Effect of MAZ gene in Ph-positive acute lymphocytic leukemia and treatment application of MAZ gene

Abstract

The invention belongs to the technical field of biomedical research, and particularly relates to promotion of malignant progression of Ph positive lymphocytic leukemia by MAZ in Ph chromosome positive acute lymphocytic leukemia through transcriptional regulation and control of expression of SIRT5. The invention aims to provide a novel signal path for determining the malignant progression mechanism of the Ph chromosome positive acute lymphoblastic leukemia, so as to provide a potential target spot for the treatment of a Ph chromosome positive acute lymphoblastic leukemia patient. According to the invention, MAZ is proposed for the first time to mediate malignant progression of Ph chromosome positive acute lymphoblastic leukemia by promoting transcription of SIRT5, so that progression of Ph chromosome positive acute lymphoblastic leukemia is promoted; by inhibiting the MAZ gene, proliferation of Ph chromosome positive acute lymphocytic leukemia cells can be remarkably inhibited, the apoptosis rate of the Ph chromosome positive acute lymphocytic leukemia cells is increased, and the sensitivity of the Ph chromosome positive acute lymphocytic leukemia cells to imatinib is enhanced.

Description

Technical Field

[0001] The present invention relates to a method for treating Ph chromosome-positive acute lymphoblastic leukemia by targeting MAZ. This study found that the MAZ gene plays an important role in Ph chromosome-positive acute lymphoblastic leukemia. MAZ can promote the proliferation of Ph chromosome-positive acute lymphoblastic leukemia cells, inhibit their apoptosis, and participate in the resistance of Ph chromosome-positive acute lymphoblastic leukemia cells to the tyrosine kinase inhibitor imatinib (Imatinib Mesylate, IM). MAZ not only plays a role in promoting the malignant phenotype of Ph chromosome-positive acute lymphoblastic leukemia, but also promotes the transcription of SIRT5, thereby driving the enhancement of oxidative phosphorylation in Ph chromosome-positive acute lymphoblastic leukemia. By inhibiting the expression of the MAZ gene, SIRT5 expression can be significantly reduced, cancer cell proliferation can be inhibited and apoptosis can be promoted, while enhancing the sensitivity of Ph+ ALL cells to IM. The present invention provides pharmaceutical compositions comprising MAZ gene inhibition, as well as MAZ gene inhibition and TKI, providing a new strategy for the treatment of Ph+ ALL. Background Art

[0002] Acute lymphoblastic leukemia (ALL) is a malignant tumor of lymphocyte precursors characterized by abnormal proliferation and differentiation, leading to failure of normal immune responses and decreased hematopoietic function. Abnormally differentiated, long-lived hematopoietic progenitor cells undergo malignant transformation and abnormal proliferation, resulting in the presence of numerous blasts in the circulation. Normal bone marrow is replaced by malignant cells, which can infiltrate the central nervous system and testes. Philadelphia chromosome-positive ALL (Ph+ALL) is the most common genetic subgroup of adult ALL. Its primary characteristic is the translocation of the Abelson (ABL) non-receptor tyrosine kinase proto-oncogene on chromosome 9 to the breakpoint cluster region (BCR) of chromosome 22, forming the BCR::ABL fusion gene. This fusion chromosome is named the Philadelphia chromosome (Ph). Currently, clinical treatment is typically intensive chemotherapy combined with a BCR::ABL tyrosine kinase inhibitor, followed by allogeneic hematopoietic stem cell transplantation after first remission. However, the 5-year overall survival rate with intensive chemotherapy combined with first- or second-generation BCR::ABL tyrosinase inhibitors is only 40-50%, and the majority of patients die from disease relapse. Therefore, there is an urgent need to find new therapeutic strategies to address these current clinical challenges. To explore more diverse treatment options and the mechanisms underlying the development and progression of Ph+ALL, our group's previous research revealed that SIRT5, which is highly expressed in Ph+ALL, modifies glutamate dehydrogenase (GDH) by desuccinylation, enhancing its enzymatic activity, promoting the conversion of glutamate to alpha-ketoglutarate (α-KG), increasing carbon flux into the tricarboxylic acid (TCA) cycle, and enhancing oxidative phosphorylation, thereby promoting the malignant progression of Ph+ALL. However, the mechanism of increased SIRT5 expression in Ph+ALL is still unclear. Therefore, this study explored the mechanism of SIRT5 regulation in Ph+ALL.

[0003] Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blot analysis of SIRT5 mRNA and protein levels revealed that SIRT5 mRNA and protein levels were significantly elevated in Ph+ALL cells compared with CML cells and other Ph-ALL cells, indicating that SIRT5 is regulated at the transcriptional level. DNA pull-down experiments combined with analysis of transcription factor prediction websites identified MAZ as a key transcription factor regulating SIRT5 transcription. Chromatin immunoprecipitation (ChIP) confirmed that MAZ is indeed a transcription factor for SIRT5 and regulates its transcription by binding to the -739 to -638 and -79 to +49 regions upstream of the SIRT5 transcription start site. Knockdown of MAZ inhibited proliferation, increased apoptosis, arrested cell cycle, and significantly increased sensitivity to imatinib (IM) in Ph+ALL cells. Therefore, targeting MAZ may become a new therapeutic target for patients with Ph+ALL. Summary of the Invention

[0004] 1. To solve the above technical problems, the purpose of the present invention is to provide a new strategy to enhance the efficacy of Ph+ ALL patients by targeting the MAZ gene.

[0005] 2. The embodiments of the present invention provide experimental verification that MAZ promotes SIRT5 transcription in Ph+ALL, thereby mediating the application of Ph+ALL malignant progression.

[0006] The present invention discovered that MAZ can regulate SIRT5 transcription, and inhibiting MAZ gene expression downregulates SIRT5 expression in Ph+ALL cells. Furthermore, inhibiting MAZ expression reduces the proliferation activity of Ph+ALL cells, increases apoptosis levels, and significantly enhances the sensitivity of Ph+ALL cells to IM.

[0007] 3. The present invention has at least the following advantages:

[0008] (1) This paper first proposed the existence of a MAZ-SIRT5 signaling axis in Ph+ALL, specifically that MAZ can bind to the SIRT5 promoter region to promote SIRT5 transcription and mediate its high expression.

[0009] (2) The present invention establishes a strategy and method for inhibiting the malignant progression of Ph+ALL. Ph+ALL cells express MAZ at levels much higher than non-Ph+ALL cell lines. By inhibiting the MAZ gene and combining it with IM, the efficacy of treatment for Ph+ALL patients can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art:

[0011] Figure 1 MAZ regulates and promotes SIRT5 transcription in Ph+ALL. (A) qRT-PCR assay shows elevated SIRT5 mRNA expression in Ph+ALL, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; (B) 2000 bp upstream and 100 bp downstream of the SIRT5 transcription start site were selected; (C, D) Intersection of the TFDB website and the specifically bound proteins; (E) Proteomic sequencing results of each transcription factor in several cell types, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0012] Figure 2 MAZ is highly expressed in the nuclei of Ph+ALL cells. (A) qRT-PCR assay detected elevated MAZ mRNA expression in Ph+ALL cells, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; (B) Western blot assay detected elevated MAZ protein expression in Ph+ALL cells, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; (C) Western blot assay detected elevated MAZ expression in the nuclei of Ph+ALL cells; (D) Western blot assay detected low MAZ expression in the cytoplasm of all cells, with no significant differences.

[0013] Figure 3 Ph+ALL cells were inoculated with MAZ after knockdown and the knockdown efficiency was verified. (A) qRT-PCR assay for MAZ knockdown efficiency: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; (B) Western blot assay for MAZ and SIRT5 protein levels after MAZ knockdown; (C) qRT-PCR assay for changes in SIRT5 mRNA levels after SIRT5 knockdown: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0014] Figure 4Knockdown of MAZ inhibited the proliferation of Ph+ALL cells. (A) Colony formation assay detected the changes in colony formation of Ph+ALL cells after MAZ knockdown; (B) Flow cytometry detected the apoptosis of Ph+ALL cells, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; (C) CCK8 proliferation assay detected the proliferation of Ph+ALL cells after MAZ knockdown, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; (D) Western blot assay detected the expression of various apoptosis-related molecules in MAZ knockdown cells; (E) Flow cytometry detected the changes in the division cycle of MAZ knockdown cells, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001;

[0015] Figure 5 Knockdown of MAZ increases the sensitivity of Ph+ALL cells to IM. (A) CCK8 proliferation assay detects the sensitivity of CML cells to IM after MAZ knockdown; (B) CCK8 proliferation assay detects the sensitivity of Ph+ALL cells to IM after MAZ knockdown; (C) Flow cytometry detection of apoptosis in CML cells, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; (D) Flow cytometry detection of apoptosis in Ph+ALL cells, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001;

[0016] Figure 6 Specific sites of MAZ binding in the SIRT5 promoter region in Ph+ALL. (A) Typical MAZ binding motifs on the JASPAR website; (B) MAZ-binding regions in the SIRT5 promoter; (C) ChIP assay results showed binding sites in the P1 and P3 regions, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; (D) Nonspecific amplification of PCR products detected by agarose gel electrophoresis. DETAILED DESCRIPTION

[0017] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0018] The following is a specific technical solution: Based on the increased expression of SIRT5 mRNA level, DNA pulldown combined with MS experiment was selected and MAZ was selected as the transcription factor regulating SIRT5 in Ph+ALL through prediction from relevant data websites (such as Figure 1 ); Based on the previous results, the expression of MAZ in Ph+ALL cell models and the subcellular distribution of MAZ were studied (e.g. Figure 2 ), knock down MAZ and further verify the expression of SIRT5 after knockdown (e.g. Figure 3 ); explore the effects of knocking down MAZ on Ph on cell proliferation, apoptosis, and cell division cycle (e.g. Figure 4 ); explore the effect of knockdown of MAZ on IM sensitivity (e.g. Figure 5 ); further explore the specific region of MAZ binding to the SIRT5 promoter region, and use ChIP experiments combined with qRT-PCR to obtain the specific binding region (such as Figure 6 ).

[0019] 1. Experimental Results

[0020] (1) MAZ is a transcription factor that regulates SIRT5 expression in Ph+ALL cells

[0021] First, the mRNA level of SIRT5 was determined in Ph+ALL cells, which was significantly upregulated compared with Ph-ALL cells and CML cells ( Figure 1 A), select the region 2000 bp upstream and 100 bp downstream of the transcription start point of the upstream gene of SIRT5 gene, i.e., the region 13572274-13574373 on chromosome 6 ( Figure 1 B) The transcription factor prediction website TFDB (AnimalTFDB4 (wchscu.cn)) was used to search for transcription factors that may regulate SIRT5. 949 transcription factors with a P value less than 0.05 and a score greater than 8 were selected from the results obtained from the transcription factor prediction website, and the promoter region was biotin-labeled as a DNA-specific probe. The transcription factors that can bind to the promoter region were pulled down by DNA pull-down experiments. Mass spectrometry sequencing analysis was performed on the pulled-down transcription factors, and 154 proteins that simultaneously met the three conditions of specific binding to the DNA probe, being located in the cell nucleus, and regulating DNA to promote transcription were selected. The intersection of the specific binding proteins predicted by the website and the specific binding proteins predicted by the website and the transcription factors predicted by the website was taken ( Figure 1 C), found the presence of ten transcription factors ( Figure 1D). Proteomic analysis was also performed on the representative cell line of Ph+ALL, SUP-B15, the representative cell line of CML, K562, and the representative cell line of Ph-B-ALL, NALM-6. The results showed that the expression level of Myc-associated zinc finger protein MAZ (MAZ) was the highest in SUP-B15, followed by NALM-6, and the lowest in K562, which was consistent with the expression level of SIRT5 mRNA obtained in the experiment ( Figure 1 E), we therefore selected MAZ as the transcription factor that regulates SIRT5 expression in Ph+ALL.

[0022] (2) Expression level of MAZ in Ph+ALL

[0023] In the qRT-PCR experiment, it was found that the mRNA level of MAZ was significantly increased compared with CML cells ( Figure 2 A), Western blot experiments showed that the protein level of MAZ was also increased compared with CML cells ( Figure 2 B). The expression of MAZ was then determined by extracting the nucleus and cytoplasm. It was found that MAZ was more highly expressed in the nucleus of Ph+ALL cells compared to CML cells ( Figure 2 C), and there was no significant difference in the expression of the two in the cytoplasm ( Figure 2 D),

[0024] (3) Ph+ALL cells after MAZ knockdown and verification of knockdown efficiency

[0025] Lentivirus was used to knock down the expression of MAZ in Ph+ALL cells and CML cells. The knockdown efficiency was first detected by qRT-PCR. Figure 3 A), Western blot assay to detect lentiviral knockdown efficiency ( Figure 3 B), and qRT-PCR experiments also found that the mRNA level of SIRT5 was significantly increased after knocking down MAZ ( Figure 3 C) and protein levels ( Figure 3 B) both declined.

[0026] (4) Knockdown of MAZ inhibited the proliferation of Ph+ALL cells

[0027] To further explore the role of MAZ in Ph+ALL cells, we performed a clone formation assay on knockdown cells and found that knockdown of MAZ significantly inhibited the clone formation of Ph+ALL cells, while no significant changes were observed in CML cells ( Figure 4A). Flow cytometry analysis of knockdown cells revealed no significant changes in the apoptosis level of CML, while a significant inhibition was observed in Ph+ALL cells ( Figure 4 B). Subsequently, the proliferation experiment of knockdown cells using CCK8 was conducted and it was found that knockdown of MAZ significantly inhibited the proliferation of Ph+ALL cells, but did not show significant inhibition on CML cells ( Figure 4 C). Detection of pro-apoptotic and anti-apoptotic proteins in Ph+ALL cells and CML cells revealed that the pro-apoptotic protein Bax in Ph+ALL cells increased while the anti-apoptotic proteins Bcl-2 and Bcl-XL decreased, while no significant changes were found in K562 cells ( Figure 4 D). Detection of the cell cycle of knockdown cells revealed that Ph+ALL cells were mainly arrested in the G2 phase, while the number of cells in the S and G1 phases, which represent division, decreased ( Figure 4 E).

[0028] (5) Knockdown of MAZ increases the sensitivity of Ph+ALL cells to IM

[0029] When used in combination with IM, a common drug for treating Ph+ALL, it was found that MAZ could significantly reduce the IC50 value of Ph+ALL cells, while CML cells did not show similar changes ( Figure 5 A, B). Flow cytometry showed no significant changes in CML cell apoptosis after combined with IM, while apoptosis of Ph+ALL cells increased significantly after IM treatment ( Figure 5 C, D).

[0030] (6) Specific sites of MAZ binding in the SIRT5 promoter region in Ph+ALL

[0031] Download the typical binding motif of MAZ from the JASPAR website ( Figure 6 A), and predicted sites where MAZ can bind to the SIRT5 promoter region ( Figure 6 B), named P1, P2, and P3, and designed specific primers. ChIP combined with Q-PCR experiments revealed that MAZ can bind to the -739 to -638 and -79 to +49 regions of SIRT5 ( Figure 6 C). To further verify, nucleic acid agarose gel electrophoresis experiments were performed on the PCR products of P1, P2 and P3 to confirm that P1 and P3 did have specific amplification ( Figure 6 D).

[0032] 2. Experimental Methods

[0033] (1) Cell culture methods for the present invention: Ba / F3, Ba / F3-p210, Ba / F3-p190, NALM6, K562, and SUP-B15 cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum in a 37°C incubator with 5% CO₂. Cells were passaged every 1-2 days on average, and cells in the logarithmic growth phase were harvested for subsequent experiments.

[0034] (2) The method of proteomic sequencing and bioinformatics analysis in the embodiment of the present invention is as follows: the sample is lysed, the protein is extracted and its concentration is determined; the protein is digested with trypsin according to the filter-assisted sample preparation (FASP digestion) procedure; the digested protein fragments are subjected to liquid chromatography-mass spectrometry to detect the fragmentation and characteristics of the protein; and bioinformatics is used to perform expression fold difference analysis and GO and KEGG functional enrichment analysis. The promoter region sequence of SIRT5 was downloaded from NCBI and predicted on the TFDB, JASPAR, and PROMO websites.

[0035] (3) qRT-PCR method in the embodiment of the present invention

[0036] (a) RNA extraction: Collect cells in the logarithmic growth phase and centrifuge at 1000 rpm for 10 min. Discard the supernatant and add 0.5 ml of Trizol reagent to the cell pellet, mixing thoroughly by pipetting. Lyse on ice for 5 min. Add 0.2 ml of chloroform to the sample and cap tightly. Manually shake the tube vigorously for 15 seconds and incubate for 2 to 3 minutes. Centrifuge at 12,000 rpm for 15 minutes at 4°C. After centrifugation, the mixture will separate into a lower, red phenol-chloroform phase, an intermediate phase, and a colorless aqueous upper phase.

[0037] (b) RNA precipitation: Pipette about 0.2 ml of supernatant into an RNase-free centrifuge tube. Add an equal volume of isopropanol to precipitate the RNA. After mixing, incubate at 15 to 30°C for 10 minutes, and centrifuge at 12,000 rpm at 4°C for 10 minutes. Remove the supernatant and add 1 ml of 75% ethanol to each 1 ml of Trizol-lyzed sample to wash the RNA precipitate. After mixing, centrifuge at 7,500 rpm at 4°C for 5 minutes. Repeat the wash once. Carefully remove most of the ethanol solution and allow the RNA precipitate to dry in air at room temperature for 5-10 minutes. Add 40 μL of RNase-free water and blow repeatedly with a gun several times to completely dissolve it. The obtained RNA solution is stored at -80°C until use.

[0038] (d) RNA concentration determination: The purity and concentration of RNA were determined using an ultra-micro spectrophotometer.

[0039] (e) Sample cDNA synthesis: Vortex the sample to mix thoroughly, centrifuge briefly, and immediately place in a PCR instrument. Run the program at 37°C for 15 min, 85°C for 5 s, and then cool to 4°C. cDNA is obtained upon removal and stored at -40°C.

[0040] (e) qRT-PCR analysis of SIRT5 mRNA levels in a / F3, BP210, BP190, NALM-6, K562, and SUP-B15 cells, and data analysis. Primers were designed using Primer5 software, and their sequences were aligned using BLAST and synthesized by BGI. Primer sequences are shown in Table 1. Primers were dissolved in ddH2O according to the manufacturer's instructions and stored at −20°C. Table 1 Related primer sequences

[0041] (f) The reagent composition and corresponding volume of the qRT-PCR reaction system (10 μL) are shown in Table 2 Table 2 qRT-PCR reaction system

[0042] (4) DNA pull-down method in the embodiment of the present invention:

[0043] (a) Total protein extraction: Wash the sample (approximately 2 × 10^7 cells) twice with 1 mL of pre-cooled PBS, absorbing as much PBS as possible during the final wash.

[0044] Add 980uL Lysis buffer, 10uL Protease inhibitors (100X), and 10μLPMSF according to the cell amount, and fully lyse on ice for 20-30 minutes, vortexing once every 5 minutes for 10 seconds / time; use an ultrasonic cell disruptor to sonicate for 5 minutes, 20% power, sonicate for 3 seconds, rest for 3 seconds, and sonicate in an ice bath; centrifuge: 4°C, 12000rpm, 10 minutes, and collect the supernatant.

[0045] (b) Magnetic bead preparation and washing: Remove the Nucleic-Acid Compatible Streptavidin Magnetic Beads from the 4°C refrigerator, invert the tubes several times to mix the magnetic bead storage solution, and transfer 30 μL of each tube into two 1.5 mL Eppendorf tubes, designated as the control group and the experimental group. Place the tubes on a magnetic rack and let them stand for 1 min to separate the magnetic beads, then discard the supernatant. Add 500 μL of Nucleic dilution buffer to each of the control and experimental groups, resuspend the magnetic beads, place the tubes on a magnetic rack for 1 min, and discard the supernatant. Repeat this step three times.

[0046] (c) Magnetic bead binding to DNA: Add 300 pmol of biotin-labeled DNA probe to the experimental tube and an equal amount of non-biotin-labeled DNA to the control tube. Add Nucleic dilution buffer to the volume of 500 μL and incubate at room temperature for 2 h on a silent mixer. Remove the control and experimental tubes from the silent mixer, place them on a magnetic stand for 1 min, and discard the supernatant. Add 500 μL of Nucleic dilution buffer to each of the control and experimental tubes, resuspend the magnetic beads, place them on a magnetic stand for 1 min, and discard the supernatant. Repeat this step 3 times.

[0047] (d) DNA-magnetic bead-bound protein: Add 450 μL of extracted protein to each of the control and experimental tubes, add protein dilution buffer to the volume to 1 mL, and incubate on a silent mixer at 4°C overnight (approximately 16 h). Reserve 100 μL of lysate as the input group. Remove the control and experimental tubes from the silent mixer, place them on a magnetic stand for 1 min, and discard the supernatant. Add 1 mL of protein dilution buffer, resuspend the magnetic beads, place them on a magnetic stand for 1 min, and discard the supernatant. Repeat this step 5 times.

[0048] (e) Elution of the complex: Add 100 μL of elution buffer to each control and experimental tubes, mix well, and incubate in a boiling water bath for 8-10 minutes. Place on a magnetic rack and let stand for 2 minutes. Transfer the supernatant to a new EP tube, which is the pull-down product. Label the control and experimental groups. Add 20 μL of 6X Loading Buffer to each tube and incubate in a boiling water bath for 8-10 minutes. Reserve 100 μL of lysate from the input group and also add 20 μL of 6X Loading Buffer. Boil in a boiling water bath for 8-10 minutes. ③ Store the control, experimental, and input samples at -20°C until needed. Silver staining and mass spectrometry analysis will be performed later.

[0049] (4) ChIP method in the embodiment of the present invention:

[0050] (a) Crosslinking of cell DNA and transcription factors: Prepare an appropriate amount of ice-cold PBS and 100mM PMSF. Warm the SDSLysis Buffer to fully dissolve the SDS and mix thoroughly. Culture the cells in a 10cm cell culture dish using 10ml of cell culture medium. Add an appropriate amount of formaldehyde to the cell culture medium and mix gently to a final concentration of 1%. Then incubate at 37°C for 10 minutes to crosslink the target protein and the corresponding genomic DNA. Add 1.1ml of Glycine Solution (10X) and mix gently. Incubate at room temperature for 5 minutes.

[0051] (b) Washing, Lysis, and Sonication: Aspirate the culture medium containing formaldehyde and glycine, minimizing residual liquid. Centrifuge at 2000 rpm for 5 minutes, discard the supernatant, and resuspend the cells in PBS containing 1 mM PMSF. Lyse the cells twice in Buffer A containing PMSF and DTT for 30 minutes, inverting several times to mix thoroughly. Centrifuge and discard the supernatant. Resuspend the cells in Buffer B containing proteasome inhibitors and DTT, centrifuge and discard the supernatant. Resuspend the cells again in Buffer B, add 0.5 μL micrococcal nuclease, and incubate at 37°C for 30 minutes, inverting several times to mix thoroughly. Centrifuge and discard the supernatant. Resuspend the cells in 1× ChIP buffer containing PMSF, and sonicate.

[0052] (c) Verification of DNA fragment size and antibody incubation: 10 μL of the sonicated DNA solution was stored at -80°C as the input group. 200 μL was taken from each of the IgG, IP, and H3 groups. A small amount of the solution was also used for nucleic acid agarose gel electrophoresis to observe the sonication effect. IgG, IP, and H3 histone antibodies were added, respectively, and the mixture was incubated overnight at 4°C on a rotary shaker.

[0053] (d) Protein digestion and PCR experiment: The transcription factor-DNA complex solution was digested with proteinase K, and the DNA was further purified before PCR amplification.

[0054] (5) Agarose gel electrophoresis method for nucleic acid in the embodiments of the present invention:

[0055] (a) Prepare nucleic acid agarose gel: Weigh 1.2 g agarose and dissolve it in 60 ml TAE solution. Heat to boiling, add nucleic acid dye, mix thoroughly, and pour into a mold to cool.

[0056] (b) Electrophoresis: Mix the lodding buffer and DNA sample and add them to the sample wells. Run electrophoresis for 30 minutes and observe the electrophoresis using a developer.

[0057] (5) Method for detecting protein expression level by western blot in the embodiment of the present invention:

[0058] (a) Extraction of cellular proteins: Collect cells by centrifugation at 1000-2000 rpm at 4°C for 5 min; wash cells 2-3 times with pre-chilled PBS; add an appropriate amount of RIPA buffer supplemented with PMSF immediately before use and sonicate to lyse the protein. Determine the protein concentration of the extracted protein by BCA assay. Simultaneously, add approximately 1 / 4 the sample volume of 5X loading buffer, denature by boiling for 5-6 min, and store at -80°C.

[0059] (b) SDS-PAGE gel electrophoresis: Prepare the two gels in the recipe in Table 3. Prepare a 10% separating gel for the lower layer. Add an appropriate amount of anhydrous ethanol to the upper layer to remove air bubbles and incubate at room temperature for 30-45 minutes. Discard the anhydrous ethanol and prepare a 5% stacking gel for the upper layer. Insert a ruler comb and incubate at room temperature for 30-45 minutes. Fill the electrophoresis tank with running buffer, remove the ruler comb, and load the sample according to the calculated protein concentration (40 μg). Table 3 SDS-PAGA protein electrophoresis gel formula

[0060] Two-step electrophoretic protein separation

[0061] Step 1: voltage 80V, current 100mA, P=50W, 30min;

[0062] Step 2: voltage 120V, current 100mA, P=50W, 1h 30min.

[0063] (c) Transfer: After electrophoresis, cut the gel according to the molecular weight of the protein to be tested. Then, cut a polyvinylidene fluoride (PVDF) membrane that matches the protein to be tested. Activate the cut PVDF membrane by soaking it in methanol for 15-30 seconds, then wash it with ddH2O for 2 minutes. Cut the appropriate gel and soak it in pre-chilled wet transfer buffer. Finally, transfer the gel to the membrane in the order of sponge, three layers of filter paper, PVDF membrane, gel, three layers of filter paper, and sponge, starting from the positive electrode and ending at the negative electrode. Transfer the gel to the membrane using a constant current of 210 mA. The transfer time is determined by the molecular weight of the protein.

[0064] (d) Blocking: After transfer, block the membrane with 5% skim milk powder on a shaker at room temperature for 1.5-2 hours.

[0065] (e) Antibody incubation: Wash the remaining blocking solution on the membrane with TBST; after absorbing the remaining liquid on the membrane with qualitative filter paper, place the membrane on a wax plate, add pre-diluted primary antibody working solution to evenly cover the PVDF membrane, and incubate at 4°C overnight; wash the membrane three times with TBST, 5 minutes each time; add pre-diluted horseradish peroxidase-conjugated secondary antibody to the PVDF membrane, incubate at room temperature for 1-2 hours, and wash the membrane three times with TBST, 5 minutes each time.

[0066] (f) Color development and imaging: In a dark room, place the PVDF membrane on a chemiluminescent plate, then add a pre-configured chemiluminescent reagent to evenly cover the PVDF membrane, and display the image on an imaging system.

[0067] (6) Immunofluorescence experimental method in the embodiment of the present invention:

[0068] (a) Fixing cells: Wash the coverslips on which cells have been grown in the culture plate three times with 1× PBS for 3 minutes each. Fix the coverslips with 4% paraformaldehyde for 15 minutes and then rinse the coverslips three times with 1× PBS for 3 minutes each. Permeabilize the cells with 0.5% Triton X-100 (prepared in 1× PBS) at room temperature for 15 minutes and then rinse the coverslips three times with 1× PBS for 3 minutes each.

[0069] (b) Blocking: Absorb 1× PBS with absorbent paper, add 5% normal serum on the slide, and block at room temperature for 1 hour.

[0070] (c) Antibody incubation: Add an adequate amount of diluted primary antibody to each slide and place in a humidified chamber for overnight incubation at 4°C. Add fluorescent secondary antibody: Rinse the slides three times with PBST for 3 minutes each. Blot the slides with absorbent paper and add diluted fluorescent secondary antibody. Incubate in a humidified chamber at 37°C for 1 hour. Rinse the slides three times with PBST for 3 minutes each.

[0071] (d) Fixation and Photography: Add DAPI and incubate in the dark for 5 minutes to stain the nuclei. Wash off excess DAPI with PBST for 5 minutes x 4. Blot the slide dry with absorbent paper, mount the slide with mounting solution containing a fluorescence quencher, and observe and capture images under a fluorescence microscope.

[0072] (7) Lentiviral knockdown experimental method in the embodiment of the present invention:

[0073] (a) Determine the optimal multiplicity of infection (MOI): Target cells were passaged normally and inoculated into 96-well plates. Virus was diluted according to the measured lentiviral titer; MOIs were set at 1, 5, 10, and 20. After overnight culture of cells in the 96-well plates, the medium was changed and virus was added, along with polybrene at a final concentration of 8 μg / ml. After 3 days, the fluorescence ratio was observed; the optimal MOI was determined when 80% of cells were fluorescent.

[0074] (b) Virus infection of target cells: According to the determined multiplicity of infection, the virus and target cells are mixed and cultured at a concentration ratio of MOI, and polybrene is added to improve the infection efficiency.

[0075] (3) Screening of infected cells: 48 hours after the cells were infected with the virus, the proportion of fluorescent cells was observed under a fluorescence microscope. The cells were passaged in a 96-well plate, and each well was divided into 3-5 wells according to the cell growth rate, and cultured overnight. At the same time, normal target cells that were not infected with the virus were also inoculated. Pressure screening was performed based on the screening antibiotics on the packaged lentiviral vector. After the test was correct, the cells were cultured and frozen or the screening of monoclonal cell lines was continued.

[0076] (8) Clone formation experimental method in the embodiment of the present invention:

[0077] (a) Cells in the exponential growth phase were collected and prepared into a cell suspension.

[0078] (b) The treated cells were seeded in 96-well plates at a density of 100 cells per well.

[0079] (c) One week later, cells were counted using an inverted microscope and photographed in typical fields.

[0080] (9) Method of CCK-8 experiment in the embodiment of the present invention:

[0081] (a) IC50 Assay: Collect and count the required cells for the experiment, seed each well in a 96-well plate at 4,000 cells per well, and set up in triplicate for each group. Add drug solutions of varying concentrations and incubate at 37°C in a 5% CO2 incubator for 48 hours. Add 10 μL of CCK-8 reagent, incubate in the dark for another 2 hours, and measure absorbance at 450 nm. Calculate the cell viability of each group based on the measured absorbance. Analyze cell proliferation using GraphPad Prism software, and determine the drug concentration required to reduce the number of surviving cells by half after drug administration, i.e., the IC50 value of the drug response.

[0082] (b) Proliferation assay: The required cells were collected and counted, seeded at 4000 cells per well in a 96-well plate, with three replicates per group and a blank control. The cells were cultured in an incubator. At 0, 12, 24, 36, and 48 h of culture, 10 μL of CCK-8 reagent was added to each well, mixed, and cultured for another 2 h. The absorbance (OD) was measured at a wavelength of 450 nm, and the cell proliferation curve was plotted with the OD450 value as the vertical axis and the culture time as the horizontal axis.

[0083] (10) Methods for cell apoptosis and cell cycle experiments in the embodiments of the present invention:

[0084] (a) Apoptosis: Collect cells for experimental purposes by centrifugation at 1500-2000 rpm and wash twice with pre-chilled PBS. Stain cells with Annexin-V and PI, and measure fluorescence using flow cytometry. Analyze results using FlowJo software.

[0085] (b) Cycle: Collect the cells needed for the experiment by centrifugation at 1500-2000 rpm, wash twice with pre-chilled PBS, and fix with 70% alcohol overnight at 4°C. Wash the cells twice with pre-chilled PBS, remove the supernatant by centrifugation, and resuspend the cell pellet in 0.5 ml of PI / RNase staining solution. Incubate at room temperature in the dark for 30 minutes. Cell cycle analysis was performed by flow cytometry and analyzed using ModFit 5.0 software.

Claims

1. The role and therapeutic application of MAZ gene in Ph-positive acute lymphoblastic leukemia.

2. The use according to claim 1, characterized in that The Ph chromosome-positive acute lymphoblastic leukemia (Ph+ALL) is a type of B-cell acute lymphoblastic leukemia having the t(9;22)(q34;q11.2) chromosomal translocation or the BCR-ABL1 fusion gene.

3. The use according to claim 1, characterized in that The MAZ gene is a MYC-related zinc finger protein that has DNA-binding transcription factor activity and is involved in multiple biological processes, including the regulation of gene expression.

4. The use according to claim 1, characterized in that The MAZ gene is used as a target in the preparation of drugs for treating Ph-positive acute lymphoblastic leukemia.

5. The use according to claim 3, characterized in that The drug targeting the MAZ gene is based on interference and inhibition of the MAZ gene, and can effectively and specifically interfere and inhibit the expression of MAZ mRNA and MAZ protein in Ph-positive acute lymphoblastic leukemia, or can effectively and specifically reduce the biological activity of MAZ protein in Ph-positive acute lymphoblastic leukemia cells.

6. The use according to claim 3 or claim 4, characterized in that: The substance that inhibits the MAZ encoding gene and its expression product and / or reduces its biological activity has the following effects in Ph-positive acute lymphoblastic leukemia: (1) Inhibit the proliferation of Ph-positive acute lymphoblastic leukemia cells; (2) Promote apoptosis of Ph-positive acute lymphoblastic leukemia cells; (3) Inducing cell cycle arrest in Ph-positive acute lymphoblastic leukemia cells; (4) Improve the sensitivity of Ph-positive acute lymphoblastic leukemia cells to treatment with the tyrosine kinase inhibitor imatinib (Imatinib Mesylate, IM).

7. The use according to claim 5, characterized in that Substances that inhibit the MAZ encoding gene and its expression products and / or reduce its activity include RNA interference molecules or antisense oligonucleotides, compound inhibitors, siRNA, shRNA targeting MAZ, substances for implementing lentiviral infection or gene knockout, and specific antibodies targeting MAZ itself or its upstream and downstream molecules.

8. The use according to claim 1, characterized in that The function of the MAZ gene in Ph-positive acute lymphoblastic leukemia cells is to promote the transcription of the SIRT5 gene.

9. The use according to claim 1, characterized in that MAZ binds to the promoter region of SIRT5 in the nucleus of Ph-positive acute lymphoblastic leukemia cells and initiates transcription, thereby promoting the increase of SIRT5 mRNA levels in Ph+ALL.

10. The use according to claim 7, characterized in that The MAZ protein can promote the transcription of SIRT5 by binding to the -739 to -638 and -79 to +49 regions upstream of the SIRT5 transcription start point and the promoter region to achieve an increase in the SIRT5 mRNA level.

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