provides the use of the nucleic acid drug combination for inducing myeloid leukemia cell apoptosis in preparing a leukemia cell apoptosis model.

[0016] On the other hand, the present invention also provides a method for preparing a nucleic acid drug-induced myeloid leukemia cell apoptosis model, comprising the following steps:

[0017] (1) Inoculating cells: Inoculate HL60 cells in the logarithmic growth phase into serum-containing RPMI1640 medium and resuspend the cells;

[0018] (2) Take another EP tube and add serum-free and antibiotic-free RPMI1640 culture medium, miRNA-5581-5Pmimic, TRIM22 siRNA, mimic NC or siNC as the control group; miRNA-5581-5Pmimic, TRIM22 siRNA, mimic NC or siNC are added in equal volumes;

[0019] (3) Transfection: Add Hiperfect transfection reagent to the EP tube in step (2), mix well, and leave at room temperature to form a transfection complex;

[0020] (4) adding the transfection complex prepared in step (3) to the cells prepared in step (1), and continuing to culture to obtain a leukemia cell apoptosis model.

[0021] Furthermore, the preparation method comprises the following steps:

[0022] (1) Inoculation of cells: HL60 cells in the logarithmic growth phase were aspirated into a clean sterile centrifuge tube, centrifuged and discarded the supernatant, and fresh serum-containing RPMI1640 was added to resuspend the cells. In a 24-well plate, 100 μl of cell suspension was added to each well to make the cell density 2×10 5 Pieces / ml;

[0023] (2) Take another 4 clean, autoclaved EP tubes, add 70 μl of serum-free and antibiotic-free RPMI1640, and add 10 μl of miRNA-5581-5P mimic, 10 μl of TRIM22 siRNA, 10 μl of mimic NC or 10 μl of siNC as control groups; the final concentration of miRNA-5581-5P mimic is 200 nM, the final concentration of TRIM22 siRNA is 100 nM, the final concentration of mimic NC is 200 nM, or the final concentration of siNC is 100 nM.

[0024] (3) Then add 10 μl of Hiperfect transfection reagent to the EP tube in step (2), mix well, and leave at room temperature for 10 min to form a transfection complex;

[0025] (4) Add 100 μl of the transfection complex to a 24-well plate seeded with cells, with each well containing 100 μl of RPMI1640, and continue culturing for 48 h for subsequent experiments.

[0026] In another aspect, the present invention also provides a use of a nucleic acid drug combination for inducing apoptosis of myeloid leukemia cells in the preparation of a drug for treating leukemia.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The present invention provides a nucleic acid drug combination, which is mainly composed of miR-5581-5P mimics and TRIM22 siRNA. This product is the first to use a nucleic acid drug combination to induce apoptosis of myeloid leukemia cells in vitro, avoiding the disadvantage of traditional chemical drugs accompanied by cytotoxic effects. It is a safer and more effective way to induce apoptosis and a potential therapeutic drug.

[0029] 2. This drug combination provides a basis for the next step of animal experiments and clinical trials on the treatment of leukemia tumors, and also for the study of the molecular mechanism of leukemia cell apoptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0031] Figure 1The figure shows the changes in the level of cell apoptosis after the action of miR-5581-5P mimics+TRIM22 siRNA in Example 4, where A: the percentage of cell apoptosis in the control group; B: mimics+si RNA (50+25nM) group; C: mimics+si RNA (100+50nM) group; D: mimics+si RNA (200+100nM) group; E: mimics NC+si NC (300+200nM) group; F: the average percentage of cell apoptosis in different drug concentration groups. ***P<0.001; ****P<0.0001.

[0032] Figure 2 The changes in cell proliferation level (CCK8) after the action of miR-5581-5P mimics+TRIM22 siRNA in Example 5. ***P<0.001, ****P<0.0001, where A: mimics NC+si NC; B: mimics+si RNA (200+100 nM); C: average level of cell apoptosis; D: average level of cell proliferation.

[0033] Figure 3 The cytotoxicity comparison between the nucleic acid drug composition and the chemical drug apoptosis-inducing agent in Example 6, wherein A: mimics + si RNA group; B: chemical reagent Oridonin group; C: average percentage of cell death. NS nosignificance, ***P<0.001.

[0034] Figure 4 This is a comparative experiment of the nucleic acid drug combination and a single nucleic acid drug against myeloid leukemia cell proliferation in Example 7, wherein A: cell proliferation level of the mimics+si RNA group and the control group; B: cell proliferation level of the si RNA group and the control group; C: cell proliferation level of the mimics group and the control group; D: mimics+si RNA group, mimics and si RNA group proliferation level comparison. NS no significance, ***P < 0.001. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0036] Example 1: Cell culture and passage

[0037] Take out the HL-60 cells from the liquid nitrogen bottle and put them into a 37℃ constant temperature water bath to thaw quickly. Add the thawed cells into three centrifuge tubes and centrifuge them at 800 rpm for 3 minutes. Discard the supernatant and add 4-5 ml of cell culture medium to each centrifuge tube again. Use a pipette to blow the cell sediment evenly, and then transfer the three types of cells to cell culture bottles or cell culture dishes respectively, and culture them in a 37℃, 5% CO2 incubator.

[0038] Example 2: Cell culture medium replacement and cell subculturing

[0039] Turn on the ultraviolet light to irradiate the clean bench forty minutes in advance. Wear a mask and hat before entering the cell room, open some of the clean bench glass windows, turn on the exhaust fan and lighting, and self-check for three minutes. Wipe the workbench with Sanisol, and disinfect the items entering and leaving the clean bench, including hands, every time. General operations are performed at the vents. When replacing the suspension cells, first transfer the old culture medium in the culture bottle to the centrifuge tube with a pipette, and centrifuge at 800 rpm for 3 minutes. Discard the supernatant, add new culture medium with a pipette, and gently blow to evenly distribute the cells. Use a pipette to transfer the culture medium with cells in the centrifuge tube to the culture bottle, and culture it in a 37°C, 5% CO2 incubator. If there are enough cells, cell subculture can be performed, and the original culture medium containing cells can be evenly distributed into two or three culture bottles, and an appropriate amount of culture medium can be added, and it can be used until the logarithmic growth period.

[0040] Example 3: Cell transfection and nucleic acid drug transfection

[0041] (1) Inoculation of cells: HL60 cells in the logarithmic growth phase were aspirated into a clean sterile centrifuge tube, centrifuged and discarded the supernatant, and fresh serum-containing RPMI1640 was added to resuspend the cells. In a 24-well plate, 100 μl of cell suspension was added to each well to make the cell density 2×10 5 Pieces / ml.

[0042] (2) Take another 4 clean, autoclaved EP tubes, add 70 μl of serum-free and antibiotic-free RPMI1640 culture medium, add 10 μl of miRNA-5581-5P mimic (final concentration of 200 nM), 10 μl of TRIM22 si RNA (final concentration of 100 nM). 10 μl of mimic NC (final concentration of 200 nM) or 10 μl of siNC (final concentration of 100 nM) were used as control groups. The sequence table information is as follows:

[0043]

[0044] (3) Then add 10 μl of Hiperfect transfection reagent to the EP tube, mix well, and leave at room temperature for 10 minutes to form a transfection complex.

[0045] (4) Add 100 μl of the transfection complex to a 24-well plate seeded with cells, with each well containing 100 μl of RPMI1640. Continue culturing for 48 h for subsequent experiments.

[0046] Example 4: Experiment on changes in apoptosis levels after cell transfection

[0047] Four experimental groups (50nM+25nM, 100nM+50nM, 200nM+100nM and 300nM+200nM, respectively) of four different concentration ratios of miRNA-5581-5P mimic and TRIM22 siRNA drug combinations were selected, and the cell nucleic acid drug transfection was performed according to the method of Example 3 above, and the cell apoptosis level was detected by PI / Annexin double staining flow cytometry. The main steps include: after the cells treated with the above method for 48 hours, a 24-well plate to be tested was taken out, and the cell apoptosis level was detected according to the Annexin V / PI method. The main steps include: the cells of the control group and the drug group were rinsed with PBS, centrifuged twice at 1000g / min, and after the cells were resuspended, anhydrous ethanol (70%) was added, protected from light, and fixed overnight at 4°C; centrifuged (1000g / min), 400μL 1×Binding Buffer was added to each tube after removing the ethanol, and the cells were resuspended. The cell apoptosis level was detected by flow cytometry within 1 hour. Three replicate wells were set up in each group, and the apoptosis level was expressed as mean ± standard deviation. Figure 1 As shown in Figures A to F, the results showed that compared with the control group, the above four drug combination groups could significantly induce apoptosis in leukemia cells, among which the apoptosis levels of the two concentration groups of 200nM+100nM and 300nM+200nM were not significantly different (t=0.6733, P=0.5376).

[0048] Example 5: Cell proliferation and apoptosis experiments of preferred drug combinations

[0049] According to the principle of obtaining the maximum apoptosis level at a lower concentration, miRNA-5581-5P mimic and TRIM22 siRNA (200nM+100nM) were selected, and after cell transfection and nucleic acid drug transfection were performed according to the method of Example 3 above, the cell apoptosis level was measured by the method of Example 4 above, and the cell proliferation level was measured by the CCK-8 counting method in order to observe the changes in the cell proliferation level after cell apoptosis. The main steps include: after 48 hours of treating the cells with the drugs according to the above method, a 96-well plate to be tested was taken out, 10 μL of CCK-8 solution (protected from light) was added to each experimental well, and after incubation at 37°C for 2-4 hours, the absorbance (A) value of each well within the wavelength range of 450nm was measured with an enzyme marker. The average level was calculated for three replicate wells. The results show that the drug composition can induce apoptosis in leukemia cells while ( Figure 2 A, B, C), the cell proliferation level was significantly inhibited ( Figure 2 D), indicating that the drug combination exerts its anti-leukemia effect by inhibiting the proliferation of leukemia cells.

[0050] Example 6: Cytotoxicity comparison experiment between the nucleic acid drug composition and chemical drug apoptosis-inducing agents

[0051] The above-screened miRNA-5581-5P mimic and TRIM22 siRNA (200nM+100nM) drug combination was selected, and the cell nucleic acid drug transfection was performed according to the method of Example 3. At the same time, the chemical inducer Oridonin (5μM) was selected for comparative experiments to observe nonspecific cytotoxicity. The cytotoxicity was tested using a conventional trypan blue staining experiment. The specific steps are: collect different groups of cells treated with drugs for 48 hours, centrifuge at 1000r / min for 5min, discard the supernatant, prepare a single cell suspension, and make appropriate dilutions. Add 0.4% trypan blue solution to the cell suspension, mix in an appropriate proportion (final concentration of trypan blue 0.4%-0.04%), and stain at room temperature for 3min. A small amount of stained cells was taken and counted with a hemocytometer. It can be observed under a microscope that dead cells are blue and swollen, dull; living cells are not colored and maintain normal morphology and are shiny. The viability of living cells was calculated, cell viability (%) = total number of living cells / (total number of living cells + total number of dead cells) × 100%. The results showed that the drug composition induced apoptosis of leukemia cells without causing significant cytotoxicity ( Figure 3 Although Oridonin can induce apoptosis, it is accompanied by some cytotoxic cell death ( Figure 3 Flow cytometry results also showed that their non-specific cell death was significantly different ( Figure 3 Middle C).

[0052] Example 7: Comparative experiment on the effect of the nucleic acid drug composition and a single nucleic acid drug on myeloid leukemia cell proliferation

[0053] Select three groups: miRNA-5581-5P mimic and TRIM22 siRNA (200nM+100nM), miRNA-5581-5Pmimic (200nM) and TRIM22 siRNA (100nM). After cell transfection and nucleic acid drug transfection were performed according to the method of Example 3 above, the cell proliferation level was determined by the above-mentioned CCK-8 counting method in order to observe the synergistic effect of the drug combination. The main steps include: after 48 hours of treating the cells with the above-mentioned drug method, take out a 96-well plate to be tested, add 10 μL of CCK-8 solution (protected from light) to each experimental well, incubate at 37°C for 2-4 hours, and then use an enzyme marker to measure the absorbance (A) value of each well within the wavelength range of 450nm. Calculate the average level of triplicate wells. Figure 4 As shown in Figures A to D, the results show that compared with the two drug groups alone, the drug combination has a more obvious anti-leukemia cell proliferation effect.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A nucleic acid drug combination for inducing apoptosis of myeloid leukemia cells, characterized in that: It includes miR-5581-5P mimics and TRIM22 siRNA. The sequence of miR-5581-5P mimics is shown in the sequence list as SEQ ID NO.1 and SEQ ID NO.2, and the sequence of TRIM22 siRNA is shown in the sequence list as SEQ ID NO.3 and SEQ ID NO.

4.

2. The nucleic acid drug combination for inducing apoptosis of myeloid leukemia cells according to claim 1, characterized in that: In the pharmaceutical composition, the molar ratio of miR-5581-5P mimics to TRIM22 siRNA is 1.5 to 2:

1.

3. The nucleic acid drug combination for inducing apoptosis of myeloid leukemia cells according to claim 1, characterized in that: In the pharmaceutical composition, the molar ratio of miR-5581-5P mimics to TRIM22 siRNA is 2:

1.

4. The nucleic acid drug combination for inducing apoptosis of myeloid leukemia cells according to claim 1, characterized in that: In the pharmaceutical composition, the concentration range of miR-5581-5P mimics is 50 to 300 nM; the concentration range of TRIM22 siRNA is 25 to 200 nM.

5. The nucleic acid drug combination for inducing apoptosis of myeloid leukemia cells according to claim 1, characterized in that: In the pharmaceutical composition, the concentration range of miR-5581-5P mimics is 200 nM; the concentration range of TRIM22 siRNA is 100 nM.

6. The nucleic acid drug combination for inducing apoptosis of myeloid leukemia cells according to claim 1, characterized in that: The pharmaceutical composition comprises Hiperfect transfection reagent, miR-5581-5P mimics and TRIM22 siRNA, and RPMI1640 culture medium.

7. Use of the nucleic acid drug combination for inducing apoptosis of myeloid leukemia cells according to claim 1 in preparing a leukemia cell apoptosis model.

8. A method for preparing a model of myeloid leukemia cell apoptosis induced by nucleic acid drugs, characterized in that: The steps include: (1) Inoculating cells: Inoculate HL60 cells in the logarithmic growth phase into serum-containing RPMI1640 medium and resuspend the cells; (2) Take another EP tube and add serum-free and antibiotic-free RPMI1640 culture medium, miRNA-5581-5Pmimic, TRIM22 siRNA, mimic NC or siNC as the control group; miRNA-5581-5Pmimic, TRIM22 siRNA, mimic NC or siNC were added in equal volumes; (3) Transfection: Add Hiperfect transfection reagent to the EP tube in step (2), mix well, and leave at room temperature to form a transfection complex; (4) adding the transfection complex prepared in step (3) to the cells prepared in step (1), and continuing to culture to obtain a leukemia cell apoptosis model.

9. The preparation method according to claim 8, characterized in that: The steps include: (1) Inoculation of cells: HL60 cells in the logarithmic growth phase were aspirated into a clean sterile centrifuge tube, centrifuged and discarded the supernatant, and fresh serum-containing RPMI1640 was added to resuspend the cells. In a 24-well plate, 100 μl of cell suspension was added to each well to make the cell density 2×10 5 Pieces / ml; (2) Take another 4 clean, autoclaved EP tubes, add 70 μl of serum-free and antibiotic-free RPMI1640, add 10 μl of miRNA-5581-5P mimic, 10 μl of TRIM22 siRNA, 10 μl of mimic NC or 10 μl of siNC as control groups; the final concentration of miRNA-5581-5P mimic is 200 nM, the final concentration of TRIM22 siRNA is 100 nM, the final concentration of mimic NC is 200 nM, or the final concentration of siNC is 100 nM; (3) Then add 10 μl of Hiperfect transfection reagent to the EP tube in step (2), mix well, and leave at room temperature for 10 min to form a transfection complex; (4) Add 100 μl of the transfection complex to a 24-well plate seeded with cells, with each well containing 100 μl of RPMI1640, and continue culturing for 48 h for subsequent experiments.

10. Use of the nucleic acid drug combination for inducing apoptosis of myeloid leukemia cells according to claim 1 in the preparation of drugs for treating leukemia.

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