CN108424967A - The application for the biomarker that IARS2 genes are detected as leukaemia - Google Patents

The application for the biomarker that IARS2 genes are detected as leukaemia Download PDF

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CN108424967A
CN108424967A CN201810522724.0A CN201810522724A CN108424967A CN 108424967 A CN108424967 A CN 108424967A CN 201810522724 A CN201810522724 A CN 201810522724A CN 108424967 A CN108424967 A CN 108424967A
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leukaemia
reagent
iars2
genes
detection
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李宏
董昌虎
田亚宁
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SECOND AFFILIATED HOSPITAL OF SHAANXI UNIVERSITY OF CHINESE MEDICINE
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q2600/118—Prognosis of disease development
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    • C12Q2600/158—Expression markers

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Abstract

Present invention discover that and confirmed the application of the biomarker that IARS2 genes are detected as leukaemia, be particularly used in the reagent for preparing detection leukaemia or the kit containing the reagent；Correspondingly, the corresponding reagent containing detection IARS2 genes in certain reagent, or include the kit of certain above-mentioned reagent, it can be used to detect leukaemia.Leukaemia is detected using the present invention, it is high sensitivity, high specificity, fast and convenient, it is expected to become the important means of leukaemia early diagnosis and prognosis prediction.

Description

The application for the biomarker that IARS2 genes are detected as leukaemia
Technical field
The invention belongs to biomedical sector, it is related to a kind of application of the marker of specific gene as detection leukaemia.
Background technology
Leukaemia is a kind of common hematopoietic stem/progenitor cells malignant clone disease, because leukaemia cell's self-renewing increases By force, proliferation out of control, dysdifferentiation, apoptosis are obstructed, and rest on the different phase of cell development.The incidence of leukaemia is in China For 3-4/10 ten thousand, the death rate caused by malignant tumour occupies the 6th (man), the 7th (female)；Children and 35 years old or less at The death rate is even more and comes out at the top in people, is one of the malignant disease for seriously endangering human health.China's acute leukemia compared with Chronic leukemia common (about 5.5:1), wherein acute myeloid leukemia incidence highest (1.62/10 ten thousand).Acute myeloid is white Blood disease (AML) is the most common acute leukemia type that adult makes a definite diagnosis, and is come second in the most common leukaemia of children, is sent out Sick rate is about 15-20%, and up to 40% AML infants can recur.
The development of cytogenetics, molecular biology and therapy target identification technology has pushed targeted therapy and layering to control Treat horizontal progress, hence it is evident that improve AML clinical efficacies and overall survival.But clinic still has the intractable case in part that can not obtain There are problems that different degrees of over-treatment again to radical cure, while to some prognosis bona persons.
With the continuous deepening of research, cytogenetics and Protocols in Molecular Biology clinical leukaemic diagnosis and Application in treatment is more and more extensive, has become one of conventional detection project of leukaemic.Disease diagnosis, control The many aspects such as treatment, the monitoring of minimal residual disease (mininal residual disease, MRD) and Index for diagnosis play Important function.
Consensus of experts is pointed out pre- in AML prognosis groupings in the AML guides and China AML (non-APL) practice guidelines of NCCN Well group cytogenetics includes t (8 afterwards；21)(q22；q22)、inv(16)(p13；q22)、t(16；16)(p13；q22)、t (15；17), to include normal karyotype be mutated molecular biology with NPM1, but in clinical treatment this kind of patient still have part refractory and Recurrent cases；Prognosis mala group molecule abnormality includes normal karyotype with individual FLT3-ITD, and high-risk FLT3-ITD sun Property AML patient still has apparent heterogeneity, and some patientss are through conventional inductive treatment up to complete incidence graph (complete Remission, CR), subsequent rows hematopoietic stem cell transplantation (hematopoietic stem cell transplantation, It HSCT) can long term survival.Therefore the influence that other not yet specific complicated factors at present need to be probed into, such as immune factor, adherency point The effect of sub and inflammatory Cytokines Expression etc..
Invention content
Applicant studies IARS2 genes, specific as follows：
IARS2 is the gene for encoding Isoleucyl-tRNA synthetase, belongs to I class aminoacyl-tRNA synthetases, is located at No. 1 dye 4th area of colour solid, 1 band, major function are the synthesis isoleucine-tRNA synzyme in endochylema, are then transported to mitochondria, are catalyzed Isoleucine is combined with tRNA, completes the translation of mtDNA.Studies have shown that aminoacyl-tRNA synthetase is in addition to its classical participation egg Outside the function of white matter synthesis, a variety of vital movements are also taken part in, including natural death of cerebral cells, new vessels are formed, RNA montages and immune Deng[17].Separately some researches show that IARS2 is a kind of promotion sensitivity gene, this gene of silence can inhibit the proliferation of tumour cell, and thin Born of the same parents' relation with apoptosis is close.The study found that the life cycle of the glioblastoma patient of IARS2 gene high expressions is significantly lower than IARS2 low expression persons.Result of study shows that IARS2 is apparently higher than the normal structure of surrounding in the expression of cancerous tissue, and when inhibition After ISARS2 gene expressions, cancer cell blocks in G1Phase, S phase Leukopenias, proliferative capacity are decreased obviously, and apoptosis rate increases.Separately Outside, also studies have found that, silence IARS2 genes can pass through lower P-Smad2, P-JUK etc. cyclin phosphorylation, make Stomach cancer cell blocks in G2/ M the phases, to achieve the purpose that inhibit cell Proliferation, and P-JUK is the important set of MAPK signal paths It is related with the generation of cell Proliferation, apoptosis and inflammation at part.
On the basis of the above research conclusion, the present invention establishes the marker that IARS2 genes can be used as detection leukaemia, And then obtain following scheme：
In a first aspect, providing a kind of purposes of IARS2 genes, it is used to prepare the reagent or kit of detection leukaemia.
Further, described to be detected as Serologic detection or peripheral blood cell counts.
Further, the reagent includes primer, probe, nucleic acid chip etc..
Second aspect, provide it is a kind of for detecting the reagent or kit of leukaemia, in the reagent or kit Corresponding reagent containing detection IARS2 genes.
Further, the corresponding reagent of the detection IARS2 genes is selected from the group：PCR detection reagents, molecule hybridization inspection Test agent etc..
Label or specification can be added in kit, it is white for detecting that the label or specification indicate the kit Blood disease.
The effect of the present invention is as follows：
Leukaemia detection is carried out using the present invention, it is high sensitivity, high specificity, fast and convenient, leukaemia early stage will be become The important means of diagnosis and prognosis prediction.
Description of the drawings
The table of Fig. 1 .IARS2 genes mRNA in tetra- kinds of leukaemia pattern cells of Jurkat, HL-60, THP-1 and K562 It reaches.
The influence that Fig. 2 .CCK8 testing goals genes are proliferated leukaemia pattern cell HL-60.
The influence of Fig. 3 .FACS detection IARS2 gene pairs leukaemia pattern cell HL-60 cell cycles.
Fig. 4 .IARS2 subtractive cdnas are on the active influences of leukaemia pattern cell HL-60Caspase3/7.
Specific implementation mode
Those skilled in the art can obtain the nucleotide sequence of IARS2 genes by conventional method, such as from The databases such as NCBI, Genebank obtain.
The expression of 1.IARS2 genes mRNA in leukaemia pattern cell
Use the RPMI-1640 culture solution cultures containing 10%FBS；Leukaemia pattern cell uses the DMEM containing 10%FBS Culture.Condition of culture is 37 DEG C, 5%CO2, saturated humidity incubator.HL-60 cells are in suspension growth, and 293TN cells are patch Wall is grown.Cell-seeding-density is every milliliter 1 × 105It is a, it is passed on 1 time per 2-3d.
Collect cell a to centrifuge tube in, then be added 1-2 times of uptake PBS mix well, room temperature 1500rpm from Heart 5min.Supernatant is abandoned after centrifugation, rejoins the cell mass that 1ml PBS are resuspended in centrifuge tube, and move to the EP of 1.5ml Guan Zhong, room temperature 1500rpm centrifuge 5min.Remaining PBS is removed as far as possible, is dispensed according to the amount of agglomerate in EP pipes.Point Not Yong Yu mRNA and protein detection.
The cell for extracting total serum IgE will be needed to be stored at room temperature 5 minutes, centrifuged 5 minutes for 4 DEG C with 12000g rotating speeds, it then will be upper It is transferred to clearly in the EP pipes of new 1.5ml RNase-free.The chloroform of 200 μ l is added in new EP pipes, vibrates mixing, room Temperature stands 5 minutes, is centrifuged 15 minutes for 4 DEG C with 12000g rotating speeds.Aspirate supernatant is transferred to the EP of new 1.5mlRNase-free The isopropanol of 600 μ l is added in Guan Zhong.It turns upside down and is stored at room temperature afterwards for several times 10 minutes, 15 points are centrifuged for 4 DEG C with 12000g rotating speeds Clock.It carefully discards supernatant, 75% ethyl alcohol of 1ml cleaning precipitation is added, centrifuged 5 minutes for 4 DEG C with 7500g rotating speeds.Gently abandon supernatant guarantor Precipitation is stayed, EP pipe lids, drying at room temperature 10 minutes or so are opened.Suitable RNase-free water dissolutions precipitation is then added.It carries out The measurement of RNA purity and concentration.
After RNA purity and concentration mensuration, you can carry out reverse transcription experiment.According to PrimeScriptTM RT Master Mix (Perfect Real Time) kit illustrate step by RNA reverse transcriptions be cDNA.The reaction condition of reverse transcription is as follows：37 DEG C, 15 minutes；85 DEG C, 5 seconds；4 DEG C, 10 seconds.Sample preservation is in -80 DEG C of refrigerators so that subsequent experimental detects.The experiment repeats three Secondary, final data does statistical analysis.
As a result：IARS2 genes are prompted to express higher (figure in pattern cell HL-60 by the QPCR results of internal reference of GAPDH 1).Upper figure ordinate is Δ Ct, Δ Ct=target gene Ct values-reference gene Ct values, the relatively large cells of Δ Ct, purpose base Because gene expression abundance is relatively low.
Influence of the 2.IARS2 genes in leukaemia pattern cell Proliferation
The Study on Molecular Mechanism that nadph oxidase subunit etc. participates in apoptosis of leukemia needs to design synthesis specificity SiRNA, transfection method is with reference to HiPerFectTMTransfection reagent kit specifications carry out.Used in this research Target gene siRNA and negative control siRNA by Invitrogen companies of the U.S. design and synthesize.Carry out siRNA transfections When, with reference to transfection reagent HiPerFectTMTransfection reagent operation instructions, by the siRNA of 50nM and 12 μ l Transfection reagent gently mixing, after being stored at room temperature 5-10 minutes, transfection reagent is dropped evenly with siRNA mixtures thin in 6 holes In born of the same parents' culture plate corresponding aperture, orifice plate mixing is jiggled, is placed in cell incubator and continues to cultivate.Through Real-time after 3 days PCR and Western blotting detect jamming effectiveness.Through detecting cell Proliferation when B-myb siRNA transfectional cell different times And the case where Apoptosis.When being tested using inhibitor, cell is stimulated with inhibitor 1 hour in advance, then transfect needle To the specific siRNA of target gene, continue to cultivate cell under conditions of containing inhibitor to appropriate time progress correlation in fact Test research.
Logarithmic growth phase acute myeloid leukemia bone marrow mononuclear cells and HL-60 cells are made 2 × 106Cell is outstanding Liquid is added in 96 orifice plates, 100 holes μ l/, and 10 holes μ l/ of various concentration bufotalien are added, and each concentration is respectively provided with 4 multiple holes, makees With 20 holes μ l/ of CCK-8 solution are added after 24,48,72 hours, continue after cultivating 4h, microplate reader surveys each hole A570nm values, and calculates IC50.Then it takes two kinds of cells of logarithmic growth to be separately added into the inhibitor of P38, JNK, ERK1/2, ERK5, adds most suitable medicine After the bufotalien effect 72h of object concentration, cell viability is detected.In triplicate, final data does statistical analysis for the experiment.
As a result：After shRNA slow-virus infections 3 days, CCK8 is continuously detected 5 days, finds the proliferation of experimental group HL-60 cells Rate is significantly inhibited.Prompt IARS2 genes and the proliferative capacity of HL-60 cells significantly correlated (Fig. 2).
Influence of the 3.IARS2 genes in the leukaemia pattern cell cycle
By acute myeloid leukemia mononuclearcell and HL-60 cells and respectively by P38, JNK, ERK1/2, ERK5 Two kinds of cells of inhibitor processing, are added optimum concentration dried venom of toads culture 24 hours, separately set blank control group and DMSO control groups, 1000rpm centrifugations 5min discards culture solution, collects cell, PBS cleanings, and adjustment each group cell number is 2 × 106/ ml takes 400 μ l Cell suspension is added 200 μ l PBS washings, abandons supernatant, 400 μ l binding buffer and 0 μ lAnnexinV-FITC are added (20 μ g/ml), mixing, 4 DEG C are protected from light incubation 15 minutes, then add 5 μ l propidium iodides (PI) (20 μ g/ml) in cell suspension, mix Even, 4 DEG C are protected from light incubation 5 minutes, 10 minutes are placed at room temperature for, using flow cytomery apoptosis rate.The experiment repeats three Secondary, final data does statistical analysis.
As a result：After shRNA slow-virus infections 4 days, experimental group is in the cytosis in the S phases, is in G2/ M the phases it is thin Born of the same parents are reduced, and prompt IARS2 genes and the period profile of HL-60 cells significantly correlated (Fig. 3).
The active influences of 4.IARS2 gene pairs leukaemia pattern cell Caspase3/7
Pass through two kinds of cells that the inhibitor of P38, JNK, ERK1/2, ERK5 are handled by leukaemia pattern cell and respectively, Culture 24 hours, separately sets blank control group and DMSO control groups, and 1000rpm centrifugations 5min discards culture solution, collects cell；With Caspase3/7 detection kits detect the activity of Caspase3/7.In triplicate, final data does statistics credit for the experiment Analysis.
As a result：Detection Caspase3/7 activity, as a result shows after shRNA slow-virus infections 4 days：Experimental group Caspase3/7 Activity increases, and prompts IARS2 genes and the apoptosis of HL-60 cells significantly correlated (Fig. 4).
The above experiment fully shows that IARS2 genes can be used as the biomarker of leukaemia detection, for facing for leukaemia The exploitation of bed molecular diagnostic markers and drug development, external diagnosis reagent and related kit.The specificity inspection being directed to The design method for surveying the primers of IARS2 genes, probe and nucleic acid chip belongs to conventional technical means.

Claims (6)

  1. The purposes of 1.IARS2 genes is used to prepare the reagent of detection leukaemia or the kit containing the reagent.
  2. 2. purposes according to claim 1, it is characterised in that：Described is detected as Serologic detection or peripheral blood cell counts.
  3. 3. purposes according to claim 1, it is characterised in that：The reagent is primer, probe or nucleic acid chip.
  4. 4. a kind of reagent for detecting leukaemia, it is characterised in that：Contain the corresponding of detection IARS2 genes in the reagent Reagent.
  5. 5. the reagent according to claim 4 for detecting leukaemia, it is characterised in that：The detection IARS2 genes Corresponding reagent be PCR detection reagents or molecule hybridization check reagent.
  6. 6. including the kit of the reagent described in claim 4 for detecting leukaemia.
CN201810522724.0A 2018-05-28 2018-05-28 The application for the biomarker that IARS2 genes are detected as leukaemia Pending CN108424967A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102186987A (en) * 2008-04-24 2011-09-14 阿肯色大学托管委员会 Gene expression profiling based identification of genomic signature of high-risk multiple myeloma and uses thereof
WO2015144929A1 (en) * 2014-03-28 2015-10-01 Erasmus University Medical Center Rotterdam Method for diagnosing and treating multiple myeloma.
CN105803056A (en) * 2014-12-30 2016-07-27 上海吉凯基因科技有限公司 Application of human IARS2 gene and related medicines thereof
US20180089373A1 (en) * 2016-09-23 2018-03-29 Driver, Inc. Integrated systems and methods for automated processing and analysis of biological samples, clinical information processing and clinical trial matching

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102186987A (en) * 2008-04-24 2011-09-14 阿肯色大学托管委员会 Gene expression profiling based identification of genomic signature of high-risk multiple myeloma and uses thereof
WO2015144929A1 (en) * 2014-03-28 2015-10-01 Erasmus University Medical Center Rotterdam Method for diagnosing and treating multiple myeloma.
CN105803056A (en) * 2014-12-30 2016-07-27 上海吉凯基因科技有限公司 Application of human IARS2 gene and related medicines thereof
US20180089373A1 (en) * 2016-09-23 2018-03-29 Driver, Inc. Integrated systems and methods for automated processing and analysis of biological samples, clinical information processing and clinical trial matching

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HAN YAN ET AL: "Association of a cytarabine chemosensitivity related gene expression signature with survival in cytogenetically normal acute myeloid leukemia", 《ONCOTARGET》 *
J.YIN ET AL: "IARS2 silencing induces non-small cell lung cancer cells proliferation inhibition, cell cycle arrest and promotes cell apoptosis", 《NEOPLASMA》 *
LING ZHONG ET AL: "Expression of IARS2 gene in colon cancer and effect of its knockdown on biological behavior of RKO cells", 《INT J CLIN EXP PATHOL》 *

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Application publication date: 20180821