CN106939340A - A kind of molecular marked compound related to adenocarcinoma of lung transfer and prognosis and its application - Google Patents
A kind of molecular marked compound related to adenocarcinoma of lung transfer and prognosis and its application Download PDFInfo
- Publication number
- CN106939340A CN106939340A CN201710208864.6A CN201710208864A CN106939340A CN 106939340 A CN106939340 A CN 106939340A CN 201710208864 A CN201710208864 A CN 201710208864A CN 106939340 A CN106939340 A CN 106939340A
- Authority
- CN
- China
- Prior art keywords
- adenocarcinoma
- lung
- rcc2
- prognosis
- expression
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/112—Disease subtyping, staging or classification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/56—Staging of a disease; Further complications associated with the disease
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Pathology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Oncology (AREA)
- General Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Food Science & Technology (AREA)
- Hospice & Palliative Care (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of molecular marked compound related to adenocarcinoma of lung transfer and prognosis and its application.Wherein, described molecular marked compound is detection chromosome condensation regulatory factor 2 (Regulator of Chromosome Condensation 2, RCC2).Research shows that RCC2 is related to tumor development, has differences expression in clinical pulmonary adenocarcinoma, and increasing for its expression occurs and poor prognosis is significantly correlated with transfer.Detected by the gene magnification to RCC2 and expression, can be achieved to shift the early diagnosis occurred and prognosis evaluation to adenocarcinoma of lung.The present invention passes through the gene magnification and the detection of expression to RCC2 in adenocarcinoma of lung, it is applied to RCC2 as tumor cells diagnostic marker in clinical adenocarcinoma of lung transfer diagnosis and prognosis evaluation, will be expected to be that adenocarcinoma of lung invasion and attack transfer sexual development and patients with lung adenocarcinoma prognosis provide New Set.
Description
Technical field
The present invention relates to a kind of molecular marked compound for having potential adenocarcinoma of lung diagnosis, belong to knubble biological diagnosis and treat
Technical field.
Background technology
Lung cancer is one of world today's fatal rate highest malignant tumour, and the newborn case in annual worldwide exceedes
1000000, wherein using adenocarcinoma of lung as topmost organization type.Liver, bone and brain metastes, or even some patients easily occur for adenocarcinoma of lung
Can occur far-end transfer at pathogenic initial stage, thus cause the cure rate of adenocarcinoma of lung extremely low, and due to recurring and shifting, Huan Zheshu
Five year survival rate is still less than 15% afterwards.As can be seen here, transfer is the lethal main cause of current patients with lung adenocarcinoma.Although in recent years
The diagnosis and treatment of adenocarcinoma of lung have been achieved for remarkable break-throughs, but not yet clear and definite on the definite molecular mechanism that adenocarcinoma of lung is shifted,
And there is no effective related molecular marker thing.
Chromosome condensation regulatory factor 2 (Regulator of Chromosome Condensation 2, RCC2) is dye
The main constitutive protein of colour solid passenger complex (CPC), spindle fits into participation mitosis and cytokinesis process
And adjust mitotic progression, recent report its also may participate in regulation adhesion molecule and cell migration campaign and then in interphase cell
Played a significant role in activity.
The present invention thinks that RCC2 gene magnifications and the rise of expression are shifted with adenocarcinoma of lung on the basis of early-stage Study and sent out
Raw and poor prognosis is closely related, can be applied to adenocarcinoma of lung as tumor marker and shift in the checkout and diagnosis occurred.
The content of the invention
The situation that the present invention lacks efficient diagnosis label for current clinical adenocarcinoma of lung and its transfer has there is provided one kind
Molecular marked compound --- (the Regulator of Chromosome of chromosome condensation regulatory factor 2 of potential adenocarcinoma of lung diagnosis
Condensation 2, RCC2), by detecting that RCC2 gene magnifications level and expression are transferred into so as to reach to adenocarcinoma of lung
Row early diagnosis and the purpose of prognosis evaluation.
In order to achieve the above object, present invention employs following technological means：
The present invention passes through RCC2 gene magnifications and mRNA expressions in the clinical adenocarcinoma of lung sample of TCGA database analysises.
As a result find, RCC2 gene magnifications level and RCC2 gene expression doses are in notable positive correlation in clinical adenocarcinoma of lung sample, and
And RCC2 mRNA expressions are significantly higher than cancer beside organism in clinical adenocarcinoma of lung sample.In order to further determine that RCC2 expression
Level and the relation of gland cancer grade malignancy, transfer generation and prognosis evaluation, the present invention is by Western Blot methods and is immunized
Groupization method have detected the adenocarcinoma of lung (T) and the egg of RCC2 in cancer side (N) tissue and pulmonary adenocarcinoma chip of 6 pairs of clinical pairings
White expression, as a result finds that RCC2 protein expression levels are significantly higher than cancer beside organism, and and lung in pairing adenocarcinoma of lung sample
Gland cancer invasive depth, lymphatic metastasis and clinical stages, are significantly correlated.Further, the present invention passes through Kaplan-Meier methods
(Log-rank inspections) analyzes the relation of the life cycle of RCC2 expressions and patients with lung adenocarcinoma, as a result shows that RCC2 expression is high
Patient's prognosis it is poor, and understood by Cox regression analyses, RCC2 can as judge the independent effect of patients with lung adenocarcinoma prognosis because
Son.
Therefore, on the basis of the studies above, the present invention proposes (the Regulator of of chromosome condensation regulatory factor 2
Chromosome Condensation 2, RCC2) as molecular marked compound prepare be used for adenocarcinoma of lung shift early diagnosis with
And the purposes in the reagent or kit of prognosis evaluation.
Further, prepared the invention allows for the reagent for detecting RCC2 gene magnifications level and expression
Purposes in the early diagnosis shifted for adenocarcinoma of lung and the reagent or kit of prognosis evaluation.
It is preferred that, the described reagent for being used to detecting RCC2 gene magnifications level and expression include from DNA level,
Rna level and the reagent of protein level detection.
Wherein, it is preferred that the reagent detected from rna level includes the primer being used for needed for RCC2PCR is detected, institute
Primer is stated to be made up of sense primer and anti-sense primer, in one particular embodiment of the present invention, the core of described sense primer
Nucleotide sequence is as shown in SEQ ID NO.1, and the nucleotide sequence of anti-sense primer is as shown in SEQ ID NO.2.
Certainly, all should be within protection scope of the present invention according to all primers designed by mankind's RCC2 gene orders.
Wherein, it is preferred that the reagent detected from protein level includes anti-RCC2 antibody.
There is copy number variation and differential expression in the present invention, and occur with transfer for RCC2 in clinical patients with lung adenocarcinoma
There is a situation where correlation with poor prognosis, by the detection to RCC2 gene magnifications level and mRNA, protein expression level, from
And play a part of prompting adenocarcinoma of lung grade malignancy, transfer and occur and prognosis evaluation.By RCC2 sequences and its associated antibodies application
Shifted in clinical adenocarcinoma of lung in diagnosis and prognosis evaluation, new early diagnosis and in advance can be provided for adenocarcinoma of lung and its transfer
Evaluation scheme, significant for the clinic diagnosis of adenocarcinoma of lung afterwards.
Brief description of the drawings
Fig. 1 is by RCC2 gene magnification levels in the clinical adenocarcinoma of lung sample of TCGA database analysises；
As a result it is in significantly just to be shown in RCC2 gene magnifications level and RCC2 gene expression doses in clinical adenocarcinoma of lung sample
It is related；
Fig. 2 is the mRNA expressions by RCC2 in the clinical adenocarcinoma of lung sample of TCGA database analysises；
As a result RCC2mRNA expressions in clinical adenocarcinoma of lung sample are shown in and are significantly higher than cancer beside organism；
Fig. 3 is that Western Blot detect the adenocarcinoma of lung (T) of 6 pairs of clinical pairings and RCC2 albumen tables in (N) tissue by cancer
Up to level；
As a result the protein expression level for being shown in RCC2 in pairing adenocarcinoma of lung sample is significantly higher than cancer beside organism；
Fig. 4 is the expression that ImmunohistochemistryMethods Methods detect RCC2 in clinical pulmonary adenocarcinoma sample pathological section；
Find that RCC2 albumen is expressed apparently higher than cancer beside organism in pulmonary adenocarcinoma through rank test analysis, and with lung gland
Cancer invasive depth, lymphatic metastasis and clinical stages, are significantly correlated；
Fig. 5 is the life cycle that Kaplan-Meier methods (Log-rank inspections) analyze patients with lung adenocarcinoma.
As a result show that RCC2 expresses high patient's prognosis poor (left：Organization chip data；In：TCGA data；It is right：GEO numbers
According to), and understand that RCC2 can be used as the independent effect factor for judging patients with lung adenocarcinoma prognosis by Cox regression analyses.
Embodiment
Following examples further illustrate present disclosure, but should not be construed as limiting the invention.Without departing substantially from
In the case of spirit and essence of the invention, the modification or replacement made to the inventive method, step or condition belong to the present invention
Scope.
Embodiment 1 passes through RCC2 gene magnification levels in the clinical adenocarcinoma of lung sample of TCGA database analysises
Pass through RCC2 gene magnifications and mRNA expressions in the clinical adenocarcinoma of lung sample of TCGA database analysises.As a result such as
Shown in Fig. 1 and Fig. 2, RCC2 gene magnifications level and RCC2 in clinical adenocarcinoma of lung sample are can be seen that from Fig. 1 and Fig. 2 results
Gene expression dose is in that the mRNA expressions of RCC2 in notable positive correlation, and clinical adenocarcinoma of lung sample are significantly higher than group by cancer
Knit.
The detection of the clinical sample of embodiment 2
1st, sample
The adenocarcinoma of lung (T) of 6 pairs of clinical pairings is cut with (N) tissue samples by cancer and clinical pulmonary adenocarcinoma sample pathology
Piece.
2nd, method
2.1. RCC2 gene magnification levels in pulmonary adenocarcinoma are detected.
2.1.1 in pulmonary adenocarcinoma DNA extraction.
(1) frozen tissue is taken, the rapid grind into powder under liquid nitrogen frozen.
(2) powder is put into 1.5ml centrifuge tubes, then adds buffer solution 750ul, fully mix.
(3) centrifuge tube is placed in 65 DEG C of water-baths 1-2 hours, it is gentle during water-bath to mix several times.
(4) centrifuge tube is taken out, often pipe adds isometric phenol-chloroform (V/V=1:1) solution 600-700ul, after mixing from
The heart, 10000g, 10min.
(5) supernatant is moved into another centrifuge tube, adds isometric chloroform, after mixing, 10000g, 6min.
(6) supernatant is moved into another centrifuge tube, adds the isopropanol of 0.6 times of volume, gently mix, when standing one section
Between, then tick DNA with pipette tips, and with 70% alcohol flushing 2 times.
(7) it is dissolved in after the DNA ticked, vacuum drying in 1 × TE of 500ul.
(8) 3ul RNase solution is added, 37 DEG C are incubated 1 hour.
(9) isometric phenol-chloroform solution is added, after gently mixing, 10000g centrifuges 6min.
(10) supernatant is taken, isometric chloroform is added, after gently mixing, 10000g centrifugations 6min.
(11) supernatant is taken, enters 1/10 volume 3M sodium acetates, after mixing, the cold absolute ethyl alcohol of 2 times of volumes is added.
(12) gently mix after standing a little while, DNA is ticked with pipette tips, after 70% alcohol flushing 2 times, vacuum drying.
(13) 20-50ul 1 × TE dissolving DNAs are added.
(14) DNA concentration is determined, sample is saved backup at 4 DEG C.
2.1.2 gene sequencing, detects RCC2 gene magnification levels.
2.2. the mRNA level in-site of RCC2 in pulmonary adenocarcinoma is detected.
2.2.1 Total RNAs extraction in pulmonary adenocarcinoma.
(1) take 2ml Eppendorf to manage, add 100mg frozen tissues, 100 μ l 0.5mm zirconium silicate pearls, 1ml
Trizol。
(2) it is placed in historrhexis's instrument, maximal rate crushes 5min.
(3) 0.2mL chloroforms are added, 30s is acutely vibrated, 3~5min is stood on ice.
(4) centrifuge, 4 DEG C, 12000g, 15min.
(5) upper strata aqueous phase is moved in new Eppendorf pipes, adds and the isometric isopropanol of supernatant, gently mix, room
Temperature places 10min.
(6) centrifuge, 4 DEG C, 12000g, 15min.
(7) supernatant, 75% ethanol washing RNA precipitate are abandoned.Centrifugation, 4 DEG C, 7 500g, 10min.
(8) appropriate DEPC water (30 μ l) is dissolved in after drying at room temperature RNA precipitate, 5~10min.
2.2.2RT-qPCR RCC2 expression is detected.
2.2.2.1 reverse transcription
According to concentration adjustment each group sample total serum IgE amount is determined, using the Transcriptor First Stand of Roche companies
CDNA System Kit Reverse Transcriptase kits, the method reverse transcription synthesis chains of cDNA first provided to specifications.
(1) cDNA reaction systems
(2) reaction condition
50℃60m in；85℃5min；16℃+∞.The chains of gained cDNA first survey concentration dilution to 50ng/ μ L, directly make
With or -20 DEG C save backup.
2.2.2.2 real-time quantitative PCR
Expanded with the cDNA of primer using the Transfected cells of amplifying target genes as masterplate, RCC2 primer sequences are such as
Under：
Sense primer：5′-TTCCTTTGGGTGCCCTGAA-3′(SEQ ID NO.1)
Anti-sense primer：5′-GGCAGAATCTGTCCATCTTTCG-3′(SEQ ID NO.2)
(1) reaction system
(2) reaction condition：
95℃6min；95 DEG C of 20s, Tm 20s, 72 DEG C of 20s, carry out the reaction of 45 circulations.
RCC2 protein expression situations in 2.3 detection pulmonary adenocarcinomas.
2.3.1 ImmunohistochemistryMethods Methods detect the expression of RCC2 in adenocarcinoma of lung cancerous tissue.
(1) prepared by histotomy：Each case drags for piece, 70 DEG C of bakings by 3-5 μm of section to precoat APES adhesiveness slides
Roasting 2h.
(2) dewaxing, aquation：38 DEG C of bakings are stayed overnight, the dewaxing of paraffin sections conventional xylene, step by step gradient (100%-80%)
Alcohol aquation.
(3) endogenous peroxydase is blocked：0.3%H202Solution is incubated at room temperature 30min.
(4) antigen retrieval：Citric acid repairs liquid Pressure method 10min.
(5) cooling is stored at room temperature, treats that temperature is identical with room temperature (about 45min), PBS is washed 3 times, each 5min.
(6) primary antibody is added：1% cow's serum antibody diluent dilutes primary antibody, the uniform tissue being added dropwise in paraffin section, 4 DEG C
Refrigerator overnight.
(7) 20min is stored at room temperature, PBS is washed 3 times, each 5min.
(8) add two step method detection reagent, be incubated at room temperature 1h.
(9) PBS is washed 3 times, each 5min.
(10) DAB develops the color, 30s or so (depending on staining conditions)；Haematoxylin redyes 20s.
(11) dehydration of alcohol gradient concentration, dimethylbenzene are transparent, neutral gum mounting.
(12) result stained positive judges：
Positive coloring degree (antigenic content)：Negative staining is designated as 0, and weakly positive is designated as 1, and the positive is designated as 2, and strong positive is designated as
3。
2.3.2Western Blot methods detect the expression of RCC2 in pulmonary adenocarcinoma.
2.3.2.1 quantification of protein.
(1) take 2ml Eppendorf to manage, add 300mg frozen tissues, 300 μ l 0.5mm zirconium silicate pearls, 600 μ l split
Solve liquid.
(2) it is placed in historrhexis's instrument, maximal rate crushes 10min.
(3) centrifuge, 4 DEG C, 13000r/min, 30min.
(4) supernatant is collected into new 1.5mL Eppendorf pipes, determines protein concentration, and with 50 μ L, often pipe is dispensed, and is protected
It is stored in standby in -80 DEG C of refrigerators.
2.3.2.2Western Blot
(1) the SDS-PAGE separation gels of preparation 10%
According to quickly with glue kit specification preparative separation glue.Separation gel buffer solution and each 4mL of separation sol solution is taken to mix
It is even, 80 μ L modified form ammonium persulfate solution is added, is mixed.Mixed liquor is injected in glue glass plate, appropriate anhydrous second is added
Alcohol is covered on separation gel.Upper strata absolute ethyl alcohol is removed after separation gel solidification, takes concentration glue buffer solution and concentration sol solution each
1.5mL is mixed, and the modified form ammonium persulfate solution for adding 30 μ L is mixed.Use or be placed in after solidification and be standby in 4 DEG C of humidity.
(2) sample is boiled
Different each 50ng of sample total protein are taken, 5 × sample-loading buffer of a quarter volume is added, 5min is boiled in heating,
It is immediately placed on ice, brief centrifugation, loading completely after cooling.
(3) SDS-PAGE electrophoresis
80V constant pressures electrophoresis after protein sample enters separation gel, uses 120V constant pressure electrophoresis 2h instead by spacer gel, separates
Albumen.
(4) transferring film
Gel is soaked into 30min in transfering buffering liquid.Shearing 1 and gel pvdf membrane of the same size, first with first
Alcohol soaks 30s, then is dipped in remove methanol, finally in transfering buffering liquid with deionized water immersion 3 to 5min and is soaked 20min.Together
When, transfer filter paper, foam pad and rotor are impregnated with completely in transfering buffering liquid.Assembling electrophoretic transfer is filled in certain sequence
Put：Anode → foam pad → 3 Whatman 3MM filter paper → pvdf membrane → gel → 3 Whatman3MM filter paper → foam pad
→ negative electrode, excludes bubble rear enclosed transfer device, 300mA, room temperature transfer 60-120min.
(5) close
After transferring film terminates, the pvdf membrane for carrying out mark is placed in hybridizing box, appropriate confining liquid (quick closure liquid 1 is added：
100 dilutions), room temperature shaker closes 2~4h.
(6) hybridize
TBS-T washes film 3 times, each 10min, adds 1:The primary antibody of 200 dilutions, 4 DEG C of hybridized overnights.TBS-T washes film 3 times,
Each 10min, then adds 1:5000 dilution secondary antibodies, room temperature hybridization 1h.TBS-T washes film 3 times, scanning imagery after each 10min.
3rd, result
RCC2 gene magnification levels in 3.1 detection pulmonary adenocarcinomas
It is sequenced by the DNA to (N) tissue by 6 pairs of clinical adenocarcinomas of lung (T) matched and cancer, as a result finds adenocarcinoma of lung
(T) the RCC2 gene magnification levels in tissue are significantly higher than (N) tissue by cancer.
RCC2 mRNA level in-site in 3.2 detection pulmonary adenocarcinomas
RT-qPCR inspections are carried out by the adenocarcinoma of lung (T) to 6 pairs of clinical pairings and the mRNA expressions of (N) tissue by cancer
Survey, as a result find that the mRNA expressions of the RCC2 genes in adenocarcinoma of lung (T) tissue are significantly higher than (N) tissue by cancer.
RCC2 protein expression situation in 3.3Western Blot methods detection pulmonary adenocarcinoma
Fig. 3 is that Western Blot detect the adenocarcinoma of lung (T) of 6 pairs of clinical pairings and RCC2 albumen tables in (N) tissue by cancer
Up to horizontal result.The RCC2 protein expression levels in pairing adenocarcinoma of lung sample are can be seen that from the result to be significantly higher than by cancer
Tissue.
RCC2 protein expression situation in 3.4 ImmunohistochemistryMethods Methods detection adenocarcinoma of lung cancerous tissue
Fig. 4 is the expression of results that ImmunohistochemistryMethods Methods detect RCC2 in clinical adenocarcinoma of lung sample pathological section, is examined through sum of ranks
Test analysis and find that RCC2 albumen is expressed apparently higher than cancer beside organism in pulmonary adenocarcinoma, and with adenocarcinoma of lung invasive depth, lymph
Carry down shifting and clinical stages it is significantly correlated.
3.5RCC2 expression and the relation of the life cycle of adenocarcinoma patients
Fig. 5 is the life cycle result that Kaplan-Meier methods (Log-rank inspections) analyze patients with lung adenocarcinoma, is as a result shown
It is poor (left that RCC2 expresses high patient's prognosis：Organization chip data；In：TCGA data；It is right：GEO data), and returned by Cox
Analysis is returned to understand that RCC2 can be used as the independent effect factor for judging patients with lung adenocarcinoma prognosis.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Harbin Medical University
<120>A kind of molecular marked compound related to adenocarcinoma of lung transfer and prognosis and its application
<130> KLPI170138
<160> 2
<170> PatentIn 3.5
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<400> 1
ttcctttggg tgccctgaa 19
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence
<400> 2
ggcagaatct gtccatcttt cg 22
Claims (6)
- Divide 1. chromosome condensation regulatory factor 2 (Regulator of Chromosome Condensation 2, RCC2) is used as Purposes of the sub- label in the reagent or kit of the early diagnosis shifted for adenocarcinoma of lung and prognosis evaluation is prepared.
- 2. preparing the early diagnosis shifted for adenocarcinoma of lung for the reagent for detecting RCC2 gene magnifications level and expression And the purposes in the reagent or kit of prognosis evaluation.
- 3. purposes as claimed in claim 2, it is characterised in that described to be used to detect RCC2 gene magnifications level and expression water Flat reagent includes the reagent detected from DNA level, rna level and protein level.
- 4. purposes as claimed in claim 3, it is characterised in that the reagent detected from rna level includes being used for Primer needed for RCC2PCR detections, the primer is made up of sense primer and anti-sense primer.
- 5. purposes as claimed in claim 4, it is characterised in that the nucleotide sequence of described sense primer such as SEQ ID NO.1 Shown, the nucleotide sequence of described anti-sense primer is as shown in SEQ ID NO.2.
- 6. purposes as claimed in claim 3, it is characterised in that the reagent detected from protein level includes the anti-of RCC2 Body.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710208864.6A CN106939340A (en) | 2017-03-31 | 2017-03-31 | A kind of molecular marked compound related to adenocarcinoma of lung transfer and prognosis and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710208864.6A CN106939340A (en) | 2017-03-31 | 2017-03-31 | A kind of molecular marked compound related to adenocarcinoma of lung transfer and prognosis and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106939340A true CN106939340A (en) | 2017-07-11 |
Family
ID=59463686
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710208864.6A Pending CN106939340A (en) | 2017-03-31 | 2017-03-31 | A kind of molecular marked compound related to adenocarcinoma of lung transfer and prognosis and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106939340A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112129938A (en) * | 2019-06-25 | 2020-12-25 | 中国科学院分子细胞科学卓越创新中心 | Application of UDP-Glc in lung cancer metastasis assessment |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604952A (en) * | 2012-03-16 | 2012-07-25 | 广州市锐博生物科技有限公司 | miRNA molecule and application thereof |
| WO2013022995A2 (en) * | 2011-08-08 | 2013-02-14 | Caris Life Sciences Luxembourg Holdings, S.A.R.L. | Biomarker compositions and methods |
| CN104789683A (en) * | 2015-05-01 | 2015-07-22 | 北京泱深生物信息技术有限公司 | Biomarker for diagnosis and treatment of cancer metastasis and application thereof |
| WO2015193738A2 (en) * | 2014-04-11 | 2015-12-23 | Oslo Universitetssykehus Hf | Methods and biomarkers for analysis of colorectal cancer |
| US20160312298A1 (en) * | 2013-12-20 | 2016-10-27 | The General Hospital Corporation | Methods and assays relating to circulating tumor cells |
| CN106282347A (en) * | 2016-08-17 | 2017-01-04 | 中南大学 | HoxC11 as biomarker preparation adenocarcinoma of lung pre-diagnostic reagent in application |
-
2017
- 2017-03-31 CN CN201710208864.6A patent/CN106939340A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013022995A2 (en) * | 2011-08-08 | 2013-02-14 | Caris Life Sciences Luxembourg Holdings, S.A.R.L. | Biomarker compositions and methods |
| CN102604952A (en) * | 2012-03-16 | 2012-07-25 | 广州市锐博生物科技有限公司 | miRNA molecule and application thereof |
| US20160312298A1 (en) * | 2013-12-20 | 2016-10-27 | The General Hospital Corporation | Methods and assays relating to circulating tumor cells |
| WO2015193738A2 (en) * | 2014-04-11 | 2015-12-23 | Oslo Universitetssykehus Hf | Methods and biomarkers for analysis of colorectal cancer |
| CN104789683A (en) * | 2015-05-01 | 2015-07-22 | 北京泱深生物信息技术有限公司 | Biomarker for diagnosis and treatment of cancer metastasis and application thereof |
| CN106282347A (en) * | 2016-08-17 | 2017-01-04 | 中南大学 | HoxC11 as biomarker preparation adenocarcinoma of lung pre-diagnostic reagent in application |
Non-Patent Citations (6)
| Title |
|---|
| CRISTIANAMOLLINARI等: "The Mammalian Passenger Protein TD-60 Is an RCC1 Family Member with an Essential Role in Prometaphase to Metaphase Progression", 《DEVELOPMENTAL CELL》 * |
| DINANA PAPINI等: "TD-60 links RalA GTPase function to the CPC in mitosis", 《NATURE COMMUNICATIONS》 * |
| HSU CH等: "Identification and Characterization of Potential Biomarkers by Quantitative Tissue Proteomics of Primary Lung Adenocarcinoma.", 《MOLECULAR & CELLULAR PROTEOMICS》 * |
| MYTHILI YENJERLA等: "TD-60 is required for interphase cell cycle", 《CELL CYCLE》 * |
| 周宝森等: "分子标记物在肺腺癌预后评估中的意义", 《中华流行病学杂志》 * |
| 第6卷: "Analysis of Microarray Data on Gene Expression and Methylation to Identify Long Non-coding RNAs in Non-small Cell Lung Cancer", 《SCIENTIFIC REPORTS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112129938A (en) * | 2019-06-25 | 2020-12-25 | 中国科学院分子细胞科学卓越创新中心 | Application of UDP-Glc in lung cancer metastasis assessment |
| CN112129938B (en) * | 2019-06-25 | 2023-08-18 | 中国科学院分子细胞科学卓越创新中心 | Use of UDP-Glc in lung cancer metastasis assessment |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107099584A (en) | A kind of molecular marked compound related to colorectal cancer transfer and prognosis and its application | |
| CN105219867B (en) | MiRNA biomarker and detection kit for diagnosing gastric cancer | |
| CN102876676B (en) | Blood serum/blood plasma micro ribonucleic acid (miRNA) marker relevant with pancreatic cancer and application thereof | |
| CN105176983A (en) | Kit for detecting esophageal squamous carcinoma associated serum miRNAs genes | |
| CA3077746A1 (en) | Biomarkers useful for detection of types, grades and stages of human breast cancer | |
| CN107312852A (en) | Myocardial infarction diagnosis mark compositions | |
| CN110408703B (en) | Colorectal cancer miRNA marker and application thereof | |
| Rao et al. | Identification of plasma exosomes long non-coding RNA HAGLR and circulating tumor cells as potential prognosis biomarkers in non-small cell lung cancer | |
| ZONG et al. | Expression and prognostic significance of long noncoding RNA AK001796 in esophageal squamous cell carcinoma. | |
| CN102839179B (en) | MicroRNA marker for identifying subtypes of lung cancer and application of microRNA marker | |
| CN105441565A (en) | miRNA regarding as osteosarcoma treatment target | |
| CN105154541A (en) | Application of miRNA to diagnosis and treatment of acute myelogenous leukemia | |
| CN106939340A (en) | A kind of molecular marked compound related to adenocarcinoma of lung transfer and prognosis and its application | |
| CN107475441A (en) | It is a kind of to predict reactive biomarker of the patient with breast cancer to AT scheme new adjuvant chemotherapies | |
| CN108721316B (en) | Application of marker miR-652-5p in medicines and kits for metastasis, prognosis and treatment of esophageal squamous carcinoma | |
| CN106191251A (en) | A kind of miRNAs heart failure mark and application thereof and heart failure primary dcreening operation detection kit | |
| CN106636334A (en) | MicroRNA marker group and applications thereof in preparing kit for detecting lymph node metastasis of gastric carcinoma | |
| CN107299129A (en) | Circle nucleic acid as breast cancer biomarker application | |
| CN105779640A (en) | miRNA biomarker and detection kit used for renal cancer diagnosis | |
| CN110257514A (en) | A kind of new cancer of the esophagus blood miRNA marker and its application | |
| CN111808966B (en) | Application of miRNA in diagnosis of breast cancer disease risk | |
| CN112430663B (en) | Biomarker for diagnosis and treatment of bladder cancer and application thereof | |
| CN103948941A (en) | Application of miR-146a-5p in non-small cell lung cancer cell lines | |
| CN108125976B (en) | Molecular marker miR-4270 for predicting lung cancer brain metastasis and application thereof in medicines and diagnostic kits | |
| CN102021169A (en) | Serum/plasma miRNA composition and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170711 |
|
| RJ01 | Rejection of invention patent application after publication |