CN110257521A - The application of miRNA-30a and its target gene in lung cancer detection - Google Patents
The application of miRNA-30a and its target gene in lung cancer detection Download PDFInfo
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Abstract
The present invention relates to the application of miR-30a and its target gene in detection lung cancer.Lung cancer is the most common malignant tumour within the scope of our times, although clinically also applying some markers in diagnosis lung cancer, but the sensibility and specificity of detection is limited, and finding the relevant tumor marker of novel pulmonary cancer diagnosis or new mark compositions seems most important for the early diagnosis of lung cancer.Inventor analyzes with high-flux sequence and data, screens good lung cancer marker, provides new lung cancer gene diagnosis target spot for clinic.
Description
Technical field
The present invention relates to molecular diagnosis fields, and in particular to the application of miR-30a and its target gene in detection lung cancer.
Background technique
It is about the non-coding microRNA of 22 nucleotide that miRNA, which is a kind of endogenous, highly conserved, length,.
MiRNA can be specifically bound by its end 5' with the end the 3' non-translational region (3'UTR) of target gene, played and adjusted in post-transcriptional level
Control effect.It can cause the degradation of said target mrna when miRNA and target gene 3'UTR are combined in a manner of the pairing of base complete complementary, and
The protein translation of mRNA can then be inhibited in such a way that non-fully complementary pairing combines.
Lung cancer is the first cause of the most common malignant tumour and cancer mortality within the scope of our times, great threat
The health and lives of the mankind.Between Past 30 Years, the death rate of the lung cancer in China is risen year by year, lung cancer lethal patient about 40 every year
Ten thousand, become China's increasing degree maximum, harm malignant tumour the most serious.Although in recent years in method of lung cancer diagnosis, operation
There is new development in terms of technology and chemotherapeutics, but the 5 years survival rates of patients with lung cancer always are still very low, only 15% is left
It is right.Why so low the overall 5 years survival rates of lung cancer are, be primarily due to most patients with lung cancer it is medical when oneself be in advanced stage, or relatively early
It occurs as soon as and sends out transfer and lose surgical radical treatment chance.Currently used method of lung cancer diagnosis includes chest x-ray, low dosage spiral
Brushing piece or biopsy, BALF cytology inspection are taken etc. under CT, expectorative cytology inspection, bronchoscope, but various
Detection methods sensibility, specificity, in terms of exist limitation.Although clinically also applying blood serum tumor markers
Diagnosing.Common marker includes carcinomebryonic antigen (CEA), cytokeratin 21-1 segment (CYFRA21-1), sugar antigen
125,153,199 (CA125, CA153, CA199), tissue polypeptide antigen (TPA) etc..But the sensitivity of these tumor-marker analyte detections
Property and specificity it is limited, find the control of the relevant tumor marker of novel pulmonary cancer diagnosis or new therapeutic strategy for lung cancer
Seem most important.
The present invention is based on high-flux sequence method, the survey of two generations is carried out to the cancerous tissue and cancer beside organism of 3 patients with lung adenocarcinoma
Sequence obtains the expression data of its miRNA and mRNA, meanwhile, in conjunction with bioinformatic analysis and literature survey, from candidate's
MiR-30a and its target gene EFNA3, CTHRC1, RTKN2, JAM2 are picked out in miRNA, carry out subsequent analysis, as the result is shown
The target gene combination of miR-30a can be very good diagnosing, and the present invention provides new lung cancer gene diagnosis marker for clinic.
Summary of the invention
The purpose of the present invention is to provide a kind of pulmonary cancer diagnosis reagent, the pulmonary cancer diagnosis reagent is able to detect in sample
The transcription of the target gene of miR-30a or expression, target gene are selected from down in the transcription situation of miR-30a, or detection sample
One or more of column gene: EFNA3, CTHRC1, RTKN2 or JAM2.
The miR-30a sequence is SEQ ID NO 1:5 '-uguaaacauccucgacuggaag-3 '.
Further, lung cancer is non-small cell lung cancer, and non-small cell lung cancer includes lung squamous cancer, adenocarcinoma of lung, lung adenosquamous carcinoma, big thin
Born of the same parents' cancer etc.；Preferably, lung cancer is adenocarcinoma of lung.
The sample is tumor tissues or peripheral blood.
Preferably, the combination of two of target gene EFNA3, CTHRC1, RTKN2 or JAM2 gene；It is furthermore preferred that target gene
For the combination of the combination of the combination of gene RTKN2 and EFNA3, RTKN2 and JAM2, EFNA3 and JAM2 or EFNA3 and CTHRC1
Combination.
Preferably, three or three combinations of target gene EFNA3, CTHRC1, RTKN2 or JAM2 gene；It is furthermore preferred that target gene
For the combination of gene RTKN2, EFNA3 and CTHRC1.
Further, pulmonary cancer diagnosis reagent is using high-flux sequence method and/or the hybridization of quantifying PCR method and/or probe
Method detects the transcription of miR-30a or its target gene in sample.
It is preferred that using two generation PCR sequencing PCRs, spectrum chip method, northern hybridizing method, ribozyme protection analytical technology, RAKE method,
The transcription of miR-30a or its target gene in situ hybridization, fluorescence quantitative PCR method detection sample.
Preferably, the quantifying PCR method includes specific amplification miR-30a or the primer of its target gene；Described
It include the probe of the nucleic acid array hybridizing with miR-30a or its target gene based on probing procedure.
Further, pulmonary cancer diagnosis reagent is the expression using the target gene of miR-30a in immunization method detection sample
Reagent.
It is preferred that the expression of the target gene of miR-30a is western blot in the immunologic detection method detection sample
And/or ELISA and/colloidal gold detection method.
Further, the ELISA method for detecting the expression of target gene EFNA3, CTHRC1, RTKN2 or JAM2 is to be examined using ELISA
Test agent box.The monoclonal antibody of commercially available EFNA3, CTHRC1, RTKN2 or JAM2 can be used in antibody in the kit.
Further, the kit includes: the solid phase carrier for being coated with the monoclonal antibody of EFNA3, CTHRC1, RTKN2 or JAM2, enzyme
Mark secondary antibody, the substrate of enzyme, protein standard substance, negative controls, dilution, cleaning solution, enzyme reaction terminate liquid etc..
Further, the colloidal gold method of detection EFNA3, CTHRC1, RTKN2 or JAM2 albumen is to use detection reagent
Commercially available EFNA3, CTHRC1, RTKN2 or JAM2 monoclonal antibody can be used in box, the antibody.Further, the colloid
Golden detection kit uses colloidal gold immunochromatographimethod technology or colloidal gold percolation.Further, the gold-immunochromatographyreagent reagent for assay
Detection zone (T) specking on box nitrocellulose filter has anti-EFNA3, CTHRC1, RTKN2 or JAM2 monoclonal antibody, quality control region
(C) specking has Immunoglobulin IgG.
The object of the invention is also to provide above-mentioned pulmonary cancer diagnosis reagents to prepare the application in pulmonary cancer diagnosis preparation.
Definition:
The method for detecting the expression of miRNA at this stage mainly includes based on high throughput sequencing technologies, based on nucleotide
The miRNA detection method of hybridization and based on PCR.MiRNA detection method based on probe hybridization technique is a kind of direct Detection Method,
It does not need to expand sample rna in advance, including northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analysis skill
The technologies such as art, RAKE method, in situ hybridization, bead-based flow-cytometry.
(1) Northern hybridizes
Also known as Northern blot is most classic detection eucaryote RNA size, estimates the experimental method of its abundance.Base
Present principles are as follows: fixing miRNA sample on carrier (such as silicon wafer, microballoon or film) first, then miscellaneous with the probe by label
It hands over, carries out signal detection after washing extra hybridization probe；It can also be first fixed complementary with target miRNA sequence on carrier
Then DNA probe hybridizes with the sample miRNA by label, then carries out signal detection.The method of signal label includes isotope
Label, fluorescent marker and nano gold mark etc..
(2) miRNA chip of expression spectrum
Principle is equally using the target molecule on label probe detection solid support.Pass through miRNA in design chips
Gene and internalcontrol sequence, can Accurate Analysis go out the expression of corresponding miRNA in sample.Genetic chip has the excellent of high throughput
Point can once detect whole expression of several hundred a genes in same sample.The liquid-phase chip that Luminex company develops
(Liquid chip) is also known as multifunctional suspending dot matrix (Multi analyte suspension array, MASA), is out new
Generation biochip technology.Liquid-phase chip system is made of many spherulas for main matrix, is fixed with not on every kind of spherula
With probe molecule, in order to distinguish different probes, each for label probe sphere matrix all have one it is unique
Color numbers constitute liquid-phase chip system as soon as these spherulas are suspended in a liquid-phase system.The system can be to same
Multiple and different molecules in one trace sample carry out quick qualitative and quantitative analysis simultaneously, and this detection technique is referred to as
FMAP (Flexible multianalyte profiling) technology.Molecule hybridization carries out in aaerosol solution, detects speed pole
Fastly.
(3) ribozyme protection analytical technology (RPA)
The detection of miRNA can also protect analytical technology using ribozyme, and the probe marked and RNA sample to be measured are mixed
Close, hybridize after thermal denaturation, non-hybridized RNA and the single-chain nucleic acid enzymic digestion of extra probe, after heat inactivation nuclease purifying by
Probe, colour developing is separated by electrophoresis finally by denaturation PAGE in the RNA molecule of protection.This new method based on solution hybridization is simple
Quickly, high sensitivity, but be also only used for analyzing known miRNA.
(4) RAKE method
RAKE method (RNA primed array based Klenow emzyme) is the base in miRNA microarray
The Klenow segment of DNA polymerase i, the method for hybridizing miRNA with fixed DNA probe are utilized on plinth.RAKE can be sensitive
MiRNA is specifically detected, suitable for largely quickly screening all known miRNA.It can be in specific cell and tumour
Detect miRNA express spectra situation.Moreover, RAKE method can also be from the tissue of the paraffin embedding secured by formalin
It isolates miRNA and analyzes it, to analyze the door that miRNA opens hope from archive sample.
(5) in situ hybridization (in situ hybridization)
Hybridization in situ technique can intuitively understand miRNA expression way, be a kind of easier of observation miRNA spatial and temporal expression
Method, normal mark mode include digoxin, biotin, fluorescent marker etc..In situ hybridization (Locked on the basis of locked nucleic acid
Nucleic Acid (LNA) based in situ hybridization (LNA-ISH)) it is the more probe side of current application
Formula.
(6) bead-based flow-cytometry (bead-based flow cytometry)
It is a kind of liquid-phase chip technology, FCM analysis is organically combined with chip technology, had concurrently by this method
The features such as flux is big, detection speed is fast, high sensitivity and specificity are good.
(7) Real-Time Fluorescent Quantitative PCR Technique (Real-time PCR, RT-PCR)
Fluorescence detection PCR instrument can be to the cumulative speed drafting dynamic changing curve of extension increasing sequence during entire PCR.Anti-
Answer the initial concentration of target sequence in mixed system bigger, it is desirable that the PCR cycle number for obtaining amplified production specific output is (general to use
Specific threshold recurring number Ct is expressed) it is fewer.Since miRNA length is only 22nt, it is such that traditional qRT-PCR is not suitable for amplification
Short segment.There are several real time quantitative PCR methods for miRNA, such as tailing method, neck ring method now.Neck ring method is a kind of
Ideal miRNA detects qRT-PCR method: special loop-stem structure primer is designed first, using miRNA to be measured as template reverse transcription
The first chain of cDNA is synthesized, which is stem Loop primer, and stem loop structure, which is opened, substantially increases the length of cDNA, then
Real-time quantitative PCR amplification is carried out by template design primer of the cDNA of synthesis.QRT-PCR has specificity height, sensitivity good, fast
A variety of advantages such as fast simple.
(8) PCR sequencing PCR
MiRNA known to major part is that discovery and identification is sequenced by cDNA clone.The method needs first to construct miRNA
CDNA library, then carry out PCR amplification, amplified production is then cloned on expression vector and is sequenced.Takada develops one kind and changes
Into amplification PCR cloning PCR (miRNA amplification profiling, mRAP), mRAP method first connects at the 3 ' ends of miRNA
Connector, then with the reverse transcription primer reverse transcription complementary with connector.Because specific reverse transcriptase has end deoxynucleotide
Enzymatic activity, some nucleotide (mainly deoxycytidylic acid) can be connected to 3 ' ends of the cDNA chain that reverse transcription goes out.When 5 ' terminations
For head with after poly (C) the cohesive end annealing of cDNA chain, a pair of of general primer, which is added, can be realized the PCR amplification to cDNA.By
It, can be directly with the expression quantity of miRNA in clone and a small amount of tissue of sequencing technologies detection in mRAP High sensitivity.Sequence label gram
Grand method is a kind of having developed the higher miRAGE of detection efficiency on the basis of serial analysis of gene expression (SAGE) technology
(miRNA SAGE) PCR cloning PCR, the method sub-series big by generation, can detect multiple miRNA by single sequencing reaction, bright
It is aobvious to improve detection efficiency.
High-flux sequence (High-throughput sequencing) is also known as next-generation sequencing technologies
(nextgeneration sequencing) is the change to tradition sequencing revolution, once to hundreds of thousands to millions of
DNA molecular carries out sequencing, greatly improves sequencing efficiency.This kind of large scale sequencing technology greatly improves multiple objects
The solution reading rate of kind hereditary information, for the sequence information for obtaining all miRNA, decryption miRNA map provides guarantee.It is high simultaneously
Flux sequencing to carry out the analysis of careful overall picture to the transcript profile and genome of species, so being otherwise known as
Deep sequencing (deep sequencing).The representative of high-flux sequence platform is 454 sequenators of Roche Holding Ag (Roche)
(Roch GSFLX sequencer), Solexa genome analysis instrument (the Illumina Genome of Illumina company
) and the SOLiD sequenator of ABI (ABI SOLiD sequencer) Analyzer.
Known antigen or antibody are adsorbed on surface of solid phase carriers by enzyme-linked immunosorbent assay (ELISA), make enzyme mark
The technology that the antigen-antibody reaction of note is carried out in solid phase surface.ELISA detection kit can according to testing goal and operating procedure
It is divided into indirect method, double-antibody method, competition law, double site one-step method, prize law and surveys IgM antibody, using Avidin and biotin
ELISA.Horseradish peroxidase (HRP) or alkaline phosphatase (AP) may be selected in chromogenic substrate in ELISA detection kit.
Common immune colloid gold detection technique: (1) immune colloid gold light microscopic decoration method cell suspension smear or peripheral blood
Slice, can be dyed with the antibody of colloid gold label, can also be enhanced with silver-colored developer solution and be marked on the basis of colloid gold label
Note, makes the silver atoms being reduced be deposited on marked gold particle surface, the sensibility of colloid gold label can be remarkably reinforced.(2)
Immune colloid gold electronic speculum decoration method can with the antibody of colloid gold label or antiantibody with negative staining Virus Sample or peripheral blood are ultra-thin cuts
Piece combines, and then carries out negative staining.(3) dot immunogold filtration assay application miillpore filter makees carrier, first by antigen or antibody point in
On film, sample to be examined is added after closing, the corresponding antigen of antibody test or antibody of colloid gold label are used after washing.(4) colloidal gold
The antigen of specificity or antibody are fixed on film, colloid gold label reagent (antibody or monoclonal by immunochromatographic method with ribbon
Antibody) it is adsorbed on bonding pad, after sample to be examined is added in the sample pad of test strips one end, through capillary action to Forward
It is dynamic, it reacts to each other after dissolving the colloid gold label reagent on bonding pad, when being moved to the region of fixed antigen or antibody, to
The conjugate of inspection object and gold marked reagent occurs specific binding therewith again and is trapped, and being gathered in detection takes, and can pass through naked eyes
Observe colour developing result.The method has developed into diagnosis test paper, and use is very convenient.
Detailed description of the invention
Fig. 1 is the ROC curve figure of the target gene combination of two diagnosis adenocarcinoma of lung of miR-30a
Fig. 2 is the ROC curve figure of three or the three combined diagnosis adenocarcinoma of lung of target gene of miR-30a
Fig. 3 is the ROC curve figure of the target gene Combining diagnosis adenocarcinoma of lung of miR-30a
Specific embodiment
The extraction of 1 sample collection of embodiment and total serum IgE
3 pulmonary adenocarcinomas and cancer beside organism are all from the sample of hospital surgical excision, and all samples are in vitro 10 points
It is put into liquid nitrogen container within clock, is subsequently transferred to store in -80 DEG C of refrigerators.
1 extracting method:
1) 80mg tissue block is taken, 800 μ l Lysis/Binding buffers are added, tissue block is carried out using homogenizer even
Slurry.Sample will be placed in and keep low-temperature condition on ice during homogenate.
2) 1/10 volume Homogenate Additive is added into the above-mentioned tissue sample being homogenized, and is put on ice
Set 10min.
3) be added with the water-saturated phenols of Lysis/Binding buffer equivalent volumes, shake 45s, 10000 × g room temperature from
Heart 5min.
4) supernatant is carefully taken out into new test tube, and the dehydrated alcohol of 1.25 times of volumes is added, after mixing, moves into purification column
In, 10,000 × g is centrifuged 15s, outwells the liquid in collecting pipe.Since the maximum volume of pillar only has 700 μ l, again
This multiple step operation, until all supernatants all filter completion.
5) 700 μ l miRNA eluents 1, room temperature are added into centrifugation pillar, 10000 × g is centrifuged 15s, outwells collection
Liquid uses new collecting pipe instead.
6) the step for being added in centrifugal column with 500 μ l eluents 2/3,10000 × g again, being centrifuged 10s, repeat is primary.
7) it is centrifuged 1min, 10000 × g, discards extra liquid.
8) aforesaid liquid is transferred to new centrifuge tube, adds DEPC processing 30s, the 10000 × g of 95 DEG C of 100 μ l preheatings,
Centrifugation.
9) using the ratio of nanodrop measurement RN A concentration and 260nm/280nm.
10) RNA obtained is stored in -80 DEG C of refrigerators.
2 extraction standards:
Measure the ratio of RNA concentration and 260nm/280nm: the purity requirement of total serum IgE is that OD260/OD280 value should be 1.8
To between 2.2；The detection of RNA integrality: with the integrality of 1% agarose gel electrophoresis detection RNA；
According to the requirement of sequencing company, 3 μ g of total amount or more is sequenced in tiny RNA, and concentration is more than 300ng/ μ l.
The sequencing of embodiment 2 and data analysis
The foundation and the sequencing of upper machine of sequencing library are carried out by sequencing company, used sequenator is Illumina company
HiSeq2000 sequenator.
Statistical analysis, FDR<0.001, log2 (FC) absolute value>1, two group are carried out according to the data that sequencing company provides
The difference of count average value is greater than 100.It is to select the apparent miRNA of differential expression in filtering to differential expression miRNA people, with lung
The extraordinary molecular marker miR-30a of cancer correlation enters our research range, and miR-30a is in adenocarcinoma of lung cancerous tissue
Expression quantity is higher than cancer beside organism.
Identification miRNA target gene is the extremely important step for studying specific organization and cell miRNA function.Using including
These algorithm forecasted variances of RNA22, miRanda, miRDB, miRWalk, PICTAR2 and Targetscan express miR-30a's
Target gene, in conjunction in sequencing mRNA differential expression as a result, picking effect more from the differential expression mRNA of target gene
Two cance high-expression genes EFNA3, CTHRC1 outstanding, two low expressions gene RTKN2, JAM2 carry out subsequent experiment.
The evaluation analysis of 3 diagnostic of embodiment
Method for the efficiency evaluation of individual molecule or diagnostic model is to establish Receiver Operating Characteristics (receiver
Operating characteristic, ROC) curve, judge to examine by area under calculated curve (Area UnderCurve)
Disconnected ability.Area value under ROC curve is between 1.0 and 0.5, and in the case where AUC > 0.5, AUC is closer to 1, explanation
Diagnosis effect is better, and AUC has lower accuracy in 0.5-0.7, and AUC has certain accuracy in 0.7-0.9, AUC 0.9 with
There is high accuracy when upper.
We download the data in TCGA database in relation to adenocarcinoma of lung, obtain data set (533 adenocarcinoma of lung cancerous tissues
MRNA data, the mRNA data of 59 cancer beside organisms), and then it is analyzed, the results show that RTKN2+EFNA3 when combination of two,
The AUC value of RTKN2+JAM2, RTKN2+CTHRC1, EFNA3+JAM2, EFNA3+CTHRC1, JAM2+CTHRC1 diagnosis adenocarcinoma of lung
Respectively 0.981,0.988,0.845,0.923,0.975,0.607 (see Fig. 1), three gene associations diagnose in adenocarcinomas of lung
RTKN2+EFNA3+JAM2, RTKN2+EFNA3+CTHRC1, EFNA3+JAM2+CTHRC1 combine AUC be 0.605,0.993,
0.647 (see Fig. 2), in four gene associations diagnosis adenocarcinomas of lung RTKN2+EFNA3+JAM2+CTHRC1 combination AUC be 0.871 (see
Fig. 3), diagnosis effect is preferably RTKN2+EFNA3, RTKN2+JAM2, EFNA3+JAM2, EFNA3+ in above-mentioned combination of two
CTHRC1, diagnosis effect preferably RTKN2+EFNA3+CTHRC1 in three or three combinations, but with the increase of gene diagnose it is quasi-
True rate is declined, and does not recommend the combination of four genes for diagnosing, and the application, which explores to find, can be used for clinical adenocarcinoma of lung diagnosis
In accessory molecule diagnosis assortment of genes object, have good potential applicability in clinical practice.
The ROC analysis of 1 difference expression gene of table combination
Specific embodiments above, the present invention is further explained, for explaining only the invention, and should not be understood as to the present invention
Limitation.It will be understood by those skilled in the art that: it without departing from the principle and spirit of the present invention can be right
These embodiments carry out a variety of change, modification, replacement and modification, and the scope of the present invention is defined by the claims and their equivalents.
Claims (10)
1. a kind of pulmonary cancer diagnosis reagent, the pulmonary cancer diagnosis reagent is able to detect the transcription situation of miR-30a in sample, Huo Zhejian
The transcription of the target gene of miR-30a or expression in test sample sheet, target gene are selected from one or more of following gene:
EFNA3, CTHRC1, RTKN2 or JAM2.
2. pulmonary cancer diagnosis reagent according to claim 1, which is characterized in that target gene EFNA3, CTHRC1, RTKN2 or
The combination of two of JAM2 gene；Preferably, target gene be the combination of gene RTKN2 and EFNA3, the combination of RTKN2 and JAM2,
The combination of EFNA3 and JAM2 or the combination of EFNA3 and CTHRC1.
3. pulmonary cancer diagnosis reagent according to claim 1, which is characterized in that target gene EFNA3, CTHRC1, RTKN2 or
Three or three combinations of JAM2 gene；It is furthermore preferred that target gene is the combination of gene RTKN2, EFNA3 and CTHRC1.
4. pulmonary cancer diagnosis reagent according to claim 1 to 3, which is characterized in that pulmonary cancer diagnosis reagent is to use
MiR-30a or its target gene in high-flux sequence method and/or quantifying PCR method and/or probing procedure detection sample
Transcription.
5. pulmonary cancer diagnosis reagent according to claim 1 to 3, which is characterized in that using two generation PCR sequencing PCRs, spectrum
Chip method, northern hybridizing method, ribozyme protection analytical technology, RAKE method, in situ hybridization, fluorescence quantitative PCR method detect sample
The transcription of miR-30a or its target gene in this.
6. pulmonary cancer diagnosis reagent according to claim 4, which is characterized in that the quantifying PCR method includes specificity
Expand miR-30a or the primer of its target gene；Described based on probing procedure includes core with miR-30a or its target gene
The probe of acid sequence hybridization.
7. pulmonary cancer diagnosis reagent according to claim 1 to 3, which is characterized in that pulmonary cancer diagnosis reagent is to use
Immunization method detects the reagent of the expression of the target gene of miR-30a in sample.
8. pulmonary cancer diagnosis reagent according to claim 7, which is characterized in that immunologic detection method be westernblot and/
Or ELISA and/colloidal gold detection method.
9. pulmonary cancer diagnosis reagent according to claim 1, which is characterized in that sample is tumor tissues or peripheral blood.
10. pulmonary cancer diagnosis reagent described in any one of claim 1-9 is preparing the application in pulmonary cancer diagnosis preparation.
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