Application of Tumor Suppressor Gene QRICH1 in Childhood Acute T Lymphoblastic Leukemia

Abstract

The present invention discloses the application of a novel tumor suppressor gene QRICH1 in childhood acute T-lymphoblastic leukemia, and the application is that the QRICH1 gene serves as a specific molecular marker for the prognosis and risk assessment of childhood acute T-lymphoblastic leukemia. On the basis of systematically studying the role and mechanism of QRICH1 in childhood T-ALL, the present invention provides a new molecular marker for the prognosis and disease condition assessment of childhood T-ALL, provides new targets and ideas for its treatment, and provides a necessary theoretical basis for subsequent research and clinical transformation.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to an application in the prognosis / risk assessment and molecular targeted therapy of childhood acute T-lymphoblastic leukemia. Background Art

[0002] Childhood acute T-lymphoblastic leukemia (T-ALL) is a group of malignant tumors derived from T-lymphocyte precursors in blood, bone marrow, and extramedullary lymphoid tissues, accounting for 10% - 15% of childhood ALL. It has a poor prognosis and is one of the important malignancies leading to child death. Although the 5-year survival rate has generally increased with the continuous development of multidisciplinary fields such as cytogenetics and molecular biology and the continuous improvement of treatment methods, some subtypes such as central nervous system leukemia, early T-cell precursors (ETPs) leukemia, refractory / relapsed (R / R) types, etc. still threaten the lives of children. Therefore, further studying the pathogenesis of childhood T-ALL at the molecular level, providing a more accurate assessment of the risk and prognosis of childhood T-ALL, and developing new therapeutic targets are still the key points of current work.

[0003] Based on this, the present invention intends to provide new molecular markers for the prognosis and disease assessment of childhood T-ALL, new targets and ideas for its treatment, and a necessary theoretical basis for subsequent research and clinical transformation on the basis of systematically studying the role and mechanism of QRICH1 in childhood T-ALL. Summary of the Invention

[0004] The object of the present invention is to address the above deficiencies of the prior art and provide an application of a novel tumor suppressor gene QRICH1 in childhood acute T-lymphoblastic leukemia, where the QRICH1 gene serves as a specific molecular marker for the prognosis and risk assessment of childhood acute T-lymphoblastic leukemia.

[0005] Preferably, the application of the QRICH1 gene in the prognosis and risk assessment of childhood acute T-lymphoblastic leukemia.

[0006] Preferably, the QRICH1 gene is used as a specific molecular marker for preparing in vitro clinical detection products for the prognosis and risk assessment of childhood acute T-lymphoblastic leukemia.

[0007] Preferably, the products include: reagents, reagent kits, and drugs.

[0008] Preferably, the reagent and kit contain specific primers for detecting the QRICH1 gene, and the specific primers are the upstream primer shown in SEQ ID NO:1 and the downstream primer shown in SEQ ID NO:2.

[0009] Preferably, the kit contains an antibody that specifically recognizes the QRICH1 protein.

[0010] Preferably, the drug is a drug for treating childhood acute T-lymphoblastic leukemia.

[0011] Preferably, the drug for treating childhood acute T-lymphoblastic leukemia is a QRICH1 agonist, an exogenous QRICH1 gene product, or an in vitro synthesized QRICH1 protein biological agent.

[0012] Preferably, the application of QRICH1 in enhancing the sensitivity of chemotherapy drugs for childhood acute T-lymphoblastic leukemia.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention confirms that QRICH1 is a tumor suppressor gene, and for the first time discovers that QRICH1 serves as a biomarker for the prognosis and risk assessment of childhood T-ALL, and is also a therapeutic target for childhood T-ALL, and can be applied to childhood acute T-lymphoblastic leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0016] Figure 1 It is the expression result diagram of QRICH1 and the survival analysis diagram of different groups of QRICH1; A is the protein expression diagram of QRICH1, and B is the survival analysis results of the QRICH1 high-expression group and the QRICH1 low-expression group;

[0017] Figure 2 It is the expression result diagram of QRICH1 and the expression result diagram of QRICH1 in different clinical subgroups; A is the expression of QRICH1 in adult T-PLL and childhood T-ALL in the GEO database, B is the mRNA expression of QRICH1 in Jurkat and HPBALL cells, C is the QRICH1 protein expression in the bone marrow of 3 cases of childhood T-ALL at the initial stage and remission stage, and D is the expression of QRICH1 in different risk subgroups of childhood T-ALL;

[0018] Figure 3It is a figure showing the functional verification results at the QRICH1 cellular level; A shows that the EdU experiment proves that the proliferation of pediatric T-ALL cells with overexpressed QRICH1 is significantly reduced, B shows that the cell cycle indicates that pediatric T-ALL cells with overexpressed QRICH1 are arrested at the G0 / G1 phase, and C shows that the apoptosis analysis indicates that the apoptosis of pediatric T-ALL cells with overexpressed QRICH1 is significantly increased;

[0019] Figure 4 It is a figure showing the functional verification results at the QRICH1 animal level; A shows the expression of human CD3+ cells in the peripheral blood and bone marrow of mice, and B shows the survival curve of mice xenografted with HPBALL cells with overexpressed QRICH1;

[0020] Figure 5 It is a figure showing the verification results of the indirect mechanism between QRICH1 and downstream GRP78; A shows that the expression of GRP78 mRNA decreases after overexpression of QRCH1, B shows that the expression of GRP78 protein decreases after overexpression of QRCH1, C shows that the expression of CHOP mRNA increases after overexpression of QRCH1, D shows that the expression of CHOP protein increases after overexpression of QRCH1, E shows that the cell viability partially recovers after overexpression of GRP78 in cells with overexpressed QRICH1, and F shows that the apoptosis partially recovers after overexpression of GRP78 in cells with overexpressed QRICH1;

[0021] Figure 6 It is a figure showing the verification results of the interaction mechanism between QRICH1 and GRP78; A shows that co-immunoprecipitation shows the binding of QRICH1 and GRP78, B shows that mass spectrometry analysis shows the binding of QRICH1 and GRP78, C shows the predicted model of the binding of QRICH1 protein and GRP78 protein, with a binding energy of -720 kcal / mol, D shows the surface of the docking residues of QRICH1 protein and GRP78 protein, and a total of 17 pairs of amino acid residues form hydrogen bonds, E shows that the ASP 212 and GLU 155 sites belong to the NBD domain of GRP78, F shows the molecular compounds positively correlated with the biological activity of QRICH1 in the CMap database, and G shows the ATP content in cells after overexpression of QRICH1;

[0022] Figure 7 It is the result of the sensitivity test of QRICH1 to chemotherapeutic drugs; A shows that QRICH1 can synergistically promote the apoptosis of Jurkat cells with tunicamycin, B shows that QRICH1 can synergistically promote the apoptosis of HPBALL cells with tunicamycin, and C shows that QRICH1 can restore the sensitivity to prednisone and synergistically promote the apoptosis of HPBALL cells with prednisone. Specific implementation manners

[0023] The technical solution of the present invention will be described below in conjunction with embodiments. However, the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0024] Example 1: RT-PCR experiment

[0025] Total RNA was extracted from bone marrow or cell samples using Trizol, and the RNA was quantified using a visible light spectrophotometer. The steps were as follows:

[0026] Collect bone marrow or cell samples, centrifuge at 1500 rpm for 5 min, and discard the supernatant;

[0027] Resuspend and wash with PBS, then continue to centrifuge at 1500 rpm for 5 min, and discard the supernatant;

[0028] Add 1 ml of Trizol, pipette to mix well, and incubate at room temperature for 10 min;

[0029] Add 200 μl of chloroform, shake vigorously for 30 s, and incubate at room temperature for 3 min;

[0030] Centrifuge the sample at 12000 g at 4 °C for 20 min;

[0031] The obtained sample was divided into 3 layers: the upper colorless aqueous phase (RNA), the middle white layer, and the lower pink organic phase. Pipette the colorless aqueous phase into a new centrifuge tube, add 600 μl of isopropanol, invert to mix well, and incubate at room temperature for 10 min;

[0032] Centrifuge the sample at 12000 g at 4 °C for 10 min to form a gelatinous precipitate at the bottom of the tube;

[0033] Discard the supernatant, slowly add 1 ml of 75% ethanol (prepared with DEPC water) to make the RNA float gently or remain in place;

[0034] Centrifuge the sample at 7500 g at 4 °C for 5 min, discard the supernatant, and air dry for a few minutes until the precipitate becomes gelatinous;

[0035] Add 20 μl of DEPC water, pipette to mix well until the precipitate dissolves, and measure the sample quality using a spectrophotometer.

[0036] Forward primer F: 5’-AACCCTATGACCCAGATGTGC-3’ (SEQ ID NO:1);

[0037] Reverse primer R: 5’-TAGCCACTCCGTGAACCGAAC-3’ (SEQ ID NO:2).

[0038] Reverse transcription was performed using the Novizan HiscripeⅡqRT superMix kit for reverse transcription. The reaction system was 20 μl, and the conditions were set as 50 °C for 15 min, 85 °C for 5 s, and 4 °C for infinity. Each real-time quantitative PCR reaction was performed in triplicate. Real-time fluorescence quantitative PCR used the SYBR Master Mix (Low ROX Premixed) kit from Novizan, and the quantitative instrument was the Q3 real-time PCR instrument from ABI. The reaction system is shown in Table 1, and the reaction conditions are shown in Table 2.

[0039]

[0040]

[0041] Figure 2 A represents the expression of QRICH1 in adult T-PLL and pediatric T-ALL in the GEO database. The GSE72623 and GSE147930 datasets from the GEO database contain transcriptome data of adult T-cell prolymphocytic leukemia (T-PLL), pediatric T-ALL, and normal T lymphocytes. After data cleaning and statistical analysis, the results showed that the expression of QRICH1 in T-PLL and T-ALL was significantly lower than that in the control group. Figure 2 B represents the mRNA expression of QRICH1 in Jurkat and HPBALL cells. The results showed that, using normal pediatric CD3+ T cells as the control and 293T helper cells as the auxiliary control, the expression of QRICH1 mRNA in Jurkat cells and HPBALL cells was significantly lower than that in the control group. Figure 1 It was consistent with the Western Blot experimental results of A, with β-actin as the control.

[0042] In this invention, paired bone marrow samples from the initial onset and remission stages of 3 T-ALL patients in Nanjing Children's Hospital Affiliated to Nanjing Medical University were randomly collected. All participants signed informed consent forms and were approved by the Ethics Committee of Nanjing Children's Hospital Affiliated to Nanjing Medical University (Ethics number: 202404028-1). Figure 2 C represents the QRICH1 protein expression in the bone marrow of 3 pediatric T-ALL patients at the initial onset and remission stages. It was found that the expression of QRICH1 was significantly decreased in all samples at the initial onset stage. Twenty-three T-ALL patients in Nanjing Children's Hospital Affiliated to Nanjing Medical University were grouped according to gender, age, 19dMRD, and 46dMRD. Figure 2 D represents the expression of QRICH1 in different risk subgroups of pediatric T-ALL. The results showed that the expression of QRICH1 showed a downward trend in all high-risk groups.

[0043] To further clarify the clinical significance of QRICH1, in this invention, 226 pediatric T-ALL patients who met the inclusion criteria in the TCGA database were divided into a QRICH1 high-expression group and a QRICH1 low-expression group. Survival analysis showed that the long-term EFS and OS of the QRICH1 low-expression group were significantly reduced, as Figure 1 shown in

[0044] Figure

[0045]

[0046]

[0047] B. Through propensity score matching analysis based on risk factors such as gender, age, initial white blood cell count, CNSL / TL status, 29d MRD, and biological characteristics, it was found that low expression of QRICH1 was an independent risk factor for poor prognosis in pediatric T-ALL, as shown in Tables 3 and 4.

[0048] Example 2: Construction of a stable gene expression cell line by lentiviral infection

[0049] Lentiviral plasmid GV341 (Ubi-MCS-3FLAG-SV40-puromycin) (purchased from Shanghai GeneChem Co., Ltd., catalog number: GOSL0369987), the vector and the QRICH1 gene sequence were digested with AgeI and NheI, and cloning was completed by In-fusion recombination method. The recombinant vector was detected by DNA sequencing. A 24-well plate was used, and 1×105 cells and 2 - 5×106 TU lentivirus were seeded in each well, and the medium was supplemented to 500 μl for transfection, and the screening concentration of puromycin was 4 μg / μl. The transfection efficiency was verified by qPCR and Western-blot. GRP78 overexpression lentivirus (GM-85763LV) and control lentivirus (GM-6946LV) were purchased from Shanghai Genomics Technology Co., Ltd. (catalog number: GM-LC-141426). The amplified sequence was inserted into PGMLV-CMV-MCS-EF1-ZsGreen1-T2A-Blasticidin according to the manufacturer's protocol. After transfection, screening was carried out with 7.5 μg / μl Blasticidin at the previously described concentration.

[0050] Example 3: In vitro functional experiment

[0051] The EdU detection kit (RiboBio Co., Ltd., Guangzhou, catalog number: C10310-1) and the cell counting kit (CCK8) (APExBIO, No.K1018) were used to detect cell proliferation. According to the instructions, Annexin V / 7AAD staining (BD Biosciences, catalog number: 559763) was used for cell apoptosis detection, and propidium iodide (PI) staining (Multi Sciences, catalog number: 70-CCS012) was used for cell cycle analysis. Samples were collected with a flow cytometer (model: Beckman Coulter CytoFLEX), and data analysis was performed using FlowJo software. Cell ATP content assay kit (purchased from Nanjing Jiancheng Bioengineering Institute, catalog number: A095-1-1).

[0052] Example 4: Animal experiment

[0053] Nineteen female BALB / c nude mice were purchased from the Animal Center of Nanjing Medical University and raised under specific pathogen-free conditions. The BALB / c nude mice were randomly divided into three groups (3:8:8), and separately injected with 200 μl of PBS alone or PBS plus 2x106 QRICH1-overexpressing HPBALL cells or 2x106 NC cells via the tail vein to establish a mouse xenograft leukemia model. Three weeks after inoculation, three mice in each group were randomly sacrificed for sampling and experimental analysis. Flow cytometry was used to detect the engraftment of human leukemia cells (hCD3+ cells) in the bone marrow and peripheral blood. The remaining nude mice were followed up and survival analysis was performed. The animal experiment was approved by the Experimental Animal Welfare Ethics Committee of Nanjing Medical University (Ethical number 2401020-1).

[0054] Figure 3 A was the Edu proliferation assay, and the results showed that the proliferation ability of cells overexpressing QRICH1 was significantly reduced. Figure 3 B was the result of cell cycle analysis. It was found that the proportion of cells in the G0 / G1 phase was significantly increased and the proportion of cells in the G2 / M phase was significantly decreased in cells overexpressing QRICH1, indicating that the cells were blocked in the early stage of the cell cycle. Figure 3 C was the result of apoptosis detection by flow cytometry after double staining with Annexin V and 7AAD. Apoptosis of cells increased significantly after overexpression of QRICH1. Figure 4 A was the expression of human CD3+ cells in the peripheral blood and bone marrow of mice. Flow cytometry was used to detect human CD3+ cells in the peripheral blood and bone marrow of mice. The results showed that the proportion of hCD3+ cells in the QRICH1 overexpression group was significantly lower than that in the Vector group. Figure 4 B was the survival curve of mice xenografted with QRICH1-overexpressing HPBALL cells. The survival analysis of 5:5 mice followed up for 90 days showed that the survival time of mice in the QRICH1 overexpression group was significantly longer (p = 0.0018).

[0055] Figure 5 A and Figure 5 B were the detection of the expression of GRP78 after overexpression of QRCH1 at the mRNA level and protein level, respectively. The results showed that the expression of GRP78 was decreased. Figure 5 C and Figure 5 D were the detection of the expression of CHOP after overexpression of QRCH1 at the mRNA level and protein level, respectively. CHOP is the C / EBP homologous protein, a terminal unfolded protein response marker factor downstream of QRCH1. The results showed that the expression of CHOP was significantly upregulated. The above results indicated that overexpression of QRICH1 downregulated the expression of GRP78, thereby activating the terminal unfolded protein response. Figure 5 E and Figure 5F is the test result of cells overexpressing GRP78 in cells overexpressing QRICH1. The lentivirus overexpressing GRP78 was transfected into cells overexpressing QRICH1, and the results showed that the phenotypic changes in cell proliferation and apoptosis caused by overexpression of QRICH1 were partially restored.

[0056] To explain the direct mechanism between QRICH1 and GRP78, the present invention found a complex form between QRICH1 and GRP78 in the co-immunoprecipitation (Co-IP) results, as Figure 6 shown in A. LC-MS / MS analysis also detected the presence of GRP78 in the proteins pulled down by the anti-QRICH1 antibody, as Figure 6 shown in B. The present invention established a rigid protein-protein docking between QRICH1 and GRP78 (https: / / gramm.compbio.ku.edu / ), selected the model with the lowest binding energy, and the Model is as Figure 6 shown in C-6E, with a binding energy of -720 kcal / mol. A total of 17 pairs of amino acid residues form hydrogen bonds between the two molecules. The docking sites of ASP 212 and GLU 155 of GRP78 belong to its nucleotide binding domain (NBD). NBD has the activity of binding and hydrolyzing ATP.

[0057] The present invention hypothesizes that the binding of QRICH1 to GRP78 inhibits the activity of GRP78. Differentially expressed genes were extracted from the QRICH1 high-expression group and the QRICH1 low-expression group, and 45 up-regulated genes and 31 down-regulated genes were obtained. The present invention performed cMap online analysis (https: / / clue.io / ) on these genes to obtain molecular compounds positively correlated with the biological activity of QRICH1. Most of these compounds belong to ATPase inhibitors, such as salermide, ouabain, and proscillaridin, as Figure 6 shown in F. Figure 6 G is the detection of the ATP content in cells after overexpression of QRICH1. The present invention found that the ATP content in cells overexpressing QRICH1 was significantly reduced, which is consistent with the above speculation.

[0058] Example 5: Experiment on reversing drug resistance by overexpressing QRICH1

[0059] Because QRICH1 will increase under endoplasmic reticulum stress conditions and is a response molecule of endoplasmic reticulum stress, endogenous QRICH1 combined with exogenous QRICH1 may increase its functional effect. The present invention further found through experiments that QRICH1 can increase the sensitivity to chemotherapeutic drugs resistant to treatment. The specific implementation is as follows.

[0060] The present invention selects tunicamycin (Tm), a classical inducer of endoplasmic reticulum stress, for research. It is hypothesized that the combination of endogenous QRICH1 and exogenous QRICH1 will enhance this pro-apoptotic effect. In fact, compared with the two groups of cells without Tm, cells overexpressing QRICHI showed a significant pro-apoptotic effect of 1+1>2 when cultured with 2.5 μM concentration of Tm, as shown in Figure 7 Figures A and 7B. Based on this effect, the present invention speculates that chemotherapeutic drugs can also induce endoplasmic reticulum stress, so overexpression of QRICH1 under the action of chemotherapeutic drugs can reverse the drug resistance of chemotherapeutic drugs. The present invention selects the HPBALL cell line resistant to prednisone for research. Figure 7 The results in Figure C show that the gain effect of overexpression of QRICH1 on apoptosis under the action of prednisone is significantly higher than the sum of the effects of prednisone alone or QRICH1 overexpression or both.

[0061] The embodiments of the present invention only introduce its specific implementation manners and do not limit its protection scope. Those skilled in the art can make some modifications inspired by this embodiment. Therefore, all equivalent changes or modifications made in accordance with the scope of the present invention are within the scope of the claims of the present invention.

Claims

1. Application of a novel tumor suppressor gene QRICH1 as a specific molecular marker in the preparation of in vitro clinical detection products for the prognosis and risk assessment of childhood acute T-lymphocytic leukemia.

2. The use according to claim 1, characterized in that: The products include: reagents, test kits, and drugs.

3. The use according to claim 2, characterized in that: The reagent or kit comprises specific primers for detecting the QRICH1 gene, wherein the specific primers are an upstream primer as shown in SEQ ID NO: 1 and a downstream primer as 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 QRICH1 protein.

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