CN102851283B - MicroRNA markers for discriminating metastatic and non-metastatic squamous cell lung carcinoma - Google Patents
MicroRNA markers for discriminating metastatic and non-metastatic squamous cell lung carcinoma Download PDFInfo
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Abstract
The invention relates to 21 microRNA markers for discriminating metastatic and non-metastatic squamous cell lung carcinoma. More specifically, the invention relates to a class of microRNA markers for discriminating metastatic and non-metastatic squamous cell lung carcinoma. It has been proved by examination that, the specific microRNA markers can effectively discriminate metastatic and non-metastatic squamous cell lung carcinoma 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 distinguishing the microRNA mark and the purposes that shift with non-transfer lung squamous cell carcinoma cancers.The invention still further relates to the chip and the test kit that detect described microRNA mark.
Background technology
Primary Pulmonary Squamous Carcinoma is one of modal malignant tumour of China, and grade malignancy is high, and rapidly, treatment is difficult in development, and case fatality rate is high.Therefore, the diagnosis as early as possible of lung squamous cancer just more seems urgent.
The generation development of anything is all under internal and external reasons acting in conjunction, interior because main, outer because auxiliary.Gene is as the hereditary medium of life, organism sick, old,, dead in status in basic internal cause.Overwhelming majority gene is by transcribing product nucleus ribosomal ribonucleic acid, then translation generates protein and brings into play biological function.
MicroRNA (miR or miRNA, Microrna) is that a class extensively exists in more high most eukaryotes, the single stranded RNA molecule of the about 18-26 of a length base.It can combine with the 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 controled.
MicroRNA derives from the initial transcription product of long-chain RNA (Pri-miRNA) that length is about 1000bp, and Pri-miRNA molecule is sheared and formed the approximately miRNA precursor (Pre-miRNA) with loop-stem structure of 60～80nt of length through Drosha enzyme in nucleus.Pre-miRNA is transported to after kytoplasm, is further cut into the double-stranded miRNA that is about 22nt by Dicer enzyme.After double-stranded miRNA unties, ripe miRNA enters the reticent mixture of RNA induced gene (RNA-induced silencing complex, RISC), with complementary mRNA completely 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 be efficiently produces regulating and controlling effects to all mRNA with target site, microRNA still can't neglect in the growth of organism and even the generation of tumour, evolution role.
But, up to now, this area is understood very few for the microRNA relevant to tumour (as lung squamous cancer), therefore 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
Object of the present invention be just to provide a class new, can be used for distinguishing and shift and the microRNA mark of non-transfer lung squamous cell carcinoma cancers.
Another object of the present invention is just to provide the purposes of 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 as shown in SEQ ID NO:n, wherein n is the positive integer that is selected from 1-21; Or
(ii) with the miRNA of sequence complementation shown in SEQ ID NO:n.
In another preference, described miRNA separates from people.
In a second aspect of the present invention, a kind of miRNA collection (set) or combination (combination) are provided, 21 kinds of miRNA as shown in SEQ ID NO:1-21 are formed by sequence for described collection or combination.
In a third aspect of the present invention, precursor miRNA a kind of separation or artificial constructed is provided, the miRNA described in 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 first aspect present invention in people's cell.
In another preference, described polynucleotide have the structure shown in formula I:
Seq
forward-X-Seq
oppositelyformula I,
In formula I,
Seq
forwardfor becoming at people's cells the nucleotide sequence of described miRNA,
Seq
oppositelyfor with Seq
forwardsubstantially 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
oppositelyit is not complementary,
And the structure shown in formula I is proceeding to after people's cell, forms the secondary structure shown in formula II:
In formula II, Seq
forward, Seq
oppositelywith the definition of X as above-mentioned,
|| be illustrated in Seq
forwardand Seq
oppositelybetween form base complementrity pair relationhip.
In a fifth aspect of the present invention, a kind of carrier is provided, it contains the miRNA described in first aspect present invention, or the polynucleotide described in fourth aspect.
In a sixth aspect of the present invention, provide the purposes of the miRNA described in first aspect present invention, for the preparation of distinguishing the chip or the test kit that shift with non-transfer lung squamous cell carcinoma cancers.
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 described solid phase carrier, described oligonucleotide probe is specifically corresponding to the part or all of sequence shown in SEQ ID NO:1-21 (as 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21 kind).
In another preference, described oligonucleotide probe contains:
Complementary land; And/or
The joining region being connected with solid phase carrier.
In a eighth aspect of the present invention, provide the purposes of above-mentioned miRNA chip, for the preparation of distinguishing the test kit shifting with non-transfer lung squamous cell carcinoma cancers.
In a ninth aspect of the present invention, a kind of test kit is provided, in described test kit, contain the present invention's miRNA chip described above.
Other side of the present invention, due to disclosure herein, 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 combining mutually between specifically described each technical characterictic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tire out and state no longer one by one at this.
Brief description of the drawings
Fig. 1 shows with transfer (light color) and the scatter diagram of non-transfer (dark color) in three-dimensional space after the conversion of MDS algorithm.
Embodiment
The inventor, through research for a long time and widely, shifts and the microRNA express spectra level of non-transfer lung squamous cell carcinoma cancers sample by detections, and use statistical method, therefrom filters out 21 specific microRNA first.Through testing identity, these specific microRNA marks can very effectively be distinguished and shift and non-transfer lung squamous cell carcinoma cancers.Complete on this basis the present invention.
Particularly, the inventor adopt the method for chip hybridization obtain lung squamous cancer have transfer sample (cancerous tissue and cancer beside organism) and without shift sample (cancerous tissue and cancer beside organism) microRNA express spectra, by comparing the express spectra of two kinds of samples, obtain the microRNA of differential expression between transfer and non-transfer lung squamous cell carcinoma cancers sample.Using these difference microRNA as candidate, use Bayesian network (BayesNet), SVMs (libSVM), feedforward neural network (RBFnetwork) and support vector regression model (SMO), wherein SVM model has adopted two kinds of kernel functions to calculate, adopt on this basis different algorithms to carry out calculating sifting to molecular marker, obtain classify accuracy and be a classifiers (containing 21 microRNA) of 74.8%.By these 21 sorters that microRNA forms, whether measurable sample has transfer, and its prediction accuracy reaches 74.8%.Based on these 21 microRNA of the present invention, can be developed to the prediction that small-sized microRNA chip or RT-PCR test kit shift for lung squamous cancer.
MiRNA and precursor thereof
The invention provides the miRNA finding that a class is new from people.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) conventionally, does not also get rid of the miRNA molecule with other number Nucleotide.MiRNA can be detected by Northern trace conventionally.
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 is natural surroundings) from its primal environment.As the polynucleotide under the native state in active somatic cell and polypeptide do not have separation and purification, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, 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 substantially complementary two sequences.Described precursor miRNA can be natural or synthetic.
Precursor miRNA can be sheared and generate miRNA, and described miRNA can be substantially complementary with at least a portion sequence of the mRNA of encoding gene.As used herein, " substantially complementary " refers to that the sequence of Nucleotide is enough complementary, can interact in the foreseeable mode of one, as formed secondary structure (as loop-stem structure).Conventionally it is complementary that, two " substantially complementary " nucleotide sequences have 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; As 98%, 99% or 100%.Usually, two enough can have maximum 40 unmatched Nucleotide between complementary molecule; Preferably, there are maximum 30 unmatched Nucleotide; Preferred, there are maximum 20 unmatched Nucleotide; Further preferred, there are maximum 10 unmatched Nucleotide, as there is 1,2,3,4,5,8,11 unmatched Nucleotide.
As used herein, " stem ring " structure is also known as " hair clip " structure, refer to a kind of nucleic acid molecule, it can form the secondary structure that one comprises double-stranded region (stem), described double-stranded region is formed by two regions (being positioned on same a part) of this nucleic acid molecule, the both sides of two double-stranded parts of region apportion; It also comprises at least one " ring " structure, comprises non-complementary nucleic acid molecule, i.e. strand region.Even if two regions 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, but these two regions still can be substantially complementary, and interact in foreseeable mode, form the double-stranded region of loop-stem structure.Loop-stem structure is well-known to those skilled in the art, is conventionally obtaining after the nucleic acid of a nucleotide sequence with primary structure, and those skilled in the art can determine whether this nucleic acid can form loop-stem structure.
MiRNA of the present invention has the sequence as shown in SEQ ID NO:n, and wherein n is the positive integer that is selected from 1-21.
In order to improve stability or other character of miRNA, also can add at least one protectiveness base at least one end of described miRNA, as " TT " etc.
Antisense oligonucleotide
According to miRNA sequence provided by the present invention, can design their antisense oligonucleotide, described antisense oligonucleotide can be lowered 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 obtain based on means 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 2 ' Sauerstoffatom of ribose and 4 ' carbon atom being coupled together by a methylene bridge.LNA can extend the serum half-life of miRNA, improves target affinity, reduces scope and the degree of the effect of missing the target.The antisense drug that modification technique based on nucleic acid chains skeleton develops 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 sulfo-method, for example, be sulfo-deoxynucleotide chain by deoxynucleotide chain thio-modification.The method is that the Sauerstoffatom of the phosphate bond on DNA skeleton sulphur atom is substituted, and can resist nuclease degradation.Should be understood that any modification most of or all activity that can keep described antisense oligonucleotide is included in the present invention.
As optimal way of the present invention, antisense oligonucleotide is carried out to nucleic acid lock and modify; More preferably also carry out thio-modification.
After antisense oligonucleotide of the present invention is transferred in human body, the expression that they can obviously lower relevant miRNA.
Polynucleotide construction
According to people's miRNA sequence provided by the present invention, can design the polynucleotide construction that can be processed to the miRNA that can affect corresponding mrna expression after being imported into, be also 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 formula I:
Seq
forward-X-Seq
oppositelyformula I,
In formula I,
Seq
forwardfor becoming at cells the nucleotide sequence of described miRNA,
Seq
oppositelyfor with Seq
forwardsubstantially complementary nucleotide sequence; Or, Seq
oppositelyfor becoming at cells the nucleotide sequence of described miRNA, Seq
forwardfor with Seq
forwardsubstantially 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 formula I is proceeding to after cell, forms the secondary structure shown in formula II:
In formula II, Seq
forward, Seq
oppositelywith the definition of X as above-mentioned;
|| be illustrated in Seq
forwardand Seq
oppositelybetween form base complementrity pair relationhip.
Conventionally, described polynucleotide construction is positioned on 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. conventionally.Method well-known to those having ordinary skill in the art can be used for building 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, to be provided for selecting the phenotypic character of the host cell transforming, as kalamycin, gentamicin, Totomycin, amicillin resistance.
Chip
MicroRNA chip of expression spectrum contains a nearly hundreds of probe conventionally, contains multiple microRNA, the principle of utilizing DNA double chain homologous complementary in genomic level entirely, detect sample in the content of contained various microRNA.Therefore, can detect the transcriptional level of the microRNA in full genome range in sample to be tested at one time.
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, it is a kind of for analyzing the chip of miRNA express spectra that the present invention also provides, and described chip can be used for distinguishing transfer and non-transfer lung squamous cell carcinoma cancers.Described miRNA chip of the present invention comprises:
Solid phase carrier; And
Be fixed in order the oligonucleotide probe on described solid phase carrier, described oligonucleotide probe is specifically corresponding to the part or all of sequence shown in SEQ ID NO:1-21 (as 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21 kind).
Particularly, can, according to miRNA of the present invention, design applicable probe, be fixed on solid phase carrier, form " oligonucleotide arrays ".Described " oligonucleotide arrays " refers to the have addressable point array of (with distinctive, the position that addressable address is feature), and a coupled characteristic oligonucleotide is all contained in each addressable point.As required, oligonucleotide arrays can be divided into multiple sub-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 slide, the plastic sheet etc. of the slide of modifying through active group (as aldehyde radical, amino etc.) or silicon chip, unmodified.
The preparation of described miRNA chip can adopt 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 to solution, then adopt point sample instrument that its point is being modified on slide or silicon chip, be arranged in predetermined sequence or array, then spend the night and fix by placement, just can obtain miRNA chip of the present invention.If nucleic acid containing amido modified, " the gene diagnosis technology-on-radiation operational manual " that its preparation method also can reference: Wang Shenwu chief editor; 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, Jiang Zhonghua chief editor. biochip. Beijing: Chemical Industry Press, 2000,1-130.
Another aspect, it is a kind of by the method for miRNA express spectra in miRNA chip detection people tissue that the present invention also provides, and comprises step:
(1) provide the RNA sample separating from people's tissue, on described RNA, marker is set;
(2) RNA of (1) is contacted with described chip, make the oligonucleotide probe generation hybridization on described RNA and solid phase carrier, thereby form " oligonucleotide probe-RNA " binary complex on 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 Trizol method.
Preferred, in step (1), isolating from people's tissue tissue after RNA sample, RNA sample is suitably processed, there is the RNA of certain length with enrichment, described length is between 10-100 (small fragment RNA) generally.After above-mentioned processing, utilize these small fragment RNAs to carry out follow-up hybridization, the accuracy that can improve chip like this and catch miRNA.Those skilled in the art can isolate the RNA with certain fragment length easily, such as adopting gel electrophoresis to separate.
It is also method well known to those skilled in the art that RNA is carried out to mark, and it can be realized by adding in when hybridization with the method for 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 derivative biomolecules (FITC etc.) thereof, other fluorescence molecule (as Cy3, Cy5 etc.), alkaline phosphatase (AP), horseradish peroxidase (HRP) etc.These marks and marking method thereof have been all 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 to prehybridization.
Solid-phase hybridization between RNA of the present invention and miRNA chip carries out according to the classical way of this area, and the general personnel in this area easily determine the optimum condition about damping fluid, probe and concentration of specimens, prehybridization temperature, hybridization temperature and time etc. according to experience.Or also can be with reference to described in " molecular cloning experiment guide ".Then according to marking signal, the acquisition of information such as position, intensity on miRNA chip is treated measurement information.If amplified production fluorophor mark, also can directly obtain and treat measurement information with fluorescence detection device (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 described test kit.Described test kit can be used for detecting the express spectra of miRNA; Or shift and non-transfer lung squamous cell carcinoma cancers for distinguishing.
Preferred, in described test kit, also contain the marker that is useful on labeled rna sample, and the substrate corresponding with described marker.
In addition, in described test kit, also can comprise 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, in described test kit, also can comprise working instructions and/or chip image analysis software.
Major advantage of the present invention is:
(a) the invention provides a class and can be used for distinguishing transfer and microRNA mark non-transfer lung squamous cell carcinoma cancers, new.
(b) sorter being made up of the new microRNA mark of the present invention, can very effectively distinguish and shift and non-transfer lung squamous cell carcinoma cancers.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition as people 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 1RNA sample
1. tissue samples:
46 pairs have the excision sample that shifts sample and come from From Lung Squamous Carcinoma Patients without transfer sample (21 transfers, 25 non-transfers), and these samples come from Shanghai chest hospital.Obtaining all by the agreement of the Ethics Committee of WHO cooperative association of Shanghai City government authorization of above-mentioned all samples.The clinical data of tissue samples comprises: sex, age, tumor size, pathological grading (TNM by stages), whether shift etc.
2. gene chip:
MicroRNA chip of expression spectrum, the brilliant core of employing Boao Biological Co., Ltd
chip of expression spectrum (single passage chip).
3. total tissue RNA is extracted
The Collection and preservation of 3.1 samples: in vitro tissue is placed in liquid nitrogen quick-frozen after cutting into fritter immediately, then can move to-80 DEG C of Refrigerator stores.
3.2 tissue block fragmentations: tissue block is put in liquid nitrogen and pulverizes, every gram of tissue adds 1ml Trizol (Invitrogen company).
3.3 total RNA extractings: add the chloroform of 0.2ml by every milliliter of Trizol, thermal agitation 15 seconds, incubated at room 2-3 minute; 4 DEG C, centrifugal 15 minutes of 10,000g; Colourless supernatant liquor is moved in new centrifuge tube, add 0.5ml Virahol, incubated at room 10 minutes, 4 DEG C of 10000g are centrifugal 10 minutes; Outwell supernatant, 0.75ml 75% washing with alcohol, 4 DEG C, centrifugal 5 minutes of 7500g; Outwell supernatant, drying at room temperature RNA precipitates 5-10 minute (not making RNA complete drying), after treatment without RNA enzyme H with DEPC
2o dissolution precipitation.
3.4 spectrophotometer standard measure RNA, and total RNA that takes a morsel carries out electrophoresis, checks whether RNA degrades.
The extraction of embodiment 2microRNA and mark
With the miRNAs extraction agent box extracting acquisition miRNA of Ambion company, concrete operations are according to respective description book.Sample is the method according to Thomson by T4RNA ligase enzyme markers step.In brief, method is as follows:
1.4 μ g miRNA and 500ng 5 '-phosphoric acid salt-cytosine(Cyt)-uridylic-cy3-3 ' (Dharmacon, Chicago, USA) and the 2 T4 RNA ligase (NEB of unit, Ipswich, USA), hatch 2 hours in 4 DEG C, miRNA is carried out to mark.Every part of miRNA sample is all established the corresponding negative control of equivalent.
2. the RNA of mark precipitates by 0.3M sodium-acetate and 2.5 volume ethanol, more resuspended containing the hybridization solution of 3 × SSC, 0.2%SDS and 15% methane amide with 15 μ l, and all hybridization repeats twice, hybridization LifterSlip
tM(Erie, PAUSA) is to ensure hybridization solution Uniform Flow between chip and cover plate.
3. hybridization chamber is placed on to hybridization instrument BioMixer
tMiI upper (CapitalBio Corp, Beijing, China) spends the night in 42 DEG C of water-bath hybridization, washes twice afterwards by washing lotion.
Embodiment 3 screens the microRNA of significant difference
1.46 routine cancer samples and the other sample of 46 routine cancers are respectively after fluorochrome label, with the probe competitive hybridization on microRNA chip of expression spectrum.The brilliant core of chip after hybridization
the scanning of 10K micro-array chip scanner obtains result images, more quantitative to results of hybridization by its random subsidiary general microarray image analysis software of LuxScan 3.0.Thus, obtain the microRNA express spectra data of the other sample of 46 pairs of cancer samples (cancer, Cancer, C) and cancer (by cancer, Pericancerous, P).
First above-mentioned 46 cancer chip results and 46 other chip results of cancer are used to LOWESS (locally weighted regression) normalization method.Afterwards, the cancer after normalization method, the other chip results of cancer are got the logarithm (log taking 2 end of as
2x), use the 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 checking 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.
Only, in above-mentioned statistical method, only has the just screened significant difference microRNA of being of meeting of microRNA of p value < 0.05.
2. screening and the checking of sorter (can distinguish two histioid microRNA)
Be used for distinguishing two class samples (shifting and non-transfer) for finding, totally five kinds of models in molecular marker screening process, are adopted: Bayesian network (BayesNet), SVMs (libSVM), feedforward neural network (RBFnetwork) and support vector regression model (SMO), wherein SVM model has adopted two kinds of kernel functions to calculate.Adopt on this basis different algorithms to carry out calculating sifting to molecular marker.
For 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 is totally divided into 10 sub part, select 1 son part as test data set at every turn, and all the other 9 son parts, as training dataset, are repeated 10 times (at every turn using different son parts as test data set).10 assays that so obtain combine and form sorter classify accuracy assessed value.
For showing intuitively on the same group similar between sample and not different between sample on the same group, the inventor introduces multidimensional scaling (multidimensional scaling, MDS) in 3 dimensions in depending on effect.MDS algorithm for basic, is determined the position of each sample in lower dimensional space to be classified similarity matrix between the sample-sample of device (one group of microRNA) definition, and makes it to be adapted to 3 dimensions and look effect.In this 3 dimension space, two samples are more approaching, and they are more similar; Otherwise, if two samples are at a distance of far away, more different between them.
3. result
Between transfer and non-transfer sample, screen altogether 30 difference microRNA.Using these difference microRNA as sorter candidate, be inserted in above-mentioned classifier algorithm and also take advantage of the crosscheck of 10 foldings to verify the classify accuracy of sorter with aforesaid 10.
Take advantage of 10 folding cross validations to obtain after sorter through 10 of 1000 secondary data displacements, for testing the predictive ability of this sorter, the inventor chooses at random the result that a part of sample obtains all sample calculation and carries out test verification, what in these several sorters, accuracy rate was the highest is 74.8%, the sorter (in table 1) that 21 microRNA that calculate that calculated by RBFNetwork-BestFirst (BP network model-optimal result first search) model form, its classify accuracy can reach 74.8%.We use this model to predict the unknown sample in 18 pairs of independent sources, rate of accuracy reached to 88.9%.
For ease of the classifying quality that shows sorter directly perceived, the inventor uses MDS algorithm 21 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 transfer and non-transfer two class samples.Can be judged that 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 shown above, hsa-miR-548e raises in cancerous tissue sample, therefore can be more than or equal to 1.4 by raising multiple (i.e. expression amount ratio compared with negative control), is decided to be hsa-miR-548e and raises.
Hsa-miR-892b lowers in cancerous tissue sample, therefore can be less than or equal to 0.656 by lowering multiple (i.e. expression amount ratio compared with negative control), is decided to be hsa-miR-892b and lowers.The rest may be inferred for other microRNA.
Embodiment 4 prepares miRNA chip
The miRNA sequence (SEQ ID NO:1-21) that table 1 is provided converts complementary sequence to, adds the catenation sequence of 10-20nt according to features such as the GC that produces sequence compare at sequence two ends; Core sequence difference, 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 probe sequence, the quantity of same Nucleotide (A, C, G, T) can not exceed 50% of sequence sum;
2) quantity of any continuous A, T or C, G can not exceed 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 in probe sequence, the length of complementary fragment can not exceed 30% of probe length.
For making stable being combined on 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 alkylation modification is carried out in the surface of slide, 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 put 3-6 hybridization point on slide.
Embodiment 5 test kit preparations
By good the Chip Packaging of preparation in embodiment 4, be placed in a box together with working instructions, form test kit.
The detection of embodiment 6 chips
To obtain the sample (comprising that 7 examples shift tissue samples and 11 routine non-transfer samples) of multiple lung squamous cancers from hospital, prepare and mark microRNA by embodiment 1 and 2 methods, prepare chip according to the method for embodiment 4, detect by double-blind method.According to the existence of 21 kinds of microRNA marks shown in table 1 whether and upper mediation downward situation carry out judgement sample.Wherein, positive control and negative control are respectively known transfer and non-transfer lung squamous cancer sample.
Result shows, the chip being formed by any 5 species specificity microRNA, and its exactness is 70%, can effectively distinguish and shift and non-transfer lung squamous cancer sample.
The chip being made up of any 10 species specific microRNA, its exactness is 80%, can very effectively distinguish and shift and non-transfer lung squamous cancer sample.
The chip being formed by 21 species specific microRNA, its exactness is 88.9%, the sample that can very effectively distinguish lung squamous cancer belongs to and shifts sample is also non-transfer sample.The expression pattern of 11 kinds of microRNA of 7 example transfer tissue samples all meets the situation of table 1.In contrast, the expression pattern of 11 routine non-transfer samples is just in time completely contrary, does not all meet the situation of table 1.
Therefore, the chip of specificity microRNA composition of the present invention can be used in combination, and whether the sample of effectively distinguishing primary lung cancer belongs to cancerous tissue or cancer beside organism.
All documents of mentioning in the present invention are all quoted as a reference in this application, are just quoted separately as a reference as each section of document.In addition should be understood that 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 (6)
1. a miRNA collection, is characterized in that, by sequence, 21 kinds of miRNA as shown in SEQ ID NO:1-21 are formed described collection.
2. a carrier, is characterized in that, it contains miRNA collection claimed in claim 1.
3. the purposes of miRNA collection claimed in claim 1, is characterized in that, for the preparation of distinguishing the chip or the test kit that shift with non-transfer lung squamous cell carcinoma cancers.
4. a miRNA chip, is characterized in that, described miRNA chip comprises:
Solid phase carrier; And
Be fixed in order the oligonucleotide probe on described solid phase carrier, described oligonucleotide probe is complementary to the full sequence shown in SEQ ID NO:1-21 specifically.
5. the purposes of miRNA chip as claimed in claim 4, is characterized in that, for the preparation of distinguishing the test kit shifting with non-transfer lung squamous cell carcinoma cancers.
6. a test kit, is characterized in that, contains miRNA chip claimed in claim 4 in described test kit.
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| CN201110183167.2A CN102851283B (en) | 2011-06-30 | 2011-06-30 | MicroRNA markers for discriminating metastatic and non-metastatic squamous cell lung carcinoma |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105603117A (en) * | 2016-03-31 | 2016-05-25 | 北京泱深生物信息技术有限公司 | Application of miR-3613 as miRNA marker in distinguishing metastasis and non-metastasis of lung squamous carcinoma |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107430588B (en) * | 2015-01-22 | 2021-12-31 | 斯坦福大学托管董事会 | Method and system for determining the proportion of different cell subsets |
| CN105603115B (en) * | 2016-03-31 | 2019-10-11 | 北京泱深生物信息技术有限公司 | Lung squamous cancer shifts diagnosis and treatment marker |
| CN105803067B (en) * | 2016-03-31 | 2019-08-13 | 北京泱深生物信息技术有限公司 | Application of the miR-1304 in metastatic lung squamous cancer diagnosis and treatment |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US7927805B2 (en) * | 2007-10-30 | 2011-04-19 | Veridex, Llc | Process for predicting the prognosis of squamous cell lung cancer |
| EP2470897A4 (en) * | 2009-08-28 | 2013-05-29 | Asuragen Inc | Mirna biomarkers of lung disease |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105603117A (en) * | 2016-03-31 | 2016-05-25 | 北京泱深生物信息技术有限公司 | Application of miR-3613 as miRNA marker in distinguishing metastasis and non-metastasis of lung squamous carcinoma |
| CN105603117B (en) * | 2016-03-31 | 2019-04-09 | 北京泱深生物信息技术有限公司 | MiR-3613 is used to distinguish lung squamous cancer transfer and non-diverting miRNA marker |
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