Application of a ZNF622 gene promoter in synergistic effect with an HDAC2 gene promoter in the preparation of a drug for treating acute myeloid leukemia cells

Abstract

This invention belongs to the field of genetic engineering, specifically relating to the application of a ZNF622 gene promoter in synergistic action with an HDAC2 gene promoter in the preparation of a drug for treating acute myeloid leukemia cells. The nucleotide sequence of the ZNF622 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the HDAC2 gene is shown in SEQ ID NO.3; the volume ratio of the ZNF622 gene promoter to the HDAC2 gene promoter is 2.5:10. This invention, through the construction of ZNF622 gene overexpression lentiviral vectors and HDAC2 gene overexpression lentiviral vectors, and subsequent transfection and detection of STAT3 protein expression and cell apoptosis, demonstrates that ZNF622 and HDAC2 can interact to reduce STAT3 protein expression and promote leukemia cell apoptosis.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to the application of a ZNF622 gene promoter in conjunction with an HDAC2 gene promoter in the preparation of a drug for treating acute myeloid leukemia cells. Background Technology

[0002] Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the abnormal proliferation of immature myeloid cells in the bone marrow and peripheral blood. This disease progresses rapidly, often leading to severe anemia, bleeding, and infection, and has a poor prognosis. Currently, the main treatments for AML include chemotherapy and bone marrow transplantation. Traditional chemotherapy drugs often cause severe side effects due to their high toxicity to normal cells, and many patients develop drug resistance. While bone marrow transplantation can achieve good results in some cases, its application is limited by the limited availability of donors and the risks of rejection and infection during the transplantation process.

[0003] Therefore, there is an urgent need to develop new, safe, and effective treatment strategies for acute myeloid leukemia in order to improve patient survival rates. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides the application of ZNF622 gene promoter in synergistic action with HDAC2 gene promoter in the preparation of a drug for treating acute myeloid leukemia cells.

[0005] This invention provides the application of a ZNF622 gene promoter in conjunction with an HDAC2 gene promoter in the preparation of a drug for treating acute myeloid leukemia cells. The nucleotide sequence of the ZNF622 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the HDAC2 gene is shown in SEQ ID NO.3.

[0006] Furthermore, the ZNF622 gene promoter is a reagent containing a lentiviral vector overexpressing the ZNF622 gene, and the HDAC2 gene promoter is a reagent containing a lentiviral vector overexpressing the HDAC2 gene.

[0007] Furthermore, the ZNF622 gene overexpression lentiviral vector construction process is as follows: the ZNF622 gene is ligated to the BamHI and AgeI restriction sites of the GV492 lentiviral vector to obtain the ZNF622 gene overexpression lentiviral vector.

[0008] Furthermore, the construction process of the HDAC2 gene overexpression lentiviral vector is as follows: the HDAC2 gene is ligated to the BamHI and AgeI restriction sites of the CV186 lentiviral vector to obtain the HDAC2 gene overexpression lentiviral vector.

[0009] Furthermore, the volume ratio of the ZNF622 gene promoter to the HDAC2 gene promoter is 2.5:10.

[0010] The present invention also provides an overexpression lentiviral vector, wherein the overexpression lentiviral vector is the ZNF622 gene overexpression lentiviral vector or the HDAC2 gene overexpression lentiviral vector.

[0011] The present invention also provides a stable cell line, which is obtained by sequentially transfecting MV4-11 cells with the ZNF622 gene overexpression lentiviral vector and the HDAC2 gene overexpression lentiviral vector.

[0012] The present invention also provides the application of the overexpression lentiviral vector or the stable cell line described herein in the preparation of a drug for treating acute myeloid leukemia cells.

[0013] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0014] Furthermore, the excipients include one or more of glucose, dextrin, mannitol, xylitol, lactose, starch, hydroxypropyl methylcellulose, microcrystalline cellulose, magnesium stearate, polyethylene glycol, and glycine.

[0015] Furthermore, the drug is formulated into various dosage forms by mixing any pharmaceutically acceptable excipients.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] This invention constructs lentiviral vectors overexpressing the ZNF622 and HDAC2 genes, and after transfection, detects STAT3 protein expression and leukemia cell apoptosis. The results show that ZNF622 and HDAC2 interact to reduce STAT3 protein expression and promote leukemia cell apoptosis, which is of significant importance in the treatment of acute myeloid leukemia. Therefore, the ZNF622 and HDAC2 gene overexpression lentiviral vectors can be used to prepare drugs for treating acute myeloid leukemia cells, specifically for reducing STAT3 protein expression and promoting leukemia cell apoptosis. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The relationship between the mRNA expression level of ZNF622 of this invention and the overall survival (OS) of AML patients; the horizontal axis: times (days) represents time (days), and the vertical axis: Percent survival represents the survival rate.

[0020] Figure 2 The relationship between HDAC2 mRNA expression level and AML patient OS is shown in this invention; the horizontal axis is times (days) representing time (days), and the vertical axis is Percent survival representing survival rate.

[0021] Figure 3 This invention relates to the interaction between the ZNF622 protein and the HDAC2 protein.

[0022] In the figure, A represents the HDAC2 protein in both the Input protein and the pull-down protein (IgG protein) of THP-1 cells, which can be detected by the HDAC2 antibody.

[0023] B is that both the input protein and pull-down protein (IgG protein) of THP-1 cells can be detected by the ZNF622 antibody;

[0024] C represents the Input protein and Pull-down protein (IgG protein) of MV4-11 cells, both of which can be detected by HDAC2 antibody;

[0025] Both Input protein and Pull-down protein (IgG protein) in MV4-11 cells can be detected by ZNF622 antibody.

[0026] Figure 4 This is a map of the GV492 viral vector of the present invention.

[0027] Figure 5 This is a CV186 viral vector map of the present invention.

[0028] Figure 6 The green fluorescence expression of MV4-11 cells transfected with ZNF622 lentivirus of this invention 72 hours later;

[0029] In the figure, A represents the white light of the Over-ctrl (idle group) (corresponding to the green fluorescence control in Figure C);

[0030] B represents white light for Over-ZNF622 (overexpression group) (corresponding to the green fluorescence control in Figure D);

[0031] C represents the green fluorescence of the Over-ctrl (idle group);

[0032] D represents the green fluorescence of Over-ZNF622 (overexpression group).

[0033] Figure 7 The present invention uses flow cytometry to detect the transfection rate of ZNF622 lentivirus transfected into MV4-11 cells;

[0034] In the figure, A represents the cell transfection rate of the Over-ctrl group;

[0035] B represents the cell transfection rate in the Over-ZNF622 group.

[0036] Figure 8 This invention uses RT-PCR and Western Blot to detect the expression of ZNF622 mRNA and protein in MV4-11 cells after ZNF622 lentivirus transfection.

[0037] In the figure, A represents the protein expression of ZNF622 mRNA detected by RT-PCR;

[0038] B represents the detection of ZNF622 mRNA protein expression by Western Blot.

[0039] C is a statistical graph of ZNF622 mRNA expression detected by Western Blot; the vertical axis is the protein expression level of ZNF622 mRNA, and ZNF622 / GAPDH indicates that the internal control is GAPDH, that is, the ratio of the gray value of ZNF622 mRNA protein to the gray value of internal control GAPDH protein.

[0040] Figure 9 This invention uses Western blotting to detect STAT3 protein expression after cell transfection;

[0041] In the figure, A represents the expression of STAT3 protein after cell transfection as detected by Western blotting.

[0042] B is a statistical graph of STAT3 protein expression detected by Western blotting; the vertical axis represents the STAT3 protein expression level.

[0043] Figure 10 This invention uses flow cytometry to detect cell apoptosis after cell transfection;

[0044] A is a flow cytometry image showing apoptosis in transfected cells (Over-ZNF622+Over-ctrl);

[0045] B is a flow cytometry diagram showing apoptosis in transfected cells (Over-ZNF622+Over-HDAC2);

[0046] C represents the apoptosis rate of each cell after transfection, as detected by flow cytometry. The vertical axis, Apoptosis rate, represents the apoptosis rate. Detailed Implementation

[0047] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0048] Example 1: Experimental materials and analytical methods.

[0049] I. Experimental Materials

[0050] Residual bone marrow samples were collected from newly diagnosed acute myeloid leukemia (AML) patients (AML group) and from patients with non-hematologic malignancies (control group) as experimental samples. All volunteers signed informed consent forms. The acquisition of all samples was approved by the Ethics Committee of the Affiliated Hospital of Zunyi Medical University.

[0051] The nucleotide sequence of the ZNF622 gene is shown in SEQ ID NO.1.

[0052] The nucleotide sequence of the HDAC2 gene is shown in SEQ ID NO.3.

[0053] II. Statistical Analysis

[0054] Statistical analysis was performed using software such as GraphPad Prism 8.0, and statistical significance was defined as the standard deviation mean (±SD). The Kaplan-Meier online analysis website was used to analyze the correlation between the expression levels of ZNF622 or HDAC2 and overall survival (OS) of patients. Cox proportional hazards regression model (Cox regression analysis) was used to analyze independent risk factors for OS in AML patients.

[0055] Example 2: Analysis of the mRNA expression levels of ZNF622 or HDAC2 in bone marrow specimens from patients with acute myeloid leukemia (AML) and their correlation with clinical prognosis.

[0056] I. Prognostic Analysis of ZNF622

[0057] 1. mRNA expression level of ZNF622 and its prognostic analysis

[0058] The remaining bone marrow specimens from newly diagnosed acute myeloid leukemia (AML) patients were divided into two groups: a low expression group and a high expression group (based on the median value of ZNF622 mRNA expression level).

[0059] Kaplan-Meier survival analysis was performed on the survival time of AML patients and the expression of ZNF622 mRNA to compare the overall survival (OS) of the two groups (low expression group and high expression group).

[0060] Depend on Figure 1 The results showed that compared with the high expression group, the low expression group had significantly reduced OS, and the difference was statistically significant (P<0.05). That is, the lower the OS of AML patients, the lower the mRNA expression level of ZNF622, indicating that the mRNA expression level of ZNF622 is significantly positively correlated with the overall survival of clinical AML patients.

[0061] 2. Cox regression analysis

[0062] As shown in Table 1, the independent risk factor affecting OS in AML patients was ZNF622 mRNA expression (P<0.05), while age, BM blasts, WBC, PB blasts, PLT, and LDH clinical characteristics were not independent risk factors for OS (P>0.05, Table 1).

[0063] Table 1 Clinical characteristics of AML patients and Cox regression survival analysis

[0064]

[0065]

[0066] Note: WBC: white blood cells; PLT: platelets; BM blasts: myelinated blasts; PB blasts: peripheral blood blasts; LDH: lactate dehydrogenase.

[0067] II. Prognostic Analysis of HDAC2

[0068] 1. HDAC2 mRNA expression level and its prognostic analysis

[0069] The remaining bone marrow specimens from newly diagnosed acute myeloid leukemia (AML) patients were divided into two groups: a low expression group and a high expression group (based on the 25th percentile level of HDAC2 mRNA expression).

[0070] Kaplan-Meier survival analysis was performed on the survival time of AML patients and HDAC2 mRNA expression to compare the overall survival (OS) between the two groups.

[0071] Depend on Figure 2 The results showed that patients in the HDAC2 low expression group had lower overall survival (OS) compared with the high expression group (P<0.05). This indicates that the lower the OS of AML patients, the lower the HDAC2 mRNA expression level, suggesting a significant positive correlation between HDAC2 mRNA expression level and overall survival in clinical AML patients.

[0072] Example 2: Interaction between ZNF622 protein and HDAC2 protein.

[0073] I. Experimental Materials

[0074] Leukemia cells: THP-1 cells and MV4-11 cells were provided by Beijing First People's Hospital and West China Hospital of Sichuan University.

[0075] II. Experimental Methods

[0076] The interaction between ZNF622 and HDAC2 proteins in THP-1 or MV4-11 cells was detected using co-immunoprecipitation (CoIP). The specific steps are as follows:

[0077] (1) Collect cells in the logarithmic growth phase (THP-1 cells or MV4-11 cells), centrifuge at 4℃ and 2500 rpm / min for 5 minutes, discard the supernatant after centrifugation, and keep the cell clumps;

[0078] (2) Wash cells twice: Add 1 mL of pre-cooled PBS to the cell clumps to resuspend the cells, mix gently, and then centrifuge at 4°C and 600 rpm / min for 5 min. Discard the supernatant after centrifugation and keep the cell clumps.

[0079] (3) Add 900 μL of lysis / wash buffer to the cell clumps, and add 18 μL of protease inhibitor CCK (1:50 ratio). Then add 9 μL of phosphatase inhibitor A (1:100 ratio) and 9 μL of phosphatase inhibitor B (1:100 ratio). Mix well by pipetting and incubate on ice for 18 minutes, vortexing once every 6 minutes for a total of 3 times to obtain the cell mixture.

[0080] (4) Centrifugation to collect supernatant: Set the centrifuge parameters to 1200 rpm and 4℃, and centrifuge the cell mixture for 5 minutes; carefully collect the supernatant after centrifugation; divide the supernatant into 3 groups: negative control IgG group, positive control Input group, and target group ZNF622 or target group HDAC2.

[0081] (5) Antigen-antibody binding: For each of the above three groups, 300 μL of supernatant was pipetted into a 1.5 mL centrifuge tube. For the Input group, 60 μL of 5× protein supernatant was added. The tubes were mixed, sealed, and boiled in a 100°C water bath for 18 minutes. After cooling, the tubes were stored in a -20°C refrigerator.

[0082] Following the principle of equal proportions, 1 μL of IgG antibody was added to the IgG group, 1 μL of ZNF622 antibody was added to the target group ZNF622, or 1.67 μL of HDAC2 antibody was added to the target group HDAC2. After mixing, the mixture was shaken at 4°C for 6 hours to form an antigen-antibody complex.

[0083] (6) Pretreatment of magnetic beads: Gently pipette and mix Protein A / G magnetic beads. Take 25 μL of magnetic bead suspension and place it in a new centrifuge tube. At the same time, add 500 μL of lysis / wash buffer, mix gently, and place on a magnetic rack for 1 minute. When the magnetic beads adhere to the side wall of the centrifuge tube, carefully aspirate the supernatant and keep the magnetic beads. Repeat twice.

[0084] (7) Binding of magnetic beads to antigen-antibody complex: Add the antigen-antibody complex from step (5) to the magnetic beads treated in step (6), mix gently, seal, and incubate overnight on a shaker at 4°C.

[0085] (8) The next day, take out the incubated centrifuge tubes containing IgG, ZNF622 or HDAC2, place them on a magnetic rack for 1 minute. When the magnetic beads adhere to the side wall of the tube, carefully aspirate the supernatant and keep the magnetic beads, which is the antigen-antibody magnetic bead complex. Add 0.5 mL of lysis / wash buffer to the antigen-antibody magnetic bead complex and fully resuspend the magnetic beads. Fix them on the magnetic rack again for 1 minute. When the magnetic beads adhere to the side wall of the tube, carefully aspirate the supernatant and keep the magnetic beads.

[0086] (9) Add 60 μL of 5× protein supernatant to the magnetic beads of IgG group, target group ZNF622 or target group HDAC2 in (8), mix gently with pipette and seal, boil in a water bath at 100°C for 18 minutes; after cooling, place it on a magnetic rack for 1 minute, and carefully collect the supernatant when the magnetic beads adhere to the side wall of the tube.

[0087] (10) Western Blot was used to detect protein expression in the supernatant of IgG group, Input group, target group ZNF622 or target group HDAC2.

[0088] The results are as follows Figure 3 As shown in Figure A, both Input and IgG proteins in THP-1 cells can be detected by HDAC2 antibody; Figure 3 As shown in B, Input protein and IgG protein in MV4-11 cells, and HDAC2 can be detected by HDAC2 antibody; Figure 3 As shown in C, both Input and IgG proteins in THP-1 cells can be detected by the ZNF622 antibody; Figure 3 As shown in Figure D, both Input and IgG proteins in MV4-11 cells can be detected by the ZNF622 antibody. These results indicate that ZNF622 and HDAC2 proteins interact in THP-1 and MV4-11 cells.

[0089] Example 3: Construction and cell transfection of ZNF622 gene overexpression lentiviral vector and HDAC2 gene overexpression lentiviral vector.

[0090] I. Construction of a lentiviral vector for ZNF622 gene overexpression

[0091] (1) The construction, packaging, and titer detection of the ZNF622 gene overexpression lentiviral vector (Table 2) were completed by Shanghai Jikai Gene Technology Co., Ltd.; the lentiviral vector used in the construction of the ZNF622 gene overexpression lentiviral vector was the GV492 lentiviral vector, as shown in the figure. Figure 4 As shown.

[0092] (2) The obtained ZNF622 gene overexpression lentiviral vector (target plasmid) was sequenced by positive clones. The sequencing results are shown in SEQ ID NO.2. The sequencing results show that it is consistent with the target sequence (SEQ ID NO.1). The ZNF622 gene overexpression lentiviral vector was successfully constructed.

[0093] (3) Titer detection: The results in Table 2 show that the lentivirus was successfully packaged. Therefore, the obtained ZNF622 gene overexpression lentiviral vector (LV-ZNF622) can be used for subsequent experiments.

[0094] Table 2 Analysis of titer test results

[0095] name Titration (TU / mL) LV-ZNF622 1E+9

[0096] II. Construction of HDAC2 gene overexpression lentiviral vector

[0097] (1) The construction, packaging, and titer detection of the HDAC2 gene overexpression lentiviral vector (Table 3) were completed by Shanghai Jikai Gene Technology Co., Ltd.; the lentiviral vector used in the construction of the HDAC2 gene overexpression lentiviral vector was the CV186 lentiviral vector, as shown in the figure. Figure 5 As shown.

[0098] (2) The obtained HDAC2 gene overexpression lentiviral vector (target plasmid) was sequenced by positive clones. The sequencing results are shown in SEQ ID NO.4. The sequencing results show that it is consistent with the target sequence (SEQ ID NO.3). The HDAC2 gene overexpression lentiviral vector was successfully constructed.

[0099] (3) Titer detection: The results in Table 3 show that the lentivirus was successfully packaged. Therefore, the obtained HDAC2 gene overexpression lentiviral vector (LV-HDAC2) can be used for subsequent experiments.

[0100] Table 3 Analysis of titer test results

[0101] name Titration (TU / mL) LV-HDAC2 5E+8

[0102] Example 4: Construction of stable cell lines and STAT3 protein expression and apoptosis.

[0103] I. Transfection of MV4-11 cells with a lentiviral vector overexpressing the ZNF622 gene

[0104] The ZNF622 gene overexpression lentiviral vector (overexpression group, Over-ZNF622) and the GV492 lentiviral vector (empty excitation group, Over-Ctrl) were transfected into MV4-11 cells, respectively. The specific steps are as follows:

[0105] ① Cell collection: Select MV4-11 cells in the logarithmic growth phase and centrifuge at 4℃ and 1200 rpm / min for 5 minutes;

[0106] ② After centrifugation, discard the supernatant and keep the MV4-11 cell clumps. Add 1 mL of complete culture medium to resuspend the MV4-11 cell clumps. Mix with a pipette tip to obtain the mixed cells. Take out 5 μL of the mixed cells and dilute the mixed cells with 95 μL of complete culture medium and mix well to obtain the MV4-11 cell suspension. Then take 10 μL of the MV4-11 cell suspension and inject it into a hemocytometer for counting.

[0107] ③ Prepare 12-well plates for virus transfection, 100 μL per well, 1×10 5 One MV4-11 cell suspension, i.e., a cell concentration of 1×10⁻⁶ cells. 6 / mL;

[0108] ④ Select the viral MOI concentration, prepare a 96-well plate, and set the multiplicity of infection (MOI) to 25 based on cell status and transfection status;

[0109] ⑤ According to the instructions for constructing the viral vector, find the viral titers of the overexpression group and the empty group, and calculate the viral volume of the overexpression group and the empty group: the viral volume of the overexpression group required for transfection into MV4-11 cells is 2.5 μL, and the viral volume of the empty group required for transfection into MV4-11 cells is 1.7 μL.

[0110] ⑥ Cell seeding: Seed 500 μL per well using the 1 / 2 volume seeding method. Select a 12-well plate and add 1×10⁻⁶ cells prepared in step ③ to each well. 5 100 μL of MV4-11 cell suspension; then add the calculated virus volume from step ⑤; next, add 20 μL of the viral transduction aid HitransGP; finally, add complete culture medium to bring the volume to 500 μL to obtain a stable ZNF622 gene overexpression cell line (Over-ZNF622 stable cell line, Over-ZNF622 group) or an empty transduction control stable cell line (Over-ctrl stable cell line, Over-ctrl group); seal the 12-well plate with sealing film to avoid contamination;

[0111] ⑦ Centrifugation: Set the centrifuge parameters to 200g, 37℃, 1 hour, place the 12-well plate in the centrifuge, and centrifuge;

[0112] ⑧ After centrifugation, remove the 12-well plate, remove the sealing film, observe the cells under an inverted phase contrast microscope, disinfect with 75% alcohol, and then place it in a CO2 incubator.

[0113] ⑨ After 12 hours, add another 1 / 2 volume, i.e., 500 μL of complete culture medium, and observe the cell state under an inverted phase contrast microscope;

[0114] ⑩ Observe cell viability under a microscope on the second day after transfection, and add culture medium as needed;

[0115] On day 3 after transfection, observe the GFP fluorescence expression of ZNF622. If the GFP expression level is above 90%, it is preliminarily determined that the virus has been successfully transfected into MV4-11 cells. Add 12 μg / mL puromycin for virus selection (select cells that have been successfully transfected with the virus, and kill untransfected cells with puromycin).

[0116] The transfection efficiency of each virus was detected by flow cytometry, and the expression of Over-ZNF622 and Over-ctrl was verified by Western blotting and RT-PCR.

[0117] After transfection and selection with puromycin, the GFP fluorescence expression results showed ( Figure 6The expression levels of GFP in both the Over-ctrl and Over-ZNF622 groups were above 95%, indicating successful transfection with ZNF622 lentivirus. Flow cytometry results showed that the transfection rates of all cells in both the Over-ctrl and Over-ZNF622 groups were greater than 98.0%. Figure 7 The results of A and B indicate successful transfection of ZNF622 lentivirus; Western blot and RT-PCR results show that both ZNF622 protein expression and mRNA expression are significantly increased (P<0.05). Figure 8 The system indicates that ZNF622 overexpression was successfully constructed and can be used for subsequent experiments.

[0118] II. Transfection of ZNF622 gene-overexpressing stable cell lines with HDAC2 gene-overexpressing lentiviral vector.

[0119] After obtaining the Over-ZNF622 and Over-ctrl stable cell lines, the HDAC2 gene overexpression lentiviral vector (Over-HDAC2) was then transformed into the Over-ZNF622 or Over-ctrl stable cell lines, respectively. The specific steps are as follows:

[0120] ① Cell collection: Select Over-ZNF622 stable cell lines and Over-ctrl stable cell lines in the logarithmic growth phase, and centrifuge at 4℃, 1200 rpm / min for 5 minutes;

[0121] ② After centrifugation, discard the supernatant and keep the Over-ZNF622 stable cell line or the Over-ctrl stable cell line. Resuspend the Over-ZNF622 stable cell line or the Over-ctrl stable cell line in 1 mL of complete culture medium, respectively. Mix the cells with a pipette tip to obtain the mixed cells. Take 5 μL of the mixed cells and dilute the mixed cells with 95 μL of complete culture medium and mix well to obtain the Over-ZNF622 stable cell suspension and the Over-ctrl stable cell suspension, respectively. Then take 10 μL of the cell suspension and inject it into a hemocytometer for counting.

[0122] ③ Prepare 12-well plates for virus transfection, 100 μL per well, 1×10 5 One Over-ZNF622 stable cell suspension and one Over-ctrl stable cell suspension were prepared, i.e., the cell suspension concentration was 1×10⁻⁶. 6 / mL;

[0123] ④ Select the viral MOI concentration, prepare a 96-well plate, and set the multiplicity of infection (MOI) to 50 based on cell status and transfection status;

[0124] ⑤ According to the instructions for constructing the viral vector, find the viral titer of the HDAC2 gene overexpression lentiviral vector and calculate the viral volume of the HDAC2 gene overexpression lentiviral vector: the viral volume of the HDAC2 gene overexpression lentiviral vector required for transfection into the Over-ZNF622 stable cell line is 10 μL, and the viral volume of the HDAC2 gene overexpression lentiviral vector required for transfection into the Over-ctrl stable cell line is 2.5 μL.

[0125] ⑥ Cell seeding: Seed 500 μL per well using the 1 / 2 volume seeding method. Select a 12-well plate and add 1×10⁻⁶ cells prepared in step ③ to each well. 5 100 μL of Over-ZNF622 or Over-ctrl stable cell suspension; then add the calculated virus volume (⑤); next, add 20 μL of the viral transfection aid HitransGP; finally, add complete culture medium to 500 μL to obtain stable cell lines overexpressing ZNF622 and HDAC2 (Over-ZNF622+Over-HDAC2) or stable cell lines transfected by empty transfection (Over-ZNF622+Over-ctrl); seal the 12-well plate with sealing film to avoid contamination;

[0126] ⑦ Centrifugation: Set the centrifuge parameters to 200g, 37℃, 1 hour, place the 12-well plate in the centrifuge, and centrifuge;

[0127] ⑧ After centrifugation, remove the 12-well plate, remove the sealing film, observe the cells under an inverted phase contrast microscope, disinfect with 75% alcohol, and then place it in a CO2 incubator.

[0128] ⑨ After 12 hours, add another 1 / 2 volume, i.e., 500 μL of complete culture medium, and observe the cell state under an inverted phase contrast microscope;

[0129] ⑩ Observe cell viability under a microscope on the second day after transfection, and add culture medium as needed;

[0130] On day 3 after transfection, observe the GFP fluorescence expression of ZNF622 cells. If the GFP expression level is above 90%, it is preliminarily determined that the virus has been successfully transfected into the Over-ZNF622 stable cell line and the Over-ctrl stable cell line. Add 12 μg / mL of puromycin for virus selection (select cells that have been successfully transfected with the virus, and kill untransfected cells with puromycin).

[0131] Flow cytometry was used to detect the transfection efficiency of each virus. Western blotting and RT-PCR were used to verify the expression of STAT3 in stable cell lines with overexpression of ZNF622 and HDAC2 genes (Over-ZNF622+Over-HDAC2) or stable cell lines with empty excitation (Over-ZNF622+Over-ctrl).

[0132] III. STAT3 protein expression and apoptosis after transfection

[0133] like Figure 9 As shown in Figures A and B, compared with the Over-ZNF622+Over-ctrl group, the STAT3 protein expression in the Over-ZNF622+Over-HDAC2 group was significantly reduced (P<0.05), indicating that the interaction between ZNF622 and HDAC2 reduces STAT3 protein expression.

[0134] like Figure 10 As shown in the AC, compared with the Over-ZNF622+Over-ctrl group, the apoptosis rate of MV4-11 cells in the Over-ZNF622+Over-HDAC2 group was significantly increased (P<0.05).

[0135] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0136] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of ZNF622 gene promoter and HDAC2 gene promoter in the preparation of drugs for treating acute myeloid leukemia cells, characterized in that, The ZNF622 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. HDAC2 The nucleotide sequence of the gene is shown in SEQ ID NO.3; The ZNF622 gene promoter is a ZNF622 gene overexpression lentiviral vector, and the HDAC2 gene promoter is an HDAC2 gene overexpression lentiviral vector.

2. The application of the ZNF622 gene promoter and HDAC2 gene promoter according to claim 1 in the preparation of a drug for treating acute myeloid leukemia cells, characterized in that, The ZNF622 gene overexpression lentiviral vector construction process is as follows: the ZNF622 gene is ligated into the GV492 lentiviral vector. BamH I and Age I restriction site was used to obtain ZNF622 gene overexpression lentiviral vector.

3. The application of the ZNF622 gene promoter and HDAC2 gene promoter according to claim 2 in the preparation of a drug for treating acute myeloid leukemia cells, characterized in that, The construction process of the HDAC2 gene overexpression lentiviral vector is as follows: the HDAC2 gene is ligated into the CV186 lentiviral vector. BamH I and Age I restriction site was used to obtain HDAC2 gene overexpression lentiviral vector.

4. The application of the ZNF622 gene promoter and HDAC2 gene promoter according to claim 1 in the preparation of a drug for treating acute myeloid leukemia cells, characterized in that, The volume ratio of the ZNF622 gene promoter to the HDAC2 gene promoter is 2.5:

10.

5. The application of the ZNF622 gene promoter and HDAC2 gene promoter according to claim 1 in the preparation of a drug for treating acute myeloid leukemia cells, characterized in that, The drug also includes pharmaceutically acceptable excipients.

6. The application of the ZNF622 gene promoter and HDAC2 gene promoter according to claim 5 in the preparation of a drug for treating acute myeloid leukemia cells, characterized in that, The excipients include one or more of glucose, dextrin, mannitol, xylitol, lactose, starch, hydroxypropyl methylcellulose, microcrystalline cellulose, magnesium stearate, polyethylene glycol, and glycine.

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