Expression and methylation detection of biomolecular marker ZNF300P1 and application of biomolecular marker ZNF300P1

Abstract

The invention relates to an expression and methylation quantitative detection method of a ZNF300P1 gene and application of the ZNF300P1 gene in assisting myeloid tumor diagnosis and prognosis evaluation. The invention provides ZNF300P1 gene expression and methylation quantitative detection, and the ZNF300P1 biomolecular marker gene is shown as SEQ ID NO.1. According to the expression and methylation detection primer provided by the invention, the ZNF300P1 biomolecular marker expression detection primer is shown as SEQ ID NO.2-3, the ZNF300P1 biomolecular marker methylation detection primer is shown as SEQ ID NO.6-9, and the ZNF300P1 biomolecular marker methylation detection primer is shown as SEQ ID NO.2-3. The kit is used for detecting the expression and methylation level of bone marrow / peripheral blood ZNF300P1 of patients with myelodysplastic syndromes and acute myelogenous leukemia, serves as a novel biomarker of the patients with myelodysplastic syndromes and acute myelogenous leukemia, is combined with other indexes, and provides support for early diagnosis and prognosis evaluation of the patients.

Description

Technical Field

[0001] The present invention relates to a biomolecular marker, and in particular to the expression and methylation detection of a biomolecular marker ZNF300P1 and applications thereof. Background Art

[0002] Myeloid neoplasms mainly include 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, manifested as ineffective hematopoiesis, refractory hematopoietic cell reduction, hematopoietic failure, and high risk of transformation to AML. MDS is a highly heterogeneous disease. Patients with medium and low risk have good survival, mainly symptomatic and supportive treatment; while high-risk patients have a poor prognosis and a high risk of AML transformation, mainly demethylation, chemotherapy, and transplantation. AML is a blood system malignancy originating from hematopoietic stem / progenitor cells, accompanied by abnormal proliferation of a large number of primitive cells. It is also highly heterogeneous. The prognosis of different patients varies greatly, and the overall survival ranges from a few months to long-term survival. Patients with medium and high risk often need intensive treatment and hematopoietic stem cell transplantation to improve their prognosis. The heterogeneity of MDS and AML is essentially manifested in cell and molecular genetics, but the biomarkers discovered so far are limited and fail to meet clinical needs. In the era of new drugs, the improvement of AML survival rate has reached a bottleneck based on traditional chemotherapy regimens, so more new targets are provided for AML precision and targeted treatment, which ultimately provides the possibility of improving patient survival and prognosis, and has a wide range of scenarios and important clinical application value. As we all know, the core of targeted / immunotherapy is to discover molecular markers closely related to the onset of the disease, so as to develop corresponding targeted therapeutic drugs to play a targeted killing role on tumors, improve treatment effects, and reduce adverse reactions.

[0003] In recent years, studies have shown that pseudogenes have a wide range of biological functions and may be involved in a variety of physiological processes in the body, as well as in the occurrence and development of a variety of diseases including tumors. ZNF300P1 (LOC134466) is a pseudogene of the human zinc finger protein ZNF300 (homology 89%). Gene structure analysis revealed that there is also a large CpG island in the promoter region of ZNF300P1. Targeted methylation sequencing revealed that it also undergoes hypermethylation changes in MDS and AML patients, leading to silenced expression, which may play a role in suppressing tumors. Therefore, the detection of ZNF300P1 expression and methylation as a new biomolecular marker for myeloid tumor patients has good application prospects. Summary of the invention

[0004] The purpose of the present invention is to provide a quantitative detection method for a biomolecular marker and its application, so as to solve the defect that the existing biomarkers discovered so far are limited and fail to meet clinical needs; by proposing a quantitative detection method for ZNF300P1 expression and methylation, the expression and methylation level differences of ZNF300P1 in myeloid tumors and normal controls are detected by quantitative PCR technology, which is used as a biomarker for myeloid tumor patients, providing support for early diagnosis and prognosis evaluation of myeloid tumor patients in clinical practice.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions: 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 comprises a forward primer as shown in SEQ ID NO.2 and a reverse primer as shown in SEQ ID NO.3.

[0007] An application of a ZNF300P1 biomolecular marker methylation detection product, wherein the ZNF300P1 methylation detection product comprises ZNF300P1 methylation and unmethylation specific primers as shown in SEQ ID NO.6-7 and SEQ ID NO.8-9.

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

[0009] Furthermore, the detection reagent is a quantitative PCR reagent for detecting ZNF300P1 expression and promoter methylation level.

[0010] On the one hand, the present invention provides a product for quantitatively detecting ZNF300P1 gene expression and methylation, and its application in assisting the diagnosis and prognosis evaluation of myeloid tumors.

[0011] The second aspect of the present invention provides detection primers for detecting ZNF300P1 gene expression and methylation in myeloid tumors, including 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 as SEQ ID NO.1, the primer sequence for ZNF300P1 expression is shown as SEQ ID NO.2-3, the primer sequence for ZNF300P1 methylation is shown as SEQ ID NO.6-7, and the primer sequence for ZNF300P1 unmethylation is shown as 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 as SEQ ID NO.4, and the reverse primer is shown as SEQ ID NO.5, the forward primer of the internal reference ALU primer is shown as SEQ ID NO.10, and the reverse primer is shown as SEQ ID NO.11.

[0013] The third aspect of the present invention provides a method for detecting the expression level of ZNF300P1 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 transcribing the total RNA from step (1) into cDNA;

[0016] (3) The cDNA of step (2) was amplified using the primer pairs shown in SEQ ID NO.2 and SEQ ID NO.3, respectively, and quantitative PCR was performed using ABL1 sequence-specific primers as quality control for each sample DNA, and 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] The fourth aspect of the present invention provides a method for detecting the methylation level of the ZNF300P1 gene promoter based on quantitative PCR, characterized in that it comprises the following steps:

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

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

[0021] (3) using the DNA sulfurized in step (2) to perform quantitative PCR using the methylation-specific and unmethylation-specific primers shown in SEQ ID NOs. 6-7 and 8-9, respectively, and analyzing the expression level of ZNF300P1 in the sample by quantitative methylation-specific PCR to obtain relevant data;

[0022] (4) Fluorescence quantitative PCR using ALU sequence-specific primers was used as quality control for the DNA of each specimen;

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

[0024] Beneficial effects of the present invention: Compared with the prior art, the present invention is applied to myeloid tumors based on the expression and methylation level of the gene marker ZNF300P1, combined with other clinical indicators, to provide a more accurate judgment for the diagnosis, treatment and prognosis of myeloid tumors. The primer set provided by the present invention is obtained by the inventors through creative labor and screening and optimization, and can amplify the target gene with high sensitivity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of ZNF300P1 expression in AML patients.

[0026] Figure 2 Schematic diagram of ZNF300P1 promoter methylation changes in MDS and AML patients.

[0027] Figure 3 Schematic diagram of methylation-specific quantitative PCR detection of changes in ZNF300P1 promoter methylation before and after AML transformation in MDS patients.

[0028] Figure 4 To investigate the value of ZNF300P1 promoter methylation changes in assisting the diagnosis of AML.

[0029] Figure 5 To investigate the effect of ZNF300P1 methylation on the overall survival of patients with MDS.

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

[0031] Figure 7 To investigate the effect of ZNF300P1 methylation on the overall survival of AML patients.

[0032] Figure 8 Effect of ZNF300P1 methylation on leukemia-free survival in AML patients. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] The SEQ ID NO.1 sequence of the ZNF300P1 biomolecular marker gene of the present 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] Embodiment 1:

[0047] In this example, primers were designed and synthesized to specifically amplify the expression of ZNF300P1.

[0048] Primers were designed for the ZNF300P1 gene mRNA sequence. This pair of primers can be used to specifically amplify the ZNF300P1 mRNA sequence for subsequent quantitative expression analysis. The specific design steps are as follows:

[0049] Find and export the ZNF300P1 mRNA sequence at 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] Primer Premier 5.0 software was used to design quantitative PCR primers for expression detection:

[0052] Implementation path: File→New→DNA sequence→Import the obtained mRNA sequence→As is→Primer→Search→OK→OK→Preferably, primers without Hairpin, Dimer, False Priming, Cross Dimer, etc. are selected→Finally, the primer sequence is obtained.

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

[0054]

[0055] Embodiment 2:

[0056] This example provides a method for detecting the expression level of ZNF300P1 based on quantitative PCR

[0057] (1) Total RNA extraction:

[0058] ① Bone marrow mononuclear cell separation: add 5 ml of lymphocyte separation solution and 5 ml of red blood cell lysis solution to the obtained bone marrow specimen, mix well by inversion, centrifuge at 1000× rpm for 5 min, and discard the supernatant.

[0059] ② Add 1 ml RNAiso Plus purchased from TaKaRa, Code No.9109, to the above precipitate. Use a pipette to repeatedly blow the liquid for 20 seconds, gently until there is no obvious precipitation.

[0060] ③ Add 200 μl of chloroform, vortex thoroughly 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 400μl of the supernatant containing RNA after centrifugation in step ③, place it into a new sterile 1.5ml EP tube, and add 400μl of isopropanol. Close the lid tightly, mix it upside down 50 times, let it stand at room temperature for 10 minutes after thorough mixing, and centrifuge it at 12000×rpm for 10 minutes. Carefully discard the supernatant. The white precipitate at the bottom of the EP tube is 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 place in a biological safety cabinet (purchased from Haier Bio, model HR900-ⅡB2) with the lid opened to air dry.

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

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

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

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

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

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

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

[0071] ② ZNF300P1 reaction conditions: pre-denaturation (95°C, 5 min), denaturation (95°C, 10 sec), annealing (60°C, 30 sec), extension (72°C, 30 sec), fluorescence collection (75°C, 30 sec), the number of cycles is 40 cycles; ABL1 reaction conditions: pre-denaturation (95°C, 5 min), denaturation (95°C, 10 sec), annealing (62°C, 30 sec), extension (72°C, 30 sec), fluorescence collection (75°C, 30 sec), the number of cycles is 40 cycles. Detection was completed on an ABI 7500 real-time fluorescence quantitative PCR instrument.

[0072] (4) Quantitative analysis of results: ZNF300P1 gene expression level was measured by 2 -ΔΔCt The specific calculation formula is as follows: N ZNF300P1 =2 ΔCT ZNF300P1(control-sample) ÷2 ΔCT ABL(control-sample) (2 -ΔΔCT ), and each parallel experiment was repeated 3 times.

[0073] Verification of the difference in ZNF300P1 expression between healthy subjects and AML patients

[0074] 38 healthy subjects and 88 newly diagnosed AML patient samples were collected, and each sample was treated with the same treatment method as in Example 2, and the expression level of ZNF300P1 in each sample was calculated. Figure 1ZNF300P1 expression in controls and AML patients is shown. Figure 1 The results showed that ZNF300P1 expression was downregulated in AML patients compared with controls.

[0075] Embodiment 3:

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

[0077] According to the CpG island information in the promoter region of the ZNF300P1 gene, two sets of primers for methylated and unmethylated sequences were designed. These two pairs of primers can specifically amplify the methylated and unmethylated sequences of the CpG island in the promoter region of the ZNF300P1 gene, respectively, and perform subsequent quantitative methylation analysis. The specific design steps are as follows:

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

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

[0080] Use Methyl primer 1.0 software to design the required MSP primers:

[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 SEQ ID NO.6-11 in the following table. The primers were synthesized by Shanghai BGI Genomics Co., Ltd.

[0083]

[0084] Embodiment 4:

[0085] This example provides a method for establishing a quantitative PCR-based method for detecting the methylation status of the GpG island in the ZNF300P1 promoter

[0086] The detection method of the present invention is based on the principle of quantitative methylation-specific PCR, and can distinguish methylated from non-methylated DNA sequences by quantitative PCR amplification containing methylation-specific and non-methylation-specific primers, respectively. The method comprises the following steps:

[0087] (1) DNA template preparation

[0088] Take 5-10 ml of heparin anticoagulated bone marrow specimens from MDS, AML and normal controls, separate mononuclear cells using Ficoll solution, and use a genomic DNA extraction kit. The genomic DNA extraction kit used in this example is purchased from QIAGEN, model number Gentra Puregene Cell Kit. The genomic DNA from the mononuclear cells is extracted in the following specific steps:

[0089] (2) DNA extraction:

[0090] ① Bone marrow mononuclear cell separation: add 5 ml of lymphocyte separation solution and 5 ml of red blood cell lysis solution to the obtained bone marrow specimen, mix them upside down, centrifuge at 1000× rpm, 4℃ for 5 min, and discard the supernatant.

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

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

[0093] ④ Add 300 μl of protein preparation solution, shake for 20 seconds, and centrifuge at 13000× rpm and 4℃ for 1 minute.

[0094] ⑤ Protein precipitation should be visible after centrifugation. If the precipitate is not dense, it should be placed on ice for 5 minutes and centrifuged again.

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

[0096] ⑦ Invert the tube 50 times until DNA floccules are visible in the tube. Centrifuge at 13,000×rpm, 4℃ for 1 min and discard the supernatant.

[0097] ⑧Add 600 μl of 70% ethanol and mix by inversion, centrifuge at 13,000×rpm, 4°C for 1 min, and discard the supernatant.

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

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

[0100] (3) DNA Sulfidation:

[0101] ①Prepare C / T conversion reagent (CT conversion Reagent): Add 900μl sterile water for injection, 300μl M-Dilution Buffer, and 50μl M-Dissolving Buffer to a tube of C / T conversion reagent and shake at room temperature for 10 minutes.

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

[0103] ③ Sample treatment: Add 130 μl CT conversion reagent, 18 μl sterile water for injection and 2 μl of the DNA sample with a concentration of 100 ng / μl in the above step (1) into 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 follow the steps: a. 98℃ / 10min; b. 64℃ / 150min; c. Store at 4℃.

[0105] ⑤ Add 600 μl M-Binding Buffer to the Zymo-Spin TMIC column, place the column in a collection tube, add the sample mixture obtained in step ④ to the column, cover the lid and invert to mix.

[0106] ⑥ Centrifuge at full speed (>10000×g) for 30 seconds, discard the liquid in the collection tube, add 100μl M-WashBuffer to the column, and centrifuge at full speed again for 30 seconds.

[0107] ⑦ Add 200 μl M-Desulphonation Buffer, incubate at room temperature (20-30°C) for 15-20 min, and centrifuge at full speed for 30 s.

[0108] ⑧Add 200μl M-Wash Buffer and centrifuge at full speed for 30s. Add 200μl M-Wash Buffer again and centrifuge at full speed for 30s.

[0109] ⑨ Place the chromatographic column in a 1.5ml EP tube and add 10μl M-Elution Buffer to the column. Centrifuge at full speed for 30s to elute. The obtained sulfurized DNA is stored at -80℃ for future use.

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

[0111] The RQ-MSP method was used to detect the methylation of the promoter region of ZNF300P1 and the internal reference gene ALU. Each PCR reaction carried a negative quality control (water) and a positive quality control (target gene plasmid). The instrument used was a fluorescent quantitative PCR instrument 7500 purchased from ABI. The RQ-MSP reagent used was SYBR Premix Ex Taq II purchased from TaKaRa, model No. RR82LR. The specific steps are as follows:

[0112] 1) System configuration

[0113] ① The reaction system of ZNF300P1 methylation PCR (M-MSP) was as follows: TB Green Premix Ex Taq II (TliRNaseH Plus) (2×) 10.0 μl, 0.8 μl 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 the total reaction volume was 20 μl.

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

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

[0116] 2) On-line (reaction conditions are set as follows):

[0117] ① ZNF300P1 methylation PCR (M-MSP) reaction conditions: pre-denaturation (95°C, 5 min), denaturation (95°C, 10 sec), annealing (60°C, 30 sec), extension (72°C, 30 sec), fluorescence collection (75°C, 30 sec), the number of cycles is 40 cycles; ALU reaction conditions: pre-denaturation (95°C, 5 min), denaturation (95°C, 10 sec), annealing (62°C, 30 sec), extension (72°C, 30 sec), fluorescence collection (75°C, 30 sec), the number of cycles is 20 cycles. Detection was completed on ABI7500 real-time fluorescence quantitative PCR instrument.

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

[0119] 3) Calculation of results:

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

[0121] 4) Result analysis

[0122] The melting curve obtained by PCR amplification was observed. It was found that a single melting temperature was obtained in each PCR tube, that is, the specificity of the amplified product was good.

[0123] Validation of ZNF300P1 promoter methylation differences in healthy subjects, MDS, primary AML (pAML) and secondary AML (sAML) patients

[0124] Samples from 46 healthy subjects, 70 MDS patients, 149 pAML patients and 11 sAML patients were collected. Each sample was treated with the same treatment method as in Example 4. The methylation level of the ZNF300P1 promoter in each sample was calculated. The results are as follows: Figure 2 shown. Figure 2The results showed that compared with the control, ZNF300P1 was hypermethylated in MDS, pAML and sAML, with the highest level 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 treated with the same treatment method as in Example 4. The methylation level of the ZNF300P1 promoter in each sample was calculated. The results are as follows: Figure 3 shown. Figure 3 The results showed that ZNF300P1 underwent hypermethylation after MDS progressed to AML.

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

[0128] Based on the results obtained from validating the differences in ZNF300P1 promoter methylation in healthy subjects, MDS, primary AML (pAML) and secondary AML (sAML) patients, the diagnostic efficacy of ZNF300P1 in AML was analyzed by receiver operating characteristic curve analysis. Figure 4 shown. Figure 4 The results showed that ZNF300P1 promoter methylation level can be used as a new molecular marker for auxiliary diagnosis of AML. The Kaplan-Meier survival curve analysis revealed its value for the prognosis of MDS and AML. Figure 5-8 shown.

[0129] Figure 4 To investigate the value of ZNF300P1 promoter methylation changes in assisting the diagnosis of AML.

[0130] Figure 5 To investigate the effect of ZNF300P1 methylation on the overall survival of patients with MDS.

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

[0132] Figure 7 To investigate the effect of ZNF300P1 methylation on the overall survival of AML patients.

[0133] Figure 8 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 the present invention can be used for auxiliary diagnosis and prognosis judgment of myeloid tumors including MDS and AML, and has good application prospects.

[0135] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily realized. Therefore, without departing from the general concept defined by the claims and equivalent scope, the present invention is not limited to the specific details and the illustrations shown and described here.

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

Claims

1. An application of a ZNF300P1 biomolecular marker expression and methylation detection product, characterized in that: The gene sequence of the ZNF300P1 biomolecular marker is shown in SEQ ID NO.

1.

2. The use of a ZNF300P1 biomolecular marker expression detection product according to claim 1, characterized in that: The ZNF300P1 biomolecular marker 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.

3. The use of a ZNF300P1 biomolecular marker methylation detection product according to claim 1, characterized in that: The ZNF300P1 biomolecular marker methylation detection product includes ZNF300P1 methylation and unmethylation specific primers as shown in SEQ ID NO.6-7 and SEQ ID NO.8-9.

4. The use of a ZNF300P1 biomolecular marker expression and methylation detection product as claimed in claim 2 or 3, characterized in that: The detection reagent is a quantitative PCR reagent, which detects the expression of ZNF300P1 and the methylation level of the promoter.

5. Application of ZNF300P1 biomolecular marker in assisting the diagnosis and prognostic evaluation of myeloid tumors.

6. The use of the ZNF300P1 biomolecular marker according to claim 5 in the preparation of auxiliary diagnosis and prognosis evaluation of myeloid tumors, characterized in that: The myeloid tumors are mainly myelodysplastic syndrome and acute myeloid leukemia.

7. A primer for detecting ZNF300P1 gene expression and methylation in myeloid tumors, characterized in that: It includes 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.

8. The primers for detecting ZNF300P1 gene expression and methylation in myeloid tumors according to claim 7, characterized in that: The primers also include an internal reference ABL1 primer and an internal reference ALU primer. The forward primer of the internal reference ABL1 is shown as SEQ ID NO.4, and the reverse primer is shown as SEQ ID NO.

5. The forward primer of the internal reference ALU primer is shown as SEQ ID NO.10, and the reverse primer is shown as SEQ ID NO.

11.

9. A method for detecting the expression level of ZNF300P1 in myeloid tumors based on quantitative PCR, characterized in that: The steps include: (1) Extract total RNA from human bone marrow / peripheral blood cell samples and perform quality testing on the extracted RNA samples; (2) reverse transcribing the total RNA from step (1) into cDNA; (3) The cDNA of step (2) was amplified using the primer pairs shown in SEQ ID NO.2 and SEQ ID NO.3, respectively, and quantitative PCR was performed using ABL1 sequence-specific primers as quality control for each sample DNA, and the expression level of ZNF300P1 in the sample was analyzed to obtain relevant data.

10. A method for detecting the methylation level of ZNF300P1 gene based on quantitative PCR, characterized in that: The steps include: (1) Extract bone marrow / peripheral blood cell genomic DNA samples and perform quality testing; (2) sulfurizing the DNA from step (1); (3) using the DNA sulfurized in step (2) to perform quantitative PCR using the methylation-specific and unmethylation-specific primers shown in SEQ ID NOs. 6-7 and 8-9, respectively, and analyzing the expression level of ZNF300P1 in the sample by quantitative methylation-specific PCR to obtain relevant data; (4) Fluorescence quantitative PCR using ALU sequence-specific primers was used as quality control for the DNA of each specimen; (5) Calculate the methylation and unmethylation levels.

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