CN102851282A - MicroRNA markers for discriminating constitutional lung cancer tissue and paracancerous tissue - Google Patents
MicroRNA markers for discriminating constitutional lung cancer tissue and paracancerous tissue Download PDFInfo
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Abstract
The invention relates to microRNA makers for discriminating constitutional lung cancer tissue and paracancerous tissue. More specifically, the invention relates to 38 microRNA markers for discriminating constitutional lung cancer tissue and paracancerous tissue. It has been proved by examination that, these specific microRNA markers can effectively discriminate constitutional lung cancer tissue and paracancerous tissue. The invention also relates to a chip and a test kit for detecting the microRNA markers.
Description
Technical field
The present invention relates to biological technical field, more specifically, the present invention relates to a class and can be used for microRNA mark of distinguishing primary lung cancer and cancer beside organism and uses thereof.The invention still further relates to the chip and the test kit that detect described microRNA mark.
Background technology
Lung cancer is one of modal malignant tumour of China, and grade malignancy is high, and development is treated difficulty rapidly, and case fatality rate is high.Therefore, the as early as possible diagnosis of lung cancer just more seems urgent.
The genesis of anything all is under the internal and external reasons acting in conjunction, and is interior because main, outer because auxiliary.Gene is as the hereditary medium of life, organism sick, old,, be in the internal cause status on basis in dead.Overwhelming majority gene is by transcribing the product nucleus ribosomal ribonucleic acid, and translation generates protein and brings into play biological function again.
MicroRNA (miR or miRNA, Microrna) is that a class extensively exists the single stranded RNA molecule of the about 18-26 of a length base in more high most eukaryotes.It can combine with target site on some mRNA specifically by basepairing rule, causes that said target mrna degraded or translation suppress, and then at post-transcriptional level target gene is regulated and control.
MicroRNA derives from the initial transcription product of long-chain RNA (Pri-miRNA) of the about 1000bp of length, and the Pri-miRNA molecule is sheared the miRNA precursor (Pre-miRNA) with loop-stem structure that forms the about 60-80nt of length through the Drosha enzyme in nucleus.After Pre-miRNA is transported to kytoplasm, further cut into the double-stranded miRNA that is about 22nt by the Dicer enzyme.After double-stranded miRNA untied, ripe miRNA entered the reticent mixture (RNA-induced silencing complex, RISC) of RNA induced gene, with complementary mRNA fully or incomplete pairing, degraded said target mrna or check its expression.
Although microRNA shared proportion in cell total rna is very little, but because it can produce regulating and controlling effect to all mRNA with target site efficiently, microRNA still can't neglect in the growth of organism and even generation, the evolution role of tumour.
Yet, up to now, this area is understood very few for the microRNA relevant with tumour (such as lung cancer), so this area is in the urgent need to separating further various microRNA, especially with generation, transfer, the recurrence of tumour or detect relevant microRNA.
Summary of the invention
Purpose of the present invention just provide a class new, can be used for microRNA mark of distinguishing primary lung cancer cancerous tissue and cancer beside organism and uses thereof.
Another object of the present invention provides chip and the test kit that detects described microRNA mark.
In a first aspect of the present invention, a kind of miRNA of separation is provided, described miRNA is selected from:
(i) miRNA of sequence shown in SEQ ID NO:38, wherein n is the positive integer that is selected from 1-38; Or
(ii) with the miRNA of sequence complementation shown in the SEQ ID NO:n.
In another preference, described miRNA separates from the people.
In a second aspect of the present invention, a kind of miRNA collection (set) or combination (combination) are provided, described collection or combination are made of 38 kinds of miRNA of sequence shown in SEQ ID NO:1-38.
In a third aspect of the present invention, precursor miRNA a kind of separation or artificial constructed is provided, the miRNA described in the first aspect present invention can be sheared and be expressed as to described precursor miRNA in people's cell.
In a fourth aspect of the present invention, a kind of polynucleotide of separation are provided, described polynucleotide can be become precursor miRNA by people's cell transcription, and described precursor miRNA can be sheared and be expressed as the miRNA described in the first aspect present invention in people's cell.
In another preference, described polynucleotide have the structure shown in the formula I:
Seq
Forward-X-Seq
OppositelyFormula I,
Among the formula I,
Seq
ForwardFor becoming at people's cells the nucleotide sequence of described miRNA;
Seq
OppositelyFor with Seq
ForwardBasically the nucleotide sequence of complementation or complete complementary;
X is for being positioned at Seq
ForwardAnd Seq
OppositelyBetween intervening sequence, and described intervening sequence and Seq
ForwardAnd Seq
OppositelyNot complementary;
And the structure shown in the formula I forms the secondary structure shown in the formula II after changing people's cell over to:
Among the formula II, Seq
Forward, Seq
OppositelyWith stating as defined above of X,
|| be illustrated in Seq
ForwardAnd Seq
OppositelyBetween the base complementrity pair relationhip that forms.
In a fifth aspect of the present invention, a kind of carrier is provided, it contains the miRNA described in the first aspect present invention, or the polynucleotide described in the fourth aspect.
In a sixth aspect of the present invention, provide the purposes of the miRNA described in the first aspect present invention, for the preparation of chip or the test kit of distinguishing primary lung cancer cancerous tissue and cancer beside organism.
In a seventh aspect of the present invention, a kind of miRNA chip is provided, described miRNA chip comprises:
Solid phase carrier; And
Be fixed in order the oligonucleotide probe on the described solid phase carrier, described oligonucleotide probe is specifically corresponding to the part or all of sequence shown in the SEQ ID NO:1-38 (such as 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38 kind).
In another preference, described oligonucleotide probe contains:
Complementary land; And/or
The joining region that links to each other with solid phase carrier.
In a eighth aspect of the present invention, provide the purposes of above-mentioned miRNA chip, for the preparation of the test kit of distinguishing primary lung cancer cancerous tissue and cancer beside organism.
In a ninth aspect of the present invention, a kind of test kit is provided, contain the present invention's miRNA chip described above in the described test kit.
Other side of the present invention is because the disclosure of this paper is apparent to those skilled in the art.In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and can making up mutually between specifically described each technical characterictic in below (eg embodiment), thus consist of new or preferred technical scheme.As space is limited, this tired stating no longer one by one.
Description of drawings
Fig. 1 shows with the scatter diagram of sample (dark color) in three-dimensional space by cancer sample (light color) and the cancer that have after the conversion of MDS algorithm.
Embodiment
The inventor by the cancerous tissue sample of detection primary lung cancer and the microRNA express spectra level of cancer beside organism's sample, uses statistical method through for a long time and widely research, therefrom filters out first 38 specific microRNA.Through testing identity, these specific microRNA marks can be distinguished primary lung cancer cancerous tissue and cancer beside organism very effectively.Finished on this basis the present invention.
Particularly, the inventor adopts the method for chip hybridization to obtain the cancerous tissue sample of former lung cancer and the microRNA express spectra of cancer beside organism's sample, by comparing the express spectra of two kinds of tissues, obtain the microRNA of differential expression between these two kinds of samples of cancerous tissue sample and cancer beside organism.As the candidate, using Bayesian network (BayesNet), SVMs (libSVM), feedforward neural network (RBFnetwork) and support vector regression model (SMO) to screen to obtain classify accuracy is a classifiers (containing 38 microRNA) of 93.42% with these difference microRNA.By these 38 sorters that microRNA forms, measurable sample is from cancerous tissue or cancer beside organism, and its prediction accuracy reaches 93.42%.Based on these 38 microRNA of the present invention, can be developed to small-sized microRNA chip or RT-PCR test kit and be used for distinguishing cancerous lung tissue and cancer beside organism.
MiRNA and precursor thereof
The invention provides the new miRNA that from the people, finds of a class.As used herein, described " miRNA " refers to a kind of RNA molecule, from forming the transcript processing of miRNA precursor.Ripe miRNA has 18-26 Nucleotide (nt) (more particularly about 19-22nt) usually, does not also get rid of the miRNA molecule with other number Nucleotide.MiRNA can be detected by the Northern trace usually.
The miRNA in people source can be separated from people's cell.As used herein, " separation " refers to that material separates (if natural substance, primal environment namely is natural surroundings) from its primal environment.Do not have separation and purification such as the polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
MiRNA can be from precursor miRNA (Precursor miRNA, Pre-miRNA) processing, and described precursor miRNA can be folded into a kind of stable stem ring (hair clip) structure, and described loop-stem structure length is generally between 50-100bp.Described precursor miRNA can be folded into stable loop-stem structure, and the stem both sides of loop-stem structure comprise basically complementary two sequences.Described precursor miRNA can be natural or synthetic.
Precursor miRNA can be sheared and generate miRNA, and described miRNA can be basically complementary with at least a portion sequence of the mRNA of encoding gene.As used herein, " basically complementary " refers to that the sequence of Nucleotide is enough complementary, can interact in a kind of foreseeable mode, as forming secondary structure (such as loop-stem structure).It is complementary that the nucleotide sequence of two usually, " basically complementary " has 70% Nucleotide between mutually at least; Preferably, it is complementary having 80% Nucleotide at least; Preferred, it is complementary having 90% Nucleotide at least; Further preferred, it is complementary having 95% Nucleotide at least; Such as 98%, 99% or 100%.Usually, two enough can have maximum 40 unmatched Nucleotide between the complementary molecule; Preferably, have maximum 30 unmatched Nucleotide; Preferred, have maximum 20 unmatched Nucleotide; Further preferred, have maximum 10 unmatched Nucleotide, as have 1,2,3,4,5,8,11 unmatched Nucleotide.
As used herein, " stem ring " structure also is known as " hair clip " structure, refer to a kind of nucleic acid molecule, it can form a kind of secondary structure that comprises double-stranded region (stem), described double-stranded region is formed by two zones (being positioned on high a part) of this nucleic acid molecule, the both sides of two double-stranded parts of regional apportion; It also comprises at least one " ring " structure, comprises non-complementary nucleic acid molecule, i.e. the strand zone.Even two zones of this nucleic acid molecule are not complete complementaries, the double-stranded part of Nucleotide also can keep double-stranded state.For example, insertion, disappearance, replacement etc. can cause not complementary or this zonule self formation loop-stem structure of a zonule or the secondary structure of other form, yet these two zones still can be basically complementary, and in foreseeable mode, interact, form the double-stranded region of loop-stem structure.Loop-stem structure is well-known to those skilled in the art, and behind the nucleic acid that has obtained a nucleotide sequence with primary structure, those skilled in the art can determine whether this nucleic acid can form loop-stem structure usually.
MiRNA of the present invention has the sequence shown in SEQ ID NO:38, and wherein n is the positive integer that is selected from 1-38.
In order to improve stability or other character of miRNA, also can add at least one end of described miRNA at least one protectiveness base, such as " TT " etc.
Antisense oligonucleotide
According to miRNA sequence provided by the present invention, can design their antisense oligonucleotide, described antisense oligonucleotide can be reduced the expression of corresponding miRNA in vivo.As used herein, " antisense oligonucleotide (antisense-oligonucleotides; AS-Ons or ASO) " is called again " antisense nucleotide ", refers to that length is about dna molecular or RNA molecule or its analogue of 18-26nt (more particularly about 19-22nt).
In the present invention, described " antisense oligonucleotide " also comprises the modified antisense nucleotide that adopts as based on means acquisitions such as nucleic acid lock or nucleic acid chains backbone modification technology, described modification does not change the activity of antisense oligonucleotide substantially, more preferably, described modification can improve stability, activity or the result for the treatment of of antisense oligonucleotide.Nucleic acid lock (locked nucleic acid, LNA) typically refers to the modification technique that 2 ' Sauerstoffatom and the 4 ' carbon atom of ribose is coupled together by a methylene bridge.LNA can prolong the serum half-life of miRNA, improves the target affinity, reduces scope and the degree of the effect of missing the target.The antisense drug that technical development goes out based on the nucleic acid chains modification of framework is in solubility, and the aspects such as nuclease-resistant degraded are improved greatly, and is easy to a large amount of synthetic.The backbone modification method of oligonucleotide has multiple, comprises the sulfo-method, is sulfo-deoxynucleotide chain with deoxynucleotide chain thio-modification for example.The method is that the Sauerstoffatom of the phosphate bond on the DNA skeleton is alternative with sulphur atom, can resist nuclease degradation.Should be understood that any can keep the most of of described antisense oligonucleotide or all active modifications be included among the present invention.
As optimal way of the present invention, antisense oligonucleotide is carried out the nucleic acid lock modify; More preferably also carry out thio-modification.
After transferring to antisense oligonucleotide of the present invention in the human body, they can obviously reduce the expression of relevant miRNA.
The polynucleotide construction
According to people's miRNA sequence provided by the present invention, can design the polynucleotide construction of the miRNA that after being imported into, can be processed to affect corresponding mrna expression, also be the amount that described polynucleotide construction can raise corresponding miRNA in vivo.Therefore, the invention provides a kind of polynucleotide (construction) of separation, described polynucleotide (construction) can be become precursor miRNA by people's cell transcription, and described precursor miRNA can and be expressed as described miRNA by people's cell shearing.
As a kind of optimal way of the present invention, described polynucleotide construction contains the structure shown in the formula I:
Seq
Forward-X-Seq
OppositelyFormula I,
Among the formula I,
Seq
ForwardFor becoming at cells the nucleotide sequence of described miRNA, Seq
OppositelyFor with Seq
ForwardBasically complementary nucleotide sequence; Perhaps, Seq
OppositelyFor becoming at cells the nucleotide sequence of described miRNA, Seq
ForwardFor with Seq
ForwardBasically complementary nucleotide sequence;
X is for being positioned at Seq
ForwardAnd Seq
OppositelyBetween intervening sequence, and described intervening sequence and Seq
ForwardAnd Seq
OppositelyNot complementary;
Structure shown in the formula I forms the secondary structure shown in the formula II after changing cell over to:
Among the formula II, Seq
Forward, Seq
OppositelyWith stating as defined above of X;
|| be illustrated in Seq
ForwardAnd Seq
OppositelyBetween the base complementrity pair relationhip that forms.
Usually, described polynucleotide construction is positioned on the expression vector.Therefore, the present invention also comprises a kind of carrier, and it contains described miRNA, or described polynucleotide construction.Described expression vector also contains promotor, replication orgin and/or marker gene etc. usually.Method well-known to those having ordinary skill in the art can be used for making up expression vector required for the present invention.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described expression vector preferably comprises one or more selected markers, with the phenotypic character of the host cell that is provided for selecting transforming, such as kalamycin, gentamicin, Totomycin, amicillin resistance.
Chip
The microRNA chip of expression spectrum contains a nearly hundreds of probe usually, contains multiple microRNA, utilizes the content of principle contained various microRNA in full genomic level detection sample of dna double chain homologous complementary.Therefore, can be at one time the transcriptional level of the microRNA in the full genome range in the sample to be tested be detected.
Utilize miRNA sequence of the present invention, can also prepare corresponding miRNA chip, and then study the regulative mode of its express spectra and miRNAs.
On the other hand, the present invention also provides a kind of chip for analyzing the miRNA express spectra, and described chip can be used for distinguishing primary lung cancer cancerous tissue and cancer beside organism.
Described miRNA chip of the present invention comprises:
Solid phase carrier; And
Be fixed in order the oligonucleotide probe on the described solid phase carrier, described oligonucleotide probe is specifically corresponding at least a kind in the sequence shown in the SEQ ID NO:1-38 (such as 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38 kind).
Particularly, can design suitable probe according to miRNA of the present invention, be fixed on the solid phase carrier, form " oligonucleotide arrays ".Described " oligonucleotide arrays " refer to have the addressable point array of (namely with distinctive, addressable address is the position of feature), a coupled characteristic oligonucleotide is all contained in each addressable point.As required, oligonucleotide arrays can be divided into a plurality of inferior battle arrays.
Described solid phase carrier can adopt the various common used materials in gene chip field, such as but not limited to nylon membrane, and the slide of the slide of modifying through active group (such as aldehyde radical, amino etc.) or silicon chip, unmodified, plastic sheet etc.
The preparation of described miRNA chip can be adopted the conventional manufacture method of biochip known in the art.For example, if what solid phase carrier adopted is to modify slide or silicon chip, 5 ' end of probe contains amido modified poly-dT string, oligonucleotide probe can be mixed with solution, then adopt point sample instrument that its point is being modified on slide or the silicon chip, be arranged in predetermined sequence or array, then spending the night by placement fixes, and just can obtain miRNA chip of the present invention.If it is amido modified that nucleic acid does not contain, then its preparation method also can reference: Wang Shenwu chief editor's " gene diagnosis technology-on-radiation operational manual "; J.L.erisi, V.R.Iyer, P.O.BROWN.Exploring the metabolic and genetic control of gene expression on a genomic scale.Science, 1997; 278:680 and Ma Li people, the Jiang Zhonghua chief editor. biochip. Beijing: Chemical Industry Press, 2000,1-130.
On the other hand, the present invention also provides a kind of method by miRNA express spectra in the miRNA chip detection people tissue, comprises step:
(1) provides the RNA sample that separates from people's tissue, at described RNA marker is set;
(2) RNA with (1) contacts with described chip, makes the oligonucleotide probe generation hybridization on described RNA and the solid phase carrier, thereby forms " oligonucleotide probe-RNA " binary complex at solid phase carrier;
(3) detect the marker of the binary complex of (2) formation, thereby determine the express spectra of corresponding miRNA in people's tissue.
The method of extracting RNA from people's tissue is method well known to those skilled in the art, comprises the Trizol method.
Preferred, in step (1), after from people's tissue tissue, isolating the RNA sample, the RNA sample is suitably processed, have the RNA of certain-length with enrichment, described length is between 10-100 (small fragment RNA) generally.Through after the above-mentioned processing, utilize these small fragment RNAs to carry out follow-up hybridization, can improve the accuracy that chip is caught miRNA like this.Those skilled in the art can isolate the RNA with certain fragment length easily, such as adopting gel electrophoresis to separate.
It also is method well known to those skilled in the art that RNA is carried out mark, and it can be by the method realization of adding when hybridizing with the marker of RNA specific binding, and described marker is such as being labelling groups.Described labelling groups includes but not limited to: digoxin molecule (DIG), biotin molecule (Bio), fluorescein and the biomolecules of deriving (FITC etc.) thereof, other fluorescence molecule (such as Cy3, Cy5 etc.), alkaline phosphatase (AP), horseradish peroxidase (HRP) etc.These marks and marking method thereof all have been routine techniques well-known in the art.
When above-mentioned RNA and miRNA chip are hybridized, can first miRNA chip and prehybridization damping fluid be carried out prehybridization.
Solid-phase hybridization between RNA of the present invention and the miRNA chip carries out according to the classical way of this area, and the general personnel in this area determine relevant damping fluid, probe and the optimum condition of concentration of specimens, prehybridization temperature, hybridization temperature and time etc. easily according to experience.Perhaps also can be with reference to described in " molecular cloning experiment guide ".
Then treat measurement information according to acquisition of informations such as the position of marking signal on the miRNA chip, intensity.If amplified production fluorophor mark also can directly obtain and treat measurement information with fluorescence detection device (such as laser confocal scanning instrument Scanarray 3000 etc.).
Detection kit
The present invention also provides a kind of test kit, contains chip of the present invention in the described test kit.Described test kit can be used for detecting the express spectra of miRNA; Or for distinguishing primary lung cancer cancerous tissue and cancer beside organism.
Preferred, also contain the marker that is useful on the labeled rna sample in the described test kit, and the substrate corresponding with described marker.
In addition, also can comprise in the described test kit for extracting the required all ingredients such as RNA, PCR, hybridization, colour developing, include but not limited to: extract, amplification liquid, hybridization solution, enzyme, contrast liquid, nitrite ion, washing lotion, antibody etc.
In addition, also can comprise working instructions and/or chip image analysis software in the described test kit.
Major advantage of the present invention is:
(a) the invention provides a class and can be used for distinguishing microRNA mark primary lung cancer cancerous tissue and cancer beside organism, new.
(b) sorter that is made of the new microRNA mark of the present invention can be distinguished primary lung cancer cancerous tissue and cancer beside organism very effectively.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
The preparation of embodiment 1 RNA sample
1. tissue samples:
The 46 pairs of cancers and cancer beside organism come from the excision sample of Patients With Primary Lung Cancer, and these samples come from Shanghai chest hospital.Above-mentioned all samples obtain all agreement by the Ethics Committee of WHO cooperative association of Shanghai City government authorization.The clinical data of tissue samples comprises: sex, age, tumor size, pathological grading (TNM by stages), whether shift, whether recur etc.
2. gene chip:
The microRNA chip of expression spectrum adopts the brilliant core of Boao Biological Co., Ltd
Chip of expression spectrum (single passage chip).
3. total tissue RNA is extracted
3.1 the Collection and preservation of sample: in vitro tissue places the liquid nitrogen quick-frozen after cutting into fritter immediately, then can move to-80 ℃ of Refrigerator stores.
3.2 tissue block is broken: tissue block is put in the liquid nitrogen and pulverizes, every gram tissue adds 1ml Trizol (Invitrogen company).
3.3 total RNA extracting: add the chloroform of 0.2ml, thermal agitation 15 seconds, incubated at room 2-3 minute by every milliliter of Trizol; 4 ℃, centrifugal 15 minutes of 10000g; Colourless supernatant liquor is moved in the new centrifuge tube, add the 0.5ml Virahol, incubated at room 10 minutes, 4 ℃ of 10000g are centrifugal 10 minutes; Outwell supernatant, 0.75ml75% washing with alcohol, 4 ℃, centrifugal 5 minutes of 7500g; Outwell supernatant, drying at room temperature RNA precipitates 5-10 minute (not making the RNA complete drying), after processing with DEPC without RNA enzyme H
2The O dissolution precipitation.
3.4 spectrophotometer standard measure RNA, and total RNA that takes a morsel carries out electrophoresis, checks whether RNA degrades.
Extraction and the mark of embodiment 2 microRNA
MiRNAs extraction agent box extracting with Ambion company obtains miRNA, and concrete operations are according to the respective description book.Sample uses T4RNA ligase enzyme markers step according to the method for Thomson.In brief, method is as follows:
1.4 the little RNA of μ g and 500ng 5 '-phosphoric acid salt-cytosine(Cyt)-uridylic-cy3-3 ' (Dharmacon, Chicago, USA) and the 2 T4RNA ligase of unit (NEB, Ipswich, USA) were hatched 2 hours in 4 ℃, and miRNA is carried out mark.Every part of miRNA sample is all established the corresponding negative control of equivalent.
2. the RNA of mark precipitates with 0.3M sodium-acetate and 2.5 volume ethanol, contains resuspended, all hybridization repetition twice of hybridization solution of 3 * SSC, 0.2%SDS and 15% methane amide with 15 μ l, hybridization LifterSlip again
TM(Erie, PA USA) is to guarantee hybridization solution Uniform Flow between chip and cover plate.
3. hybridization chamber is placed on hybridization instrument BioMixer
TMII upper (CapitalBio Corp, Beijing, China) spends the night in 42 ℃ of water-bath hybridization, washes twice with washing lotion afterwards.
The microRNA of embodiment 3 screening significant differences
1.46 routine cancer sample and the other sample of 46 routine cancers are respectively behind fluorochrome label, with the probe competitive hybridization on the microRNA chip of expression spectrum.The brilliant core of chip after the hybridization
TMThe scanning of 10K micro-array chip scanner obtains result images, and is quantitative to results of hybridization by its LuxScan 3.0 general microarray image analysis softwares that attach at random again.Thus, obtain the microRNA express spectra data of the other sample of 46 pairs of cancer samples (cancer, Cancer, C) and cancer (by the cancer, Pericancerous, P).
Above-mentioned 46 cancer chip results and 46 other chip results of cancer are at first used LOWESS (locally weighted regression) normalization method.Afterwards, the cancer after normalization method, the other chip results of cancer are got take the 2 logarithm (log the end of as
2X), use the at random Tobin's mean variance model (RVM) after the pairing T method of inspection (because cancerous tissue and cancer beside organism derive from same patient) is proofreaied and correct to screen the significant difference microRNA that obtains cancer/cancer other (C/P).
For the significance of verifying microRNA difference is not caused by coincidence, the inventor introduces after the above-mentioned T method of inspection data random rearrangement 1000 times again, detect a microRNA tested false positive rate (FDR, False Discovery Rate) that is decided to be significant difference microRNA after screening by preceding method.
In above-mentioned statistical method, only have the microRNA of p value＜0.05 just can screenedly be significant difference microRNA.
2. screening and the checking of sorter (can distinguish two histioid microRNA)
Totally five kinds of models in the molecular marker screening process, have been adopted: Bayesian network (BayesNet), SVMs (libSVM), feedforward neural network (RBFnetwork) and support vector regression model (SMO), wherein the SVM model has adopted two kinds of kernel functions to calculate, and has adopted on this basis different algorithms that molecular marker is carried out calculating sifting.
Be the classify accuracy of inspection-classification device, the inventor selects in all crosscheck methods the most stable 10 to take advantage of 10 folding crosscheck methods.10 folding crosscheck methods are that sample totally is divided into 10 sons part, select 1 son part as test data set at every turn, and all the other 9 sons are part as training dataset, repeat 10 times (at every turn with different son parts as test data set).10 assays that so obtain combine and form a assessed value to the sorter classify accuracy.
For showing intuitively on the same group similar between sample and not on the same group different between sample, the inventor looks in 3 dimensions and introduces multidimensional scaling (multidimensional scaling, MDS) in the effect.The MDS algorithm for the basis, is determined the position of each sample in lower dimensional space take similarity matrix between the sample-sample that is classified device (one group of microRNA) definition, and makes it to be adapted to 3 and tie up and look effect.In this 3 dimension space, two samples are more approaching, and then they are more similar; Otherwise, if two samples are at a distance of far away, then more different between them.
3. result
Screen altogether 155 difference microRNA between cancerous tissue and cancer beside organism.With these difference microRNA as the sorter candidate.Be inserted in above-mentioned classifier algorithm and take advantage of 10 crosschecks of rolling over to verify the classify accuracy of sorter with aforesaid 10.
After 10 of 1000 secondary data displacement took advantage of 10 folding cross validations to obtain sorter, for testing the predictive ability of this sorter, inventor's random choose part sample carried out test verification to the result that all sample calculation obtain.What accuracy rate was the highest in these several sorters is 93.42%, the sorter (seeing Table 1) that 38 microRNA that namely calculated by SMO-BestFirst (support vector regression model-optimal result first search) model form, its classify accuracy can reach 93.42%.Use this model that 18 pairs of independent sample source place unknown sample are predicted rate of accuracy reached to 94.4444%.
For ease of the classifying quality that shows sorter directly perceived, the inventor uses the MDS algorithm 38 microRNA signal values of each sample to be converted to the eigenvector of 3 dimensions, and they are located in 3 dimension spaces, be depicted as the three-dimensional scatter diagram (seeing Fig. 1) of the other two class samples of cancer and cancer.Can be judged by Fig. 1 whether the distance between any two samples of different group is larger than the distance between any two samples on the same group.
Table 1
As above shown in the table, hsa-miR-1308 raises in the cancerous tissue sample, therefore can will raise multiple (the expression amount ratio of namely comparing with negative control) more than or equal to 1.3, is decided to be hsa-miR-1308 and raises.
Hsa-miR-1234 reduces in the cancerous tissue sample, therefore downward modulation multiple (the expression amount ratio of namely comparing with negative control) can be less than or equal to 0.731, is decided to be the hsa-miR-1234 downward modulation.The rest may be inferred for other microRNA.
Embodiment 4 preparation miRNA chips
The miRNA sequence (SEQ ID NO:1-38) that table 1 is provided converts complementary sequence to, adds the catenation sequence of 10-20nt at the sequence two ends according to features such as the GC that produces sequence compare; Core sequence is different, and catenation sequence is also different.Catenation sequence can be produced at random by program, and the probe that catenation sequence and core sequence form meets the following conditions:
1) in the probe sequence, the quantity of same Nucleotide (A, C, G, T) can not surpass 50% of sequence sum;
2) any continuous A, T or the quantity of C, G can not surpass 25% of sequence sum;
3) G, C content account for the 40%-60% of sequence sum;
4) probe sequence can not be from hybridization, and namely the length of complementary fragment can not surpass 30% of probe length in the probe sequence.
For making stable being combined on the slide of synthetic probe, adopt conventional method to carry out glycosyl modified at 5 ' end of synthetic rear probe.
The point system of chip: first the surface of slide is carried out alkylation and modify, to improve binding ability.Adopt conventional chip point sample method to carry out point sample, in order to detect the repeatability of cross experiment, each probe is at slide point 3-6 hybridization point.
The preparation of embodiment 5 test kits
The Chip Packaging of preparation among the embodiment 4 is good, place a box with working instructions, consist of test kit.
The detection of embodiment 6 chips
To the sample (comprising 36 routine cancerous tissue samples and the other sample of 36 routine cancers) that obtains a plurality of primary lung cancers from hospital, press the preparation of embodiment 1 and 2 methods and mark microRNA, press the chip of the method preparation of embodiment 4, detect with double-blind method.According to the existence of the microRNA mark shown in the table 1 whether and the upper downward modulation situation that is in harmonious proportion come judgement sample.Wherein, positive control and negative control are respectively known cancerous tissue sample and the known other sample of cancer.
The result shows that by the chip that any 5 species specificity microRNA consist of, its exactness is 80%, and the sample that can effectively distinguish primary lung cancer belongs to cancerous tissue or cancer beside organism.
By the chip that any 10 species specific microRNA consist of, its exactness is 90%, and the sample that can very effectively distinguish primary lung cancer belongs to cancerous tissue or cancer beside organism.
By the chip that whole 38 species specific microRNA consist of, its exactness is 94%, and the sample that can very effectively distinguish primary lung cancer belongs to cancerous tissue or cancer beside organism.The expression pattern of 38 kinds of microRNA of 36 tissue samples all meets the situation of table 1.In contrast, the expression pattern of 36 other samples is just in time fully opposite, does not all meet the situation of table 1.
Therefore, the chip that specificity microRNA of the present invention forms can be used in combination, and whether the sample of effectively distinguishing primary lung cancer belongs to cancerous tissue or cancer beside organism.
All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. the miRNA of a separation is characterized in that, described miRNA is selected from:
(i) miRNA of sequence shown in SEQ ID NO:n, wherein n is the positive integer that is selected from 1-38; Or
(ii) with the miRNA of sequence complementation shown in the SEQ ID NO:n.
2. a miRNA collection is characterized in that, described miRNA collection is made of 38 kinds of miRNA of sequence shown in SEQ ID NO:1-38.
3. a precursor miRNA separation or artificial constructed is characterized in that, miRNA claimed in claim 1 can be sheared and be expressed as to described precursor miRNA in people's cell.
4. the polynucleotide of a separation is characterized in that, described polynucleotide can be become precursor miRNA by people's cell transcription, and described precursor miRNA can be sheared and be expressed as miRNA claimed in claim 1 in people's cell.
5. polynucleotide as claimed in claim 4 is characterized in that, described polynucleotide have the structure shown in the formula I:
Seq
Forward-X-Seq
OppositelyFormula I,
Among the formula I,
Seq
ForwardFor becoming at people's cells the nucleotide sequence of described miRNA;
Seq
OppositelyFor with Seq
ForwardBasically the nucleotide sequence of complementation or complete complementary;
X is for being positioned at Seq
ForwardAnd Seq
OppositelyBetween intervening sequence, and described intervening sequence and Seq
ForwardAnd Seq
OppositelyNot complementary;
And the structure shown in the formula I forms the secondary structure shown in the formula II after changing people's cell over to:
Among the formula II, Seq
Forward, Seq
OppositelyWith stating as defined above of X,
|| be illustrated in Seq
ForwardAnd Seq
OppositelyBetween the base complementrity pair relationhip that forms.
6. a carrier is characterized in that, it contains miRNA claimed in claim 1, or polynucleotide claimed in claim 4.
7. the purposes of miRNA claimed in claim 1 is characterized in that, for the preparation of chip or the test kit of distinguishing primary lung cancer cancerous tissue and cancer beside organism.
8. a miRNA chip is characterized in that, described miRNA chip comprises:
Solid phase carrier; And
Be fixed in order the oligonucleotide probe on the described solid phase carrier, described oligonucleotide probe is specifically corresponding to the part or all of sequence shown in the SEQ ID NO:1-38.
9. the purposes of miRNA chip as claimed in claim 8 is characterized in that, for the preparation of the test kit of distinguishing primary lung cancer cancerous tissue and cancer beside organism.
10. a test kit is characterized in that, contains miRNA chip claimed in claim 8 in the described test kit.
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