Use of hoxb3 gene methylation in preparing acute myeloid leukemia prognosis product

Abstract

The present application provides a kit for quantitatively detecting HOXB3 gene methylation and its application in preparing reagents for prognosis evaluation and auxiliary diagnosis of leukemia patients. The kit of the present application comprises HOXB3 methylation specific primers and HOXB3 unmethylation specific primers, the sequence of the HOXB3 methylation specific primers is shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the sequence of the HOXB3 unmethylation specific primers is shown as SEQ ID NO. 3 and SEQ ID NO. 4. The method for detecting AML biomarkers of the present application is based on the level of gene marker HOXB3 methylation, which, in combination with other clinical indicators, provides more accurate judgment for the diagnosis, treatment and prognosis of leukemia.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to the application of HOXB3 gene methylation in the preparation of prognostic products for acute myeloid leukemia. Background Technology

[0002] Acute myeloid leukemia (AML) is a malignant hematopoietic system disease originating from myeloid progenitor cells. Its pathogenesis involves abnormal changes in multiple aspects, including cell differentiation, proliferation, and apoptosis. Non-random chromosomal abnormalities, oncogene and tumor suppressor gene mutations play important roles in the development and progression of AML. Due to the high heterogeneity of AML, there is an urgent clinical need for individualized and precise treatment plans based on the patient's biological characteristics, in addition to standardized treatment. Recent studies have found that epigenetic alterations involving methylation regulation, histone modification, and RNA splicing are another important pathological mechanism in the development of leukemia, and demethylating drugs have shown good efficacy in the treatment of AML.

[0003] Studies have shown that DNA methylation is associated with the occurrence and development of AML and may serve as an effective molecular marker for the auxiliary diagnosis, prognosis, and disease monitoring of AML, while also providing more possibilities for molecularly targeted therapies for 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, a member of the HOXB family, encodes a DNA-binding transcription factor that plays a central role in development and disease by regulating gene expression networks. Aberrant HOXB3 expression (such as high expression in AML) may become a therapeutic target or prognostic biomarker. Currently, there is no protocol for detecting HOXB3 gene methylation in AML, and the significance of HOXB3 in leukemia remains to be explored. Summary of the Invention

[0005] To address the aforementioned technical problems, the purpose of this invention is to propose a method for detecting HOXB3 methylation levels in patients with acute myeloid leukemia (AML) based on real-time quantitative methylation-specific PCR technology. This method serves as a biomarker for AML patients, providing support for prognostic assessment and auxiliary diagnosis in clinical practice.

[0006] To achieve the objectives of this invention, the present invention is implemented through the following technical solution:

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

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

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

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

[0011] Preferably, the HOXB3 methylation is HOXB3 methylation of the M0 subtype, the M1 subtype, the M2 subtype, the M4 subtype, the M5 subtype, the M6 ​​subtype, and / or the 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] A second aspect of the invention provides the use of the above-described kit in the preparation of reagents for prognostic assessment and auxiliary diagnosis of leukemia patients.

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

[0017] A third aspect of the invention provides the use of the biomolecular marker HOXB3 gene methylation in the preparation of reagents for prognostic assessment and auxiliary diagnosis of leukemia patients.

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

[0019] A 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 comprising the following steps:

[0020] (1) Extract genomic DNA from human tissue or body fluid samples and perform quality testing 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, quantitative PCR was performed using HOXB3 methylation-specific primers and HOXB3 unmethylation-specific primers, respectively.

[0023] (4) ALU-specific primers were used for real-time PCR as a quality control measure for DNA from each sample.

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

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

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

[0027] Preferably, the sequences of the ALU-specific primers in step (4) are shown in 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] The beneficial effects of this invention are:

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

[0033] 2. The HOXB3 methylation and unmethylation specific primers provided by this invention were obtained through the inventor's creative efforts in optimization and screening. These primers can amplify target genes with high sensitivity and high specificity. Attached Figure Description

[0034] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in 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 a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 This is a schematic diagram illustrating the screening and identification of the HOXB3 gene as a biomolecular biomarker for the prognosis of AML patients based on the TCGA database. Figure 1 (a) The effect 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 KM analysis for molecules with prognostic value;

[0036] Figure 2 This diagram illustrates the downregulation of HOXB3 methylation in newly diagnosed AML patients detected by real-time quantitative methylation-specific PCR and its diagnostic value. (a) shows the downregulation of HOXB3 methylation in newly diagnosed AML patients detected by real-time quantitative methylation-specific PCR; (b) shows the ROC curve analysis of HOXB3 methylation in AML differential controls. Detailed Implementation

[0037] The embodiments of the present invention are illustrated below using examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Improvements can be made to the materials, methods, and reaction conditions disclosed in this invention, and all such improvements should fall within the spirit and scope of the invention.

[0038] Example 1: Screening and identification of HOXB3 methylation as a potential molecular marker for AML based on public databases.

[0039] 1. Test Methods

[0040] Based on all AML (NEJM 2013) case data from the Cancer Genome Atlas (TCGA) public database, clinical information and HOXB family methylation data related to AML patients were downloaded, ultimately including 194 AML patients. All patient clinical information data were imported into the Social Science Statistical Suite (SPSS). The impact of HOXB family methylation on the prognosis of AML patients was analyzed by grouping patients according to the median methylation value of each HOXB family molecule. The specific steps are as follows:

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

[0042] Methylation data of HOXB family genes (HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXB7, HOXB8 and HOXB9) were screened, and the genes were grouped into high-methylation and low-methylation groups based on the median methylation level of each gene as the cutoff value.

[0043] First, the analysis module was accessed via Analysis → Survival Functions → Kaplan-Meier. Survival time was categorized into the time frame, survival status into the status frame, and gene methylation (high and low methylation groups) into the factor frame. Kaplan-Meier survival curves were used to analyze the impact of gene methylation on patient prognosis. The results showed that HOXB3, HOXB5, and HOXB6 gene methylation had statistically significant differences in all AML cases (i.e., they affected patient prognosis). The results are shown below. Figure 1 .

[0044] Because the M3 subtype is clinically classified as acute promyelocytic leukemia, and over 90% of patients can be cured with the standard regimen of arsenic trioxide combined with all-trans retinoic acid, the impact of the aforementioned genes on the prognosis of AML patients was further analyzed after excluding the M3 subtype. The analysis was conducted via Data → Select Cases → If Conditions Meet → FAB Subtype Not Equal to M3 → Analysis → Survival Function → Kaplan-Meier. Survival time was categorized into time and survival status into status categories. Simultaneously, gene methylation (high and low methylation groups) was categorized into factors. Kaplan-Meier survival curve analysis was used to analyze the impact of gene methylation on patient prognosis. The results showed that only HOXB3 gene methylation had a statistically significant difference in AML excluding the M3 subtype (i.e., it affected patient prognosis). The results are shown in [Figure number missing]. Figure 1 .

[0045] Secondly, the analysis module was accessed via Analysis → Survival Function → Cox Regression. Survival time was included in the time frame, survival status in the status frame, and gene methylation (high and low methylation groups) in the covariate frame. Cox univariate regression analysis was performed to analyze the impact of all variables on the prognosis of all AML patients. Next, the analysis module was accessed via Data → Select Cases → If Conditions Meet → FAB Subtype Not Equal to M3 → Analysis → Survival Function → Cox Regression. Survival time was included in the time frame, survival status in the status frame, and gene methylation (high and low methylation groups) in the covariate frame. Cox univariate regression analysis was performed to analyze the impact of all variables on the prognosis of M3 AML patients. The results are shown in Table 1.

[0046] Based on the results of the univariate Cox regression analysis, variables with P < 0.2 were included in the multivariate analysis. The analysis module was accessed via Analyze → Survival Function → Cox Regression, with survival time included in the time categorization and survival status in the status categorization. Variables with P < 0.2 in the univariate Cox regression analysis were also included in the covariate categorization. The impact of all variables on patient prognosis was then analyzed via Cox multivariate regression analysis. The analysis module was accessed via Data → Select Cases → If Conditions Meet → FAB Classification Not Equal to M3 → Analyze → Survival Function → Cox Regression, with survival time included in the time categorization and survival status in the status categorization. Variables with P < 0.2 in the univariate Cox regression analysis were also included in the covariate categorization. The impact of all variables on the prognosis of patients excluding M3 AML was then analyzed via Cox multivariate regression analysis. The results are shown in Table 2.

[0047] 2. Test Results

[0048] Table 1. Univariate analysis of Cox family genes in AML patients.

[0049]

[0050]

[0051] Table 2. Multivariate analysis of Cox family genes in AML patients.

[0052]

[0053] Based on the above results, HOXB3 methylation was ultimately selected as a potential biomolecular marker for AML for further investigation.

[0054] Example 2: Design and synthesis of primers specifically amplifying the CpG islands in the HOXB3 gene promoter region.

[0055] To target the CpG island information in the HOXB3 gene promoter region, two sets of primers were designed for methylated and unmethylated sequences. These two primer pairs can specifically amplify the methylated and unmethylated sequences of the CpG islands in the HOXB3 promoter region, respectively, for subsequent quantitative methylation analysis. The specific sequence information is shown in SEQ ID NO.1-6 in the table below. The primers were synthesized by BGI Genomics in Shanghai.

[0056]

[0057]

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

[0059] The detection method of this invention is based on the principle of real-time quantitative methylation-specific PCR. Methylated and unmethylated DNA sequences can be distinguished by quantitative PCR amplification using primers specifically for methylation and unmethylation, and then quantitatively calculated. The method includes the following steps:

[0060] (1) DNA template preparation

[0061] Take 5-10 ml of heparin-anticoagulated bone marrow specimens from one AML and one normal control. Isolate mononuclear cells using Ficoll solution. Extract genomic DNA from the 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 buffer) to the cells and mix thoroughly until a watery sample is obtained;

[0063] ② Add 200 μl of chloroform, tighten the cap, and vortex thoroughly (30-60 s) to mix. Let stand for 10 min. Centrifuge at 12000 rpm for 10 min to separate the layers into three layers: upper, middle, and lower. Transfer the middle white membrane layer (containing DNA) to a new EP tube, add 300 μl of 100% anhydrous ethanol, invert to mix, let stand for 10 min, and then centrifuge at 12000 rpm for 10 min. Discard the supernatant.

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

[0065] ④ Add 1 ml of 75% ethanol, invert to mix, let stand for 5 min, centrifuge at 12000 r / min for 10 min, and discard the supernatant;

[0066] ⑤ Add 1 ml of 100% anhydrous ethanol again, gently invert to mix, let stand for 1 min, centrifuge at 12000 r / min for 10 min and discard the supernatant.

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

[0068] ⑦ After incubation, DNA integrity was detected by agarose gel electrophoresis and quantified using a UV spectrophotometer. The 260 / 280 ratio was between 1.7 and 2.1, and the DNA sample concentration was 250 ng / μl. The sample was stored at 4℃ for later use.

[0069] (2) Sulfite modification

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

[0071] ② Preparation of M-Wash Buffer: M-Wash buffer concentrate and anhydrous ethanol were mixed in a ratio of 1:4.

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

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

[0074] ⑤ Add 600 μl of M-Binding Buffer to the Zymo-SpinTMIC column, place the column into the collection tube, add the sample mixture obtained in step ④ to the column, cap it, and invert it to mix.

[0075] ⑥ Centrifuge at full speed (>10000×g) for 30s, discard the liquid in the collection tube, add 100μl of M-WashBuffer to the column, and centrifuge at full speed again for 30s.

[0076] ⑦ Add 200 μl of M-Desulphonation Buffer, incubate at room temperature (20-30℃) 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 seconds, then add another 200 μl of M-Wash Buffer and centrifuge at full speed for 30 seconds.

[0078] ⑨ Place the chromatographic column into a 1.5 ml EP tube and add 10 μl M-Elution Buffer to the column. Centrifuge at full speed for 30 s to elute. Store the obtained sulfurized DNA at -80℃ for later use.

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

[0080] The promoter methylation of HOXB3 and the internal reference gene ALU was detected using the RQ-MSP method. Each PCR reaction carried a negative control (water) and a positive control (target gene plasmid). A 7500 real-time PCR instrument (ABI, USA) was used. The RQ-MSP reagent used was SYBR Premix ExTaqI (TaKaRa, Japan). The specific steps are as follows:

[0081] 1) System configuration:

[0082] ①The HOXB3 methylation PCR (M-MSP) reaction system consisted of 0.8 μL each of 10 μM M-MSP forward and reverse primers, and 10 μL LTB Green. TM Premix Ex Taq TM II. 0.4 μL ROX Reference Dye II (Takara), 2 μL DNA template and 6 μL sterile water.

[0083] ②The HOXB3 unmethylated PCR (U-MSP) reaction system consisted of 0.8 μL each of 10 μM U-MSP forward and reverse primers, and 10 μL LTB Green. TM Premix Ex Taq TM II. 0.4 μL LROX Reference Dye II (Takara), 2 μL DNA template and 6 μL sterile water.

[0084] ③The ALU methylation reaction system consisted of: 0.5 μL each of the forward and reverse primers (both at 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 DNase, 2.0 μL of DNA template, and 14.3 μL of sterile water.

[0085] 2) Start-up (reaction conditions are set as follows):

[0086] ① HOXB3 methylation PCR (M-MSP) reaction conditions: pre-denaturation 95℃ 5min → 40 cycles (denaturation 95℃ 10s; annealing 56℃ 30s; extension 72℃ 30s; fluorescence collection 75℃ 30s) → 95℃ 15s → 60℃ 1min.

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

[0088] ③ALU reaction conditions: pre-denaturation 95℃ 5min → 20 cycles (denaturation 95℃ 30s; annealing 61℃ 30s; extension 72℃ 30s; fluorescence collection 75℃ 30s) → 95℃ 15s → 60℃ 1min.

[0089] 3) Results Analysis:

[0090] Observation of the melting curves obtained from PCR amplification revealed that each PCR tube exhibited a single melting temperature, indicating good specificity of the amplified products.

[0091] (4) Calculation of results

[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 2) -ΔΔCT Law)

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

[0094] Example 4: Verifying the difference in HOXB3 gene promoter methylation between healthy individuals and newly diagnosed acute myeloid leukemia patients.

[0095] Twenty healthy individuals and 54 newly diagnosed AML samples were used as analysis samples. Each sample was processed using the same method as in Example 3. The HOXB3 promoter methylation level in each sample was calculated, and the results are as follows: Figure 2 As shown. Figure 2The results showed that, compared with the healthy group, the HOXB3 promoter methylation level in newly diagnosed AML samples was significantly increased. ROC analysis also revealed that HOXB3 promoter methylation has certain value in assisting the diagnosis of AML.

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

[0097] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Other modifications can be readily made by those skilled in the art. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and examples shown and described herein.

Claims

1. A reagent kit for use in preparing reagents to assist in the diagnosis of patients with non-M3 subtype acute myeloid leukemia, characterized in that, The kit includes HOXB3 methylation-specific primers and HOXB3 unmethylation-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 unmethylation-specific primers are shown in SEQ ID NO.3 and SEQ ID NO.

4.

2. The application according to claim 1, characterized in that, The kit also includes ALU-specific primers as internal controls, the sequences of which are shown in SEQ ID NO.5 and SEQ ID NO.

6.

3. The application according to claim 1, characterized in that, The kit is a quantitative PCR detection kit.

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