CN110468203A - A kind of marker, detection primer sequence and its application for predicting Gliblastoma patient prognosis - Google Patents

A kind of marker, detection primer sequence and its application for predicting Gliblastoma patient prognosis Download PDF

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CN110468203A
CN110468203A CN201910729866.9A CN201910729866A CN110468203A CN 110468203 A CN110468203 A CN 110468203A CN 201910729866 A CN201910729866 A CN 201910729866A CN 110468203 A CN110468203 A CN 110468203A
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marker
seq
gliblastoma
prognosis
patient
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周佳
施玲飞
厉民
王峥
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Zhejiang Provincial Peoples Hospital
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Abstract

The present invention relates to a kind of marker, detection primer sequence and its applications for predicting Gliblastoma patient prognosis, comprising: the marker of this prediction Gliblastoma patient prognosis, marker CXXC4-mRNA；For detecting the detection primer of the marker of the prediction Gliblastoma patient prognosis, the sequence of the detection primer is respectively as shown in SEQ ID NO:1 and SEQ ID NO:2；For predicting that the detection primer of the marker of Gliblastoma patient's prognosis is preparing the application in detection kit；Kit contains PCR reaction system reagent, and the real-time fluorescence quantitative PCR reaction system reagent containing primer sequence SEQ ID NO:1 and SEQ ID NO:2.The beneficial effects of the present invention are: present invention firstly discovers that CXXC4 expression can be used as the index of glioma prognosis, and it can be used as the potential target spot that therapeutic intervention is carried out to GBM, not only with the innovation of scientific meaning, but also has exploitation at tumor prognosis Testing index and prepare the huge clinical value of related targeted drug.

Description

It is a kind of to predict the marker of Gliblastoma patient prognosis, detection primer sequence And its application
Technical field
The invention belongs to tumor biotherapy fields, and in particular to a kind of mark for predicting Gliblastoma patient prognosis Will object, detection primer sequence and its application.
Background technique
Glioma is to be primary in the malignant tumour of encephalic, can derive from a variety of histological types, including astrocyte source, few Prominent cell origin, dash forward less astrocyte source, ependymal epithelium source etc..Wherein, the pleomorphism colloid in astrocyte source Blastoma (GBM) WHO is classified 4 grades, is most common, the most fatal primary malignant tumor of central nervous system (CNS).It suffers from The prognosis of crowd is poor, and median survival interval is 12-16 months, and survival rate is only 5.5% or so within 5 years.
In recent years the study found that GBM genome change have important clinical meaning, comprehensive diagnos is to prognosis and essence Really treatment has important value.Since there is, with heterogeneity in tumour, cause the complexity of its occurrence and development between tumour by GBM Property and its cause the root of various malignant activities, it is therefore desirable to constantly accumulation GBM patient clinical and molecular biology data, The mechanism of action that the gene of further investigation research anomalous variation may play in tumor development.As people are to glioma Glioma pathological diagnosis, is combined carry out comprehensive analysis, for disease by gradually going deep into for disease cognitive with molecular diagnosis The judgement of feelings and treatment, the prognosis of disease are very necessary.
Currently, a few molecules label such as methylation state of only IDH-1 mutation, TP53, RB, MGMT promoter can be used In prediction glioblastoma patient prognosis or therapeutic effect.However, often being deposited due to the unstability of tumor cell gene group In the exception of multiple genes.Therefore, it is necessary to which it is different to obtain gene expression from the clinical data and related sample of GBM patient The related data often changed, and therefrom find and lead to the suspicious clue of disease development, carry out it is more extensive, deeper into grind Study carefully, so that discovery more more has the molecular labeling of clinical value.
For glue mother patient, gene therapy or targeted therapy are carried out using the molecular labeling for influencing its prognosis of discovery, Tumour may be implemented precisely to treat, treatment validity is greatly improved, reduce chemotherapy dosage, reduce chemical therapy toxic side effect, improve and suffer from Person's quality of life extends patient survival, final that patient is helped to realize that high quality has the life of dignity.
Summary of the invention
It is an object of the invention to overcome the shortage of prior art, a kind of prediction Gliblastoma patient prognosis is provided Marker, detection primer sequence and its application.
The marker of this prediction Gliblastoma patient prognosis, marker CXXC4-mRNA.
For detecting the detection primer of the marker of the prediction Gliblastoma patient prognosis, the detection primer Sequence respectively as shown in SEQ ID NO:1 and SEQ ID NO:2.
For predict Gliblastoma patient's prognosis marker detection primer preparation detection kit in Using.
Kit contains PCR reaction system reagent, and contains primer sequence SEQ ID NO:1 and SEQ ID NO:2 Real-time fluorescence quantitative PCR reaction system reagent.
As preferred: PCR reaction system reagent, the 10mM deoxidation core including 500 5 × RT Buffers of μ l, 250 μ l Thuja acid, the 10U/ μ l RNase inhibitor of 200 μ l, the Rever Tra Ace reverse transcriptase of 200 μ l, 200 μ l random primer； Real-time fluorescence quantitative PCR reaction system reagent, PCR premixed liquid Sybr Green Master's Mix, 100 μ g including 5ml SEQ ID NO:1 and SEQ ID NO:2.
The beneficial effects of the present invention are: present invention firstly discovers that CXXC4 expression can be used as the finger of glioma prognosis Mark, and can be used as to GBM carry out therapeutic intervention potential target spot, not only with scientific meaning innovation, but also have exploitation at Tumor prognosis Testing index and the huge clinical value for preparing related targeted drug.
Detailed description of the invention
Fig. 1 is expression of the CXXC4 in GBM, different type glioma and normal tissue.
Fig. 2 is influence of the CXXC4 overexpression to A172, U251 cell line proliferation ability.
Fig. 3 is influence of the CXXC4 overexpression to A172, U251 cell line apoptosis.
Fig. 4 is influence of the CXXC4 expression to GBM patient survival.
Specific embodiment
Illustrate embodiments of the present invention below by way of specific embodiment, unless otherwise stated, disclosed in this invention Experimental method be all made of the art routine techniques, all primer synthetic works are by the raw work biotechnology service in Shanghai Co., Ltd completes, and used reagent and raw material are available on the market in embodiment.It is related in following embodiments Cell is purchased from Shanghai Inst. of Life Science, CAS cell resource center.
Embodiment 1
1.qRT-PCR detects expression of the CXXC4 in GBM different subtype and normal tissue；
1) the normal of GBM, Astrocytic glioma, the tumor tissues of mesoglioma patient and remains donations acquisition is obtained Brain tissue sample, Trizol cracking, extracts RNA.
2) CDs is found with DNAMAN and guard domain, with OLIGO design primer SEQ.ID.NO.1 and SEQ.ID.NO.2.
3) reverse transcription system:
4) qRT-PCR detection and analysis:
Mix configuration:
* cDNA solution can be by the volume ddH of 1:10~1:202O dilution
PCR program:
5) data are analyzed: 2-△△CTMethod is analyzed, as a result such as Fig. 1.It can be seen that the mRNA of GBM patient CXXC4 is with respect to table Up to horizontal with normal cerebral tissue's no significant difference (Fig. 1, p=0.976), but in GBM CXXC4 mRNA relative expression levels it is obvious Lower than Astrocytoma (p < 0.0001) and Oligodendroglioma (p < 0.0001).
The detection of 2.MTT method is overexpressed the variation of the glioblastoma cell line proliferative capacity of CXXC4:
1) A172, U251 cell for taking P2 to be overexpressed for A172, U251 cell and CXXC4, adjustment cell density is 4 × 105/ ml, it is spare；
2) for A172 cell, 4 groups are set altogether, and 1 group is zeroing hole, remaining 3 groups are respectively as follows: common A172 groups of cells, transfection The A172 groups of cells that the A172 groups of cells of empty plasmid, CXXC4 are overexpressed；For U251 cell, 4 groups are set altogether, and 1 group is tune Zero hole, remaining 3 groups are respectively as follows: common U251 groups of cells, the U251 groups of cells for having transfected empty plasmid, CXXC4 and are overexpressed U251 cell is added in 96 well culture plates, 100 μ l suspensions of every hole addition, every group of 3 holes,；
3) respectively at 0h, for 24 hours, 48h, 72h and 96h when, first washed twice with without the DMED culture solution of FBS, then existed 100 μ l fresh cultures are added in each group culture plate, 10 μ l MTT solution are then added, continue to be incubated in cell incubator 1h；
4) with spectrophotometric determination 450nm absorbance: making Detection wavelength with 450-490nm, 600-650nm makees reference wave It is long；
5) cell growth curve is drawn according to the OD value of measurement, as a result such as Fig. 2.It can see and work as according to MTT testing result A172 and U251 cell line after CXXC4 gene overexpression, the increased rate of cell quantity will lower than empty plasmid control group (p < 0.05)。
The bis- staining for flow cytometry detections of 3.Annexin V/PI are overexpressed CXXC4 to glioblastoma cell line apoptosis Influence
1) by A172, U251 cell for being overexpressed CXXC4 and corresponding blank control cell dissociation, count, be configured to it is dense Degree is 5 × 104The cell suspension of a/ml is inoculated in 6 orifice plates with 2ml volume, if 3 multiple holes, are placed in 37 DEG C, 5%CO2Culture 48h is cultivated in case；
2) it digests and cell (1000 × g of revolving speed) is collected by centrifugation and abandon supernatant afterwards, addition is washed 2 times, after centrifugation without calcium PBS 1ml Abandon supernatant；
3) 100 μ l binding buffer, 5 μ L Annexin V-FITC and 5 μ l PI solution are separately added into, room temperature is kept away Light is incubated for 15min；
4) it is added after 400 μ l binding buffer with standardization program flow cytomery, mercury excitation wavelength 488nm counts 10,000 cells, is as a result analyzed with ModFit software, such as Fig. 3.The bis- dye test prompts of Annexin V/PI, In It is overexpressed CXXC4 gene and when 48h, the apoptosis rate of overexpression group cell wants bright after the A172 cell passage of transfection empty plasmid Aobvious to be higher than empty plasmid group (Fig. 3 is left, p=0.012), the U251 apoptosis rate of CXXC4 overexpression group will be apparently higher than blank Plasmid group (Fig. 3 is right, p=0.0034).
The GBM survival of patients curve of 4.CXXC4 difference expression compares；
It can be accessed from Betastasis (http://betastasis.com) data platform and download to TCGA data The related gene expression chip of GBM specimens and normal cerebral tissue donor in library and REMBRANDT database Detection data is to be detected to obtain with Affymetrix HT HG U133A chip.Meanwhile it is accessible and download to TCGA number According to the patient survival of the GBM in library and REMBRANDT database, tumor classification and use Affymetrix HT HG U133A The data such as the detected mRNA expression of gene associated level of chip.In addition, the mRNA expression of different glioma cell lines Data can also be downloaded to therefrom.According to CXXC4 expression height, survivorship curve is drawn, as a result such as Fig. 4.In GBM patient The mRNA expression of CXXC4 significantly affects patient survival.CXXC4mRNA high expresses the middle position of patient in TCGA database Life cycle is 442 days, hence it is evident that is longer than 422 days (Fig. 4 is left, p=0.006) of low expression patient；In REMBRANDT database The median survival interval that CXXC4mRNA high expresses patient is 856 days, hence it is evident that be longer than low expression patient 425 days (Fig. 4 is right, p < 0.0001)。
Sequence table
<110>Zhejiang Provincial People's Hospital
<120>a kind of marker, detection primer sequence and its application for predicting Gliblastoma patient prognosis
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
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<212> DNA
<213>people (Homo sapiens)
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<213>people (Homo sapiens)
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caggaaacag ctatgacc 18

Claims (5)

1. a kind of marker for predicting Gliblastoma patient prognosis, it is characterised in that: marker CXXC4-mRNA.
2. it is a kind of for detecting the detection primer for predicting the marker of Gliblastoma patient prognosis described in claim 1, It is characterized by: the sequence of the detection primer is respectively as shown in SEQ ID NO:1 and SEQ ID NO:2.
3. as claimed in claim 2 for predicting prepared by the detection primer of the marker of Gliblastoma patient's prognosis Application in detection kit.
4. a kind of kit, it is characterised in that: contain PCR reaction system reagent, and contain primer sequence SEQ ID NO:1 With the real-time fluorescence quantitative PCR reaction system reagent of SEQ ID NO:2.
5. kit according to claim 4, it is characterised in that: PCR reaction system reagent, including 500 5 × reverses of μ l Record the Rever Tra Ace of buffer, the 10mM deoxynucleotide of 250 μ l, the 10U/ μ l RNase inhibitor of 200 μ l, 200 μ l The random primer of reverse transcriptase, 200 μ l；Real-time fluorescence quantitative PCR reaction system reagent, the PCR premixed liquid Sybr including 5ml The SEQ ID NO:1 and SEQ ID NO:2 of Green Master Mix, 100 μ g.
CN201910729866.9A 2019-08-08 2019-08-08 A kind of marker, detection primer sequence and its application for predicting Gliblastoma patient prognosis Withdrawn CN110468203A (en)

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Cited By (1)

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CN116564421A (en) * 2023-06-08 2023-08-08 苏州卫生职业技术学院 Method for constructing prognosis model related to copper death of acute myelogenous leukemia patient

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CN107574247A (en) * 2017-09-28 2018-01-12 郑州大学第附属医院 A kind of the glioblastoma auxiliary diagnosis based on CLCF1 genes, prognostic evaluation kit and its application method
CN109182528A (en) * 2018-10-22 2019-01-11 中国医科大学附属第医院 A kind of glioblastoma auxiliary diagnosis based on ITGB5 gene, prognostic evaluation kit and its application method

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Publication number Priority date Publication date Assignee Title
CN107574247A (en) * 2017-09-28 2018-01-12 郑州大学第附属医院 A kind of the glioblastoma auxiliary diagnosis based on CLCF1 genes, prognostic evaluation kit and its application method
CN109182528A (en) * 2018-10-22 2019-01-11 中国医科大学附属第医院 A kind of glioblastoma auxiliary diagnosis based on ITGB5 gene, prognostic evaluation kit and its application method

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天高云淡的博客: "常用的通用引物序列", 《新浪微博》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116564421A (en) * 2023-06-08 2023-08-08 苏州卫生职业技术学院 Method for constructing prognosis model related to copper death of acute myelogenous leukemia patient
CN116564421B (en) * 2023-06-08 2024-01-30 苏州卫生职业技术学院 Method for constructing prognosis model related to copper death of acute myelogenous leukemia patient

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CN110468203A (en) A kind of marker, detection primer sequence and its application for predicting Gliblastoma patient prognosis

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Application publication date: 20191119