CN107779509A - A kind of mark of pulmonary cancer diagnosis and its application - Google Patents
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Abstract
The invention belongs to technical field of biomedical detection, is related to mark and its application of a kind of pulmonary cancer diagnosis, and the invention discloses a kind of pulmonary cancer diagnosis mark, the pulmonary cancer diagnosis mark is UHRF1 genes.The biomarker has very high application value, and detection method is simple, simple to operate, and new direction is provided for the research of pulmonary cancer diagnosis reagent, is provided fundamental basis for clinical pharmacy.
Description
Technical field
The invention belongs to technical field of biomedical detection, is related to mark and its application of a kind of pulmonary cancer diagnosis, specifically
It is related to mark UHRF1 genes and its application of a kind of pulmonary cancer diagnosis.
Background technology
Epidemiology points out that lung cancer is one of fastest-rising malignant tumour of morbidity and mortality, in the world because tumour causes
The reason for dead, is central, and lung cancer ranks first position.Non-small cell lung cancer (non-small cell lung cancer, NSCLC) is about
The 85% of the primary lung cancer totality made a definite diagnosis is accounted for, ED-SCLC (SCLC) accounts for the 20%-25% of lung cancer.In recent years, section
Grind the research that worker increasingly payes attention to oncomolecularbiology, it was found that many tumor markerses can prompt tumour source and
Histological type.Tumor-marker analyte detection has important in the examination of lung cancer, diagnosis, examination of curative effect and Index for diagnosis etc.
Clinical value.
The albumen of UHRF1 gene codes is the member of RING finger-type E3 ubiquitin ligase subfamilies, the albumen and specific DNA
Sequence combines, and raises histone deacetylase and is expressed with controlling gene.UHRF1 genes reach highest in the G1 phases of cell cycle,
And continue to express in the G2 phases of cell cycle and M phases.It is acted as in G1/S conversions and in p53 dependent DNAs damage inspection point
With, it is considered to be the maincenter protein that epigenetic information is integrated.UHRF1 genes raise in kinds cancer, therefore are considered as
It is therapeutic targets.
The A of CN 10588660 disclose a kind of gene marker, and the gene marker is UHRF1, and UHRF1 can be used for judging
Whether subject has the risk for suffering from carcinoma of endometrium or judges whether subject suffers from carcinoma of endometrium.CN 102305747
A discloses a kind of biomarker reagent for being used to detect breast cancer status, the biomarker reagent behaviour UHRF1 bases
The DISTANT METASTASES IN ability of cause, biomarker reagent UHRF1 genes and breast cancer is closely related, can utilize UHRF1 genes
Biomarker as prediction breast cancer DISTANT METASTASES IN ability.But above-mentioned patent is all without open UHRF1 genes and lung cancer phase
Close.
It is the genes such as EGFR, KRAS, BRAF, ALK, MET that the genetic test product of lung cancer, which is related to more, in the market,
UHRF1 genes are not included substantially, it is related to lung cancer also to disclose UHRF1 genes without any prior art.The generation of lung cancer
Development is closely related with genetic mutation, and therefore, there is an urgent need to find the pulmonary cancer diagnosis mark of accuracy height and high specificity.
The content of the invention
The defects of present invention is in order to overcome the above method, there is provided a kind of mark of pulmonary cancer diagnosis and its application, it is described
UHRF1 genes are a kind of very high lung cancer tumor marks of Sensitivity and Specificity, can preferably to tumour make diagnosis and
Differentiate, accomplish early discovery, early treatment and the progression free survival phase for extending patient.
To reach the purpose of this invention, the present invention uses following technical scheme：
In a first aspect, the present invention provides a kind of pulmonary cancer diagnosis mark, the pulmonary cancer diagnosis mark is UHRF1 genes.
In the present invention, inventor analyzes the operation of 378 lung cancer patients in China acquisitions using immunohistochemical method
Sample, have been surprisingly found that, UHRF1 genes specific stain in neoplastic cell nuclei, and do not dyed in other cells, therefore,
UHRF1 gene expressions can be used for the pathological diagnosis of lung cancer.
Second aspect, the pulmonary cancer diagnosis mark that the present invention provides as described in relation to the first aspect are used to prepare examining for screening lung cancer
Application in disconnected and/or medicine.
The third aspect, the pulmonary cancer diagnosis mark that the present invention provides as described in relation to the first aspect are used for the detection examination for preparing lung cancer
Application in agent.
According to the present invention, the detection reagent is also included for Immunohistochemical Method and/or PCR methods detection biomarker
The related reagent of UHRF1 genes.
Fourth aspect, the present invention provide a kind of pulmonary cancer diagnosis kit, and the kit includes one or more special
Property detection UHRF1 gene expression doses primer and/or probe.
According to the present invention, the kit detects the primer of UHRF1 genes using real-time quantitative PCR method, described to draw
The nucleotide sequence of thing is as shown in SEQ ID NO.1-2：
Sense primer (SEQ ID NO.1):5'-CCA GCA GAG CAG CCT CAT C-3'；
Anti-sense primer (SEQ ID NO.2):5'-TCC TTG AGT GAC GCC AGG A-3'.
According to the present invention, the kit also includes GAPDH reference gene primers, the GAPDH reference genes primer
Nucleotide sequence is as shown in SEQ ID NO.3-4：
Sense primer (SEQ ID NO.3):5'-CAG GAG GCA TTG CTG ATG AT-3'；
Anti-sense primer (SEQ ID NO.4):5'-GAA GGC TGG GGC TCA TTT-3'.
Preferably, the kit also includes buffer solution, dNTPs and archaeal dna polymerase.
5th aspect, the present invention provide a kind of kit as described in fourth aspect and are used to prepare answering for pulmonary cancer diagnosis reagent
With.
6th aspect, the present invention provide a kind of detection method of UHRF1 genes, and the detection method is including the use of the such as the 4th
Kit described in aspect.
According to the present invention, comprise the following steps：
(1) sample to be checked is gathered, carries out SABC detection and/or using in the kit as described in claim 5 or 6
Primer carries out qRT-PCR amplifications；
(2) expression of UHRF1 genes is analyzed.
According to the present invention, the reaction condition of step (1) the qRT-PCR amplifications is：
(a) 48-52 DEG C of 1-3min, 93-98 DEG C of 8-12min, 1-2 are circulated；(b)92-98℃10-30s、58-62℃1-
3min, 25-35 circulations；(c) 4 DEG C of preservations.
Preferably, the reaction condition of step (1) the qRT-PCR amplifications is：
(a) 50 DEG C of 2min, 95 DEG C of 10min, 1 circulation；(b) 95 DEG C of 15s, 60 DEG C of 1min, 30 circulations；(c) 4 DEG C of preservations.
In a specific embodiment of the invention, the specific reactions steps of the PCR amplifications are as follows：
Specific reaction system is as follows；
One group of negative control using water as sample is set.
Reaction condition is as follows：
As optimal technical scheme, the detection method of the UHRF1 genes, comprise the following steps：
(1) SABC detects：Sample tissue is extracted, FFPE is simultaneously cut into slices, and using UHRF1 antibody, carries out immune group
Change experiment；
(2) expression of UHRF1 genes is analyzed.
As optimal technical scheme, the detection method of the UHRF1 genes, comprise the following steps：
(1) qRT-PCR is expanded：Sample tissue is extracted, RNA is extracted, is entered using the primer of kit as described in fourth aspect
Row qRT-PCR amplified reactions：(a) 50 DEG C of 2min, 95 DEG C of 10min, 1 circulation；(b) 95 DEG C of 15s, 60 DEG C of 1min, 30 circulations；
(c) 4 DEG C of preservations；
(2) expression of UHRF1 genes is analyzed.
To those skilled in the art, even if not understanding the Cleaning Principle of the present invention, it can equally implement, reproduce this
Whether invention, i.e. Cleaning Principle of the invention are cheer and bright, do not affect the implementation and reproduction of the present invention.
Compared with prior art, the present invention has the advantages that：
(1) present invention analyzes the specimens from pri of patients with lung cancer by inquiry, it is found that UHRF1 genes are the new of pulmonary cancer diagnosis
Mark, and using the expression of SABC and PCR method detection UHRF1 genes, accuracy is high, and specificity is good, false sun
Property false negative rate it is low, it is simple to operate, instrument and equipment require low, wide adaptability；
(2) detection kit of the present invention can be used for UHRF1 genetic tests, and lung cancer situation is predicted, and find to deposit ahead of time
Risk, as early as possible to treat and preparation is made in preventing and treating；
(3) detection kit and its detection method provided by the present invention for being used to detect UHRF1 genes, has very high
Application value, it is simple to operate, new direction is provided for the research of pulmonary cancer diagnosis reagent, theoretical base is provided for clinical pharmacy
Plinth.
Brief description of the drawings
Fig. 1 is that SABC detects expression of the UHRF1 genes in lung cancer, wherein, figure (A) is normal lung tissue, is schemed (B)
For non-small cell lung cancer (female, large cell carcinoma, T2N0M0), figure (C) is non-small cell lung cancer (female, adenosquamous carcinoma, T2N1M0), figure
(D) it is non-small cell lung cancer (man, adenosquamous carcinoma, T2N1M0), figure (E) is ED-SCLC, and without by stages, figure (F) is cellule lung
Cancer (man, squamous carcinoma, T2N0M0), figure (G) are non-small cell lung cancer (man, squamous carcinoma, T2N0M0), and figure (H) is non-small cell lung cancer
(man, squamous carcinoma, T2N0M0), figure (I) are non-small cell lung cancer (man, squamous carcinoma, T2N0M0), figure (J) be non-small cell lung cancer (man,
Squamous carcinoma, T2N1M0), figure (K) is non-small cell lung cancer (man, squamous carcinoma, T3N0M0), figure (L) be non-small cell lung cancer (man, squamous carcinoma,
T3N0M0)；
Fig. 2 is that SABC detects expression of the UHRF1 in non-small cell lung cancer, wherein, figure (A) is normal lung tissue,
It is non-small cell lung cancer (female, gland cancer, T2N0M0) to scheme (B), and figure (C) is non-small cell lung cancer (female, gland cancer, T2N0M0), is schemed (D)
For non-small cell lung cancer (female, gland cancer, T2N0M0), figure (E) is non-small cell lung cancer (female, gland cancer, T2N0M0)；
Fig. 3 is detection of expression of the UHRF1mRNA in cancerous lung tissue.
Embodiment
Further to illustrate the technological means and its effect of the invention taken, below in conjunction with being preferable to carry out for the present invention
Example further illustrates technical scheme, but the present invention is not limited in scope of embodiments.
In the examples where no specific technique or condition is specified, according to the technology or condition described by document in the art,
Or carried out according to product description.Agents useful for same or the unreceipted production firm person of instrument, be can be by regular channel commercially available from
The conventional products of acquisition.
Material：
Primary antibody：Anti- UHRF1 mouse monoclonal antibodies (1：1000, BD Bioscience) or normal mouse IgG (1:100,
Santa Cruz, Biotechnology, Santa Cruz, CA, USA)；
Secondary antibody：HRP coupling secondary antibodies (Dako Cytomation).
The assembling of the qRT-PCR detection kits of embodiment 1
Specifically, the main agents in the detection kit of the present invention include：
PCR amplifing reagents：10 × buffer solution, dNTPs, archaeal dna polymerase, pure water, comprising for detecting UHRF1 gene locis
Pcr amplification primer thing and GAPDH reference gene primers, wherein,
The nucleotide sequence of the UHRF1 gene primers is as shown in SEQ ID NO.1-2：
Sense primer (SEQ ID NO.1):5'-CCA GCA GAG CAG CCT CAT C-3'；
Anti-sense primer (SEQ ID NO.2):5'-TCC TTG AGT GAC GCC AGG A-3'.
The nucleotide sequence of the GAPDH reference genes primer is as shown in SEQ ID NO.3-4：
Sense primer (SEQ ID NO.3):5'-CAG GAG GCA TTG CTG ATG AT-3'；
Anti-sense primer (SEQ ID NO.4):5'-GAA GGC TGG GGC TCA TTT-3'.
Embodiment 2：UHRF1 SABC detection example
(1) SABC detects：Extract the tissue samples of 1 normal tissue samples and 11 lung cancer patients, paraffin bag
Bury and cut into slices, using UHRF1 antibody, carry out immunohistochemical experiment, specific experiment step is as follows：
(a) materials tissue samples, it is 25px × 25px × 12.5px to take size, is cut into slices, and is placed on 40 DEG C of constant temperature roasters
In, dry standby；
(b) dewaxing and aquation：In 65 DEG C of baking boxs, 30min is toasted, xylene soak 15min × 2, absolute ethyl alcohol 5min,
95% ethanol 5min, 85% ethanol 5min, PBS 5min × 2, H2O 1min；
(c) antigen retrieval：High-temperature resistance plastice slide holding frame is placed in microwave box, and is added antigen retrieval buffers, waits to seethe with excitement
The histotomy being put into afterwards after dewaxing aquation, first heat 5 seconds, then suspend 15 seconds, so circulation, microwave box is taken out after 15min,
Rinsed under running water, after it is cool but afterwards take out slide, cleaned in distilled water 2 times, then clean 3 times in 1 × PBS, every time
2min；
(d) unnecessary moisture is got rid of, residual liquid around tissue, 3% hydrogen peroxide incubation at room temperature are blotted with filter paper
20min, H2O are cleaned, PBS 2min × 3；
(e) 5% hyclone closing 20min；
(f) primary antibody is added dropwise, every 50 μ l, 4 DEG C overnight, PBS 2min × 3；
(g) 37 DEG C of secondary antibody, 20min is added dropwise；
(h) DAB develops the color, and developing time is longer than 20 seconds, and is less than 7min, and clear water rinses；
(i) haematoxylin dyeing, developing time are longer than 20 seconds, and are less than 3min, the anti-blue 3-5min of PBS；
(j) it is dehydrated, is sequentially placed into 85%, 95% and absolute ethyl alcohol, each 5min, transparent 5min × 2 of dimethylbenzene；
(k) resin mounting drips neutral gum, cover glass in pressure, dries；
(l) the use of fluorescence microscope eyepiece it is 10 times, object lens are 40 times, carry out result statistics；
(2) expression of UHRF1 genes is analyzed.
The analysis result of SABC detection is as shown in figure 1, it will be seen from figure 1 that UHRF1 is in normal pneumonocyte not table
Reach, and the high expression of the non-small cell lung cancer 66%, large cell carcinoma and the nucleus of adenosquamous carcinoma and 92% ED-SCLC.
Embodiment 3：UHRF1 SABC detection example
(1) SABC detects：1 normal tissue samples and the tissue samples of 4 non-small cell lung cancer patients are extracted,
FFPE is simultaneously cut into slices, and using UHRF1 antibody, carries out immunohistochemical experiment；
The specific method of SABC detection is the same as embodiment 1.
(2) expression of UHRF1 genes is analyzed.
The analysis result of SABC detection is as shown in Fig. 2 figure it is seen that UHRF1 is in normal pneumonocyte not table
Reach, and the high expression of nucleus of the non-small cell lung cancer and gland cancer 59%.
Embodiment 4：UHRF1 qRT-PCR detection examples
(1) sample is extracted：1 normal tissue samples and 2 pulmonary adenocarcinoma samples are extracted, extract RNA；
(2) qRT-PCR is expanded：Carried out using the UHRF1 primers and GAPDH reference genes primer of kit in embodiment 1
QRT-PCR amplified reactions, reaction system is as follows,
One group of negative control using water as sample is set.
Reaction condition is as follows：
(3) expression of UHRF1 genes is analyzed.
QRT-PCR testing result is as shown in figure 3, from figure 3, it can be seen that compared with normal lung tissue, pulmonary adenocarcinoma
In UHRF1 mRNA expressions significantly raise (* * represent p<0.01).
In summary, the present invention analyzes the specimens from pri of patients with lung cancer by inquiry, it is found that UHRF1 genes are pulmonary cancer diagnosis
Novel marker, and the expression of the method detection UHRF1 genes using SABC and PCR, accuracy is high, specificity
Good, false positive false negative rate is low, simple to operate, and instrument and equipment requires low, wide adaptability.
Applicant states that the present invention illustrates the method detailed of the present invention, but not office of the invention by above-described embodiment
It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implemented.Art
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., within the scope of all falling within protection scope of the present invention and disclosing.
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Claims (10)
1. a kind of pulmonary cancer diagnosis mark, it is characterised in that the pulmonary cancer diagnosis mark is UHRF1 genes.
2. pulmonary cancer diagnosis mark according to claim 1 is used to prepare in the diagnosis for screening lung cancer and/or medicine
Application.
3. pulmonary cancer diagnosis mark according to claim 1 is used to prepare the application in the detection reagent of lung cancer.
4. application according to claim 3, it is characterised in that the detection reagent also include be used for Immunohistochemical Method and/
Or the related reagent of PCR methods detection biomarker UHRF1 genes.
5. a kind of pulmonary cancer diagnosis kit, it is characterised in that the kit includes one or more specific detection UHRF1
The primer and/or probe of gene expression dose.
6. kit according to claim 5, it is characterised in that the kit is examined using real-time quantitative PCR method
Survey the primer of UHRF1 genes；
Preferably, the nucleotide sequence of the primer is as shown in SEQ ID NO.1-2；
Preferably, the kit also includes GAPDH reference gene primers；
Preferably, the nucleotide sequence of the GAPDH reference genes primer is as shown in SEQ ID NO.3-4；
Preferably, the kit also includes buffer solution, dNTPs and archaeal dna polymerase.
7. a kind of kit as described in claim 5 or 6 is used for the application for preparing pulmonary cancer diagnosis reagent.
8. a kind of detection method of UHRF1 genes, it is characterised in that the detection method is including the use of such as institute of claim 5 or 6
The kit stated.
9. detection method according to claim 9, it is characterised in that comprise the following steps：
(1) sample to be checked is gathered, carries out SABC detection and/or using the primer in the kit as described in claim 5 or 6
Carry out qRT-PCR amplifications；
(2) expression of UHRF1 genes is analyzed.
10. detection method according to claim 9, it is characterised in that the reaction bar of step (1) the qRT-PCR amplifications
Part is：
(a) 48-52 DEG C of 1-3min, 93-98 DEG C of 8-12min, 1-2 are circulated；(b) 92-98 DEG C of 10-30s, 58-62 DEG C of 1-3min,
25-35 circulation；(c) 4 DEG C of preservations；
Preferably, the reaction condition of step (1) the qRT-PCR amplifications is：
(a) 50 DEG C of 2min, 95 DEG C of 10min, 1 circulation；(b) 95 DEG C of 15s, 60 DEG C of 1min, 30 circulations；(c) 4 DEG C of preservations.
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| CN201711268409.1A CN107779509A (en) | 2017-12-05 | 2017-12-05 | A kind of mark of pulmonary cancer diagnosis and its application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108660212A (en) * | 2018-05-25 | 2018-10-16 | 武汉科技大学 | Application of the WDR1 genes in preparing Treatment for Non-small Cell Lung and detecting product |
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| CN105886660A (en) * | 2016-06-29 | 2016-08-24 | 北京泱深生物信息技术有限公司 | Application of UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) as endometrial cancer diagnosis and treatment marker |
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2017
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