CN112760379A

CN112760379A - Application of TOX3 in preparation of AML prognosis prediction kit

Info

Publication number: CN112760379A
Application number: CN202110102809.5A
Authority: CN
Inventors: 陈少华; 梁朝峰; 李扬秋; 查显丰; 黄舒欣; 赵玉洁; 谭家雄
Original assignee: Jinan University
Current assignee: Jinan University; University of Jinan
Priority date: 2021-01-26
Filing date: 2021-01-26
Publication date: 2021-05-07

Abstract

The invention provides an application of a TOX3 gene in preparing a kit for predicting AML prognosis, which is based on the first discovery by the inventor that the expression condition of the TOX3 gene in peripheral blood of AML patients is related to the prognosis of the AML patients. When the expression level of the TOX3 gene is high, it indicates that there is a high possibility that the clinical prognosis of AML patients is poor. The expression level of the TOX3 gene has important guiding significance for formulating an AML early diagnosis and treatment scheme and an effective comprehensive layered system, can provide more clinical prognosis research data for the target treatment of AML patients, and has wide application prospect.

Description

Application of TOX3 in preparation of AML prognosis prediction kit

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to an application of TOX3 in preparation of an AML prognosis prediction kit.

Background

Acute Myeloid Leukemia (AML) is the most common type of adult leukemia, with a rate of incidence of 3.7/100000. The conventional treatment of AML is based on the induction of remission and post-remission treatment with maximum tolerance, and although some curative effects have been achieved in the treatment of AML patients in recent years with the improvement of chemotherapy regimens and the development of hematopoietic stem cell transplantation technology, there are problems of high risk of relapse after remission treatment and low disease-free survival rate. Studies have shown that 5-year overall survival rates are approximately 40% in patients under 60 years of age, while 10-20% in patients over 60 years of age, Complete Remission (CR) is not achieved in 20% of first-visit patients, recurrence is maintained in 50-70% of patients with CR, and survival is more than five years in a few patients with post-CR recurrence.

Different prognoses of AML patients suggest that other key factors (e.g. cellular immunity) may also affect the therapeutic efficacy. It is well known that immune evasion of cancer and abnormal immune monitoring play a crucial role in the development and progression of cancer. In recent years, studies show that TOX is closely related to the occurrence and development of tumors and promotion of T cell depletion. The protein encoded by the TOX (Thymycete selection-associated HMG BOX) gene belongs to a member of the high mobility group BOX (HMG-BOX) protein superfamily, which is a transcription regulator bound to DNA. The human TOX gene comprises 4 subfamilies: TOX, TOX2, TOX3, and TOX4 are located on different human chromosomes. The four members of the TOX family each have different functions. In recent years, scholars have reported that TOX3 may be involved in the formation of various malignant tumors. TOX3, also known as TNRC9, is located on human chromosome 16, q12.1, originally found in the brain and is considered an important protective factor for neurons. TOX3 does not function equally well in different tumors. In breast cancer, high expression of TOX3 is closely correlated with the risk of breast cancer patients. The overall survival rate (OS) and the remote metastasis-free survival rate (DMFS) of breast cancer patients with high TOX3 expression are lower than those of breast cancer patients with low TOX3 expression. In lung cancer patients, TOX3 exhibited the function of a tumor suppressor gene. Lung cancer patients with high expression of TOX3 have a higher survival rate than lung cancer patients with low expression. Although TOX3 is closely related to the prognosis of the above-mentioned tumors, the role of TOX3 in hematological tumors, especially leukemia, is still unclear.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides the application of TOX3 in preparing a kit for predicting AML prognosis. When TOX3 highly expressed in peripheral blood is detected, it indicates that the probability of poor prognosis of AML patients is high, and it can be used as an index for predicting the prognosis evaluation of AML patients.

It is another object of the present invention to provide a kit for predicting the prognosis of AML.

The purpose of the invention is realized by the following technical scheme:

the application of TOX3 in preparing the kit for predicting AML prognosis is based on the first discovery by the inventor of the invention that the expression of TOX3 in peripheral blood of AML patients is related to the prognosis of AML patients.

In the application, the expression level of the TOX3 gene in the peripheral blood of a clinical patient is detected, and statistical analysis is performed by combining the expression level of an internal reference gene to obtain the gene differential expression fold of the TOX3 of the clinical patient compared with a healthy adult, so that the AML prognosis is predicted.

The reference gene is preferably beta microsphere gene (beta 2M).

The statistical analysis method is preferably a delta CT method; the gene differential expression fold is 2^ (-Delta CT), and the Delta CT is Delta CT_{Clinical patients}–ΔCT_{Healthy adult}，ΔCT_{Clinical patients}＝(CT_Gene-CT_{Internal reference})，ΔCT_{Healthy adult}＝(CT_Gene-CT_{Internal reference})，CT_GeneAnd CT_{Internal reference}The fluorescent probe is obtained by detecting through a real-time fluorescent quantitative PCR method.

The prediction specifically refers to:

(1) when the gene differential expression multiple of TOX3 is more than or equal to 0.1820 times, the possibility of poor clinical prognosis of patients is high;

(2) when the gene differential expression fold of TOX3 is <0.1820 fold, the likelihood of a good clinical prognosis for the patient is greater.

The poor prognosis specifically means that the 3-year OS rate is not higher than 4%.

The good prognosis specifically means that the 3-year OS rate is not less than 61%.

A kit for predicting AML prognosis comprising primers for amplifying TOX3 cDNA.

The primers used for amplifying the TOX3 cDNA were as follows:

TOX3-F:5'-TATGCCTCACACATCTCCTTCA-3'(SEQ ID NO.1)；

TOX3-R:5'-ATGGCTCTGTTGGCTTCATC-3'(SEQ ID NO.2)。

the kit also comprises primers for amplifying the internal reference, and the primers are as follows:

β2M-F:5'-TACACTGAATTCACCCCCAC-3'(SEQ ID NO.3)；

β2M-R:5'-CATCCAATCCAAATGCGGCA-3'(SEQ ID NO.4)。

the kit also comprises any one or at least two of a reagent for separating peripheral blood mononuclear cells, a reagent for extracting total RNA, a reagent for Reverse transcription polymerase chain reaction (RT-PCR) and a reagent for real-time quantitative PCR (qRT-PCR).

The reagent for separating peripheral blood mononuclear cells is preferably lymphocyte separation solution (Ficoll).

The reagent for extracting the total RNA is preferably TRIZOL.

The kit for predicting AML prognosis is applied to non-disease diagnosis detection of TOX3 expression.

The application comprises the following steps:

(1) taking a peripheral blood sample to be detected, and separating to obtain mononuclear cells;

(2) adding a reagent for extracting total RNA into the mononuclear cells obtained in the step (1), and uniformly mixing;

(3) adding chloroform, mixing, and centrifuging;

(4) sucking the upper layer liquid, adding isopropanol, and centrifuging to remove the supernatant;

(5) washing with ethanol, and drying to obtain an RNA sample;

(6) reverse transcribing the RNA sample obtained in step (5) into cDNA;

(7) and (4) detecting the expression quantity of the target gene by using the cDNA obtained in the step (6) by using the kit.

The proportion of the mononuclear cells, the total RNA extraction reagent and the chloroform is preferably 5-10 multiplied by 10⁶And (2) cell: 1 ml: 0.1-0.3 ml.

The reagent for extracting total RNA in the step (2) is preferably TRIZOL.

The conditions for the centrifugation in step (3) are preferably: the temperature is 2-8 ℃, the rotating speed is 10000-15000 g, and the time is 15-30 minutes.

The dosage of the isopropanol in the step (4) is preferably as follows: calculating the volume ratio of isopropanol to isopropanol being 1-2: 1-2; preferably calculated as 1: 1.

The conditions for the centrifugation in the step (4) are preferably: the temperature is 2-8 ℃, the rotating speed is 10000-15000 g, and the time is 10-20 minutes;

the number of ethanol washes described in step (5) is preferably two; more preferably, the first time is preferably washed by 75% ethanol at the temperature of minus 20 ℃, the second time is preferably washed by 100% ethanol at the temperature of minus 20 ℃, after each time of uniform mixing for 30s, the mixture is centrifuged for 5 to 10 minutes at the temperature of 2 to 8 ℃ and 8000 to 12000 g.

The drying in the step (5) is preferably vacuum centrifuge drying; the conditions are preferably 2-8 ℃ and 5-10 minutes.

The reverse transcription described in the step (6) is carried out by a reagent for reverse transcription PCR.

The detection of the expression level of the target gene in the step (7) is realized by a reagent for real-time quantitative PCR.

Compared with the prior art, the invention has the following advantages and effects:

the invention adopts a real-time quantitative polymerase chain reaction (qRT-PCR) to detect the expression condition of the TOX3 gene in the peripheral blood of AML patients, and discovers the prognostic relation of the TOX3 and the AML patients for the first time. When TOX3 is highly expressed, the prognosis of AML patients is poor.

The invention uses qRT-PCR method to determine the TOX3 gene expression in peripheral blood of AML patients, the method is simple and easy to implement, and the method is stable. When the TOX3 gene with high expression is detected, the probability of poor prognosis of AML patients is suggested to be high, and the probability can be used as an index for predicting the prognosis evaluation of AML patients.

The invention analyzes the relationship between clinical outcome and prognosis of the TOX3 and AML patients, verifies and identifies the possibility of using the TOX3 as a new molecular biomarker for prognosis, lays an experimental foundation for further exploring the TOX3 as a potential biomarker and a target point for AML immunotherapy, provides more clinical prognosis research data for the AML patients, and has important guiding significance and good application prospect for prognosis judgment of the AML patients and formulation of clinical treatment schemes.

Drawings

FIG. 1 is a graph showing the effect of the expression level of TOX3 gene on the prognosis of AML naive patients. When TOX3 is highly expressed, the prognosis of AML patients is poor.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The reagent information used in the examples is specifically as follows:

lymphocyte isolate Ficoll (available from GE Healthcare Life Sciences);

TRIZOL (available from Invitrogen);

reverse transcription PCR kit (purchased from Promega);

real-time quantitative PCR kit (purchased from TIANGEN);

example 1

1. Peripheral blood was collected with the patient signed an informed consent. 40 primary AML peripheral blood and 29 healthy adult samples were collected from the first hospital department of hematology, subsidiary of river-south university, Guangzhou, all of which were anticoagulated with ethylenediaminetetraacetic acid (EDTA) at the time of admission, a section of the research protocol that was approved by the ethical Committee of the Unit. Clinical data, such as survival time and survival status of AML patients, were also collected (as shown in table 1).

2. Isolation of mononuclear cells from peripheral blood. 4mL of lymphocyte separation medium (Ficoll, density 1.077) is added into a 15mL centrifuge tube, the diluted anticoagulated peripheral blood specimen suspension is laid on the separation medium, and the sample is centrifuged for 15 minutes at 1500rpm by a horizontal centrifuge. Sucking the middle single core layer, transferring to another 15mL centrifuge tube, adding appropriate amount of 1 XPhosphate buffer saline (PBS), gently blowing, centrifuging at 1000rpm for 10 minutes, discarding supernatant, adding 1 XPBS solution to 2mL, mixing, counting, washing twice with the same method, and washing with 5-10 × 10⁶1 ml of TRIZOL reagent was added to each mononuclear cell to prepare a single cell suspension for use.

3. Total RNA extraction

3.1 placing the single cell suspension on ice, adding 0.2 ml of chloroform after 5 minutes, fully and uniformly mixing (15 seconds), placing on ice for 2-3 minutes, and centrifuging at low temperature and high speed (2-8 ℃, 12000g) for 15-30 minutes;

3.2 carefully sucking the supernatant (about 50% of the total volume) and transferring to a 1.5 ml new tube, adding 0.5 ml isopropanol and mixing uniformly, standing on ice for 10 minutes, and centrifuging at low temperature and high speed (2-8 ℃, 12000g) for 10-20 minutes;

3.3 removing the supernatant and washing twice with 1 ml of 75% ethanol (-20 ℃), mixing evenly for 30 seconds each time, and centrifuging for 5-10 minutes (2-8 ℃, 10000 g);

3.4 removing the supernatant, centrifuging again, and removing the residual ethanol;

3.5 drying in a vacuum centrifuge at the low temperature of 2-8 ℃ for 5-10 minutes;

3.6 Add ultra-clean water (Biotecx BL-5700) 50. mu.l (optionally increased or decreased) and mix well to obtain RNA sample.

3.7 taking 2 microliter RNA sample and diluting to 400 microliter with ultra-clean water, detecting the optical density of the sample at A260nm wavelength in an ultraviolet spectrophotometer, and estimating the purity and content (1 OD)_A260nm40 μ g/ml), total amount of RNA OD × 400 μ g;

3.8 RNA samples were stored at-70 ℃ for future use after addition of 0.1 volume (5. mu.l) of 3mol/L NaAC (sodium acetate) and 2 volumes (100. mu.l) of ethanol.

4.RT-PCR

4.1 Add 500ng RNA, 0.5. mu.l oligo (dT) (0.5. mu.g/reaction), 0.5. mu.l random primer (0.5. mu.g/reaction) and double distilled water without RNase (ddH2O) (maximum 5. mu.l) together and incubate at 70 ℃ for 5 minutes followed by rapid cooling on ice for 5 minutes;

4.2 Add 4.0. mu.l of GoScript^TM5 Xreaction buffer, 1.7. mu.l MgCl₂(final concentration 2.0mmol/L), 1.0. mu.l 0.5mmol/L dNTP, 0.3. mu.l RNase inhibitor (20U), 1. mu.l reverse transcriptase, ddH₂O is complemented to 15 mu l;

4.3 after mixing, 20 microliter of the sample was incubated at 42 ℃ for 60 minutes and then inactivated at 70 ℃ for 15 minutes;

4.4 addition of 80. mu.l of ddH₂And (4) diluting with oxygen.

5.qRT-PCR

5.1 qRT-PCR 20 microliter system was formulated as: 10. mu.l Mix, 0.5. mu.l forward primer (10. mu. mol/L), 0.5. mu.l reverse primer (10. mu. mol/L), 1. mu.l cDNA, ddH₂O is added to 8 mu l;

wherein, the sequences of the primers are as follows:

TOX3-F:5'-TATGCCTCACACATCTCCTTCA-3'；

TOX3-R:5'-ATGGCTCTGTTGGCTTCATC-3'。

internal reference primers:

β2M-F:5'-TACACTGAATTCACCCCCAC-3'；

β2M-R:5'-CATCCAATCCAAATGCGGCA-3'。

5.2 qRT-PCR the program settings were as follows: 15 minutes at 95 ℃; 10s at 95 ℃, 30s at 60 ℃ and 45 cycles;

5.3 Gene expression level is shown to be 2^ (- Δ Δ CT).

Gene expression quantity CT obtained by qRT-PCR determination_TOX3Further performing statistical analysis by a delta CT method to obtain the gene differential expression level multiple, wherein:

ΔCT_TOX3＝(CT_TOX3-CT_β2M)；

ΔΔCT＝ΔCT_{incipient AML patient}–ΔCT_{Healthy adult}；ΔCT_{Healthy adult}Obtained by calculating the average value of 29 healthy adult peripheral blood samples;

the fold of gene differential expression is 2^ (-Delta CT).

The gene expression profile and clinical prognosis data of AML patients are shown in Table 1.

The expression of TOX3 was analyzed in combination with clinical prognosis data of AML patients, and the optimal prognostic cutoff value of TOX3 gene expression was obtained in R language by inputting gene expression level fold, survival status and survival time using the surfminer R package (version 0.4.8) to obtain 0.1820. As shown in FIG. 1, the prognosis was found to be worse in AML patients with 2^ (- Δ Δ CT) ≧ 0.1820 times.

The above experimental results show that the combination of TOX3 has important significance in predicting the clinical prognosis of AML, and provides prognostic research data for clinical combined application of immune checkpoint inhibitors.

TABLE 1 differential TOX3 Gene expression and clinical data for AML patients

Note: 0 in the state represents survival and 1 represents death.

The gene expression level measured as described above, although not directly leading to future diagnosis and health condition, can be used as an intermediate result as one of reference information for the clinical treatment planning of patients.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> river-south university

Application of <120> TOX3 in preparation of AML prognosis prediction kit

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tatgcctcac acatctcctt ca 22

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggctctgt tggcttcatc 20

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tacactgaat tcacccccac 20

<210> 4

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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catccaatcc aaatgcggca 20

Claims

Use of TOX3 in the preparation of a kit for predicting AML prognosis.
2. Use of TOX3 in the preparation of a kit for the prognosis of AML according to claim 1, wherein: by detecting the expression level of TOX3 in peripheral blood of clinical patients and combining the expression level of internal reference genes, statistical analysis is carried out to obtain the gene differential expression multiple of TOX3 of clinical patients compared with healthy adults, thereby predicting the AML prognosis.
3. The TOX3 of claim 2 in the preparation of predictive AML prognosesThe application of the kit is characterized in that: the internal reference gene is beta 2M; the statistical analysis method is a delta CT method; the fold of gene differential expression is 2^ (-Delta CT), and the Delta CT is Delta CT_{Clinical patients}–ΔCT_{Healthy adult}，ΔCT_{Clinical patients}＝(CT_Gene-CT_{Internal reference})，ΔCT_{Healthy adult}＝(CT_Gene-CT_{Internal reference})；CT_GeneAnd CT_{Internal reference}Detecting by a real-time fluorescent quantitative PCR method; the prediction specifically refers to:

(1) when the gene differential expression multiple of TOX3 is more than or equal to 0.1820 times, the possibility of poor clinical prognosis of patients is high;

(2) when the gene differential expression fold of TOX3 is <0.1820 fold, the likelihood of a good clinical prognosis for the patient is greater.
4. A kit for predicting AML prognosis, comprising: primers for amplification of the TOX3 cDNA were included.
5. The kit for predicting AML prognosis as claimed in claim 4, characterized in that:

the primers used for amplifying the TOX3 cDNA were as follows:

TOX3-F:5'-TATGCCTCACACATCTCCTTCA-3'；

TOX3-R:5'-ATGGCTCTGTTGGCTTCATC-3'。
6. the kit for predicting AML prognosis as claimed in claim 5, characterized in that: the kit also comprises primers for amplifying the internal reference, and the primers are as follows:

β2M-F:5'-TACACTGAATTCACCCCCAC-3'；

β2M-R:5'-CATCCAATCCAAATGCGGCA-3'。
7. kit for predicting the prognosis of AML according to claim 5 or 6, characterized in that: the kit also comprises any one or at least two of a reagent for separating peripheral blood mononuclear cells, a reagent for extracting total RNA, a reagent for reverse transcription PCR and a reagent for real-time quantitative PCR.
8. Use of a kit according to any one of claims 4 to 7 for the detection of the expression of TOX3 in a non-disease diagnostic test.
9. Use according to claim 8, characterized in that: the method comprises the following steps:

(1) taking a peripheral blood sample to be detected, and separating to obtain mononuclear cells;

(2) adding a reagent for extracting total RNA into the mononuclear cells obtained in the step (1), and uniformly mixing;

(3) adding chloroform, mixing, and centrifuging;

(4) sucking the upper layer liquid, adding isopropanol, and centrifuging to remove the supernatant;

(5) washing with ethanol, and drying to obtain an RNA sample;

(6) reverse transcribing the RNA sample obtained in step (5) into cDNA;

(7) and (4) detecting the expression quantity of the target gene by using the cDNA obtained in the step (6) by using the kit.
10. Use according to claim 9, characterized in that:

the ratio of the mononuclear cells, the total RNA extraction reagent and chloroform is 5-10 multiplied by 10⁶And (2) cell: 1 ml: calculating 0.1-0.3 ml;

the reagent for extracting the total RNA in the step (2) is TRIZOL;

the centrifugation conditions in the step (3) are as follows: the temperature is 2-8 ℃, the rotating speed is 10000-15000 g, and the time is 15-30 minutes;

the dosage of the isopropanol in the step (4) is as follows: calculating the volume ratio of isopropanol to isopropanol being 1-2: 1-2;

the centrifugation conditions in the step (4) are as follows: the temperature is 2-8 ℃, the rotating speed is 10000-15000 g, and the time is 10-20 minutes;

washing with ethanol twice in the step (5), washing with 75% ethanol at-20 ℃ for the first time, washing with 100% ethanol at-20 ℃ for the second time, uniformly mixing for 30s each time, and centrifuging at 2-8 ℃ and 8000-12000 g for 5-10 minutes;

the drying in the step (5) is vacuum centrifuge drying, and the conditions are that the temperature is 2-8 ℃ and the time is 5-10 minutes;

the reverse transcription in the step (6) is realized by a reagent for reverse transcription PCR;

the detection of the expression level of the target gene in the step (7) is realized by a reagent for real-time quantitative PCR.