CN114634983A

CN114634983A - Marker for early diagnosis, risk stratification and prognosis risk stratification of acute myelogenous leukemia and application thereof

Info

Publication number: CN114634983A
Application number: CN202210086976.XA
Authority: CN
Inventors: 陈莹; 牧启田; 牛婷婷; 邹多兵; 石淙; 吴莹; 张昭艺
Original assignee: Ningbo First Hospital
Current assignee: Ningbo First Hospital
Priority date: 2022-01-25
Filing date: 2022-01-25
Publication date: 2022-06-17

Abstract

The invention discloses a marker for early diagnosis, risk stratification and prognosis risk stratification of acute myelogenous leukemia and application thereof. The patentee firstly discovers that the expression level of SSBP4 in the bone marrow of an Acute Myelogenous Leukemia (AML) patient is remarkably increased compared with that of a normal person, meanwhile, the expression level of an SSBP4 gene is remarkably related to the prognosis of the AML (non-APL) patient, and the high-expression AML (non-APL) patient of SSBP4 has worse overall survival and no event survival, which indicates that the prognosis is poor; high expression of SSBP4 also suggests a poor prognosis in the risk group (cytogenetic risk stratification) of AML, high expression of SSBP4 has a very important role in particular in suggesting early death of patients; this indicates that the SSBP4 gene can be used as an early diagnosis, risk stratification and prognostic risk stratification marker of acute myeloid leukemia.

Description

Marker for early diagnosis, risk stratification and prognosis risk stratification of acute myelogenous leukemia and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an early diagnosis, risk stratification and prognosis risk stratification marker for acute myeloid leukemia and application thereof.

Background

Acute Myeloid Leukemia (AML) is a hematopoietic malignancy characterized by a block in differentiation and clonal proliferation of hematopoietic stem and progenitor cells with high heterogeneity. Karyotyping is often used to assess the prognosis of AML patients, and mutations and rearrangements of genes have been widely used clinically as the level of molecular biology techniques has increased. Some molecular mutations such as NPM1, FLT3-ITD, CEBPA, c-KIT, etc. are included in the NCCN (national Comprehensive Cancer network) and ELN (European Leukemia Net) guidelines as markers for the prognosis evaluation system for the stratification of AML risk.

With the increasing level of medical care, due to the discovery of arsenic trioxide and all-trans retinoic acid, most Acute Promyelocytic Leukemia (APL) patients can be completely cured by the combination therapy of the two drugs. Despite significant advances in prognostic risk stratification, supportive therapy, multiple chemotherapeutic drug-intensive therapy, autologous or allogeneic hematopoietic stem cell transplantation (auto/allo-HSCT), etc., the prognosis of adult non-M3 AML patients remains unattractive due to characteristics of AML relapse and refractory.

In recent years, the emergence of targeted inhibitors against gene mutation and multi-kinase has been desired, but long-term follow-up data is lacking, so that the exploration of the mechanism of AML molecular pathogenesis, the search of new therapeutic targets and the discovery of potential markers of AML stratification are urgently needed.

Single-stranded DNA binding protein (SSBPs) family genes are widely existed in nature, wherein the SSBP4 gene is located on chromosome 19p13.11, and no report about the association of the SSBP4 gene with acute myeloid leukemia exists at present.

Disclosure of Invention

The invention aims to provide a novel marker for early diagnosis, risk stratification and prognosis risk stratification of acute myelogenous leukemia and application thereof.

In order to achieve the above purpose, the technical solution of the present application is as follows:

an early diagnosis, risk stratification and prognosis risk stratification marker of acute myelogenous leukemia is SSBP4 gene.

After multiple experiments, patentee discovers that the expression level of SSBP4 in the bone marrow of a patient with acute myelogenous leukemia (AML for short) is obviously up-regulated compared with a normal person for the first time, and the SSBP4 gene can be used as an early diagnosis marker of AML; meanwhile, patentees also find that the expression level of the SSBP4 gene is in significant correlation with the prognosis of AML (non-APL) patients, and the AML (non-APL) patients with high SSBP4 expression have poorer overall survival and no-event survival, which indicates that the prognosis is poor; high expression of SSBP4 also suggests a poor prognosis in the risk group (cytogenetic risk stratification) of AML, high expression of SSBP4 has a very important role in particular in suggesting early death of patients; this also indicates that the SSBP4 gene can be used as a risk stratification marker or a prognostic risk stratification marker for acute myeloid leukemia.

Based on the above, the invention also provides the application of the SSBP4 gene in the preparation of acute myeloid leukemia early diagnosis products, acute myeloid leukemia risk stratification products and acute myeloid leukemia disease prognosis stratification products.

Preferably, the acute myeloid leukemia early diagnosis product, the acute myeloid leukemia risk stratification product or the acute myeloid leukemia prognosis risk stratification product can be a specific detection primer of SSBP4 gene or a specific detection probe of SSBP4 gene; the kit may contain 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 SSBP4 gene, wherein the specific detection primer of SSBP4 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 SSBP4 gene includes:

a forward primer: 5'-TACGTTTATGAGTACCTGCTGCACATC-3', respectively;

reverse primer: 5'-TTCTTCTCCCATCGGATCTCAGACAG-3' is added.

The invention also provides a kit for early diagnosis, risk stratification and prognosis risk stratification of acute myeloid leukemia, which contains the specific detection primer of the SSBP4 gene.

The kit of the invention is preferably a real-time quantitative PCR kit, which is convenient for comparing the expression level of the SSBP4 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 SSBP4 in the bone marrow of AML patients is obviously up-regulated compared with normal people for the first time; meanwhile, the expression level of the SSBP4 gene is significantly related to the prognosis of AML (non-APL) patients, and the AML (non-APL) patients with high SSBP4 expression have worse overall survival and no event survival, which indicates that the prognosis is poor; high expression of SSBP4 also suggests a poor prognosis in the risk group (cytogenetic risk stratification) of AML, high expression of SSBP4 has a very important role in particular in suggesting early death of patients; the SSBP4 gene can be used as an early diagnosis, risk stratification and prognosis 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 SSBP4 gene in the bone marrow and normal bone marrow of patients with acute myelogenous leukemia;

wherein SSBP4 mRNA expression (Fold change) indicates the relative expression level (Fold difference) of SSBP4 gene, AML (n 148) indicates the bone marrow specimen (specimen number 148) of acute myelogenous leukemia patient, and Control (n 40) indicates the normal human bone marrow specimen (specimen number 40);

FIG. 2 is the results of correlation analysis of SSBP4 gene expression with overall survival of AML (non-APL) patients;

wherein, Overall survival indicates Overall survival, time (days) indicates time after treatment (days), Low SSBP4(n ═ 67) indicates samples with Low SSBP4 expression (number of samples is 67), High SSBP4(n ═ 22) indicates samples with High SSBP4 expression (number of samples is 22), and p ═ 0.029 indicates that there is a difference; the same applies below;

FIG. 3 is the results of correlation analysis of SSBP4 gene expression and event-free survival in AML (non-APL) patients;

wherein, Event free survival indicates that no Event exists, and p is 0.052 indicates that the difference trend exists;

FIG. 4 is the results of correlation analysis of SSBP4 gene expression with overall survival in risk group patients among AML (non-APL) patients;

here, Low SSBP4(n ═ 39) indicates a sample with Low SSBP4 expression (sample number 39), High SSBP4(n ═ 10) indicates a sample with High SSBP4 expression (sample number 10), and p ═ 0.04 indicates that there is a significant difference.

Detailed Description

The technical scheme of the invention is further explained in detail by combining the drawings and the detailed description.

Example 1

Specific detection primers are designed aiming at the SSBP4 gene, and the reliability of the specific detection primers is verified by amplifying the SSBP4 gene in cells through RT-qPCR and sequencing the gene.

The specific detection primer of the SSBP4 gene comprises:

a forward primer: 5'-TACGTTTATGAGTACCTGCTGCACATC-3' (SEQ ID No. 1);

reverse primer: 5'-TTCTTCTCCCATCGGATCTCAGACAG-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 Shanghai Biotech company was entrusted with the synthesis of the above two pairs of specific primers.

The RT-qPCR amplification method comprises the following steps:

(1) cell collection and pretreatment

The in vitro cultured MV4-11 cell strain was collected, washed 2 times with PBS, 1mL of RNAioso Plus reagent (purchased from TaKaRa) was added to the obtained cell pellet, and after repeated pipetting and mixing, the RNAioso Plus lysate was stored at-80 ℃ for further 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; placing the supernatant in a new EP tube, adding isopropanol with the same volume, reversing, mixing uniformly, standing for 10min, and centrifuging at 4 ℃ at 12000g for 10 min; pouring out the supernatant, adding 1ml of 75% fresh ethanol, reversing and cleaning, and centrifuging at 4 ℃ for 5min at 12000 g; pouring out the supernatant, adding 1ml of absolute ethyl alcohol, 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 amplification product is confirmed to be SSBP4 gene (SEQ ID No.5) through sequence Blast comparison, which shows that the primer designed in the embodiment can specifically amplify the SSBP4 gene.

Example 2

The specific detection primer is adopted to detect and analyze the expression condition of the SSBP4 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

148 AML bone marrow specimens which are newly issued in 2016, 6-2021, 8 months in Ningbo city in the first hospital are collected, 40 normal control bone marrow specimens are collected, and all participants sign informed consent; adopting a bone marrow cell culture bottle to collect a bone marrow sample, 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 SSBP4 Gene expression level

The MV4-11 cell line in example 1 was used as a control, and 2 was used^-ΔΔCtThe SSBP4 gene expression quantity is calculated by the method, and delta Ct ═ Ct_SSBP4-Ct_GAPDH]_Sample(s)-[Ct_SSBP4-Ct_GAPDH]_{Normal control}. The data were statistically analyzed using SPSS 24.0 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 in survival data between groups (P < 0.05 considered for statistical differences), and the results are shown in FIG. 1.

As can be seen from FIG. 1, the SSBP4 gene was abnormally highly expressed in the bone marrow of the initially diagnosed AML patient.

(6) Association analysis of SSBP4 with AML prognosis

The correlation between the expression level of SSBP4 gene and the prognosis of AML (non-APL) patients was analyzed, and the analysis results are shown in FIG. 2 and FIG. 3.

As can be seen in fig. 2 and 3, AML (non-APL) patients with high expression of SSBP4 had worse overall survival and no-event survival, suggesting poor prognosis.

(7) Multifactorial analysis of AML patient prognosis

The correlation between the five indicators of age, cytogenetics, treatment pattern, gene mutation and SSBP4 gene expression and the 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, determines a new acute myeloid leukemia prognosis diagnostic marker, namely SSBP4 gene, and finds that the relative expression level of the SSBP4 gene has good independent prediction value (particularly after being combined with clinical indexes) through multi-factor regression analysis, and particularly has very important function in prompting early death of patients, so that the invention can be applied to the preparation of acute myeloid leukemia prognosis evaluation reagents or kits.

Sequence listing

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Claims

1. An early diagnosis, risk stratification or prognostic risk stratification marker for acute myeloid leukemia, which is SSBP4 gene.

Application of SSBP4 gene in preparing early diagnosis products of acute myeloid leukemia.

Application of SSBP4 gene in preparing hierarchical products of acute myeloid leukemia risk.

Application of SSBP4 gene in preparing a stratification product for prognosis risk of acute myelogenous leukemia.

5. The use of the SSBP4 gene in the preparation of an acute myelogenous leukemia prognosis risk stratification product according to claim 4, wherein the acute myelogenous leukemia prognosis risk stratification product is a specific detection primer of SSBP4 gene or a specific detection probe of SSBP4 gene.

6. The use of the SSBP4 gene in the preparation of an acute myeloid leukemia prognosis risk stratification product according to claim 4, wherein the acute myeloid leukemia prognosis risk stratification product is a kit comprising at least one of SSBP4 gene specific detection primers and specific detection probes, or a gene chip carrying SSBP4 gene specific detection probes.

The specific detection primer of the SSBP4 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 the specific detection of SSBP4 gene according to claim 7, comprising:

a forward primer: 5'-TACGTTTATGAGTACCTGCTGCACATC-3', respectively;

reverse primer: 5'-TTCTTCTCCCATCGGATCTCAGACAG-3' are provided.

9. An early diagnosis, risk stratification and prognostic risk stratification kit for acute myeloid leukemia, comprising the primers for detecting SSBP4 gene of claim 7 or 8 specifically.

10. The kit for the early diagnosis, risk stratification and prognostic risk stratification of acute myeloid leukemia according to claim 9, further comprising a primer for detecting the specificity of the reference gene GAPDH, wherein the primer for detecting the specificity of the reference gene GAPDH comprises:

a forward primer: 5'-ATGGGGAAGGTGAAGGTCG-3', respectively;

reverse primer: 5'-GGGTCATTGATGGCAACAATATC-3' are provided.