Application of cancer suppressor gene SLC26A11 in hyperleukocyte acute B lymphocytic leukemia

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a cancer suppressor gene SLC26A11 in hyperleukocyte acute B lymphocytic leukemia. The invention discloses an application of a cancer suppressor gene SLC26A11 as a specific molecular marker in preparation of induction treatment reaction, prognosis and risk assessment of high leukocyte acute B lymphocytic leukemia of children, and an application in improvement of sensitivity of chemotherapeutic drugs for high leukocyte acute B lymphocytic leukemia of children. The SLC26A11 is used as a potential target, so that a personalized treatment scheme is provided for HALL patients, the toxic and side effects of chemotherapeutic drugs are reduced, and the treatment effect is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of the tumor suppressor gene SLC26A11 in hyperleukocytic acute B lymphoblastic leukemia. Background Art

[0002] Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, accounting for approximately 25%-30% of all childhood malignancies and a leading cause of death in children. Over the past three decades, standard chemotherapy has significantly improved the prognosis of childhood ALL, with a 5-year survival rate exceeding 80%. Hyperleukocytic acute lymphoblastic leukemia (HALL), a specific type of ALL characterized by a significantly elevated peripheral white blood cell count (WBC ≥ 50 x 10^9 / L) at diagnosis, accounts for approximately 10.2% to 19.2% of childhood ALL and carries a higher risk of induction therapy failure, relapse, and mortality. In developing countries, the 5-year overall survival rate for children with HALL is only 37.2%, significantly lower than that of children without HALL. Long-term quality of life is also poor, necessitating an urgent need for new treatment strategies to improve the prognosis of children with HALL.

[0003] The reasons for failure of induction therapy in childhood HALL (HALL) remain unclear. ALL is a clonal proliferation disorder. Chemotherapy can suppress or eliminate the dominant ALL clone, leaving behind subclones with primary or acquired mutations, which can lead to drug resistance. Further expansion of resistant subclones increases the risk of ALL relapse. Current studies indicate that chemotherapy resistance in ALL is closely linked to genetic mutations or deletions. For example, the TP53 R248Q mutation induces resistance to multiple drugs in ALL cells, including vincristine, daunorubicin, and cytarabine. In mismatch repair (MMR)-deficient leukemia, thiopurine therapy may induce drug-resistant relapse in ALL cells through specific mutational signatures, including the hotspot TP53 R248Q mutation. Although these studies provide important molecular insights into drug resistance mechanisms in ALL, the specific resistance mechanisms in children with HALL remain unclear.

[0004] Therefore, revealing the molecular mechanism of HALL-induced drug resistance and finding new therapeutic targets have become the key to the current precision treatment of ALL. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of the tumor suppressor gene SLC26A11 in hyperleukocytic acute B-lymphocytic leukemia, so as to provide personalized treatment plans for HALL patients, reduce the toxic and side effects of chemotherapy drugs, and improve the treatment effect.

[0006] To this end, the present invention provides the following technical solutions.

[0007] The first aspect of the present invention provides a use of the tumor suppressor gene SLC26A11 as a specific molecular marker in the preparation of an in vitro clinical detection product for induction treatment response, prognosis and risk assessment of childhood hyperleukocytic acute B lymphoblastic leukemia.

[0008] In a preferred embodiment of the present invention, the product comprises a reagent, a kit or a medicine.

[0009] In a preferred embodiment of the present invention, the reagent or kit comprises a specific primer pair for detecting the SLC26A11 gene, wherein the specific primer pair comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2.

[0010] In a preferred embodiment of the present invention, the kit comprises an antibody that specifically recognizes the SLC26A11 protein.

[0011] A second aspect of the present invention provides a pharmaceutical composition for treating childhood hyperleukocytic acute B lymphoblastic leukemia, wherein the pharmaceutical composition comprises an agent for overexpressing SLC26A11.

[0012] In a preferred embodiment of the present invention, the SLC26A11 overexpression agent includes a SLC26A11 agonist, an exogenous SLC26A11 gene product, or an in vitro synthesized SLC26A11 protein biological preparation.

[0013] In a preferred embodiment of the present invention, the SLC26A11 overexpression agent is a SLC26A11 overexpression lentivirus.

[0014] In a preferred embodiment of the present invention, the pharmaceutical composition further comprises pegaspargase.

[0015] A third aspect of the present invention provides a use of an SLC26A11 overexpression reagent in the preparation of a drug for improving the sensitivity of childhood hyperleukocytic acute B lymphoblastic leukemia to chemotherapy drugs.

[0016] In a preferred embodiment of the present invention, the SLC26A11 overexpression agent includes a SLC26A11 agonist, an exogenous SLC26A11 gene product, or an in vitro synthesized SLC26A11 protein biological preparation.

[0017] In a preferred embodiment of the present invention, the SLC26A11 overexpression agent is a SLC26A11 overexpression lentivirus.

[0018] In a preferred embodiment of the present invention, the drug comprises pegaspargase.

[0019] A fourth aspect of the present invention provides a kit for detecting the sensitivity of childhood hyperleukocytic acute B lymphoblastic leukemia to pegaspargase treatment, the kit comprising at least an antibody that specifically binds to the SLC26A11 protein.

[0020] In a preferred embodiment of the present invention, the kit is selected from any one of a qPCR kit, an immunoblotting detection kit, a flow cytometry analysis kit, and an ELISA kit.

[0021] By means of the above technical solution, the present invention has at least the following advantages: Through a series of experiments, this study systematically reveals the direct association between SLC26A11 and the failure of induction therapy for HALL, filling a gap in the research on the molecular mechanisms of drug resistance to induction therapy for HALL and laying a theoretical foundation for further research. This study also reveals the use of the tumor suppressor gene SLC26A11 as a specific molecular marker for the preparation of induction therapy responses, prognosis, and risk assessment for childhood hyperleukocytic acute B-lymphoblastic leukemia, as well as for enhancing the sensitivity of chemotherapeutic drugs for childhood hyperleukocytic acute B-lymphoblastic leukemia. As a potential target, SLC26A11 not only provides personalized treatment options for HALL patients, but also reduces the toxic side effects of chemotherapy drugs and improves treatment efficacy.

[0022] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The differential expression analysis method was used to identify candidate genes that were significantly abnormally expressed in children with HALL who failed induction therapy. Figure 2 The expression of SLC26A11 in bone marrow samples of children with HALL and non-HALL is shown; Figure 3 The map of the recombinant plasmid overexpressing SLC26A11 is shown; Figure 4 The expression of SLC26A11 in the Nalm6 control group (NC) and overexpression group (OE) is shown; Figure 5 Shown is the WB validation of SLC26A11 in cell lines; Figure 6The proliferation of SLC26A11-Nalm6 cells at 0 h, 24 h, 48 h, and 72 h is shown; Figure 7 The cell cycle of SLC26A11-Nalm6 cells is shown; Figure 8 Apoptosis of SLC26A11-Nalm6 cells is shown; Figure 9 SLC26A11-Nalm6 cell migration is shown; Figure 10 The effect of SLC26A11 on the sensitivity of pegaspargase drug treatment is shown; Figure 11 The expression of SLC26A11 in mouse peripheral blood and bone marrow is shown; Figure 12 Shown are the staining images of mouse liver, spleen, and sternum tissues, from top to bottom: liver, spleen, sternum; Figure 13 Shown are immunohistochemistry images of mouse spleen. DETAILED DESCRIPTION

[0024] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0025] The present invention is based on existing research and has the following main deficiencies: 1. Functional loss of SLC26A11: Although SLC26A11 has been identified as a tumor-related gene in multiple cancers, its specific role in ALL has not been studied, especially its function in HALL-induced drug resistance is unknown. 2. Lack of target research: The main research on HALL resistance has focused on traditional gene mutations and microenvironmental regulation, and lacks in-depth exploration of new potential therapeutic targets. 3. Lack of clinical translation: Current research on HALL has not yet formed a clear treatment strategy, and there is a lack of new molecular targets that can guide precision treatment, resulting in limited treatment effects and a high recurrence rate. Therefore, those skilled in the art have proposed the use of the SLC26A11 gene in inhibiting B-HALL-induced drug resistance, providing a candidate molecular target for HALL-induced drug resistance treatment, and providing important clues for the exploration of new auxiliary diagnostic indicators and prognostic markers for childhood ALL.

[0026] This study collected data from consecutive children with HALL and non-HALL matched by age, gender, and immunophenotype who were admitted to Nanjing Children's Hospital. Through analysis of clinical data, induction therapy responses, and whole transcriptome data, and combined with external data verification from St. Jude Children's Research Hospital in the United States, it was found that the gene SLC26A11 was closely associated with the failure of induction therapy for hyperleukocytic B-acute lymphoblastic leukemia (B-HALL). In vitro drug sensitivity data predicted that children with low SLC26A11 expression were resistant to pegaspargase. Cell experiments confirmed that overexpression of SLC26A11 increased the drug sensitivity of ALL cells to pegaspargase and reduced the malignant proliferation ability of leukemia cells. This discovery provides a new genetic basis for the mechanism of resistance to induction therapy in HALL.

[0027] Whole-transcriptome analysis not only comprehensively captures changes in gene expression but also identifies the role of key genes in pathological processes. This study, using whole-transcriptome analysis combined with large-scale external data validation, systematically reveals a direct association between SLC26A11 and the failure of induction therapy for HALL. This external data validation further confirms the broad applicability of SLC26A11 as a core regulatory gene. Through mechanistic research, this study fills a gap in the understanding of the molecular mechanisms of resistance to induction therapy for HALL and lays a theoretical foundation for further research.

[0028] In one embodiment, the use of the tumor suppressor gene SLC26A11 as a specific molecular marker in the preparation of an in vitro clinical test product for evaluating the response to induction therapy, prognosis, and risk of childhood B-HALL is provided.

[0029] As used herein, the SLC26A11 gene (NCBI ID: 284129) is located on chromosome 17q25.3 and encodes SLC26A11 / KBAT (kidney-brain anion transporter), a member of the SLC26 anion transporter family whose main functions are maintaining chloride homeostasis, oxalate excretion and kidney stone formation, vascular volume and blood pressure regulation, and acid-base balance.

[0030] Preferably, the in vitro clinical detection product includes a reagent, a kit or a drug.

[0031] Preferably, the reagent or kit comprises a specific primer pair for detecting the SLC26A11 gene, wherein the specific primer pair comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2.

[0032] Preferably, the kit comprises an antibody that specifically recognizes the SLC26A11 protein.

[0033] In another embodiment, a pharmaceutical composition for treating childhood hyperleukocytic acute B-lymphoblastic leukemia is provided, comprising a SLC26A11 overexpression agent. Preferably, the SLC26A11 overexpression agent comprises a SLC26A11 agonist, an exogenous SLC26A11 gene product, or an in vitro synthesized SLC26A11 protein biologic. More preferably, the SLC26A11 overexpression agent is a SLC26A11 overexpression lentivirus.

[0034] As a pharmaceutical composition for treating childhood hyperleukocytic acute B-lymphocytic leukemia, the composition also includes the commercially available therapeutic drug pegaspargase. By combining the SLC26A11 overexpression lentivirus with pegaspargase, the drug resistance of pegaspargase can be significantly improved, further enhancing the therapeutic effect.

[0035] This study, based on the theory that targeted gene therapy strategies can significantly enhance the sensitivity of therapeutic drugs to drug-resistant cells, thereby avoiding the extensive toxicity of traditional chemotherapy to normal cells, clarified the pathogenic role of SLC26A11 in drug resistance in leukemia cells through gene function validation experiments and proposed its feasibility as a precision therapy target. Further experiments confirmed that increasing SLC26A11 expression can enhance the sensitivity of leukemia cells to the chemotherapy drug pegaspargase. This result suggests that SLC26A11, as a potential target, not only provides personalized treatment options for HALL patients, but also reduces the toxic side effects of chemotherapy drugs and improves treatment efficacy.

[0036] Therefore, in yet another embodiment, a use of an SLC26A11 overexpression reagent in the preparation of a drug for improving the sensitivity of childhood hyperleukocytic acute B-lymphocytic leukemia to chemotherapy drugs is provided.

[0037] Preferably, the SLC26A11 overexpression agent comprises a SLC26A11 agonist, an exogenous SLC26A11 gene product, or an in vitro synthesized SLC26A11 protein biological preparation. Preferably, the SLC26A11 overexpression agent is a SLC26A11 overexpression lentivirus. Preferably, the drug further comprises pegaspargase.

[0038] In another embodiment, a kit for detecting the sensitivity of childhood hyperleukocytic acute B-lymphocytic leukemia to pegaspargase treatment is provided, wherein the kit comprises at least an antibody that specifically binds to the SLC26A11 protein.

[0039] Preferably, the kit is selected from any one of a qPCR kit, an immunoblotting kit, a flow cytometry kit, and an ELISA kit. When it is a qPCR kit, the kit comprises a specific primer pair for detecting the SLC26A11 gene, wherein the specific primer pair comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2; when it is an ELISA kit, the kit comprises an antibody that specifically recognizes the SLC26A11 protein.

[0040] In specific examples of the present invention, analysis of clinical data, induction therapy responses, and whole transcriptome data, combined with external data validation from 805 cases at St. Jude Children's Research Hospital in the United States, identified the gene SLC26A11 as closely associated with induction therapy failure in B-HALL, and children with low SLC26A11 expression had a worse prognosis. Further validation of the significant association between significantly low SLC26A11 gene expression and failure of induction therapy for HALL and, through gene-in vitro drug sensitivity comparison, predicted resistance to pegaspargase in HALL children with low SLC26A11 expression. The present invention is further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit its scope. Conventional reagents used in the following examples are commercially available, and the biological experiments performed are conventional in the art and can be performed according to the instructions in the corresponding laboratory manual or kit instructions.

[0041] Example 1: Screening of biomarkers and clinical sample collection and verification 1.1 Sample Source All samples in this invention were obtained from Nanjing Children's Hospital and St. Jude Children's Research Hospital in the United States.

[0042] Specific inclusion criteria: 1) Children diagnosed and treated at Nanjing Children's Hospital between July 2019 and March 2023 with confirmed acute B-lineage lymphoblastic leukemia by bone marrow cell morphology and immunophenotyping; 2) Age at diagnosis was 0-17 years old, regardless of gender; Specific exclusion criteria: patients with other chronic diseases and congenital diseases, such as Down syndrome and systemic lupus erythematosus.

[0043] Group information: Development cohort: 87 consecutive HALL patients and 96 age-, sex-, and immunophenotype-matched non-HALL patients admitted to Nanjing Children's Hospital.

[0044] Validation cohort: 805 ALL patients from a public database at St. Jude Children's Research Hospital, USA.

[0045] 1.2 Screening of markers Bone marrow specimens from newly diagnosed children with HALL and clinically residual bone marrow samples from non-HALL patients were collected from the Children's Hospital Affiliated to Nanjing Medical University. All participants signed informed consent. Whole-transcriptome RNA-Seq sequencing was used to sequence the bone marrow samples from 87 children with HALL and 96 children without HALL. Combined with the results of minimal residual disease (MRD) after induction therapy, differential expression analysis methods (such as DESeq2) were used to identify candidate genes that were significantly abnormally expressed in children with HALL induction therapy failure: SERPINI2, ARTN, LTK, MX1, DSG2, AXIN2, SLC26A11, and TMPRSS15 (see ). Figure 1 Initial screening revealed that the gene SLC26A11 was closely associated with the failure of induction therapy for B-HALL, and that children with low SLC26A11 expression had a worse prognosis.

[0046] 1.3 External Database Verification The public database of 805 ALL patients from St. Jude Research Hospital in the United States (sequencing and in vitro drug sensitivity test) was analyzed. The results of sequencing and in vitro drug sensitivity test showed that the LC50 of the low expression group of the gene SLC26A11 was significantly higher than that of the high expression group (LC50 低 =0.494 IU / ml, LC50 高 =0.392 IU / ml, p =0.013), suggesting that high expression of SLC26A11 gene leads to higher drug sensitivity to pegaspargase.

[0047] 1.4 Human sample verification—RT-PCR experiment 1) RNA extraction and RT-PCR Total RNA was extracted from bone marrow samples of children with B-ALL (obtained from Nanjing Children's Hospital) using Trizol and quantified using a visible light spectrophotometer. The RNA was then amplified using reverse transcription polymerase chain reaction (RT-PCR). β-Actin was used as an internal reference gene, and the primer sequences used are shown in Table 1.

[0048] Table 1 Primer sequences used in RT-PCR Reverse transcription PCR (RT-PCR) was performed using the system shown in Table 2 and the procedure shown in Table 3, specifically including: according to the description of the reverse transcription kit instructions (Aikuer biological, item number: AG11706), the following 20 μL reverse transcription system shown in Table 2 was configured on ice. The mixed system was shaken well and mixed, and then centrifuged to collect the mixture at the bottom of the tube. Finally, it was placed in a PCR instrument for reverse transcription. According to the reverse transcription kit instructions, the following Table 3 shows the program for the reaction. After the reaction, the reaction product cDNA was collected and stored at -20°C for short-term storage or -80°C for long-term storage.

[0049] Table 2 RT-PCR reaction system Table 3 RT-PCR reaction program 2) qRT-PCR Then, the obtained cDNA was subjected to quantitative real-time PCR (qRT-PCR) reaction using the system shown in Table 4 and the procedure shown in Table 5, specifically including: first, according to the description of the kit instructions (Aikuer biological, item number: AG11718), the following 10 μL PCR system shown in Table 4 was configured on ice. Add the above mixed system to the 96-well PCR plate, all steps are carried out on ice, then centrifuge in the plate rack, and then perform amplification reaction in Roche LightCycler96 real-time fluorescence quantitative PCR instrument, qRT-PCR reaction program is set as shown in Table 5. After the reaction, the relative quantification-2 -△△CT value method calculation results are shown in Figure 2 . As Figure 2 shown, compared with the high white group (HALL patient group), the expression of SLC26A11 in the non-high white group (non-HALL patient) group was significantly increased.

[0050] Table 4 qRT-PCR reaction system Table 5 qRT-PCR reaction program Example 2: Ex vivo cell experiment Lentivirus packaging plasmid GV703 (CMV enhancer-MCS-3FLAG-EF1a-ZsGreen1-T2A-puromycin) (purchased from Shanghai Jikai Gene Medical Technology Co., Ltd., item number: GOSL0392138). First, the coding sequence of SLC26A11 gene (NCBI ID: 284129) was connected to the lentivirus expression vector GV703 to obtain a recombinant vector. The recombinant vector was detected by DNA sequencing, and a recombinant plasmid overexpressing SLC26A11 was constructed, and the plasmid map is shown in Figure 3 . The lentivirus liquid overexpressing SLC26A11 was obtained by the steps of plasmid transformation, lentivirus packaging and concentration, lentivirus titration detection, etc.

[0051] Lentivirus transfection: 24-well plates were used, and two groups were set in the experiment--control group (NC group) and overexpression group (OE group), 5x10 6 Nalm6 cells (a B-ALL cell line, purchased from the Shanghai Cell Biology Research Institute of the Chinese Academy of Sciences) were inoculated in each well, and the cell volume in each well was 500 μL. Before operation, the cell state and cell density of each group were observed under a microscope to ensure that the cells were in the appropriate state and quantity. According to the lentivirus transfection steps of Jikai, the appropriate MOI value (16) was selected, 2.5 μL of SLC26A11 control group virus liquid (blank plasmid GV703, titer: 2E+9 TU / mL) was added to the NC group well, and 2.5 μL of SLC26A11 overexpression virus liquid (titer: 2E+9 TU / mL) was added to the OE group well. After completion, the cells in each group were shaken in the shape of 8, and then placed in a cell incubator at 37.0°C and 5% CO2. After 12 hours, the cell state was observed under a microscope, and after 48 hours, the fluorescence was observed under a fluorescence microscope to determine whether the transfection was successful and the transfection efficiency.

[0052] Stable strain screening: Before the experiment, the concentration gradient of puromycin was set to determine the puromycin screening concentration of 4 ug / mL. After the cells were transduced to grow to about 60%, the screening medium was added for screening.

[0053] Transfection efficiency verification: The stable strains selected were plated into 6-well plates, 3 replicates per group. Total RNA was extracted from each group of cells and reverse transcribed into cDNA for PCR amplification. Total protein was extracted from each group of cells for Western blotting experiments.

[0054] 2.1 qPCR verification The cells in the NC and OE groups were collected in 5 mL centrifuge tubes, resuspended and washed once with PBS, and the PBS was discarded. The cells were resuspended and lysed with Trizol, and the RNA in each group was extracted using an RNA extraction kit, and the results are shown in Figure 4 .

[0055] like Figure 4 As shown in the data, in Nalm6 cells (NC group, blank control group), the mRNA level of SLC26A11 was significantly lower than that in the overexpression group (OE group, SLC26A11 overexpression group).

[0056] 2.2 Protein Verification 2.2.1 Total protein extraction and protein concentration determination of cell lines (1) Total protein extraction: Pre-cool the centrifuge at 4°C. Collect the Nalm6 cell pellets from the NC and OE groups transfected with the SLC26A11 gene and place them in 1.5 mL EP tubes. Place the pellets on ice. Add an appropriate amount of lysis buffer to each tube based on the cell count, pipette and mix thoroughly. Place the pellets on ice for 25 minutes. Ultrasonicate on ice at 50W until foam is just formed. Centrifuge at 4°C and 14,000g for 20 minutes. Pipette the supernatant into a new 1.5 mL EP tube. Place the pellets on ice and immediately determine the protein concentration.

[0057] (2) Protein concentration determination: Prepare BCA working solution and keep it away from light. Prepare a protein standard with a stock concentration of 0.5 mg / mL. Use RIPA to dilute the protein standard in a concentration gradient of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL. Add 20 μL to each well of a 96-well plate to draw a standard curve. Add 2 μL of protein sample and 18 μL of RIPA to each well, and then add 200 μL of BCA working solution to each well. Be careful to avoid bubbles during the addition process. After the addition is completed, incubate in a 37°C oven for 30 minutes. Set the microplate reader to 562 nm to detect the absorbance value, draw a standard curve, and use it if the fit is >0.9. Calculate the sample protein concentration according to the formula.

[0058] (3) Protein denaturation: The obtained protein stock solution and 4X SDS were mixed in a ratio of 3:1, vortexed and centrifuged immediately, then heated in a 100℃ metal bath for 10 minutes, quickly cooled on ice after heating, and finally aliquoted and stored in a -80℃ deep freezer for later use.

[0059] 2.2.2 Western blotting (1) Gel preparation: According to the experimental requirements and the molecular weight of the target protein, prepare separation gel (concentration 10%) and stacking gel (concentration 5%). The preparation ratios are shown in Table 6 below: Table 6 Composition of separation gel and stacking gel Note: 1. The reagents must be returned to room temperature before use; 2. TEMED should be added last.

[0060] Clean the glass plate, rinse thoroughly with ddH2O, and dry in a 37°C oven. Check the glass plate and gel rack to prevent gel leakage. Prepare the separating gel solution according to Table 6 above. Mix gently and quickly to avoid bubbles. Add the solution to the clamped glass plate and flatten the separating gel with anhydrous ethanol. After the separating gel solidifies, discard the anhydrous ethanol. After evaporation, add the prepared stacking gel and slowly insert the comb to prevent splashing. Once the stacking gel has solidified, remove it and set aside.

[0061] (2) Electrophoresis: Load the PAGE gel from the previous step into the electrophoresis tank, pour in the electrophoresis solution, and check for leaks. Pull out the comb vertically, and add the protein sample and protein marker. Set the electrophoresis conditions: constant voltage 80V, 30 minutes, constant voltage 120V, 60 minutes. The electrophoresis conditions can be adjusted according to the molecular weight of the target protein.

[0062] (3) Transfer: After electrophoresis, activate the PVDF membrane of appropriate size in methanol for at least 30 seconds. Soak the filter paper completely in the pre-cooled transfer solution and set aside. Make a "sandwich" by placing the black plate (-, negative electrode), sponge, 2 layers of filter paper, glue, PVDF membrane, 2 layers of filter paper, black sponge, (+, positive electrode) white plate in this order, remove all bubbles, and tighten the clamp. Insert the clamp into the tank according to the positive and negative poles, put an ice box in the tank, pour in the transfer solution, and place it in an ice water bath. Set the transfer conditions: constant current, 300mA, 90min. The transfer conditions can be adjusted according to the molecular weight of the target protein.

[0063] (4) Blocking: After the transfer is completed, place the PVDF membrane in 5% milk blocking solution and block for 2 hours.

[0064] (5) Primary antibody incubation: Dilute the target antibody stock solution with primary antibody diluent according to the antibody instructions, place the PVDF membrane in the incubation box, pour in the primary antibody (Leading Biology, catalog number: APR23601N), and incubate on a shaker at 4°C overnight.

[0065] (6) Secondary antibody incubation: Place the PVDF prepared in the previous step in a clean incubation box, add 1X TBST buffer, and wash the membrane on a horizontal shaker. Change the buffer every 5 minutes. Repeat the washing process 5 times, then add the appropriate secondary antibody (Proteintech, catalog number: 60004-1-Ig) and incubate on a shaker at room temperature for 1-2 hours.

[0066] (7) Development: After washing the membrane 5 times on a shaker with 1X TBST, prepare an appropriate volume of ECL chemiluminescent solution, use it immediately, and evenly cover it on the PVDF. Save the image after exposure on the gel imaging system, and use Image J for quantitative analysis of grayscale values. The results are shown in Figure 5 .like Figure 5As shown, compared with the blank control group NC group, the expression of SLC26A11 protein in the B-ALL cells in the OE group transfected with the SLC26A11 overexpressing lentivirus was significantly increased.

[0067] 2.3 Cell function experiments 2.3.1 Cell proliferation assay (1) Cell counting: Collect Nalm6 cells stably transfected with SLC26A11 lentivirus (OE group) and cells with control virus (NC group) in good logarithmic growth phase into appropriate centrifuge tubes, add appropriate amount of PBS to wash the cells, aspirate 100 μL of cells, add PBS to dilute the appropriate multiple, aspirate 10 μL of cells and inoculate them onto a cell counting plate, let it stand for about 1 minute, and count under a microscope.

[0068] (2) Plating and OD value detection: Each group of cells (5×10³ / well) was inoculated into a 96-well culture plate. Before each cell was taken, the cells were blown to mix, and shaken vigorously to evenly. Six replicate wells were set. 1640 culture medium was added around the culture plate to prevent the influence of water evaporation and culture medium color on the accuracy of OD value. After 0, 24, 48, and 72 hours of culture, 10 μL CCK8 reagent was added to avoid the generation of bubbles. The cells were incubated in an incubator for 2-3 hours. After the incubation, the absorbance (OD value) was measured with a microplate reader to reflect the cell proliferation ability. The wavelength was set to 450 nm. The results are shown in the table. Figure 6 .like Figure 6 As shown in Figure 3, CCK8 experiments demonstrated that the proliferation of B-ALL cells in the SLC26A11 overexpression group (OE group) was significantly reduced compared with that in the NC group.

[0069] 2.3.2 Cell cycle assay Collect 1×10 6 Cells stably transfected with SLC26A11 overexpression lentivirus (OE group) and control virus (NC group) in good growth phase were placed in appropriate centrifuge tubes, washed once with PBS, centrifuged at 1500rpm for 5 minutes, and the supernatant was discarded. Add 500 μL of pre-cooled 75% ethanol for fixation (2 hours to overnight), store at 4°C, and wash off the fixative with PBS before staining. Add 100 μL of RNase A and incubate at 37°C in a water bath for 30 minutes, or place in a 37°C incubator for 30 minutes. Then add 400 μL of PI staining solution and react at 4°C in the dark for 30 minutes. Save the data after detection on the machine and analyze it using FlowJo software. The results are shown in the figure. Figure 7 .like Figure 7 As shown, compared with the OE group, the number of S+G2 phase cells, i.e., division phase cells, in the B-ALL cells in the NC group was significantly increased.

[0070] 2.3.3 Cell apoptosis assay (flow cytometry) Collect 1×10 6 Cells stably transfected with SLC26A11 overexpression lentivirus (OE group) and control virus (NC group) in a good growth phase were placed in a suitable centrifuge tube, washed twice with PBS, and centrifuged at 1500 rpm for 5 minutes. The cells were resuspended to 1×10 6 100 μL of cell suspension was pipetted into a flow cytometry sample tube. 5 μL of PE Annexin V was added and incubated in the dark for 10 minutes. 5 μL of 7-AAD was then added and incubated in the dark for 5 minutes. 400 μL of 1X Binding Buffer was added and the data was saved after testing on the flow cytometry machine. Analyzed using FlowJo software, the results are shown in the table. Figure 8 .like Figure 8 As shown in Figure 3, the apoptosis of B-ALL cells in the OE group overexpressing SLC26A11 was significantly increased compared with that in the NC group.

[0071] 2.3.4 Cell migration assay (Transwell) (1) Chamber pretreatment: Prepare a 24-well plate and several Transwell chambers in advance. The day before the experiment, turn the chamber upside down and apply 50 μL of FN dilution to the bottom of each chamber. Place the chamber in a clean bench for 2 hours to allow it to air dry naturally. After air drying, place it in a cell culture incubator overnight.

[0072] (2) Cell inoculation: On the day of the experiment, the cells stably transfected with SLC26A11 overexpression lentivirus (OE group) and control group virus (NC group) were suspended in serum-free culture medium (1640 culture medium), and the cells were counted and the cell density was adjusted to 1×10 6 Cells were plated at 400 μL / mL. 100 μL was injected into the upper layer of the Transwell chamber. 600 μL of culture medium containing 10% FBS was added to the lower layer of the chamber (i.e., the 24-well plate). The cells may be starved the day before the experiment, depending on the conditions. The plated cells were cultured in a cell incubator for 24-48 hours. Adjust the incubation time based on the cell type.

[0073] (3) Fixation + staining: Remove the Transwell chamber and fix the cells on the chamber membrane with 4% paraformaldehyde for about 20-30 minutes. Wash the chamber three times with PBS and let it dry naturally. Then stain with crystal violet for about 20 minutes. Wash off the crystal violet with PBS and gently wipe off the cells in the upper chamber with a cotton swab. After the chamber is air-dried, place the chamber on a slide and observe and photograph it under a microscope. Take a typical field of view at the top, bottom, left, right, and center of each chamber. Use Image J to count cells and perform statistical analysis. The results are shown in Figure 9 .like Figure 9As shown in Figure 3, the migration ability of B-ALL cells in the OE group overexpressing SLC26A11 was significantly reduced compared with that in the NC group.

[0074] 2.3.5 Drug sensitivity test Nalm6 cells (NC group) and Nalm6 cells overexpressing SLC26A11 (OE group) were treated with different concentrations of pegaspargase (purchased from Hengrui Medicine Co., Ltd., trade name: Aiyang) for 72 hours. Cell proliferation was then detected using CCK8, and the drug concentration that achieved 50% proliferation of B-ALL cells (IC50) was calculated. Overexpression of SLC26A11 increased the sensitivity of Nalm6 cells to pegaspargase. The results are shown in Figure 10 .like Figure 10 As shown, the half-maximal inhibitory concentration of B-ALL pegaspargase in the SLC26A11 control group (NC group) was 0.004931 IU / mL, and the half-maximal inhibitory concentration of B-ALL pegaspargase in the SLC26A11 overexpression group (OE group) was 0.0020821 IU / mL.

[0075] The above experiments show that overexpression of SLC26A11 in B-ALL cells can significantly inhibit the proliferation and migration of acute B lymphocytes, inhibit the cell cycle, promote cell apoptosis, and reduce the migration ability of B-ALL cells. Drug sensitivity results show that overexpression of SLC26A11 can increase the sensitivity of tumor cells to the drug pegaspargase, providing a potential molecular target for the chemotherapy sensitivity treatment of acute B lymphocytes.

[0076] Example 3: Nalm6 cell tail vein injection experiment in nude mice Ten M-NSG nude mice (purchased from Shanghai Model Organisms Science Co., Ltd.) were provided with ample sterile feed and water. They were housed in an environment with a temperature of 23 ± 2°C, a humidity of 45-65%, and a 12-hour day / night cycle for one week of acclimatization. After weighing, the mice were randomly divided into two groups: the NC group and the OE group, with 5 mice in each group. The mice in each group were treated as follows: NC control group (5 rats): Nalm6 cells of the control group (NC group) in Example 2 were injected into the tail vein; OE intervention group (5 mice): Nalm6 cells overexpressing SLC26A11 (OE group) as described in Example 2 were injected into the tail vein; Ceturegel™ high-concentration matrix gel and cell suspension were diluted in a 1:1 ratio and inoculated into the tail vein of 4-5 week-old female mice. The specific experimental procedures are as follows: 1. Prepare Nalm6 cells in logarithmic phase growth with a cell density of about 80-90%. Replace the culture medium with fresh medium the night before cell collection.

[0077] 2. Collect the cells by centrifugation, add serum-free medium (Gibco product number: C11875500BT) and resuspend the cells to a final concentration of 5×10 7 cells / mL to obtain a cell suspension.

[0078] 3. Combine the obtained cell suspension and Ceturegel TM High concentration matrix gel was diluted at a ratio of 1:1 at 4°C to prepare a final concentration of 5 × 10 7 cells / mL.

[0079] 4. Grasp and fix the nude mice with the left hand, and inject into the tail vein of each group of mice. The inoculation volume is 100 μL (cell density is 5×10 7 / mL). (This process should be completed within half an hour. The cell suspension should be placed on ice during the process to slow down cell apoptosis and prevent gelation.)

[0080] 5. After the injection is completed, the mice are returned to the cage and continued to be raised. The nude mice are observed daily for their general condition (mental state, diet and activity, hair and urine and feces). They are weighed twice a week after 1 week of inoculation. When signs of disease appear (mental depression, reduced diet and activity, erect hair, 20% weight loss), the test is strengthened. After about 3 weeks, the nude mice are bled from their orbits. After euthanasia, the mice are dissected and the bone marrow, liver, and spleen are taken for further experiments and photographed. The expression levels of SLC26A11 in the peripheral blood and bone marrow of the two groups of mice are statistically analyzed. The results are shown in Figure 11 .like Figure 11 As shown in the figure, the expression of SLC26A11 gene in the peripheral blood and bone marrow of mice. Compared with the NC group, the expression level of SLC26A11 mRNA in the peripheral blood and bone marrow of OE group mice was significantly higher.

[0081] The results of organ staining of each group of mice are shown in Figure 12 and 13 .like Figure 12As shown, in the NC group, in the liver, tumor cells showed infiltrative growth, primarily distributed in the portal areas, around the central veins, and in the hepatic sinusoids. The tumor cells were polygonal in shape, with weakly eosinophilic cytoplasm. The nuclei were reniform, lobed, or oval, with prominent nucleoli. Numerous pathological mitotic figures were observed, accompanied by mild cellular necrosis. In the spleen, tumor cells showed infiltrative growth, with the red pulp filled with tumor cells and the white pulp almost invisible. The tumor cells were polymorphic, with nuclei that were round, reniform, lobed, oval, or annular, with prominent nucleoli. Numerous tumor cells of varying stages of differentiation, including erythroid, megakaryocyte, and granulocyte lineages, were observed. Pathological mitotic figures were prominent, accompanied by mild cellular necrosis. In the sternum, tumor cells filled the bone marrow cavity and infiltrated outward. The polygonal tumor cells had bright cytoplasm, with nuclei that were round, oval, reniform, lobed, or annular, with prominent nucleoli. These cells were primarily granulocyte lineage cells of varying stages of differentiation. A moderate increase in mitotic figures can be seen, accompanied by moderate cell necrosis. OE group mice: Liver: Tumor cells grow in an infiltrative manner, mainly distributed around the central vein, and scattered tumor cells can also be seen in the hepatic sinusoids. Tumor cells are oval or kidney-shaped, with round, oval, and kidney-shaped nuclei, obvious nucleoli, and many pathological mitotic figures can be seen. Spleen: Tumor cells grow in an infiltrative manner, with tumor cells scattered in the red pulp, and a small amount of residual white pulp tissue can be seen. Tumor cells are polymorphic, with kidney-shaped, lobed, or oval nuclei, etc., mainly tumor cells of granulocyte origin at different stages of differentiation. Many pathological mitotic figures can be seen; Sternum: Tumor cells fill the bone marrow cavity and grow infiltratively outward. They are polygonal tumor cells with bright cytoplasm, round, oval, kidney-shaped, lobed, ring-shaped nuclei, obvious nucleoli, and mainly tumor cells of granulocyte origin at different stages of differentiation. A mild increase in mitotic figures can be seen, accompanied by mild cell necrosis. As Figure 13 As shown, Ki67 immunohistochemistry of the SLC26A11 gene in mouse liver showed significant differences between the control group (NC group) and the overexpression group (OE group) of mice.

[0082] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with this profession can make slight changes or modifications to equivalent embodiments of the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. Application of the tumor suppressor gene SLC26A11 as a specific molecular marker in the preparation of in vitro clinical detection products for induction therapy response, prognosis and risk assessment of childhood hyperleukocytic acute B-lymphoblastic leukemia.

2. The use according to claim 1, characterized in that The product includes a reagent, a kit or a drug.

3. The use according to claim 2, characterized in that The reagent or kit comprises a specific primer pair for detecting the SLC26A11 gene, wherein the specific primer pair comprises an upstream primer whose nucleotide sequence is shown in SEQ ID NO: 1 and a downstream primer whose nucleotide sequence is shown in SEQ ID NO:

2.

4. The use according to claim 2, characterized in that The kit comprises an antibody that specifically recognizes the SLC26A11 protein.

5. A pharmaceutical composition for treating childhood hyperleukocytic acute B lymphoblastic leukemia, characterized in that: The pharmaceutical composition includes an SLC26A11 overexpression agent, which includes a SLC26A11 agonist, an exogenous SLC26A11 gene product, or an in vitro synthesized SLC26A11 protein biological preparation.

6. Application of SLC26A11 overexpression reagent in the preparation of drugs to improve the sensitivity of chemotherapeutic drugs in children with hyperleukocytic acute B-lymphocytic leukemia.

7. The use according to claim 6, characterized in that The SLC26A11 overexpression reagent includes a SLC26A11 agonist, an exogenous SLC26A11 gene product, or an in vitro synthesized SLC26A11 protein biological preparation.

8. The use according to claim 6, characterized in that The drug includes pegaspargase.

9. A kit for detecting the sensitivity of children's hyperleukocytic acute B lymphoblastic leukemia to pegaspargase treatment, characterized in that: The kit at least contains an antibody that specifically binds to the SLC26A11 protein.

10. The kit according to claim 9, characterized in that The kit is selected from any one of a qPCR kit, an immunoblotting detection kit, a flow cytometry analysis kit, and an ELISA kit.

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