CN107058579A - Adenocarcinoma of lung related miRNA, composition and its application - Google Patents
Adenocarcinoma of lung related miRNA, composition and its application Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The present invention relates to adenocarcinoma of lung related miRNA, composition and its application, the application of miR 6087, miR 7976, miR 7705, the 5p of miR 6744 and combinations thereof in adenocarcinoma of lung diagnostic preparation is prepared is more particularly related to.Invention carries out transcript profile sequencing to cancerous tissue and the sample of cancer beside organism, and analyzed with bioinformatics method, it is found that several molecular markers related to adenocarcinoma of lung being never reported before, analyzed by Molecular and ROC curve, show that these newfound miRNA can turn into potential adenocarcinoma of lung molecular marker, basis is provided for clinical diagnosis.
Description
Technical field
The present invention relates to biology field, adenocarcinoma of lung related miRNA, composition and its application are specifically related to,
More particularly relate to miR-6087, miR-7976, miR-7705, miR-6744-5p and combinations thereof and prepare adenocarcinoma of lung diagnosis
Application in preparation.
Background technology
Lung cancer is one of higher malignant tumour of the death rate, mainly includes ED-SCLC and non-small cell lung cancer, clinical
It is in the majority with non-small cell lung cancer in morbidity, 80% is accounted for, non-small cell lung cancer can further be subdivided into gland cancer and squamous carcinoma two is big
Type.Continued to develop recently as accurate medical, the individualized treatment requirement of lung cancer carries out the essence of molecular level to lung cancer
Quasi- parting, although there are many reports to disclose the diagnosis molecular markers of part lung cancer, but can not still meet demand, it is necessary to
Molecule parting, targeted therapy and the Index for diagnosis of more molecular marker power-assisted lung cancer.
MiRNA is the non-coding endogenous tiny RNA of about 22 nucleotides of a class length, right with various biological function
Grow, physiological function especially produces significant impact, compared with mRNA, miRNA in processes such as the generation development of malignant tumour
More stable in vitro, the metabolism cycle is longer in vivo, thus is more suitable for tumor molecular marker.Multinomial research is found
The generation development of miRNA and lung cancer has substantial connection, and the research such as Yanaihara N shows that patients with lung cancer there are about 66%
People at least more than 2 times of miR-21 expressions up-regulation, the research such as Weiss finds that the lung cancer of miRNA-128b missings is thin
Born of the same parents are very sensitive to epidermal growth factor recipient tyrosine kinase inhibitor Gefitinib.
The present invention carries out transcript profile sequencing to 4 pairs of cancerous tissues and the sample of cancer beside organism, and uses bioinformatics method
Analyzed, it was found that the molecular marker related to adenocarcinoma of lung being never reported before several, wherein, miR-6087,
MiR-7976 is expressed in cancerous tissue less than cancer beside organism, and the expression quantity of miR-7705, miR-6744-5p in cancerous tissue is high
In cancer beside organism, further, the analysis of high-flux sequence of fluorescent quantitative PCR experiment result verification shows miR-6087, miR-
7976th, miR-7705, miR-6744-5p probably turn into new adenocarcinoma of lung diagnosis marker, and the present invention is clinical adenocarcinoma of lung
Accurate diagnosis provide potential molecular marker, with important actual application value.
The content of the invention
It is an object of the invention to provide a kind of adenocarcinoma of lung diagnostic reagent, the adenocarcinoma of lung diagnostic reagent can detect lung gland
The expression of miRNA and/or its precursor in cancer sample, or the target gene of miRNA regulation and control expression, detect the miRNA
Selected from one of the following or several：miR-6087、miR-7976、miR-7705、 miR-6744-5p.
It is preferred that, the miRNA is miR-7705 or miR-6744-5p or combination.
It is preferred that, the miRNA is miR-7705, miR-6744-5p and miR-6087 combination.
The miR-6087 sequences are shown in sequence table SEQ ID NO 1, and its precursor mir-6087 sequences are shown in sequence table SEQ ID
NO 2；The miR-7976 sequences are shown in sequence table SEQ ID NO 3, and its precursor mir-7976 sequences are shown in sequence table SEQ ID NO
4；The miR-7705 sequences are shown in sequence table SEQ ID NO 5, and its precursor mir-7705 sequences are shown in sequence table SEQ ID NO 6；
The miR-6744-5p sequences are shown in sequence table SEQ ID NO 7, and its precursor miR-6744-5p sequences are shown in sequence table SEQ ID NO
8。
Described sample is tumor tissues or peripheral blood.
Further, adenocarcinoma of lung diagnostic reagent and/or based on high-flux sequence method and/or based on quantifying PCR method be based on
Probing procedure detection adenocarcinoma of lung sample in miRNA and/or its precursor transcription or based on immunization method detect adenocarcinoma of lung sample
The expression of the target gene of the ripe miRNA regulation and control of its in this.
It is preferred to use northern hybridizing methods, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE methods, original
The transcription of miRNA and/or its precursor in position hybridization, bead-based flow-cytometry detection adenocarcinoma of lung sample.
It is preferred that, the described quantifying PCR method that is used for includes specific amplification miRNA and/or the primer of its precursor；It is described
Probe with miRNA and/or the nucleic acid array hybridizing of its precursor is included based on probing procedure.
Further, amplification miR-6087 primer is sequence SEQ ID NO 9；The primer for expanding miR-7976 is sequence
SEQ ID NO 10；The primer for expanding miR-7705 is sequence SEQ ID NO 11；The primer for expanding miR-6744-5p is sequence
Arrange SEQ ID NO 12.
The present invention also aims to provide above-mentioned miRNA and/or its precursor answering in adenocarcinoma of lung diagnostic tool is prepared
With.
Adenocarcinoma of lung pharmaceutical composition is treated it is an object of the invention to provide one kind, it is characterised in that the drug regimen
Thing is included：
(a) compound or composition, the compound lower miR-7705 or miR-6744-5p transcription and/or suppression
MiR-7705 or miR-6744-5p maturations miRNA activity；
(b) receptible carrier in pharmacy.
Further, using ASON, antagomiRs, miRNA sponge, miRNA Erasers, Target
The method of Masking and/or Mutiple Targets ASON lowers miR-7705 or miR-6744-5p transcription and/or blocking
MiR-7705 or miR-6744-5p activity.
Adenocarcinoma of lung pharmaceutical composition is treated it is an object of the invention to provide one kind, it is characterised in that the drug regimen
Thing is included：
(a) compound or composition, the transcription of the compound up-regulation miR-6087 or miR-7976 and/or promotion miR-
6087 or miR-7976 maturations miRNA activity；
(b) receptible carrier in pharmacy.
Further, using the microRNA gain-of-functions technology based on RNA and/or gene specific miR Mimics skills
Art up-regulation miR-6087 or miR-7976 transcription and/or the activity for promoting its ripe miRNA.It is preferred that artificial synthesized miR-6087
MiR-7976 maturations miRNA short hairpin RNA (short hairpin RNA, shRNA) or by regulate and control promoter raise
MiR-6087 or miR-7976.
Definition：
The method of detection miRNA expression is mainly included based on high throughput sequencing technologies, based on nucleotides at this stage
Hybridization and the miRNA detection methods of PCR-based.MiRNA detection methods based on probe hybridization technique are a kind of direct Detection Methods,
Sample rna need not in advance be expanded, including northern hybridizing methods, miRNA chip of expression spectrum, ribozyme protection analysis skill
The technologies such as art, RAKE methods, in situ hybridization, bead-based flow-cytometry.
(1) Northern hybridizes
Also known as Northern blot is most classical detection eucaryote RNA sizes, estimates the experimental method of its abundance.Base
Present principles are as follows：MiRNA samples are fixed on carrier (such as silicon chip, microballoon or film) first, then it is miscellaneous with the probe of process mark
Hand over, signal detection is carried out after washing unnecessary hybridization probe；Can also the first fixed and target miRNA sequence complementation on carrier
DNA probe, then with the sample miRNA hybridization by mark, then carries out signal detection.The method of signal mark includes isotope
Mark, fluorescence labeling and nano gold mark etc..
(2) miRNA chip of expression spectrum
Principle is equally that the target molecule on solid support is detected using label probe.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 high-throughout excellent
Point, can once detect whole expression of hundreds of genes in same sample.The liquid-phase chip that Luminex companies develop
(Liquid chip) is also known as multifunctional suspending dot matrix (Multi analyte suspension array, MASA), is new
Generation biochip technology.Liquid-phase chip system is made up of many spherulas for main matrix, is fixed with not on every kind of spherula
With probe molecule, in order to distinguish different probes, sphere matrix that each is used for label probe is all unique with one
Color numbers, these spherulas are suspended in a liquid-phase system, liquid-phase chip system is just constituted.The system can be to same
Multiple different moleculars 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 is carried out in aaerosol solution, detection speed pole
It hurry up.
(3) ribozyme protection analytical technology (RPA)
MiRNA detection can also protect analytical technology using ribozyme, and the probe marked and RNA samples to be measured are mixed
Close, hybridize after thermal denaturation, non-hybridized RNA and the single-chain nucleic acid enzymic digestion of unnecessary probe, purify after heat inactivation nuclease by
The RNA molecule of protection, finally by denaturation PAGE electrophoretic separation probes, colour developing.This new method based on solution hybridization is simple
Quickly, sensitivity is high, but is also only used for analyzing known miRNA.
(4) RAKE methods
RAKE methods (RNA primed array based Klenow emzyme) are the bases in miRNA microarray
The Klenow fragments of DNA polymerase i are utilized on plinth, make miRNA and the method for fixed DNA probe hybridization.RAKE can be sensitive
Specifically detect miRNA, it is adaptable to largely quickly screen all miRNA that oneself knows.Can be in specific cell and tumour
Detect miRNA express spectra situations.Moreover, RAKE methods can also be from the tissue of the FFPE secured by formalin
In isolate miRNA and it analyzed, be the door that analysis miRNA opens hope from sample is achieved.
(5) in situ hybridization (in situ hybridization)
Hybridization in situ technique can intuitively understand miRNA expression ways, be a kind of easier of observation miRNA spatial and temporal expressions
Method, normal mark mode includes digoxin, biotin, fluorescence labeling 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, had concurrently by this method with chip technology
Flux is big, detection speed is fast, sensitivity is high and it is specific good the features such as.
(7) Real-Time Fluorescent Quantitative PCR Technique (Real-time PCR, RT-PCR)
Fluoroscopic examination PCR instrument can draw dynamic changing curve to the cumulative speed of extension increasing sequence during whole PCR.Anti-
Answer the initial concentration of target sequence in mixed system bigger, it is desirable to which the PCR cycle number for obtaining amplified production specific output is (general
Expressed with specific threshold period Ct) it is fewer.Because miRNA length is only 22nt, traditional qRT-PCR is not suitable for amplification such as
This short fragment.There are several real time quantitative PCR methods for miRNA, such as tailing method, neck ring method now.Neck ring method is one
Plant preferable miRNA detections qRT-PCR methods：Special loop-stem structure primer is designed first, it is inverse using miRNA to be measured as template
The transcription synthesis chains of cDNA first, the cDNA one end is stem Loop primer, and stem loop structure, which is opened, substantially increases cDNA length
Degree, then carries out real-time quantitative PCR amplification by template design primer of the cDNA of synthesis.QRT-PCR has specificity high, sensitive
Spend, a variety of advantages such as quick and easy.
(8) PCR sequencing PCR
Most of known miRNA is to be sequenced to find and identify by cDNA clone.The method needs first to build miRNA
CDNA library, then enter performing PCR amplification, amplified production be then cloned on expression vector be sequenced.Takada develops one kind and changed
The amplification PCR cloning PCR (miRNA amplification profiling, mRAP) entered, mRAP methods are first connected at miRNA 3 ' ends
Joint, then with the reverse transcription primer reverse transcription complementary with joint.Because specific reverse transcriptase has end deoxynucleotide
Enzymatic activity, some nucleotides (mainly deoxycytidylic acid) can be connected to 3 ' ends of the cDNA chains that reverse transcription goes out.When 5 ' terminations
Head is the achievable PCR amplifications to cDNA with after poly (C) cohesive end annealing of cDNA chains, adding a pair of general primers.By
In mRAP High sensitivities, the expression quantity of miRNA in a small amount of tissue can be directly detected with clone and sequencing technologies.Sequence label gram
Grand method is that one kind is developing 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 generating 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 sequencing technologies (next of future generation
Generation sequencing) it is the change to tradition sequencing revolution, once to hundreds of thousands to millions of DNA
Molecule carries out sequencing, greatly improves sequencing efficiency.This kind of large scale sequencing technology greatly improves multiple species and lost
The solution reading rate of information is passed, to obtain all miRNA sequence information, decryption miRNA collection of illustrative plates, which is provided, to be ensured.While high pass
The analysis that sequence to carry out the transcript profile and genome of a species in careful overall picture is measured, so the depth that is otherwise known as
Degree sequencing (deep sequencing).The representative of high-flux sequence platform is 454 sequenator (Roch of Roche Holding Ag (Roche)
GSFLX sequencer), the Solexa genome analysises instrument (Illumina Genome Analyzer) of Illumina companies
With ABI SOLiD sequenators (ABI SOLiD sequencer).
MicroRNA gain-of-functions technology based on RNA be by exogenous supplement miRNAs synthesis precursor substance come
Raise miRNAs level.For example, can be with the artificial synthesized bob folder sample RNA (short consistent with endogenous miRNA sequence
Hairpin RNA, shRNA), promoter is done by polymerase II or III, with virus for carrier transfectional cell, by Dicer enzyme modifications
RISC is loaded into afterwards to play a role, it is lasting equivalent to rise pre-miRNA level, action effect stabilization.
This technique avoids the nonspecific action of miRNA and gene for gene specific miR Mimics technologies.This people
The specific oligonucleotide chain combined with the UTR of target gene 3 ' complementations of work synthesis, can be played after being transcribed with miRNA identicals
Adjustment effect.
The carrier permitted in the pharmacy of the pharmaceutical composition of the present invention is the load generally utilized in preparation
Body, the carrier includes lactose (lactose), dextrose (dextrose), sucrose (sucrose), sorbierite (sorbitol), sweet
Reveal alcohol (mannitol), starch, acacia gum, calcium phosphate, alginates (alginate), gel (gelatin), calcium silicates,
Microcrystalline cellulose, polyvinylpyrrolidone (polyvinylpyrrolidone), cellulose (cellulose), water, syrup, first
Base cellulose (methyl cellulose), methyl hydroxybenzoate (methyl hydroxybenzoate), propyl hydroxy benzene
Propyl formate (propyl hydroxybenzoate), talcum, magnesium stearate (stearic acid magnesium) and mineral
Oily (mineral oil) etc., but it is not limited to this.
The pharmaceutical composition of the present invention can easily be implemented according to general technical staff of the technical field of the invention
Method, carried out using receptible carrier in pharmacy and/or excipient it is formulation, so as to unit dose form
Prepare or interior prepare in the multicapacity container.Now, formulation be the solution of oiliness or aqueous medium, suspension or
Emulsion form, or can also be extract, powder agent, granule, tablet or capsule form, it can also include scattered
Agent or stabilizer.
Brief description of the drawings
Fig. 1 is the ROC curve figure that miR-6087, miR-7976 diagnose adenocarcinoma of lung respectively
Fig. 2 is the ROC curve figure that miR-7705, miR-6744-5p diagnose adenocarcinoma of lung respectively
Fig. 3 is the ROC curve figure of miR-7705 and miR-6744-5p Combining diagnosis adenocarcinomas of lung
Fig. 4 is the ROC curve figure of three kinds of miRNA Combining diagnosis adenocarcinomas of lung
Embodiment
With reference to specific embodiment, the present invention is expanded on further, is only used for explaining the present invention, and it is not intended that to this
The limitation of invention.It will be understood by those skilled in the art that：Can in the case where not departing from the principle and objective of the present invention
So that these embodiments are carried out with a variety of change, modification, replacement and modification, the scope of the present invention is limited by claim and its equivalent
It is fixed.The experimental method of unreceipted actual conditions in the following example, generally according to normal condition or according to the bar proposed by manufacturer
Part examinations.
The collection of the sample of embodiment 1
4 pulmonary adenocarcinomas and cancer beside organism are all from Beijing Friendship Hospital's surgery excision in July, -2016 in January, 2015
Sample, all samples were put into liquid nitrogen container within vitro 10 minutes, were subsequently transferred to store in -80 DEG C of refrigerators.
The Total RNAs extraction of embodiment 2
1 extracting method
1) 80mg tissue blocks are taken, 800 μ l Lysis/Binding buffer solutions are added, tissue block are 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 are added into the above-mentioned tissue sample being homogenized, are put on ice
Put 10min.
3) water-saturated phenol with Lysis/Binding buffer solution equivalent volumes is added, 45s, 10,000 × g room temperatures is shaken
Centrifuge 5min.
4) the careful supernatant that takes out adds the absolute ethyl alcohol of 1.25 times of volumes into new test tube, after mixing, moves into purification column
In, 10,000 × g centrifuges 15s, outwells the liquid in collecting pipe.Because the maximum volume of pillar only has 700 μ l, therefore again
This multiple step operation, until all supernatants all filter completion.
5) 700 μ l miRNA eluents 1 are added into centrifugation pillar, room temperature, 10,000 × g centrifuges 15s, outwells collection
Liquid, uses new collecting pipe instead.
6) added again with 500 μ l eluents 2/3 in centrifugal column, 10,000 × g, centrifuge 10s, the step for repeating is once.
7) 1min, 10,000 × g are centrifuged, unnecessary liquid is discarded.
8) aforesaid liquid is transferred to new centrifuge tube, plus 100 μ l, 95 DEG C of preheatings DEPC processing 30s, 10,000 ×
G, centrifugation.
9) RN A concentration and 260nm/280nm ratio are determined using nanodrop.
10) RNA obtained is stored in -80 DEG C of refrigerators.
2 extraction standards
Determine RNA concentration and 260nm/280nm ratio：The purity requirement of total serum IgE is that OD260/OD280 values should be 1.8
To between 2.2；The detection of RNA integralities：RNA integrality is detected with 1% agarose gel electrophoresis；
According to the requirement of sequencing company, tiny RNA sequencing more than the μ g of total amount 3, concentration is in more than 300ng/ μ l.
Embodiment 3 is sequenced and data analysis
The foundation and the sequencing of upper machine of sequencing library are carried out by sequencing company, used sequenator is Illumina companies
HiSeq2000 sequenators.
The data provided according to sequencing company carry out statistical analysis, fdr<0.001, log2 (FC) absolute value>1, two groups
The difference of count average values is more than 100.It is several to differential expression miRNA people to select the obvious miRNA of differential expression in filtering
The molecular marker related to adenocarcinoma of lung being never reported before enter our research range, wherein miR-6087,
MiR-7976 is expressed in cancerous tissue less than cancer beside organism, and the expression quantity of miR-7705, miR-6744-5p in cancerous tissue is high
In cancer beside organism.
MiRNA expression in embodiment 4Real-time PCR detection adenocarcinoma of lung peripheral blood samples
1 sample collection：
32 adenocarcinoma of lung patient peripheral blood samples and 24 normal healthy controls peripheral blood samples are all from Beijing Friendship Hospital, suffer from
Disease group is made a definite diagnosis by pathological examination.
2 miRNA are extracted：
Use the GenElute of sigma companiesTMPlasma/serum RNA small scale purification kits (production code member RNB500),
Concrete operations are with reference to kit operational manual.
3 miRNA reverse transcriptions
The RT systems of table 1
| Component | Concentration | Volume (μ l) |
| Total RNA | - | 1μg |
| miScript HiSpec Buffer | 5× | 4 |
| Nucleics Mix | 10× | 2 |
| miScript Reverse Transcriptase Mix | - | 2 |
| Nuclease-free H2O | - | Filling-in is to 20 |
After 37 DEG C of insulation 60min make reverse transcription reaction complete in the type PCR instruments of ABI 9700,95 DEG C of 5min terminating reactions.Plus
Enter 80 μ l Nuclease-free H2O is diluted to 100 μ l and is stored in -20 DEG C of refrigerators, for subsequent experimental.
4 quantitative fluorescent PCRs
The RT-PCR systems of table 2
MiRNAs detection of expression sets 3 parallel tube reactions every time, and microRNA-specific primer primers are shown in
Table 3, internal reference is used as using general snRNA U6.
The microRNA-specific primer primers of table 3
| Primer | Numbering | Sequence |
| MiR-6087 primers | SEQ ID NO 9 | TGAGGCGGGG GGGCGAGC |
| MiR-7976 primers | SEQ ID NO 10 | TGCCCTGAGA CTTTTGCTC |
| MiR-7705 primers | SEQ ID NO 11 | AATAGCTCAG AATGTCAGTT CTG |
| MiR-6744-5p primers | SEQ ID NO 12 | TGGATGACAG TGGAGGCCT |
PCR programs：95℃10min；40 circulations (95 DEG C of 10s, 60 DEG C of 30s).Circulation utilizes melting curve inspection after terminating
Survey product specificities：97 DEG C, 5 fluorescence signals of every DEG C of collection are to slowly warm up to from 60 DEG C.
5 statistical analysis
Analyzed using OriginPro8.1 softwares.Compare between statistical method mean and examined using t, P<0.05 (difference
Significantly) and P<0.01 (difference highly significant) is set to statistically significant.As a result show compared with normal healthy controls, adenocarcinoma of lung group
MiR-7705 and miR-6744-5p are significantly raised, and respectively may be about 2.5 times and 4.7 times of control group, and miR-6087 and miR-
7976 slightly reduce, respectively the 0.65 of control group times and 0.6 times.RT-PCR results are consistent with high flux data results.
The evaluation analysis of the diagnostic of embodiment 5
It is to set up Receiver Operating Characteristics for single miRNA molecule or the method for the efficiency evaluation of diagnostic model
(receiver operating characteristic, ROC) curve, passes through (the Area Under of area under calculated curve
Curve) come judge diagnosis ability.We download the data in TCGA databases about adenocarcinoma of lung, acquisition data set (including
46 control groups and 519 case groups), and then analyzed, as a result shown, miR-6087, miR-7976, miR-7705 and
The AUC of miR-6744-5p diagnosis adenocarcinomas of lung is 0.452,0.460,0.755 and 0.318 (see Fig. 1 and Fig. 2), except miR-7705
AUC with miR-6744-5p Combining diagnosis adenocarcinomas of lung is 0.813 outer (see Fig. 3), other miR-7705 and other miRNA group
Close the AUC that AUC is respectively less than independent miR-7705, miR-7705, miR-6744-5p and miR-7976 Combining diagnosis lung gland
The AUC of cancer is 0.744 (see on Fig. 4), and the AUC of miR-7705, miR-6744-5p and miR-6087 Combining diagnosis adenocarcinoma of lung is
0.768 (see under Fig. 4).The AUC of miR-6087, miR-7976, miR-7705 and miR-6744-5p Combining diagnosis adenocarcinoma of lung is
0.725.Analysis shows miR-7705 and miR-6744-5p Combining diagnosis adenocarcinoma of lung has Overlay.
Although describing the present invention with reference to various preferred embodiments, it will be appreciated by those skilled in the art that can carry out each
Change is planted, and available equivalents substitute base region of its component without departing from the present invention.Come in addition, many changes can be carried out
Particular case or material is set to be suitable for the teachings of the present invention without departing from its base region.
Therefore, the present invention is not intended to be defined in the particular disclosed herein for being used to carry out the present invention；On the contrary,
This invention is intended to all embodiments including falling within the scope of the appended claims.
SEQUENCE LISTING
<110>Beijing Yang Shen biology information technologies Co., Ltd
<120>Adenocarcinoma of lung related miRNA, composition and its application
<160> 12
<170> PatentIn version 3.3
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Claims (10)
1. a kind of adenocarcinoma of lung diagnostic reagent, the adenocarcinoma of lung diagnostic reagent can detect in adenocarcinoma of lung sample miRNA and/or its before
The expression of body, or the target gene of miRNA regulation and control expression, detect that the miRNA is selected from one of the following or several：
miR-6087、miR-7976、miR-7705、miR-6744-5p。
2. adenocarcinoma of lung diagnostic reagent according to claim 1, it is characterised in that the miRNA be miR-7705 and/or
miR-6744-5p。
3. adenocarcinoma of lung diagnostic reagent according to claim 1, it is characterised in that the miRNA is miR-7705, miR-
6744-5p and miR-6087 combination.
4. adenocarcinoma of lung diagnostic reagent according to claim 1, it is characterised in that adenocarcinoma of lung diagnostic reagent is measured based on high pass
Sequence method and/or based on quantifying PCR method and/or based on probing procedure detection adenocarcinoma of lung sample in miRNA and/or its before
The transcription of body or the expression that the target gene that the ripe miRNA of its in adenocarcinoma of lung sample regulates and controls is detected based on immunization method, preferably
Using northern hybridizing methods, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE methods, in situ hybridization, based on micro-
The transcription of miRNA and/or its precursor in the Flow cytometry adenocarcinoma of lung sample of ball.
5. adenocarcinoma of lung diagnostic reagent according to claim 4, it is characterised in that specificity is included based on quantifying PCR method
Expand miRNA and/or the primer of its precursor；Based on probing procedure including miscellaneous with the nucleotide sequence of miRNA and/or its precursor
The probe of friendship, it is preferred that amplification miR-6087 primer is list SEQ ID NO 9；The primer for expanding miR-7976 is list
SEQ ID NO 10；The primer for expanding miR-7705 is list SEQ ID NO 11；The primer for expanding miR-6744-5p is list
SEQ ID NO 12。
6. adenocarcinoma of lung diagnostic reagent according to claim 1, it is characterised in that sample is tumor tissues or peripheral blood.
7. application of the adenocarcinoma of lung diagnostic reagent in adenocarcinoma of lung diagnostic tool is prepared described in claim 1-6.
8. one kind treats adenocarcinoma of lung pharmaceutical composition, it is characterised in that described pharmaceutical composition is included：(a) compound or combination
Thing, the compound lower miR-7705 or miR-6744-5p transcription and/or suppress miR-7705 or miR-6744-5p into
Ripe miRNA activity, or raise miR-6087 or miR-7976 transcription and/or promote miR-6087 or miR-7976 ripe
MiRNA activity；(b) receptible carrier in pharmacy.
9. pharmaceutical composition according to claim 8, it is characterised in that using ASON, antagomiRs,
MiRNA sponges, miRNA Erasers, the method for Target Masking and/or Mutiple Targets ASON lower miR-
7705 or miR-6744-5p transcription and/or the activity for blocking miR-7705 or miR-6744-5p.
10. pharmaceutical composition according to claim 8, it is characterised in that use the microRNA gain-of-functions based on RNA
Property technology and/or gene specific miR Mimics technologies up-regulation miR-6087 or miR-7976 transcription and/or promote its into
Ripe miRNA activity.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109161596A (en) * | 2018-10-10 | 2019-01-08 | 四川大学华西医院 | The application of miR-129 and its target gene in detection adenocarcinoma of lung |
| CN111424093A (en) * | 2020-03-30 | 2020-07-17 | 中国医学科学院肿瘤医院 | Kit, device and method for lung cancer diagnosis |
| CN113322327A (en) * | 2021-08-02 | 2021-08-31 | 北京泱深生物信息技术有限公司 | Biomarker-based product for predicting lung cancer prognosis and related application |
-
2017
- 2017-06-01 CN CN201710405001.8A patent/CN107058579A/en not_active Withdrawn
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109161596A (en) * | 2018-10-10 | 2019-01-08 | 四川大学华西医院 | The application of miR-129 and its target gene in detection adenocarcinoma of lung |
| CN109161596B (en) * | 2018-10-10 | 2019-05-24 | 四川大学华西医院 | The application of miR-129 and its target gene in detection adenocarcinoma of lung |
| CN111424093A (en) * | 2020-03-30 | 2020-07-17 | 中国医学科学院肿瘤医院 | Kit, device and method for lung cancer diagnosis |
| CN111424093B (en) * | 2020-03-30 | 2021-10-15 | 中国医学科学院肿瘤医院 | Kit, device and method for lung cancer diagnosis |
| CN113322327A (en) * | 2021-08-02 | 2021-08-31 | 北京泱深生物信息技术有限公司 | Biomarker-based product for predicting lung cancer prognosis and related application |
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