CN113637750A - Auxiliary diagnosis, prognosis diagnosis or risk stratification marker for acute myeloid leukemia and application thereof - Google Patents
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Abstract
The invention discloses an auxiliary diagnosis, prognosis diagnosis or risk degree layering marker for acute myeloid leukemia and application thereof. After multiple experiments, the patentee discovers for the first time that the expression level of ARF3 in the bone marrow of a patient with acute myelogenous leukemia (AML for short) is obviously up-regulated compared with that of a normal person, and the ARF3 gene can be used as an auxiliary diagnostic marker of AML; meanwhile, the patentees also find that the expression level of the ARF3 gene is significantly related to the prognosis of AML patients, and the AML patients with high ARF3 expression have worse overall survival and no event survival, which indicates that the prognosis is poor, particularly in the critical group in cytogenetic risk stratification; this also indicates that the ARF3 gene can be used as a prognostic diagnosis or a risk stratification marker for acute myeloid leukemia.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an auxiliary diagnosis, prognosis diagnosis or risk degree layering marker for acute myeloid leukemia and application thereof.
Background
Acute Myeloid Leukemia (AML) is a malignant clonal disease with high heterogeneity in cell morphology, molecular biology, cytogenetics, immunophenotyping, clinical presentation, etc. In recent years, although the complete remission rate and 5-year disease-free survival rate of AML patients have improved over the past; however, due to the problems of difficult matching of bone marrow transplant patients, high price of molecular targeted therapy, tolerance of chemotherapeutic drugs, strong side effects and the like in the existing therapeutic means, the search for new therapeutic targets and prognostic biomarkers is increasingly urgent.
Altered or mutated forms of expression of various genes involved in the proliferation or survival of leukemic cells are not only associated with the pathogenesis of the disease, but are also important for predicting treatment response and survival. Although mutations in genes such as NPM1, FLT3, C-kit, C/EBPA and RUNX1 have been incorporated into the 2016 World Health Organization (WHO) AML classification reference, accurate risk stratification or prognostic diagnosis of AML remains difficult. Therefore, identification of new prognostic indicators is crucial to improve the current poor survival outcome of AML.
With the innovation of tumor molecular biology technology, in recent years, the ARF family of small GTPases belonging to the RAS superfamily can participate in various physiological processes such as cell proliferation, motility, and differentiation, etc. by regulating the membrane vesicle trafficking and binding to the cytoskeleton. An increasing number of studies have also shown that the ARF family is involved in the development of tumors, and ARF3 is an important member of the ARF family, and its expression is also associated with various tumors. Studies have shown that ARF3 is not only positively correlated with clinical staging of breast cancer, but that 92.8% of patients with malignant breast cancer have higher expression of ARF3 than patients with benign breast cancer. In addition, the over-expression of ARF3 can inhibit the activity of FOXO1 transcription factor, regulate the G1/S transition of cell cycle, and promote the proliferation of breast cancer cells. These studies suggest that ARF3 plays an important role in the development of tumor, and is expected to provide a new aid for the prognosis diagnosis of acute myeloid leukemia.
Currently, prognosis evaluation of AML is mostly judged by changes in age, secondary leukemia and cytogenetics, and risk stratification is performed by combining changes in molecular genetics. However, cytogenetically-based risk stratification for AML is not perfect, as most patients are at moderate cytogenetic risk. Therefore, finding a faster, more sensitive and more specific prognostic diagnostic index is of great clinical significance.
Disclosure of Invention
The invention aims to provide a novel auxiliary diagnosis, prognosis diagnosis or risk stratification marker for acute myeloid leukemia and application thereof.
In order to achieve the above purpose, the technical solution of the present application is as follows:
an auxiliary diagnostic, prognostic or risk stratification marker for acute myeloid leukemia is ARF3 gene.
After multiple experiments, the patentee discovers for the first time that the expression level of ARF3 in the bone marrow of a patient with acute myelogenous leukemia (AML for short) is obviously up-regulated compared with that of a normal person, and the ARF3 gene can be used as an auxiliary diagnostic marker of AML; meanwhile, the patentees also find that the expression level of the ARF3 gene is significantly related to the prognosis of AML patients, and the AML patients with high ARF3 expression have worse overall survival and no event survival, which indicates that the prognosis is poor, particularly in the critical group in cytogenetic risk stratification; this also indicates that the ARF3 gene can be used as a prognostic diagnosis or a risk stratification marker for acute myeloid leukemia.
Based on the above, the invention also provides an application of the ARF3 gene in the preparation of an acute myeloid leukemia auxiliary diagnosis product, an application of the ARF3 gene in the preparation of an acute myeloid leukemia prognosis diagnosis product, and an application of the ARF3 gene in the preparation of an acute myeloid leukemia risk stratification product.
Preferably, the acute myeloid leukemia auxiliary diagnosis product, the acute myeloid leukemia prognosis diagnosis product and the acute myeloid leukemia risk stratification product are specific detection primers of ARF3 gene or specific detection probes of ARF3 gene; or a kit containing at least one of the specific detection primer and the specific detection probe, or a gene chip carrying the specific detection probe.
As an example of a specific embodiment, the present invention provides a specific detection primer of ARF3 gene, the specific detection primer of ARF3 gene comprises a forward primer having a nucleotide sequence shown as SEQ ID No.1 and a reverse primer having a nucleotide sequence shown as SEQ ID No. 2.
The nucleotide sequences of the forward primer and the reverse primer can be shown as SEQ ID No.1 and SEQ ID No.2, and a plurality of bases can be added on the basis of the nucleotide sequences shown as SEQ ID No.1 and SEQ ID No. 2.
Preferably, the primer for detecting the specificity of the ARF3 gene comprises:
a forward primer: 5'-GGAAGAGCTGATGAGAATGCTGG-3', respectively;
reverse primer: 5'-ACCAGTTACGGTGACGAAGGGA-3' are provided.
The invention also provides an auxiliary diagnosis, prognosis diagnosis or risk stratification kit for acute myeloid leukemia, which contains the specific detection primer of the ARF3 gene.
The kit of the invention is preferably a real-time quantitative PCR kit, which is convenient for comparing the expression level of the ARF3 gene, and the kit also contains a specific detection primer of an internal reference gene GAPDH, and the specific detection primer of the internal reference gene GAPDH comprises:
a forward primer: 5'-ATGGGGAAGGTGAAGGTCG-3', respectively;
reverse primer: 5'-GGGTCATTGATGGCAACAATATC-3' are provided.
Compared with the prior art, the invention has the beneficial effects that:
after multiple experiments, patentees find that the expression level of ARF3 in the bone marrow of patients with acute myelogenous leukemia (AML for short) is obviously up-regulated compared with normal people for the first time; meanwhile, the patentees also find that the expression level of the ARF3 gene is significantly related to the prognosis of AML patients, and the AML patients with high ARF3 expression have worse overall survival and no event survival, which indicates that the prognosis is poor, particularly in the critical group in cytogenetic risk stratification; the ARF3 gene can be used as an auxiliary diagnosis, prognosis diagnosis or risk stratification marker of acute myeloid leukemia, provides a new detection index for AML diagnosis or prognosis analysis, and provides a new theoretical basis for AML prognosis analysis.
Drawings
FIG. 1 shows the expression of ARF3 gene in bone marrow and normal bone marrow of patients with acute myelogenous leukemia;
wherein ARF3 mRNA expression (Fold change) indicates the relative expression level (Fold difference) of ARF3 gene, AML (n 116) indicates the bone marrow sample (sample number 116) of acute myeloid leukemia patient, and Control (n 15) indicates the normal bone marrow sample (sample number 15);
FIG. 2 is the results of correlation analysis of ARF3 gene expression and overall survival of patients with acute myeloid leukemia;
wherein, Overall survival indicates Overall survival, time (days) indicates time after treatment (days), Low ARF3(n ═ 34) indicates samples with Low ARF3 expression (number of samples is 34), High ARF3(n ═ 34) indicates samples with High ARF3 expression (number of samples is 34), and p ═ 0.015 indicates that there is a difference; the same applies below;
FIG. 3 is a graph showing the correlation between the expression of ARF3 gene and the survival rate of patients with acute myeloid leukemia without events;
wherein, Event free survival indicates that no Event survives, and p is 0.012 indicates that there is a difference;
FIG. 4 is a graph showing the correlation between the expression of ARF3 gene and the overall survival rate of critical patients among patients with acute myelogenous leukemia;
wherein, Low ARF3(n ═ 24) indicates a sample with Low ARF3 expression (sample number 24), High ARF3(n ═ 18) indicates a sample with High ARF3 expression (sample number 18), and p ═ 0.006 indicates that there is a significant difference; the same applies below;
FIG. 5 is a graph showing correlation analysis results of ARF3 gene expression and event-free survival rate of critical patients among acute myelogenous leukemia patients;
where p ═ 0.005 indicates a significant difference.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the accompanying drawings and the detailed description.
Example 1
Specific detection primers are designed aiming at the ARF3 gene, and the reliability of the specific detection primers is verified by amplifying the ARF3 gene in cells through RT-QPCR and sequencing the ARF3 gene.
The specific detection primer of the ARF3 gene comprises:
a forward primer: 5'-GGAAGAGCTGATGAGAATGCTGG-3' (SEQ ID No. 1);
reverse primer: 5'-ACCAGTTACGGTGACGAAGGGA-3' (SEQ ID No. 2);
a specific detection primer of an internal reference gene GAPDH is designed, and comprises the following components:
a forward primer: 5'-ATGGGGAAGGTGAAGGTCG-3' (SEQ ID No. 3);
reverse primer: 5'-GGGTCATTGATGGCAACAATATC-3' (SEQ ID No. 4).
The above two pairs of specific primers were synthesized by Onychoma corporation (Hangzhou).
The RT-QPCR amplification method comprises the following steps:
(1) cell collection and pretreatment
Collecting in vitro cultured THP-1 cells, washing with PBS for 2 times, adding 1mL of RNAioso Plus reagent (purchased from TaKaRa) into the obtained cell mass, repeatedly beating and uniformly mixing, and storing the RNAioso Plus lysate at-80 ℃ for later use.
(2) RNA extraction
Adding 200 μ l chloroform directly into the RNAioso Plus lysate obtained in step (1), shaking and mixing for 15s with force from top to bottom, standing for 10min, and centrifuging at 4 ℃ and 12000g for 15 min; taking the supernatant to a new EP tube, adding isopropanol with the same volume to mix evenly, standing for 10min, and centrifuging for 10min at 12000g at 4 ℃; removing supernatant, adding 1ml of 75% ethanol, washing by inversion, centrifuging at 4 deg.C and 12000g for 5 min; removing supernatant, adding 1ml of anhydrous ethanol, reversing and cleaning, and centrifuging at 4 ℃ and 12000g for 5 min; the supernatant was removed and dried at room temperature. The RNA concentration was determined by adding a suitable volume (20-50. mu.l) of DEPC water to the RNA pellet and dissolving the RNA (A260/A280: 1.8-2.1).
(3) Reverse transcription of cDNA
Reverse transcription of cDNA was carried out using the RevertAid First Strand cDNA Synthesis Kit (purchased from Thermo), and the First reaction was carried out according to the reaction system and reaction program shown in Table 1, and the second reaction was carried out according to the reaction system and reaction program shown in Table 2.
TABLE 1
TABLE 2
(4) Quantitative PCR
Quantitative PCR amplification is carried out on the cDNA obtained in the step (3) by adopting a TB GreenTM Premix Ex TaqTM II (Tli RNaseH Plus) kit (purchased from TaKaRa) and the specific primers, and the reaction system and the reaction program of the quantitative PCR amplification are shown in Table 3.
TABLE 3
(5) Agarose electrophoresis
GAPDH is used as an internal reference, a PCR amplification product is detected through a 3% agarose gel electrophoresis test, the PCR amplification product is sent to Shanghai Producer sequencing company for sequencing, and the ARF3 gene (SEQ ID No.5) is confirmed through sequence Blast comparison, so that the primer designed in the embodiment can specifically amplify the ARF3 gene.
Example 2
The specific detection primer is adopted to detect and analyze the expression condition of the ARF3 gene in the bone marrow of the patient with acute myelogenous leukemia, and the detection and analysis method comprises the following steps:
(1) bone marrow specimen collection and processing
116 AML bone marrow specimens newly issued in 2016-2020 in 6 months in Ningbo City were collected in the first hospital, 15 normal control bone marrow specimens were collected, and all participants signed informed consent; adopting a bone marrow cell culture bottle to collect a bone marrow specimen and recording the information of a patient; collecting mononuclear cells by lymphocyte separation liquid (Ficoll) density gradient centrifugation, adding RNAioso Plus reagent into the collected mononuclear cell mass, repeatedly beating and uniformly mixing, and storing at-80 ℃ for later use.
(2) RNA extraction: same as example 1;
(3) reverse transcription of cDNA: same as example 1;
(4) quantitative PCR: same as in example 1.
(5) Calculation of ARF3 Gene expression level
Using the THP-1 cell line of example 1 as a control, 2 was used-ΔΔCtThe method calculates the ARF3 gene expression quantity, delta Ct ═ Ct [, inARF3-CtGAPDH]Sample(s)-[CtARF3-CtGAPDH]Normal control. The data were statistically analyzed using SPSS 24 software, the Fisher's exact test was used to compare the differences between groups, the Kaplan-Meier method and the Log-rank test were used to analyze the differences between groups for survival data (P < 0.05 considered for statistical differences), and the results are shown in FIG. 1.
As can be seen from FIG. 1, the ARF3 gene was abnormally highly expressed in the bone marrow of the initially diagnosed AML patient.
(6) Correlation analysis of ARF3 expression level and AML clinical characteristics
The correlation between the expression level of ARF3 and the clinical characteristics of AML was analyzed, and the analysis results are shown in Table 4.
TABLE 4
As can be seen from Table 4, the correlation between the expression level of ARF3 gene and white blood cells, platelets, bone marrow primary cells, hemoglobin and the like has no statistical significance, but the complete remission rate of the low-expression group (74.1%) of ARF3 is obviously higher than that of the high-expression group (48.7%) of ARF 3; this suggests that ARF3 may play a role in the diagnosis of AML assistance as an independent indicator and may be prognostic.
(7) Correlation analysis of ARF3 with AML prognosis
The correlation between the expression level of ARF3 gene and the prognosis of AML was analyzed, and the results are shown in FIG. 2, FIG. 3, FIG. 4 and FIG. 5.
As can be seen from fig. 2 and 3, ARF3 highly expressed AML patients had worse overall survival and no-event survival, suggesting poor prognosis; particularly in intermediate-risk cytogenetic stratification (as shown in fig. 4 and 5), high expression of ARF3 is more correlated with poor prognosis.
(8) Multifactorial analysis of AML patient prognosis
The correlation between age, leukocyte count, cytogenetics and ARF3 gene expression and overall survival rate of AML patients was analyzed, and the analysis results are shown in Table 5.
TABLE 5
As can be seen from Table 5, the invention uses clinical samples to verify that a novel acute myeloid leukemia prognosis diagnostic marker, namely the ARF3 gene, is determined, and the relative expression level of the ARF3 gene is found to have a good predictive value (particularly after being combined with clinical indexes) by multi-factor regression analysis, so that the invention can be applied to the preparation of acute myeloid leukemia prognosis diagnostic reagents or kits.
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Claims (10)
1. An auxiliary diagnosis, prognosis or risk stratification marker for acute myeloid leukemia, which is ARF3 gene.
Application of ARF3 gene in preparation of acute myeloid leukemia auxiliary diagnosis product.
Application of ARF3 gene in preparation of acute myelogenous leukemia prognosis diagnosis product.
4. The use of the ARF3 gene in the preparation of acute myeloid leukemia prognosis diagnostic product of claim 3, wherein the acute myeloid leukemia prognosis diagnostic product is a specific detection primer of ARF3 gene or a specific detection probe of ARF3 gene.
5. The use of ARF3 gene in the preparation of acute myeloid leukemia prognosis diagnostic product as claimed in claim 4, wherein the acute myeloid leukemia prognosis diagnostic product is a kit comprising at least one of the specific detection primer and the specific detection probe, or a gene chip carrying the specific detection probe.
Application of ARF3 gene in preparing hierarchical products of acute myeloid leukemia risk.
The specific detection primer of the ARF3 gene is characterized by comprising a forward primer with a nucleotide sequence shown as SEQ ID No.1 and a reverse primer with a nucleotide sequence shown as SEQ ID No. 2.
8. The primer for detecting ARF3 gene according to claim 7, which comprises:
a forward primer: 5'-GGAAGAGCTGATGAGAATGCTGG-3', respectively;
reverse primer: 5'-ACCAGTTACGGTGACGAAGGGA-3' are provided.
9. An acute myelogenous leukemia auxiliary diagnosis, prognostic diagnosis or risk stratification kit comprising the primer for detecting the ARF3 gene according to claim 7 or 8.
10. The acute myeloid leukemia aided diagnosis, prognosis diagnosis or risk stratification kit of claim 9, further comprising specific detection primers for internal reference gene GAPDH, wherein the specific detection primers for internal reference gene GAPDH comprise:
a forward primer: 5'-ATGGGGAAGGTGAAGGTCG-3', respectively;
reverse primer: 5'-GGGTCATTGATGGCAACAATATC-3' are provided.
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| CN103502473A (en) * | 2011-03-01 | 2014-01-08 | 耶鲁大学 | Predicting gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) |
| CA3076613A1 (en) * | 2017-09-22 | 2019-03-28 | Kymera Therapeutics, Inc. | Protein degraders and uses thereof |
| US20220301656A1 (en) * | 2021-03-18 | 2022-09-22 | Washington University | Genome sequencing as an alternative to cytogenetic analysis |
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2021
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103502473A (en) * | 2011-03-01 | 2014-01-08 | 耶鲁大学 | Predicting gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) |
| CA3076613A1 (en) * | 2017-09-22 | 2019-03-28 | Kymera Therapeutics, Inc. | Protein degraders and uses thereof |
| US20220301656A1 (en) * | 2021-03-18 | 2022-09-22 | Washington University | Genome sequencing as an alternative to cytogenetic analysis |
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