Expression and methylation detection of biomarker ZNF300P1 and application thereof

Abstract

The present application relates to the expression and methylation quantitative detection method of ZNF300P1 gene and its application in assisting myeloid tumor diagnosis and prognosis evaluation. The present application provides ZNF300P1 gene expression and methylation quantitative detection, the ZNF300P1 biomarker gene is shown as SEQ ID NO. 1, the present application provides expression and methylation detection primer, the ZNF300P1 biomarker expression detection primer is shown as SEQ ID NO. 2-3, the ZNF300P1 biomarker methylation detection primer is shown as SEQ ID NO. 6-9, which is used for detecting the level of ZNF300P1 expression and methylation in bone marrow / peripheral blood of myelodysplastic syndrome and acute myeloid leukemia patients, so as to provide support for early diagnosis and prognosis evaluation of the patients by taking the level as a new biomarker of myelodysplastic syndrome and acute myeloid leukemia patients in combination with other indexes.

Description

Technical Field

[0001] This invention relates to a biomolecular marker, and more particularly to the expression and methylation detection of the biomolecular marker ZNF300P1 and its applications. Background Technology

[0002] Myeloid tumors mainly include diseases such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS is a group of heterogeneous myeloid clonal diseases originating from hematopoietic stem cells, characterized by abnormal differentiation and development of myeloid cells, manifesting as ineffective hematopoiesis, refractory cytopenia, hematopoietic failure, and a high risk of transformation to AML. MDS is a highly heterogeneous disease; low- and intermediate-risk patients have good survival and are mainly treated with symptomatic and supportive care; while high-risk patients have a poor prognosis and a high risk of AML transformation, and are mainly treated with demethylation, chemotherapy, and transplantation. AML is a hematologic malignancy originating from hematopoietic stem / progenitor cells, accompanied by a large number of abnormal proliferations of primitive cells. It is also highly heterogeneous, with extremely different prognoses among different patients, ranging from several months to long-term survival. Intermediate- and high-risk patients often require intensive therapy and hematopoietic stem cell transplantation to improve their prognosis. The heterogeneity between MDS and AML is fundamentally manifested in cellular and molecular genetics; however, the number of biomarkers discovered to date is limited and fails to meet clinical needs. In the era of new drugs, improvements in AML survival rates based on traditional chemotherapy regimens have reached a bottleneck. Therefore, providing more new targets for precision and targeted therapy of AML is crucial, ultimately offering the possibility of improving patient survival and prognosis, and possessing broad application value and significant clinical implications. As is well known, the core of targeted / immunotherapy is the discovery of molecular markers closely related to disease pathogenesis, thereby developing corresponding targeted therapeutic drugs that exert a directed killing effect on tumors, improving treatment efficacy and reducing adverse reactions.

[0003] Recent studies have shown that pseudogenes possess a wide range of biological functions, potentially participating in various physiological processes and the occurrence and development of multiple diseases, including tumors. ZNF300P1 (LOC134466) is a pseudogene of the human zinc finger protein ZNF300 (89% homology). Gene structure analysis revealed large CpG islands in the ZNF300P1 promoter region. Targeted methylation sequencing showed that it also undergoes hypermethylation alterations in MDS and AML patients, leading to expression silencing and potentially playing a tumor-suppressive role. Therefore, the detection of ZNF300P1 expression and methylation has promising applications as a novel biomarker for myeloid tumor patients. Summary of the Invention

[0004] The purpose of this invention is to provide a quantitative detection method for biomolecular markers and their applications, in order to address the shortcomings of existing biomarkers, which are limited and fail to meet clinical needs. By proposing a quantitative detection method for ZNF300P1 expression and methylation, the method uses quantitative PCR technology to detect the differences in ZNF300P1 expression and methylation levels in myeloid tumors and normal controls, thereby serving as a biomarker for myeloid tumor patients and providing support for early diagnosis and prognostic assessment of myeloid tumor patients in clinical practice.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: an application of a biomolecular marker expression and methylation detection product, wherein the gene sequence of the ZNF300P1 biomolecular marker is shown in SEQ ID NO.1.

[0006] An application of a ZNF300P1 biomolecular marker expression detection product, wherein the ZNF300P1 expression detection product includes a forward primer as shown in SEQ ID NO.2 and a reverse primer as shown in SEQ ID NO.3.

[0007] Application of a ZNF300P1 biomolecular marker methylation detection product, wherein the ZNF300P1 methylation detection product includes ZNF300P1 methylated and unmethylated specific primers as shown in SEQ ID NO.6-7 and SEQ ID NO.8-9.

[0008] Furthermore, the myeloid tumors mentioned are mainly myelodysplastic syndromes and acute myeloid leukemia.

[0009] Furthermore, the detection reagent is a quantitative PCR reagent used to detect ZNF300P1 expression and promoter methylation levels.

[0010] This invention provides an application of a product for quantitative detection of ZNF300P1 gene expression and methylation in assisting the diagnosis and prognostic assessment of myeloid tumors.

[0011] A second aspect of the present invention provides a detection primer for detecting ZNF300P1 gene expression and methylation in myeloid tumors, comprising a forward primer and a reverse primer for detecting ZNF300P1 gene expression, an upstream primer and a downstream primer for detecting ZNF300P1 methylation, and an upstream primer and a downstream primer for detecting ZNF300P1 unmethylation. The ZNF300P1 gene sequence is shown in SEQ ID NO.1, the primer sequences for ZNF300P1 expression are shown in SEQ ID NO.2-3, the primer sequences for ZNF300P1 methylation are shown in SEQ ID NO.6-7, and the primer sequences for ZNF300P1 unmethylation are shown in SEQ ID NO.8-9.

[0012] Furthermore, the detection primers also include an internal reference ABL1 primer and an internal reference ALU primer. The forward primer of the internal reference ABL1 is shown in SEQ ID NO.4, and the reverse primer is shown in SEQ ID NO.5. The forward primer of the internal reference ALU primer is shown in SEQ ID NO.10, and the reverse primer is shown in SEQ ID NO.11.

[0013] A third aspect of the present invention provides a method for detecting ZNF300P1 expression levels in myeloid tumors based on quantitative PCR, comprising the following steps:

[0014] (1) Extract total RNA from human bone marrow / peripheral blood cell samples and perform quality testing on the extracted RNA samples;

[0015] (2) Reverse transcribe the total RNA from step (1) into cDNA;

[0016] (3) The cDNA from step (2) was amplified using the primer pairs shown in SEQ ID NO.2 and SEQ ID NO.3 respectively. Quantitative PCR with ABL1 sequence-specific primers was used as the quality control of DNA for each sample. The expression level of ZNF300P1 in the sample was analyzed to obtain relevant data.

[0017] Furthermore, the myeloid tumors are myelodysplastic syndrome and acute myeloid leukemia.

[0018] A fourth aspect of the present invention provides a method for detecting the methylation level of the ZNF300P1 gene promoter based on quantitative PCR, characterized by comprising the following steps:

[0019] (1) Extract genomic DNA samples from human bone marrow / peripheral blood cells and perform quality testing;

[0020] (2) Sulfide the DNA from step (1);

[0021] (3) The DNA after sulfurization in step (2) was subjected to quantitative PCR using the methylation- and non-methylation-specific primers shown in SEQ ID NO.6-7 and SEQ ID NO.8-9, respectively. The expression level of ZNF300P1 in the sample was analyzed by quantitative methylation-specific PCR to obtain relevant data.

[0022] (4) ALU sequence-specific primers were used for real-time PCR as a quality control method for DNA samples.

[0023] (5) Calculate the methylation and unmethylation levels.

[0024] The beneficial effects of this invention are as follows: Compared with the prior art, this invention, applied to myeloid tumors, is based on the expression and methylation levels of the gene marker ZNF300P1, combined with other clinical indicators, to provide a more accurate assessment of the diagnosis, treatment, and prognosis of myeloid tumors. The primer set provided by this invention was obtained through the inventors' creative efforts and screening and optimization, and it can amplify the target gene with high sensitivity and specificity. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the expression of ZNF300P1 in AML patients.

[0026] Figure 2 This is a schematic diagram of promoter methylation alterations of ZNF300P1 in patients with MDS and AML.

[0027] Figure 3 A schematic diagram illustrating the changes in ZNF300P1 promoter methylation before and after AML transformation in MDS patients, as detected by methylation-specific quantitative PCR.

[0028] Figure 4 The value of ZNF300P1 promoter methylation alteration in the auxiliary diagnosis of AML.

[0029] Figure 5 The impact of ZNF300P1 methylation on overall survival in MDS patients.

[0030] Figure 6 The effect of ZNF300P1 methylation on leukemia-free survival in MDS patients.

[0031] Figure 7 The impact of ZNF300P1 methylation on overall survival in AML patients.

[0032] Figure 8 The effect of ZNF300P1 methylation on leukemia-free survival in AML patients. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] The SEQ ID NO.1 sequence of the ZNF300P1 biomolecular marker gene described in this invention is as follows:

[0035] Homo sapiens zinc finger protein 300pseudogene 1(ZNF300P1),non-codingRNA

[0036] NCBI Reference Sequence:NR_026867.1

[0037]

[0038]

[0039]

[0040] 2101ctgaacacag aaaagccttc agggtcagtt caagccttaa tagatagtgc aacaaccaat

[0041] 2161ggatttgatg attttgggga ctacatcttt gttgataaaa ttttacaagt gaagtcatgt

[0042] 2221tcctaatgta tttcattctt tatcaaagat aatagagaag tcaatacgta aatgatggac

[0043] 2281attttcacta tggcatataa aagtttttaa attgagaaat gaatgattag cataacagaa

[0044] 2341cgaattgcat gtacatctct tttgaagtta tgtgctcctg attatactac ataacaatca

[0045] 2401gatatgtgta agattgttaa

[0046] Example 1:

[0047] In this embodiment, primers specifically amplifying ZNF300P1 expression were designed and synthesized.

[0048] Primers were designed targeting the ZNF300P1 gene mRNA sequence. These primers can specifically amplify the ZNF300P1 mRNA sequence for subsequent quantitative expression analysis. The specific design steps are as follows:

[0049] The ZNF300P1 mRNA sequence was found and exported from NCBI (https: / / www.ncbi.nlm.nih.gov / ):

[0050] Path: NCBI homepage → Nucleotide → search (ZNF300P1 homo) → go → select Homosapiens zinc finger protein 300pseudogene 1 (ZNF300P1), non-coding RNA → export sequence.

[0051] Quantitative PCR primers for expression detection were designed using Primer Premier 5.0 software:

[0052] Implementation path: File→New→DNA sequence→Import the obtained mRNA sequence→As is→Primer→Search→OK→OK→Select primers without hairpin, dimer, false priming, cross dimer, etc.→Finally obtain the primer sequence.

[0053] The specific sequence information is shown in the table below as SEQ ID NO.2-5. The primers were synthesized by BGI Genomics in Shanghai.

[0054]

[0055] Example 2:

[0056] This embodiment provides a method for establishing a quantitative PCR-based method for detecting ZNF300P1 expression levels.

[0057] (1) Total RNA extraction:

[0058] ① Bone marrow mononuclear cell isolation: Add 5ml of lymphocyte separation solution and 5ml of erythrocyte lysis solution to the obtained bone marrow specimen, mix by inverting, centrifuge at 1000×rpm for 5min, and discard the supernatant.

[0059] ② Add 1 ml of RNAiso Plus (purchased from TaKaRa, Code No. 9109) to the above precipitate, and gently pipette the liquid for 20 seconds until no obvious precipitate remains.

[0060] ③ Add 200 μl of chloroform, vortex for 30 seconds, let stand at room temperature for 5 minutes, and centrifuge at 12000 × rpm for 10 minutes. At this time, the liquid can be seen to be divided into three layers: upper (containing RNA), middle (DNA), and lower (organic phase).

[0061] ④ Take 400 μl of the supernatant containing RNA after centrifugation in step ③, place it into a new sterile 1.5 ml EP tube, add 400 μl of isopropanol, tighten the cap, invert and mix 50 times, after thorough mixing, let stand at room temperature for 10 min, then centrifuge at 12000×rpm for 10 min, carefully discard the supernatant, the white precipitate at the bottom of the EP tube is the RNA.

[0062] ⑤ Add 1 ml of 75% ethanol, invert to mix and rinse the precipitate, centrifuge at 12000 rpm for 5 min, and carefully discard the supernatant.

[0063] ⑥ Add 1 ml of 100% ethanol, invert to mix and rinse the precipitate, centrifuge at 12000 rpm for 10 min, carefully discard the supernatant, and air dry in a biosafety cabinet (purchased from Haier Bio, model HR900-ⅡB2).

[0064] ⑦ Add 10 μl of DEPC and dissolve the RNA on ice.

[0065] ⑧ After mixing, take 2 μl of RNA and use a NanoDrop spectrophotometer to detect the concentration and purity of the extracted RNA sample, and adjust it to a uniform concentration.

[0066] (2) cDNA reverse transcription, the reverse transcription reagent was purchased from TaKaRa, Code No. RR047A:

[0067] ① Prepare the Master Mix on ice. The reagents and amounts used are as follows: 2 μl 5×gDNA Eraser Buffer, 1 μl gDNA Eraser, 2 μl Total RNA and RNase Free dH2O up to 10 μl. Prepare the mixture according to the reaction number + 2, and then aliquot it into each 0.2 ml EP tube and place it in a regular PCR instrument (37℃, 5 min).

[0068] ② Add 1 μl PrimeScript RT Enzyme Mix I, 1 μl RT Primer Mix*4, 4 μl 5×PrimeScript Buffer 2 (for Real Time), and 4 μl RNase Free dH2O to the EP tube from the previous step, and place it in a standard PCR instrument (37℃ for 15 min, 85℃ for 5 sec). After completion, store the cDNA product at -20℃ for later use.

[0069] (3) Real-time quantitative PCR detection, the quantitative reagent was purchased from TaKaRa, Code No. RR82LR:

[0070] ① Using ABL1 as an internal control, the reaction system of ZNF300P1 and ABL1 was as follows: 10.0 μl of TB Green Premix Ex Taq II (Tli RNaseH Plus) (2×), 0.8 μl each of upstream and downstream expression primers, 0.4 μl of ROX Reference Dye or Dye II (50×), 6.0 μl of ddH2O, 2.0 μl of cDNA template, and the total reaction volume was 20 μl.

[0071] ②ZNF300P1 reaction conditions: pre-denaturation (95℃, 5 min), denaturation (95℃, 10 sec), annealing (60℃, 30 sec), extension (72℃, 30 sec), fluorescence collection (75℃, 30 sec), 40 cycles; ABL1 reaction conditions: pre-denaturation (95℃, 5 min), denaturation (95℃, 10 sec), annealing (62℃, 30 sec), extension (72℃, 30 sec), fluorescence collection (75℃, 30 sec), 40 cycles. Detection was performed using an ABI 7500 real-time quantitative PCR instrument.

[0072] (4) Quantitative analysis of results: The expression level of ZNF300P1 gene was determined by 2... -ΔΔCt The calculation is performed using the following formula: N ZNF300P1 =2 ΔCT ZNF300P1(control-sample) ÷2 ΔCT ABL(control-sample) (2 -ΔΔCT Each parallel experiment was repeated 3 times.

[0073] Validating the difference in ZNF300P1 expression between healthy individuals and AML patients

[0074] Samples were collected from 38 healthy individuals and 88 newly diagnosed AML patients. Each sample was processed using the same method as in Example 2, and the ZNF300P1 expression level was calculated for each sample. Results are as follows: Figure 1ZNF300P1 expression was shown in the control group and AML patients. Figure 1 The results showed that ZNF300P1 was downregulated in AML patients compared with the control.

[0075] Example 3:

[0076] In this embodiment, primers were designed and synthesized to specifically amplify the CpG islands in the ZNF300P1 promoter region.

[0077] To target the CpG island information in the promoter region of the ZNF300P1 gene, 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 ZNF300P1 promoter region, respectively, for subsequent quantitative methylation analysis. The specific design steps are as follows:

[0078] The ZNF300P1 promoter CpG island sequence was located and exported from UCSC (http: / / genome.ucsc.edu / ):

[0079] Path: UCSC homepage → Tools → gene sorter → Genome (Human) → search (ZNF300P1) → go → select ZNF300P1 → Genomic Sequence → select the upstream 2000bp and downstream 500bp 5'UTR → export the obtained sequence.

[0080] The required MSP primers were designed using Methyl primer 1.0 software:

[0081] Implementation path: Import the obtained promoter sequence → Design Primers → Find CpG Islands → NEXT → NEXT → select MSP or BSP and click NEXT (Design MSP Primers) → Obtain methylated and unmethylated primer sequences.

[0082] The specific sequence information is shown in the table below as SEQ ID NO.6-11. The primers were synthesized by BGI Genomics in Shanghai.

[0083]

[0084] Example 4:

[0085] This embodiment provides a method for establishing a quantitative PCR-based detection method for the methylation status of GpG islands in the ZNF300P1 promoter.

[0086] The detection method of this invention is based on the principle of quantitative methylation-specific PCR. Methylated and unmethylated DNA sequences can be distinguished by quantitative PCR amplification using primers specifically for methylation and unmethylation, respectively. The method includes the following steps:

[0087] (1) DNA template preparation

[0088] Take 5-10 ml of heparin-anticoagulated bone marrow specimens from one MDS, AML, and normal control sample. Isolate mononuclear cells using Ficoll solution and then use a genomic DNA extraction kit. The genomic DNA extraction kit used in this example was purchased from QIAGEN, model number Gentra Puregene Cell Kit. The specific steps for extracting genomic DNA from mononuclear cells are as follows:

[0089] (2) DNA extraction:

[0090] ① Bone marrow mononuclear cell isolation: Add 5ml of lymphocyte separation solution and 5ml of erythrocyte lysis solution to the obtained bone marrow specimen, mix by inverting, centrifuge at 1000×rpm, 4℃ for 5min, and discard the supernatant.

[0091] ② Resuspend the precipitate in 1×PBS, centrifuge at 800×g for 2 min at 4℃, and discard the supernatant.

[0092] ③ Add 900 μl of cell lysis solution to the above precipitate and blow until it is no longer viscous. If clumps are deposited, incubate at 37°C for 15 min and then incubate on ice for 1 min.

[0093] ④ Add 300 μl of protein precipitaton solution, shake for 20 seconds, centrifuge at 13000 × rpm for 1 min at 4 °C.

[0094] ⑤ After centrifugation, protein precipitation should be visible. If the precipitation is not dense, place it on ice for 5 minutes and then centrifuge again.

[0095] ⑥ Take a new 1.5ml EP tube, add 600μl of isopropanol and 600μl of the supernatant from the above centrifugation.

[0096] ⑦ Invert the tube 50 times until DNA flocculent precipitate is visible in the tube. Centrifuge at 13000 rpm for 1 min at 4℃ and discard the supernatant.

[0097] ⑧ Add 600 μl of 70% ethanol and mix by inverting. Centrifuge at 13000 rpm for 1 min at 4°C and discard the supernatant.

[0098] ⑨ Dry for 5 minutes. After the precipitate becomes transparent, add 100 μl of DNA dissolving solution, mix well, shake, and incubate at 65°C for 5 minutes.

[0099] ⑩ After mixing, take 2 μl of DNA and use a NanoDrop spectrophotometer to detect the concentration and purity of the extracted sample DNA, and adjust it to a uniform concentration. In this experiment, the concentration was adjusted to 100 ng / μl.

[0100] (3) DNA sulfurization:

[0101] 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.

[0102] ② Preparation of M-Wash Buffer: M-Wash buffer concentrate and anhydrous ethanol were mixed in a ratio of 1:4.

[0103] 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).

[0104] 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℃.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] (4) Quantitative methylation-specific PCR:

[0111] The promoter methylation of ZNF300P1 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 was used, purchased from ABI. The RQ-MSP reagent used was SYBR Premix Ex Taq II, purchased from TaKaRa, model code No. RR82LR. The specific steps are as follows:

[0112] 1) System Configuration

[0113] ①The ZNF300P1 methylation PCR (M-MSP) reaction system is as follows: 10.0 μl of TB Green Premix Ex Taq II (TliRNaseH Plus) (2×), 0.8 μl each of upstream and downstream expression primers, 0.4 μl of ROX Reference Dye or Dye II (50×), 6.0 μl of ddH2O, 2.0 μl of cDNA template, and the total reaction volume is 20 μl.

[0114] ②The ZNF300P1 unmethylated PCR (U-MSP) reaction system is as follows: 10.0 μl of TB Green Premix Ex Taq II (TliRNaseH Plus) (2×), 0.8 μl each of upstream and downstream expression primers, 0.4 μl of ROX Reference Dye or Dye II (50×), 6.0 μl of ddH2O, 2.0 μl of cDNA template, and the total reaction volume is 20 μl.

[0115] ③The ALU methylation reaction system was as follows: 10.0 μl of TB Green Premix Ex Taq II (Tli RNaseH Plus) (2×), 0.8 μl each of upstream and downstream primers, 0.4 μl of ROX Reference Dye or Dye II (50×), 6.0 μl of ddH2O, 2.0 μl of cDNA template, and a total reaction volume of 20 μl.

[0116] 2) Start-up (reaction conditions are set as follows):

[0117] ① ZNF300P1 methylation PCR (M-MSP) reaction conditions: ZNF300P1 methylation PCR (M-MSP) reaction conditions: pre-denaturation (95℃, 5 min), denaturation (95℃, 10 sec), annealing (60℃, 30 sec), extension (72℃, 30 sec), fluorescence collection (75℃, 30 sec), 40 cycles; ALU reaction conditions: pre-denaturation (95℃, 5 min), denaturation (95℃, 10 sec), annealing (62℃, 30 sec), extension (72℃, 30 sec), fluorescence collection (75℃, 30 sec), 20 cycles. Detection was performed using an ABI 7500 real-time quantitative PCR instrument.

[0118] ②ZNF300P1 unmethylated PCR (U-MSP) reaction conditions: pre-denaturation (95℃, 5 min), denaturation (95℃, 10 sec), annealing (60℃, 30 sec), extension (72℃, 30 sec), fluorescence collection (75℃, 30 sec), for a total of 40 cycles; ALU reaction conditions: pre-denaturation (95℃, 5 min), denaturation (95℃, 10 sec), annealing (62℃, 30 sec), extension (72℃, 30 sec), fluorescence collection (75℃, 30 sec), for a total of 20 cycles. Detection was performed using an ABI 7500 real-time quantitative PCR instrument.

[0119] 3) Result Calculation:

[0120] N M-ZNF300P1 =2 ΔCT M-ZNF300P1(control-sample) ÷2 ΔCT ALU(control-sample) (2 -ΔΔCT )

[0121] 4) Results Analysis

[0122] 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.

[0123] To validate differential ZNF300P1 promoter methylation in healthy individuals, patients with myocardial infarction (MDS), primary AML (pAML), and secondary AML (sAML).

[0124] Samples were collected from 46 healthy individuals, 70 MDS patients, 149 pAML patients, and 11 sAML patients. Each sample was processed using the same method as in Example 4. The ZNF300P1 promoter methylation level was calculated for each sample. The results are as follows: Figure 2 As shown. Figure 2The results showed that, compared with the control, ZNF300P1 exhibited high methylation in MDS, pAML, and sAML, with the highest methylation in sAML, followed by pAML, and the lowest in MDS.

[0125] Verify the difference in ZNF300P1 promoter methylation before and after AML progression in MDS patients.

[0126] Specimens were collected from 11 MDS patients before and after AML progression. Each pair of samples was processed using the same method as in Example 4. The ZNF300P1 promoter methylation level was calculated in each sample. The results are as follows: Figure 3 As shown. Figure 3 The results showed that ZNF300P1 underwent hypermethylation changes after MDS progressed to AML.

[0127] Validating the diagnostic and prognostic value of ZNF300P1 methylation

[0128] Based on the results obtained from the differences in ZNF300P1 promoter methylation in healthy individuals, MDS, primary AML (pAML), and secondary AML (sAML), the diagnostic efficacy of ZNF300P1 in AML was analyzed using receiver operating characteristic curves. The results are as follows: Figure 4 As shown. Figure 4 The results indicate that ZNF300P1 promoter methylation level can serve as a novel molecular marker for the auxiliary diagnosis of AML. Kaplan-Meier survival curve analysis revealed its prognostic value for MDS and AML, as shown in the following results. Figure 5-8 As shown.

[0129] Figure 4 The value of ZNF300P1 promoter methylation alteration in the auxiliary diagnosis of AML.

[0130] Figure 5 The impact of ZNF300P1 methylation on overall survival in MDS patients.

[0131] Figure 6 The effect of ZNF300P1 methylation on leukemia-free survival in MDS patients.

[0132] Figure 7 The impact of ZNF300P1 methylation on overall survival in AML patients.

[0133] Figure 8 The effect of ZNF300P1 methylation on leukemia-free survival in AML patients.

[0134] In summary, the method for detecting ZNF300P1 expression and promoter methylation level based on quantitative PCR established in this invention can be used for the auxiliary diagnosis and prognosis of myeloid tumors including MDS and AML, and has good application prospects.

[0135] 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.

[0136] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a product for detecting the methylation level of the ZNF300P1 gene promoter in the preparation of products for diagnosing acute myeloid leukemia, characterized in that, The detection products for the methylation level of the ZNF300P1 gene promoter include ZNF300P1 methylation primers as shown in SEQ ID NO. 6-7 and ZNF300P1 unmethylation primers as shown in SEQ ID NO. 8-9.

2. The application according to claim 1, characterized in that, The detection product also includes an internal reference ALU primer, the forward primer of which is shown in SEQ ID NO.10 and the reverse primer is shown in SEQ ID NO.11.

Images