Application of HOXB3 gene methylation in preparation of acute myelogenous leukemia prognosis product

Abstract

The invention provides a kit for quantitatively detecting HOXB3 gene methylation and application of the kit in preparation of reagents for prognosis evaluation and auxiliary diagnosis of leukemia patients. The kit provided by the invention comprises an HOXB3 methylation specific primer and an HOXB3 unmethylation specific primer, the sequences of the HOXB3 methylation specific primer are as shown in SEQ ID NO. 1 and SEQ ID NO. 2, and the sequences of the HOXB3 unmethylation specific primer are as shown in SEQ ID NO. 3 and SEQ ID NO. 4. The method for detecting the AML biomarker is based on the methylation level of the gene marker HOXB3, is combined with other clinical indexes, and provides more accurate judgment for diagnosis, treatment and prognosis of leukemia.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and particularly to the application of HOXB3 gene methylation in the preparation of products for predicting the prognosis of acute myeloid leukemia. Background Art

[0002] Acute myeloid leukemia (AML) is a type of malignant hematological disease originating from myeloid progenitor cells. Its onset involves abnormal changes in multiple aspects such as cell differentiation, proliferation, and apoptosis. Non-random chromosomal abnormalities, oncogene and tumor suppressor gene mutations, etc. play important roles in the occurrence and development of AML. Due to the large heterogeneity of AML, there is an urgent clinical need for individualized precision diagnosis and treatment plans based on the biological characteristics of patients on the basis of standardized treatment. In recent years, studies have found that epigenetic changes involving methylation regulation, histone modification, RNA splicing, etc. are another pathological mechanism that plays an important role in the occurrence of leukemia, and demethylating drugs have good efficacy in the treatment of AML.

[0003] Research shows that DNA methylation is related to the occurrence and development of AML, and may be used as an effective molecular marker for the auxiliary diagnosis, prognosis judgment, and disease monitoring of AML, and provides more possibilities for the molecular targeted therapy of AML. Clinically, there is an expectation to discover more specific and sensitive DNA methylation modifications and apply them to the clinical diagnosis and treatment of AML.

[0004] HOXB3 is a member of the HOXB family, encoding a DNA-binding transcription factor that plays a central role in development and diseases by regulating gene expression networks. Abnormal expression of HOXB3 (such as high expression in AML) may become a therapeutic target or a prognostic marker. Currently, there is no protocol for detecting HOXB3 gene methylation in AML, and the significance of HOXB3 in leukemia also needs to be discovered urgently. Summary of the Invention

[0005] In view of the above technical problems, the object of the present invention is to propose a method for detecting the methylation level of HOXB3 in patients with acute myeloid leukemia based on real-time quantitative methylation-specific PCR technology, which is used as a biomarker for patients with acute myeloid leukemia to provide support for the prognosis judgment and auxiliary diagnosis of patients with acute myeloid leukemia clinically.

[0006] To achieve the object of the invention, the present invention is realized by the following technical solutions:

[0007] The first aspect of the present invention provides a kit for quantitatively detecting a biomarker, wherein the biomarker is HOXB3 gene methylation, and the kit is used for prognostic evaluation and auxiliary diagnosis of leukemia patients.

[0008] Preferably, the kit includes HOXB3 methylation-specific primers and HOXB3 unmethylated-specific primers. The sequences of the HOXB3 methylation-specific primers are shown in SEQ ID NO.1 and SEQ ID NO.2, and the sequences of the HOXB3 unmethylated-specific primers are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0009] Preferably, the kit further includes ALU-specific primers as internal references. The sequences of the ALU-specific primers are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0010] Preferably, the HOXB3 methylation is HOXB3 methylation of non-M3 subtype.

[0011] Preferably, the HOXB3 methylation is HOXB3 methylation of M0 subtype, HOXB3 methylation of M1 subtype, HOXB3 methylation of M2 subtype, HOXB3 methylation of M4 subtype, HOXB3 methylation of M5 subtype, HOXB3 methylation of M6 subtype, and / or HOXB3 methylation of M7 subtype.

[0012] Preferably, the kit is a quantitative PCR detection kit.

[0013] Preferably, the kit is used to detect the methylation or unmethylation level of the HOXB3 promoter.

[0014] Preferably, the leukemia patient is an acute myeloid leukemia patient.

[0015] The second aspect of the present invention provides the application of the above kit in the preparation of reagents for prognostic evaluation and auxiliary diagnosis of leukemia patients.

[0016] Preferably, the leukemia patient is an acute myeloid leukemia patient.

[0017] The third aspect of the present invention provides the application of the biomolecular marker HOXB3 gene methylation in the preparation of reagents for prognostic evaluation and auxiliary diagnosis of leukemia patients.

[0018] Preferably, the leukemia patient is an acute myeloid leukemia patient.

[0019] The fourth aspect of the present invention provides a method for detecting the methylation level of the HOXB3 gene promoter based on quantitative PCR. The method includes the following steps:

[0020] (1) Extract genomic DNA from human tissue or body fluid samples and perform quality detection on the extracted DNA samples;

[0021] (2) Modify genomic DNA with sodium bisulfite and recover the modified DNA product;

[0022] (3) Using the recovered modified DNA as a template, perform quantitative PCR respectively with HOXB3 methylation-specific primers and HOXB3 unmethylated-specific primers;

[0023] (4) Perform fluorescence quantitative PCR with ALU-specific primers as the quality control of DNA for each specimen;

[0024] (5) Calculate the methylation level of the HOXB3 gene.

[0025] Preferably, the sequences of the HOXB3 methylation-specific primers described in step (3) are shown as SEQ ID NO.1 and SEQ ID NO.2.

[0026] Preferably, the sequences of the HOXB3 unmethylated-specific primers described in step (3) are shown as SEQ ID NO.3 and SEQ ID NO.4.

[0027] Preferably, the sequences of the ALU-specific primers described in step (4) are shown as SEQ ID NO.5 and SEQ ID NO.6.

[0028] Preferably, the method is for non-diagnostic and non-therapeutic purposes.

[0029] Preferably, the formula for calculating methylation in step (5) is as follows:

[0030] N HOXB3甲基化 =2 ΔCT HOXB3甲基化(对照-目的) ÷2 ΔCT ALU(对照-目的) 。

[0031] Advantages of the present invention:

[0032] 1. Compared with the prior art, the method for detecting AML biomarkers in the present invention is based on the methylation level of the gene biomarker HOXB3, combined with other clinical indicators, providing a more accurate judgment for the diagnosis, treatment and prognosis of leukemia.

[0033] 2. The HOXB3 methylation and unmethylated-specific primers provided by the present invention are obtained by the inventor's creative labor for optimization and screening, and the above primers can amplify the target gene with high sensitivity and high specificity. Description of the Drawings

[0034] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0035] Figure 1 Schematic diagram for screening and identifying HOXB3 gene as a prognostic biomarker for AML patients based on the TCGA database, where Figure 1 (a) shows the impact of HOXB family genes on the prognosis of AML patients, where ns indicates no significance, * indicates P < 0.05, and ** indicates P < 0.01; Figure 1 (b) is a schematic diagram of the KM analysis of molecules with prognostic value;

[0036] Figure 2 Schematic diagram for detecting the downregulation of HOXB3 methylation in newly diagnosed AML patients by real-time quantitative methylation-specific PCR and its diagnostic value, where (a) shows the detection of the downregulation of HOXB3 methylation in newly diagnosed AML patients by real-time quantitative methylation-specific PCR; (b) is the ROC curve analysis of HOXB3 methylation in AML differential controls. Detailed implementation manners

[0037] The embodiments of the present invention will be specifically described below by way of illustrative examples. It should be understood that these examples are only for illustrative purposes and are not used to limit the scope of the present invention. Improvements can be made to the disclosed content of the present invention simultaneously in terms of materials, methods, and reaction conditions, and all these improvements should fall within the spirit and scope of the present invention.

[0038] Example 1: Screening and identifying HOXB3 methylation as a potential biomarker for AML based on public databases

[0039] 1. Test method

[0040] Based on all case data of AML (NEJM 2013) in the Cancer Genome Atlas (TCGA) public database, the clinical information data and HOXB family methylation data related to AML patients were downloaded. Finally, 194 AML patients were included, and all the clinical information data of the patients were imported into the Statistical Package for the Social Sciences (SPSS). Grouped by the median value of methylation of each HOXB family molecule, the impact of methylation of HOXB family molecules on the prognosis of AML patients was analyzed respectively. The specific steps are as follows:

[0041] Through the cBioPortal website (https: / / www.cbioportal.org / ), first search for "acute myeloid leukemia" on the Query page, select the AML data from NEJM in 2013, click "view clinical and genomic data of this study" to enter, and download all the clinical data of the patients. Search for "acute myeloid leukemia" on the Data Sets page, select the AML data from NEJM in 2013, and download all the sequencing results of the patients.

[0042] Screen the methylation data of HOXB family genes (HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXB7, HOXB8, and HOXB9), and use the median value of the methylation level of each gene as the cut-off value to group and define them as high- and low-methylation groups respectively.

[0043] First, enter the analysis module through Analyze → Survival function → Kaplan-Meier. Enter the survival time into the Time box, enter the survival status into the Status box, and at the same time enter the methylation of each gene (high- and low-methylation groups) into the Factor box respectively. Analyze the impact of gene methylation on the prognosis of patients through the Kaplan-Meier survival curve. The results show that there are statistical differences in the methylation of HOXB3, HOXB5, and HOXB6 genes in all AMLs (that is, it has an impact on the prognosis of patients). The results are shown in Figure 1 .

[0044] Since M3 subtype is acute promyelocytic leukemia clinically, and more than 90% of the patients can be cured after treatment with the standard regimen of arsenic trioxide combined with all-trans retinoic acid, the impact of the above genes on the prognosis of AML patients was further analyzed after excluding the M3 subtype. Enter the analysis module through Data → Select Cases → If condition is met → FAB classification is not equal to M3 → Analyze → Survival function → Kaplan-Meier. Enter the survival time into the Time box, enter the survival status into the Status box, and at the same time enter the methylation of each gene (high- and low-methylation groups) into the Factor box respectively. Analyze the impact of gene methylation on the prognosis of patients through the Kaplan-Meier survival curve. The results show that only the methylation of HOXB3 gene has statistical differences in AML after excluding the M3 subtype (that is, it has an impact on the prognosis of patients). The results are shown in Figure 1 .

[0045] Secondly, through analysis → survival function → Cox regression to enter the analysis module, incorporate the survival time into the time slot, the survival status into the status slot, and at the same time incorporate the methylation of each gene (high and low methylation groups) into the covariate slot respectively, and conduct a Cox univariate regression analysis on the impact of all variables on the prognosis of all AML patients; through data → select cases → if condition is met → FAB classification is not equal to M3 → analysis → survival function → Cox regression to enter the analysis module, incorporate the survival time into the time slot, the survival status into the status slot, and at the same time incorporate the methylation of each gene (high and low methylation groups) into the covariate slot respectively, and conduct a Cox univariate regression analysis on the impact of all variables on the prognosis of AML patients excluding M3, and the results are shown in Table 1.

[0046] Based on the above Cox univariate analysis results, incorporate the variables with P < 0.2 into the multivariate analysis. Through analysis → survival function → Cox regression to enter the analysis module, incorporate the survival time into the time slot, the survival status into the status slot, and at the same time incorporate all the variables with P < 0.2 in the univariate Cox analysis into the covariate slot, and conduct a Cox multivariate regression analysis on the impact of all variables on the prognosis of the patients; through data → select cases → if condition is met → FAB classification is not equal to M3 → analysis → survival function → Cox regression to enter the analysis module, incorporate the survival time into the time slot, the survival status into the status slot, and at the same time incorporate all the variables with P < 0.2 in the univariate Cox analysis into the covariate slot, and conduct a Cox multivariate regression analysis on the impact of all variables on the prognosis of AML patients excluding M3, and the results are shown in Table 2.

[0047] 2. Test results

[0048] Table 1 Cox univariate analysis of HOXB family genes in AML patients

[0049]

[0050]

[0051] Table 2 Cox multivariate analysis of HOXB family genes in AML patients

[0052]

[0053] Based on the above results, finally select HOXB3 methylation as a potential biomarker for AML for further research.

[0054] Example 2: Design and synthesize primers for specifically amplifying the CpG island of the HOXB3 gene promoter

[0055] For the CpG island information in the promoter region of the HOXB3 gene, two sets of primers targeting methylated and unmethylated sequences were designed. Using these 2 pairs of primers, the methylated and unmethylated sequences of the CpG island in the HOXB3 promoter region can be specifically amplified respectively for subsequent methylation quantitative analysis. The specific sequence information is shown as SEQ ID NO.1 - 6 in the following table, and the primers were synthesized by Shanghai BGI.

[0056]

[0057]

[0058] Example 3: Establish a method for detecting the methylation status of the GpG island in the HOXB3 gene promoter based on quantitative PCR

[0059] The detection method of the present invention is based on the principle of real-time quantitative methylation-specific PCR. By quantitative PCR amplification with methylation- and non-methylation-specific primers respectively, methylated and non-methylated DNA sequences can be distinguished and quantitatively calculated. The method includes the following steps:

[0060] (1) DNA template preparation

[0061] Take 5 - 10 ml of heparinized bone marrow specimens from AML and normal controls, separate mononuclear cells using Ficoll solution, and extract genomic DNA from mononuclear cells using a genomic DNA extraction kit (purchased from Gentra). The specific steps are as follows:

[0062] ① Add 1 ml of TRIZOL (lysis solution) to the cells and mix well until it becomes a watery solution;

[0063] ② Add 200 μl of chloroform, tightly cap the tube, and mix well on a vortex oscillator (30 - 60 s), then let it stand for 10 min; Centrifuge at 12000 r / min for 10 min to separate it into upper, middle, and lower layers; Pipette the middle white film layer (containing DNA) into a new EP tube, add 300 μl of 100% anhydrous ethanol, mix well by inverting up and down, let it stand for 10 min, then centrifuge at 12000 r / min for 10 min, and discard the supernatant;

[0064] ③ Add 1 ml of trisodium citrate, mix well by inverting up and down, let it stand for 15 min, centrifuge at 12000 r / min for 10 min, and discard the supernatant;

[0065] ④ Add 1 ml of 75% ethanol, mix well by inverting up and down, let it stand for 5 min, centrifuge at 12000 r / min for 10 min, and discard the supernatant;

[0066] ⑤ Add 1 ml of 100% absolute ethanol again, gently invert the tube up and down to mix well, let it stand for 1 min, centrifuge at 12,000 r / min for 10 min, and then discard all the supernatant.

[0067] ⑥ Dry the remaining residue in the lower layer thoroughly in a 37°C metal bath, add an appropriate amount of DNA lysis solution (TE), incubate in a 65°C metal bath for 5 min, and incubate overnight at 4°C.

[0068] ⑦ After incubation, perform agarose gel electrophoresis to detect the integrity of DNA, and quantify it using a UV spectrophotometer. The 260 / 280 ratio is between 1.7 - 2.1, the DNA sample concentration is 250 ng / μl, and store it at 4°C for later use.

[0069] (2) Bisulfite modification

[0070] ① Prepare the C / T conversion reagent: Add 900 μl of sterile injection water, 300 μl of M-Dilution Buffer, and 50 μl of M-Dissolving Buffer to a tube of C / T conversion reagent, and shake at room temperature for 10 min.

[0071] ② Prepare M-Wash Buffer: Mix the M-Wash buffer concentrate and absolute ethanol in a ratio of 1:4.

[0072] ③ Sample treatment: Add 130 μl of CT conversion Reagent, 18 μl of sterile injection water, and 2 μl of the DNA sample with a concentration of 100 ng / μl in the above step (1) (i.e., the total DNA sample loading is 200 μg) to each 200 μl EP tube.

[0073] ④ Place the sample tube in a qualitative PCR instrument and perform the steps: a. 98°C / 10 min; b. 64°C / 150 min; c. Store at 4°C.

[0074] ⑤ Add 600 μl of M-Binding Buffer to the Zymo-SpinTM IC column, place the chromatography column in the collection tube, then add the sample mixture obtained in step ④ to the column, cover the lid and invert to mix well.

[0075] ⑥ Centrifuge at full speed (>10,000×g) for 30 s, discard the liquid in the collection tube, add 100 μl of M-Wash Buffer to the column, and centrifuge at full speed again for 30 s.

[0076] ⑦ Add 200 μl of M-Desulphonation Buffer, incubate at room temperature (20 - 30°C) for 15 - 20 min, and centrifuge at full speed for 30 s.

[0077] ⑧ Add 200 μl of M-Wash Buffer, centrifuge at full speed for 30 s, add another 200 μl of M-Wash Buffer, and centrifuge at full speed for 30 s again.

[0078] ⑨ Place the chromatography column into a 1.5 ml EP tube, add 10 μl of M-Elution Buffer to the column, centrifuge at full speed for 30 s for elution, and store the sulfonated DNA obtained at -80 °C for later use.

[0079] (3) Real-time quantitative methylation-specific PCR (RQ-MSP) amplification

[0080] Detect the methylation of the promoter regions of HOXB3 and the internal reference gene ALU by the RQ-MSP method. Each PCR reaction is carried with a negative control (water) and a positive control (plasmid of the target gene); the fluorescence quantitative PCR instrument 7500 (ABI, USA) is used; the RQ-MSP reagent SYBR Premix ExTaqI (TaKaRa, Japan) is used. The specific steps are as follows:

[0081] 1) System preparation:

[0082] ① The reaction system for HOXB3 methylation PCR (M-MSP) is: 0.8 μL each of the M-MSP forward primer and reverse primer with a concentration of 10 μM, 10 μL of TB Green TM Premix Ex Taq TM Ⅱ, 0.4 μL of ROX Reference Dye Ⅱ (Takara), 2 μL of DNA template, and 6 μL of sterilized water.

[0083] ② The reaction system for HOXB3 unmethylated PCR (U-MSP) is: 0.8 μL each of the U-MSP forward primer and reverse primer with a concentration of 10 μM, 10 μL of TB Green TM Premix Ex Taq TM Ⅱ, 0.4 μL of ROX Reference Dye Ⅱ (Takara), 2 μL of DNA template, and 6 μL of sterilized water.

[0084] ③ The reaction system for ALU methylation is: 0.5 μL each of the ALU forward primer and reverse primer with a concentration of 10 μM, 2.5 μL of 10×Buffer, 2.0 μL of MgCl2 (25 mmol / L), 0.5 μL of dNTP (10 mmol / L), 1.2 μL of 20×EvaGreen, 0.5 μL of 50×ROX, 1.0 μL of Taq DNA polymerase, 2.0 μL of DNA template, and 14.3 μL of sterilized water.

[0085] 2) Upper machine (reaction conditions are set as follows):

[0086] ① Reaction conditions for HOXB3 methylation PCR (M-MSP): Pre-denaturation at 95°C for 5 min → 40 cycles (denaturation at 95°C for 10 s; annealing at 56°C for 30 s; extension at 72°C for 30 s; fluorescence collection at 75°C for 30 s) → 95°C for 15 s → 60°C for 1 min.

[0087] ② Reaction conditions for HOXB3 unmethylated PCR (U-MSP): Pre-denaturation at 95°C for 5 min → 40 cycles (denaturation at 95°C for 10 s; annealing at 54°C for 30 s; extension at 72°C for 30 s; fluorescence collection at 75°C for 30 s) → 95°C for 15 s → 60°C for 1 min.

[0088] ③ Reaction conditions for ALU: Pre-denaturation at 95°C for 5 min → 20 cycles (denaturation at 95°C for 30 s; annealing at 61°C for 30 s; extension at 72°C for 30 s; fluorescence collection at 75°C for 30 s) → 95°C for 15 s → 60°C for 1 min.

[0089] 3) Result analysis:

[0090] Observe the melting curve obtained by PCR amplification. It is found that a single melting temperature is obtained in each PCR tube, indicating good specificity of the amplification product.

[0091] (4) Result calculation

[0092] N HOXB3甲基化 = 2 ΔCT HOXB3甲基化(对照-目的) ÷ 2 ΔCT ALU(对照-目的) (equivalent to the 2 -ΔΔCT method)

[0093] N HOXB3甲基化 represents the relative copy value of HOXB3 methylation; 2 ΔCT HOXB3甲基化(对照-目的) represents the copy number of HOXB3 methylation, where ΔCT represents the difference in the CT values of PCR amplification of HOXB3 methylation in the set control and the target sample; 2 ΔCT ALU(对照-目的) represents the copy number of ALU methylation, where ΔCT represents the difference in the CT values of PCR amplification of ALU methylation in the set control and the target sample.

[0094] Example 4. Verification of the difference in HOXB3 gene promoter methylation between healthy people and newly diagnosed acute myeloid leukemia patients

[0095] Take 20 healthy people and 54 newly diagnosed AML samples as analysis samples. Each sample is processed using the same treatment method as in Example 3, and the HOXB3 promoter methylation level in each sample is calculated respectively. The results are as Figure 2 shown. Figure 2The results showed that, compared with the healthy control group, the methylation level of the HOXB3 promoter in newly diagnosed AML samples was significantly increased. At the same time, ROC analysis found that the methylation of the HOXB3 promoter had certain value in assisting the diagnosis of AML.

[0096] In summary, the method established in the present invention for detecting the expression and promoter methylation level of HOXB3 based on quantitative PCR can be used for the auxiliary diagnosis, prognosis judgment and disease monitoring of AML and other tumors, and has good application prospects.

[0097] Although the embodiments of the present invention have been disclosed as above, it is not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those skilled in the art, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the examples shown and described herein.

Claims

1. A kit for quantitatively detecting biomarkers, characterized in that, The biomarker is HOXB3 gene methylation, and the kit is used for prognostic evaluation and auxiliary diagnosis of leukemia patients.

2. The kit according to claim 1, wherein The kit includes HOXB3 methylation-specific primers and HOXB3 unmethylation-specific primers. The sequences of the HOXB3 methylation-specific primers are shown as SEQ ID NO.1 and SEQ ID NO.2, and the sequences of the HOXB3 unmethylation-specific primers are shown as SEQ ID NO.3 and SEQ ID NO.

4.

3. The kit according to claim 1, characterized in that, The kit further includes ALU-specific primers as internal references, and the sequences of the ALU primers are shown as SEQ ID NO.5 and SEQ ID NO.

6.

4. The kit according to claim 1, characterized in that, The HOXB3 methylation is the HOXB3 methylation of non-M3 subtypes.

5. The kit according to claim 1, wherein The kit is a quantitative PCR detection kit.

6. The kit according to claim 1, characterized in that, The leukemia patients are acute myeloid leukemia patients.

7. Use of the kit according to any one of claims 1-6 in the preparation of reagents for prognostic evaluation and auxiliary diagnosis of leukemia patients.

8. The application according to claim 7, wherein The leukemia patients are acute myeloid leukemia patients.

9. A method for detecting the methylation level of the HOXB3 gene promoter based on quantitative PCR, characterized in that, The method includes the following steps: (1) Extract genomic DNA from human tissue or body fluid samples, and perform quality detection on the extracted DNA samples; (2) Modify genomic DNA with sodium bisulfite and recover the modified DNA product; (3) Using the recovered modified DNA as a template, perform quantitative PCR with HOXB3 methylation-specific primers and HOXB3 unmethylation-specific primers respectively; (4) Perform fluorescence quantitative PCR with ALU-specific primers as the quality control of DNA for each specimen; (5) Calculate the methylation level of the HOXB3 gene.

10. The method according to claim 9, wherein In step (3), the sequences of the HOXB3 methylation-specific primers are shown as SEQ ID NO.1 and SEQ ID NO.2, and the sequences of the HOXB3 unmethylation-specific primers are shown as SEQ ID NO.3 and SEQ ID NO.4.

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