CN104784705A

CN104784705A - Cancer metastasis inhibitor and application thereof

Info

Publication number: CN104784705A
Application number: CN201510243490.2A
Authority: CN
Inventors: 杨承刚; 边洋; 任静
Original assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Current assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Priority date: 2015-05-13
Filing date: 2015-05-13
Publication date: 2015-07-22

Abstract

The invention relates to a cancer metastasis inhibitor and an application thereof, and particularly relates to a novel application of mir-518a-1 and/or manure miRNA thereof in diagnosis and treatment of lung adenocarcinoma metastasis. According to sequencing analysis of a lung adenocarcinoma metastasis group and a lung adenocarcinoma non-metastasis group, mir-518a-1 in lung adenocarcinoma metastasis group is remarkable, and target genes MKI67, FOXM1 and MID1 of miR-518a-1-5p are found. According to the further molecular biology verification results, the miR-518a-1 is closely related to lung adenocarcinoma metastasis, and the expression of proteins of the target genes MKI67, FOXM1 and MID1 of miR-518a-1 can be effectively reduced by excessively expressing miR-518a-1-5p, and therefore, the cancer metastasis inhibitor can be used for clinical diagnosis and prevention/detection, and is very high in practical application value.

Description

A kind of cancer transfer inhibitor and application thereof

Technical field

The present invention relates to biology field, be specifically related to a kind of cancer transfer inhibitor and application thereof, relate to the novelty teabag of mir-518a-1 and/or its ripe miRNA in the transfer of diagnosis and treatment adenocarcinoma of lung more specifically.

Background technology

MiRNA transcribes generation by rna plymerase ii (Pol II) usually.After Pol II is combined in formed hairpin structure neck ring DNA sequence near.The transcript modified interpolation 5 ' cap sequence generated and 3 ' end polyadenylic acid tail structure, and shear, the product generated is called elementary miRNA (pri-miRNA), and this product may reach thousands of or hundreds of nucleotide, may comprise multiple miRNA ring structure.

Single pri-miRNA may contain one to six miRNA precursors.The each nucleotide by about about 70nt of these hairpin structures forms.Each hairpin structure is accompanied by partial sequence and is beneficial to effective shear treatment.Double stranded hairpin RNA structure in pri-miRNA is called nucleoprotein (the DiGeorge Syndrome CriticalRegion 8) identification of DGCR8, and DGCR8 forms micro-process (microprocessor) complex together with Drosha enzyme.In this complex, DGCR8 organizes the RNase III domain of Drosha albumen to make it at distance hairpin structure about 11 nucleotide place cutting pri-miRNA, makes it discharge hairpin structure.The hairpin structure of release is front miRNA (pre-miRNA), and pre-miRNA exists two unsettled nucleotide 3 ', and pre-miRNA 5 ' is phosphoric acid group, and 3 ' is hydroxy groups.

In endochylema, pre-miRNA hairpin structure is through RNase III Dicer cutting process.3 ' of this endogenous rnase (endoribonuclease) and hairpin structure interacts and at 3 ' and 5 ' of ring, arm completes cutting, and generation is about the miRNA:miRNA* duplex structure of 22nt not Perfect Matchings.

The typical effect mode of miRNA and said target mrna mainly contains two kinds.In most of the cases, the incomplete complementary pairing of 3 ' UTR of the strand miRNA in complex and said target mrna, blocks the translation of target gene, thus regulator gene is expressed.This mode major effect protein expression level, does not affect the stability of mRNA.Recently, research is had to query to Translational repression theory, find that repressed said target mrna s and miRNAs is gathered in endochylema the region being called as P corpusculum (processing bodies, P-bodies) jointly, this region also concentrates the enzyme of many participation mRNA degraded.P corpusculum may be the container carrying out temporary transient reversible storage as untranslated mRNA, and the expression reducing some specific P corpusculum constitutive proteins can relax the gene expression inhibition effect of miRNA mediation.P corpusculum is the certain area in endochylema, it comprises the multiple protein of transcribing rear process of participation, such as: mRNA degraded (mRNA degradation), nonsense mediation mRNA decline (nonsense-mediated mRNA decay, NMD), the gene silencing (RNA-mediated gene silencing) of Transcription inhibition and RNA mediation.

Another kind of model of action is similar with siRNA, and when miRNA and mRNA complete complementary matches, Ago2 albumen passes through to cut mRNA and directly causes it to degrade, and realizes gene silencing.Participate in for siRNA RNAi: siRNA can be combined with RISC, and as template identification mRNA target, by base pair complementarity principle, the antisense strand in mRNA and siRNA combines, and displaces positive-sense strand.Double-strand mRNA produces the siRNA of about 22nt under Dicer enzyme, ATP and unwindase combined effect, and siRNA continues to form complex with RISC, is combined, makes mRNA by RNA enzymatic lysis with the mRNA of siRNA complementation.This process is also referred to as PTGS (PTGS).

In a word, currently think that miRNA is relevant with the pairing degree of genes of interest effect and miRNA and genes of interest in which way.When miRNA and genes of interest match incomplete, miRNA is just to suppress the expression of genes of interest to play a role; When miRNA and genes of interest section sequence are matched complete, genes of interest just may be caused to cause gene silencing in the fracture of complementary district.In addition, miRNAs sometimes also causes the DNA methylation of histidine modification and promoter region, thus affects the expression of target gene.In addition, recently find that de-polyadenylation (accelerated deadenylation) is the new mechanism of miRNA inhibition of gene expression fast.In mammalian cell, find that miR-125b and let-7 can promote the removal of mRNA poly A tail (polyA tail).Poly A tail is replaced with 3 ' histone stem-ring structure, not only can eliminate the impact of miR-125b on mRNA content, can also reduce the effect to protein synthesis, visible miRNA carrys out inhibition of gene expression by the concentration reducing translation efficiency and polyadenylation mRNA.

Adenocarcinoma of lung (lung adenocarcinoma) belongs to nonsmall-cell lung cancer, easily betides women and nonsmoker.The normal comparatively periphery in the position of pulmonary, the speed comparatively slow (about 120 days doubling times) of tumor enlargement.Early stage without sign, be late period when being usually diagnosed.Nonsmall-cell lung cancer accounts for the 75%-80% of pulmonary carcinoma sum.Death in China caused by pulmonary carcinoma accounts for about 23% of whole tumor associated death, the death of the malignant tumor of about 90% is relevant with neoplasm metastasis, most patients with lung cancer is due to the local challenge of tumor cell and metastasis, and when making a definite diagnosis, oneself is through losing surgical engine meeting.Therefore, understand the factor and possible mechanism that participate in invasion of lung cancer and transfer in depth, provide guidance, to the prognosis of pulmonary carcinoma can be improved for the early intervention of pulmonary carcinoma and individualized treatment.

The present invention is by carrying out retrospective analysis to patients with lung adenocarcinoma, be divided into two groups: adenocarcinoma of lung transfer group and non-diverting group, sequencing analysis is carried out by Illumina platform, obtain miRNA expression data, and then carry out bioinformatic analysis, find that the low expression of mir-518a-1 is remarkable in adenocarcinoma of lung transfer group, finds target gene MKI67, FOXM1 and MID1 of miR-518a-1-5p simultaneously.Further molecular biology the result display, mir-518a-1 and adenocarcinoma of lung shift closely related, process LAN miR-518a-1-5p effectively can reduce the expression of the albumen of its target gene MKI67, FOXM1 and MID1, can be used for clinical diagnosis and prevention detection, there is good actual application value.

Summary of the invention

The present invention also aims to provide the application of mir-518a-1 and/or its ripe miRNA in preparation prevention, diagnosis and/or treatment adenocarcinoma of lung transfering reagent.The sequence of mir-518a-1 is shown in sequence table SEQ ID NO 1.The ripe miRNA of mir-518a-1 is that its sequence of miR-518a-1-5p and miR-518a-1-3p is shown in sequence table SEQ ID NO 2 (miR-518a-1-5p) and SEQ ID NO 3 (miR-518a-1-3p).

Further, described prevention, Diagnosis of pulmonary adenocarcinoma transfering reagent comprises based on high-flux sequence method and/or based on quantifying PCR method and/or the expression transcribing or detect based on immunologic detection method the target gene of its ripe miRNA regulation and control in adenocarcinoma of lung sample detecting mir-518a-1 and/or its ripe miRNA in adenocarcinoma of lung sample based on probing procedure, preferred employing northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, based on transcribing of mir-518a-1 and/or its ripe miRNA in the Flow cytometry adenocarcinoma of lung sample of microsphere, ELISA and/or colloidal gold strip is adopted to detect the expression of the target gene of its ripe miRNA regulation and control in adenocarcinoma of lung sample.The target gene of the ripe miRNA regulation and control of preferred described mir-518a-1 is FOXM1, MID1, MKI67.

Preferably, the described primer comprising specific amplification mir-518a-1 and/or its ripe miRNA based on quantifying PCR method, preferably specific amplification mir-518a-1 primer sequence is the ripe miRNA primer sequence of SEQ ID NO 4 and SEQ ID NO 5, specific amplification mir-518a-1 is further SEQ ID NO 6; Described comprises the probe with the nucleic acid array hybridizing of mir-518a-1 and/or its ripe miRNA based on probing procedure; Described immunologic detection method comprises the antibody that miRNA regulate gene expression protein-specific ripe with it is combined, and the target gene of preferably regulation and control is further the antibody that FOXM1, MID1, MKI67 protein-specific combines.

Further, described treatment adenocarcinoma of lung transfering reagent comprises the reagent of activity that transcribing and/or promoting its ripe miRNA raising mir-518a-1 and/or its ripe miRNA.Preferably, the activity transcribing and/or promote its ripe miRNA raising mir-518a-1 and/or its ripe miRNA based on the microRNA gain-of-function technology of RNA and/or gene specific miR Mimics technology is adopted.More preferably the ripe miRNA of synthetic mir-518a-1 short hairpin RNA (short hairpin RNA, shRNA) or raise mir-518a-1 by regulation and control promoter.

The object of the present invention is to provide one to treat adenocarcinoma of lung diversion medicaments compositions, it is characterized in that, described pharmaceutical composition comprises:

A () is raised transcribing of mir-518a-1 and/or its ripe miRNA and/or is promoted the reagent of activity of its ripe miRNA;

Receptible carrier on (b) pharmaceutics.

Further, the activity transcribing and/or promote its ripe miRNA raising mir-518a-1 and/or its ripe miRNA based on the microRNA gain-of-function technology of RNA and/or gene specific miRMimics technology is adopted.The short hairpin RNA (short hairpin RNA, shRNA) of the ripe miRNA of preferred synthetic mir-518a-1 or raise mir-518a-1 by regulation and control promoter.

The object of the present invention is to provide a kind of adenocarcinoma of lung to shift diagnostic reagent, described adenocarcinoma of lung transfer diagnostic reagent can detect the expression that transcribing of mir-518a-1 and/or its ripe miRNA in adenocarcinoma of lung sample or immunologic detection method detect the target gene of its ripe miRNA regulation and control in adenocarcinoma of lung sample.

Further, described adenocarcinoma of lung transfer diagnostic reagent is based on high-flux sequence method and/or based on quantifying PCR method and/or the expression transcribing or detect based on immunization method the target gene of its ripe miRNA regulation and control in adenocarcinoma of lung sample detecting mir-518a-1 and/or its ripe miRNA in adenocarcinoma of lung sample based on probing procedure, preferred employing northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, based on transcribing of mir-518a-1 and/or its ripe miRNA in the Flow cytometry adenocarcinoma of lung sample of microsphere, ELISA and/or colloidal gold strip is adopted to detect the expression of the target gene of its ripe miRNA regulation and control in adenocarcinoma of lung sample.The target gene of the ripe miRNA regulation and control of preferred described mir-518a-1 is FOXM1, MID1, MKI67.

Preferably, the described primer comprising specific amplification mir-518a-1 and/or its ripe miRNA for quantifying PCR method; Described comprises the probe with the nucleic acid array hybridizing of mir-518a-1 and/or its ripe miRNA based on probing procedure; Described immunologic detection method comprises the antibody that miRNA regulate gene expression protein-specific ripe with it is combined, and the target gene of preferably regulation and control is further the antibody that FOXM1, MID1, MKI67 protein-specific combines.

Definition:

Present stage, the method for expression that detects miRNA mainly comprised based on high throughput sequencing technologies, miRNA detection method based on nucleotide hybridization and PCR-based.MiRNA detection method based on probe hybridization technology is a kind of direct Detection Method; do not need to increase in advance to sample rna, comprise northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, the technology such as flow cytometry based on microsphere.

(1) Northern hybridization

Also known as the detection eukaryote RNA size that RNA engram technology is the most classical, estimate the experimental technique of its abundance.Ultimate principle is as follows: first at the upper fixing miRNA sample of carrier (as silicon chip, microsphere or film etc.), then with the probe hybridization through labelling, carry out signal detection after washing unnecessary hybridization probe; Also can first fix the DNA probe with the complementation of target miRNA sequence on carrier, then hybridize with the sample miRNA through labelling, then carry out signal detection.The method of signal labelling comprises isotopic labeling, fluorescent labeling and nano gold mark etc.

(2) miRNA chip of expression spectrum

Principle is the target molecule on usage flag probe in detecting solid support equally.By miR-96 gene in design chips and internal reference sequence, Accurate Analysis the expression of corresponding miRNA in sample can be gone out.Gene chip has high-throughout advantage, once can detect whole expression of a hundreds of gene in same sample.The liquid-phase chip (Liquid chip) of Luminex company development, also known as multifunctional suspending dot matrix (Multi analytesuspension array, MASA), is the biochip technology of new generation.Liquid-phase chip system is that main matrix is formed by many spherulas, often kind of spherula is fixed with different probe molecules, in order to distinguish different probes, each sphere matrix for label probe is all with a unique color numbers, these spherulas are suspended in a liquid-phase system, just constitute liquid-phase chip system.This system can carry out qualitative and quantitative analysis fast to the multiple different moleculars in same trace sample simultaneously, and this detection technique is called as FMAP (Flexible multianalyte profiling) technology.Molecular hybridization carries out in aaerosol solution, and detection speed is exceedingly fast.

(3) ribozyme protection analytical technology (RPA)

The detection of miRNA can also adopt ribozyme to protect analytical technology; the probe that labelling is good and RNA sample to be measured mixing; hybridize after thermal denaturation; the RNA of not hybridizing and unnecessary probe single-chain nucleic acid enzymic digestion; the shielded RNA molecule of purification after heat inactivation nuclease; finally by degeneration PAGE electrophoretic separation probe, colour developing.This new method based on solution hybridization is simple and quick, highly sensitive, but also can only be used for analyzing known miRNA.

(4) RAKE method

RAKE method (RNA primed array based Klenow emzyme) is the Klenow fragment utilizing DNA polymerase i on the basis of miRNA microarray, makes the method that miRNA is hybridized with fixing DNA probe.RAKE sensitivity can detect miRNA specifically, is applicable to screen all miRNA that oneself knows fast in a large number.MiRNA express spectra situation can be detected in specific cell and tumor.Moreover, RAKE method can also be isolated miRNA and analyze it from the paraffin-embedded tissue secured by formalin, for analyzing the door that miRNA opens hope from file specimen.

(5) in situ hybridization (in situ hybridization)

Hybridization in situ technique can intuitively understand miRNA expression way, and be a kind of easier method of observation miRNA spatial and temporal expression, normal mark mode comprises digoxin, biotin, fluorescent labeling etc.In situ hybridization (Locked Nucleic Acid (LNA) based in situ hybridization (LNA-ISH)) on locked nucleic acid basis is the more probe mode of current application.

(6) based on the flow cytometry (bead-based flow cytometry) of microsphere

Be a kind of liquid-phase chip technology, FCM analysis organically combines with chip technology by the method, has that flux is large, detection speed is fast concurrently, a feature such as highly sensitive and specificity is good.

(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 in whole PCR process.In reaction mixture, the initial concentration of target sequence is larger, requires that the PCR period (generally expressing with specific threshold period Ct) obtaining amplified production specific output is fewer.Because miRNA length is only 22nt, traditional qRT-PCR is not suitable for increasing so short fragment.There is several real time quantitative PCR method for miRNA now, as tailing method, neck ring method etc.Neck ring method is that a kind of desirable miRNA detects qRT-PCR method: first design special loop-stem structure primer, with miRNA to be measured for template reverse transcription synthesis cDNA first chain, this cDNA one end is stem Loop primer, stem circulus is opened and substantially increases the length of cDNA, carries out real-time quantitative PCR amplification for template design primer subsequently with the cDNA of synthesis.QRT-PCR has that specificity is high, sensitivity good, the multiple advantage such as simple fast.

(8) sequencing

The known miRNA of major part is found by cDNA cloning and sequencing and identifies.This method needs the cDNA library first building miRNA, then carries out pcr amplification, and amplified production is cloned into subsequently on expression vector and checks order.Takada develops a kind of amplification cloning (miRNA amplification profiling, mRAP) of improvement, and mRAP method first connects joint, then with the reverse transcription primer reverse transcription with joint complementation at 3 ' of miRNA end.Because specific reverse transcription has terminal deoxynucleotidyl transferase activity, some nucleotide (mainly dCMP 1beta-Deoxyribofuranosylcytosine-5'-phosphate) can be connected to 3 ' end of the cDNA chain that reverse transcription goes out.After poly (C) sticky end of 5 ' end connector and cDNA chain is annealed, add the pcr amplification that a pair general primer can realize cDNA.Due to mRAP High sensitivity, the expression of miRNA in can directly organizing on a small quantity by Cloning and sequencing technology for detection.Sequence label cloning is that one has developed the higher miRAGE of detection efficiency (miRNA SAGE) cloning on the basis in serial analysis of gene expression (SAGE) technology, this method is by generating large sub-series, multiple miRNA can be detected by single sequencing reaction, significantly improve detection efficiency.

High-flux sequence (High-throughput sequencing) is the change to tradition order-checking revolution also known as sequencing technologies of future generation (nextgeneration sequencing), once to millions of DNA moleculars, sequencing is carried out to hundreds of thousands, greatly improve order-checking efficiency.This kind of large scale sequencing technology greatly improves the solution reading rate of multiple species hereditary information, and for obtaining the sequence information of all miRNA, deciphering miRNA collection of illustrative plates provides guarantee.Simultaneously high-flux sequence makes the analysis transcript profile of species and genome being carried out to careful overall picture become possibility, so degree of depth order-checking (deep sequencing) that is otherwise known as.The representative of high-flux sequence platform is 454 sequenators (Roch GSFLX sequencer) of Roche Holding Ag (Roche), the Solexa gene element analyzer (Illumina Genome Analyzer) of Illumina company and the SOLiD sequenator (ABI SOLiD sequencer) of ABI.

Immunologic detection method is using a kind of antibody or Multiple Antibodies as analytical reagent, carries out quantitatively or the detection method of qualitative analysis determinand.Its ultimate principle is the interaction between antibody and antigen.For improving the sensitivity of antigen and antibody test, by the material of easily display in known antibodies or antigenic mark, by certification mark thing, reflect with or without antigen antibody reaction, thus indirectly measure antigen or the antibody of trace.Conventional label has enzyme, fluorescein, radiosiotope, gold colloidal and electron dense substances etc.The specific reaction that on this antigen or antibody labeling, display object is carried out is called immunolabelling technique (immunolabelling technique).Immunoassay technology most widely used at present mainly contains: elisa (enzyme-linked immunosorbentassay, ELISA), colloidal gold immunity chromatography etc.

Elisa principle is combined antigen or antibody and substrate (enzyme), makes it keep the activity of immunoreation and enzyme.The antigen of labelling or antibody and the ligand binding that is coated on solid phase carrier, then make it and corresponding colorless substrate effect and Show Color, measure OD value result of determination according to the range estimation of colour developing depth degree or by microplate reader.

Colloidal gold strip is generally made up of sample pad, gold mark pad, chromatographic film, adsorptive pads four part.Chromatographic material has nitrocellulose membrane (NC), polyester film, nylon membrane and pvdf membrane etc., need to select the different film required according to test, wherein NC film is the most conventional, can determine whether to need activation or process according to test concrete condition before using, in most cases without the need to process, can directly use.Gold is marked protein solution even application on gold mark pad, dry for subsequent use under room temperature.NC film can catch a certain amount of bag by (antibody) and two anti-as detection line and nature controlling line.Finally sample pad, gold mark pad, NC film and absorbent paper are fixed on PVC board successively, test strips.

Namely microRNA gain-of-function technology based on RNA raises the level of miRNAs by the precursor substance of exogenous supplementary miRNAs synthesis.Such as, sample RNA (short hairpin RNA can be pressed from both sides by the synthetic bob consistent with endogenous miRNA sequence, shRNA), promoter is done by polymerase II or III, take virus as vector-transfected cell, being loaded into RISC after Dicer enzyme modification plays a role, and is equivalent to raise the level of pre-miRNA, and action effect is stable and lasting.

Gene specific miR Mimics technology this technique avoids the nonspecific action of miRNA and gene.This synthetic with the specific oligonucleotide chain that combines of target gene 3 ' UTR complementation, can play and identical with miRNA transcribe rear regulating action.

Being included in the carrier that the pharmaceutics of pharmaceutical composition of the present invention is permitted is the carrier usually utilized when preparation, this carrier comprises lactose (lactose), dextrose (dextrose), sucrose (sucrose), sorbitol (sorbitol), mannitol (mannitol), starch, acacia gum, calcium phosphate, alginate (alginate), gel (gelatin), calcium silicates, microcrystalline Cellulose, polyvinylpyrrolidone (polyvinylpyrrolidone), cellulose (cellulose), water, syrup, methylcellulose (methyl cellulose), methyl hydroxybenzoate (methyl hydroxybenzoate), propyl hydroxy propyl benzoate (propyl hydroxybenzoate), Talcum, magnesium stearate (stearic acid magnesium) and mineral oil (mineral oil) etc., but it is not limited thereto.

Pharmaceutical composition of the present invention can also comprise lubricant, wetting agent, sweeting agent, flavouring agent, emulsifying agent, suspending agent, antiseptic etc. except mentioned component.The carrier be applicable to that pharmaceutics is permitted and preparation are recorded in Lei Mingdengshi pharmacy pandect in detail.

Pharmaceutical composition of the present invention by oral or parenterally carry out administration, during as non-oral administration, by intravenous injection, intranasal injection, local injection, intracerebral ventricle injection, spinal cavity is injected, subcutaneous injection, lumbar injection, the modes such as percutaneous dosing carry out administration.

The dosage be applicable to of pharmaceutical composition of the present invention can carry out multiple prescription according to the factor of age of preparation ways, administering mode, patient, body weight, sex, morbid state, food, administration time, route of administration, drainage rate and being quick on the draw property and so on, usually, skilled doctor can easily determine and prescription to desired treatment or prevent effective dosage.

The method that pharmaceutical composition of the present invention can easily be implemented according to general technical staff of the technical field of the invention, to utilize on pharmaceutics receptible carrier and/or excipient formulation to carry out, thus can with the preparation of unit dose form or in be contained in multicapacity container and prepare.Now, dosage form is solution, suspension or emulsion form in oiliness or aqueous medium, or also can be extractum, powder agent, granule, tablet or capsule form, can also comprise dispersant or stabilizing agent.

Detailed description of the invention

Below in conjunction with specific embodiment, setting forth the present invention further, only for explaining the present invention, and can not limitation of the present invention be interpreted as.Those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.The experimental technique of unreceipted actual conditions in the following example, the usually conveniently conditioned disjunction condition examinations of advising according to manufacturer.

The collection of embodiment 1 sample

10 routine adenocarcinoma of lung tumor tissues are all from the specimen of Beijing Friendship Hospital's in January, 2009 in December ,-2009 excision, all specimen all put into liquid nitrogen container within vitro 10 minutes, be transferred to subsequently in-80 DEG C of refrigerators and store, followed up a case by regular visits to through 3 years, wherein 3 routine patients shift, and 7 routine patients shift.

Embodiment 2 Total RNAs extraction

1 extracting method

1) get 80mg piece of tissue, add 800 μ l Lysis/Binding buffer, use homogenizer to carry out homogenate to piece of tissue.In the process of homogenate, sample will be placed in and keep low temperature state on ice.

2) add 1/10 volume Homogenate Additive again in the tissue sample of above-mentioned homogenate, place 10min on ice.

3) water-saturated phenol with Lysis/Binding buffer equivalent volumes is added, concussion 45s, the centrifugal 5min of 10,000 × g room temperature.

4) the careful supernatant that takes out is in new test tube, adds the dehydrated alcohol of 1.25 times of volumes, after mixing, moves in purification column, and 10,000 × g, centrifugal 15s, outwell the liquid in collecting pipe.Maximum volume due to pillar only has 700 μ l, therefore repeats this step operation, until all supernatants have all filtered.

5) in centrifugal pillar, add 700 μ l miRNA eluents 1, room temperature, 10,000 × g, centrifugal 15s, outwell collection liquid, use new collecting pipe instead.

6) add in centrifugal column with 500 μ l eluents 2/3 again, 10,000 × g, centrifugal 10s, repeat this step once.

7) centrifugal 1min, 10,000 × g, discard unnecessary liquid.

8) aforesaid liquid is transferred to new centrifuge tube, add the DEPC process 30s of 100 μ l, 95 DEG C of preheatings, 10,000 × g, centrifugal.

9) nanodrop is used to measure the ratio of RN A concentration and 260nm/280nm.

10) RNA obtained is stored in-80 DEG C of refrigerators.

2 extract standard

Measure the ratio of RNA concentration and 260nm/280nm: the purity requirement of total serum IgE is that OD260/OD280 value should between 1.8 to 2.2; The detection of RNA integrity: the integrity detecting RNA with 1% agarose gel electrophoresis;

According to the requirement of order-checking company, tiny RNA order-checking total amount 3 more than μ g, concentration is at 300ng/ more than μ l.

Embodiment 3 checks order and data analysis

Foundation and the order-checking of upper machine of sequencing library is carried out, the HiSeq2000 sequenator that the sequenator used is Illumina company by order-checking company.

Data results according to company provides: transfer group (7 example) and non-diverting group (3 example) carry out statistical analysis, P value is less than 0.05, and the difference of transfer group and non-diverting group at least will differential expression miRNA more than more than 2 times, is select in filtration to lower to express the research range that obvious mir-518a-1 enters us to differential expression miRNA people.

The relation that embodiment 4 electronically validating mir-518a-1 and adenocarcinoma of lung shift

Adenocarcinoma of lung (transfer group vs non-diverting group) data screening

A. according in TCGA data base to patients with lung adenocarcinoma AJCC cancer staging record, patients with lung adenocarcinoma is divided into two groups: adenocarcinoma of lung transfer group, non-diverting group.Filter out 212 examples altogether not occur to shift patient; There is lymphatic metastasis or far-end transfer patient in 186 examples.

B. adenocarcinoma transfer group and non-diverting group patient mrna expression data, microRNA expression data in TCGA data base is downloaded.Following data carry out high flux mRNA and miRNA expression data confluence analysis.

The selection of table 1 data

By transcript profile data analysis software, adenocarcinoma of lung in TCGA data base (transfer group vs non-diverting group) miRNA and mRNA initial data is carried out to t-test after carrying out background correction and standardization and obtains P value, screening differential expression miRNA and mRNA, setting P value < 0.05, filter out the miRNA of 58 differential expressions altogether, the wherein gene 44 of expression rise, the gene 14 that expression is lowered.In the gene lowered, mir-518a-1 lowers and expresses obviously.

Embodiment 5 Real-time PCR detects the expression of mir-518a-1 in pulmonary adenocarcinoma

1 sample collecting:

104 routine adenocarcinoma of lung tumor tissues are all from the specimen of Beijing Friendship Hospital's in June, 2009 in December ,-2011 excision, all specimen all put into liquid nitrogen container within vitro 10 minutes, be transferred to subsequently in-80 DEG C of refrigerators and store, followed up a case by regular visits to through 3 years, wherein 21 routine patients shift, and 83 routine patients shift.

2 miRNA extract:

MiRNA is used to extract test kit (article No.: CW0627, health is century) concrete operations reference reagent box operating instruction.

3 design of primers:

4 quantitative fluorescent PCRs

Quantitative fluorescent PCR adopts UltraSYBR one-step method PCR kit for fluorescence quantitative (With ROX) (article No.: CW0660, health is century), and concrete operations are with reference to operating instruction.Internal reference uses actin.

5 statistical analysis

OriginPro8.1 software is adopted to analyze.Compare between statistical method mean and adopt t inspection, P<0.05 (significant difference) and P<0.01 (difference highly significant) is decided to be statistical significance, analyze the mir-518a-1 expression of transfer group and non-diverting group, in result display transfer group tissue, the expression of mir-518a-1 is starkly lower than non-diverting group tissue.

Embodiment 6 one kinds of adenocarcinoma of lung transfer detection kit

Test kit comprises: UltraSYBR one-step method PCR kit for fluorescence quantitative (With ROX) (article No.: CW0660, health is century) internal reference uses actin.

Primer:

MiRNA can also be comprised further and extract test kit (article No.: CW0627, health is century)

Embodiment 7 one kinds of adenocarcinoma of lung transfer detection kit

MiRNA reverse transcription

The preparation of RT system:

Component	Concentration	Volume (μ l)
			Total RNA	-	1μg
miScript HiSpec Buffer	5×	4
			Nucleics Mix	10×	2
miScript Reverse Transcriptase Mix	-	2
			Nuclease-free H ₂O	-	Up to 20
Total Volume	-	20

After in ABI 9700 type PCR instrument, 37 DEG C of insulation 60min make reverse transcription reaction completely, 95 DEG C of 5min cessation reactions.

Add 80 μ l Nuclease-free H ₂o is diluted to 100 μ l and is stored in-20 DEG C of refrigerators, for subsequent experimental.

Quantitative fluorescent PCR

The preparation of RT-PCR system:

The detection of expression of miRNAs arranges 3 parallel pipe reactions, using snRNA U6 as internal reference at every turn.

PCR program:

95 DEG C of 10min; 40 circulations (95 DEG C of 10s, 60 DEG C of 30s).Utilize melting curve to detect product specificities after loop ends: to be slowly warming up to 97 DEG C from 60 DEG C, every DEG C gathers 5 fluorescence signals.

The cultivation of embodiment 8 cell and transfection

A549 cultivates in RPMI1640 culture fluid and 10% hyclone, and SEAS-2B cultivates in BEGM culture fluid and 10% hyclone, is placed in 37 degrees Celsius, 5%CO ₂in incubator.Use Lipofectamine 2000 to carry out transfection in 6 well culture plates, step to specifications carries out cell transfecting.Transfection is carried out when Cell binding degree is about 50%-70%.

The target gene that embodiment 9 miR-518a-1-5p regulates and controls in adenocarcinoma of lung transfer process

Standby target gene:

1)BIRC5(gene ID:332)；2)FOXM1(gene ID:2305)；3)MID1(gene ID:4281)；4)MKI67(gene ID:4288)；5)CALU(gene ID:813)；6)CCNA2(geneID:890)；

Experiment material:

Antibody: abcam antibody A nti-Survivin antibody [EP2880Y] (ab76424); Anti-FOXM1 antibody [263C2a] (ab58675), antibody A nti-MID1antibody (ab55444); Anti-Ki67 antibody (ab833); Anti-Calumenin antibody [EPR9075] (ab137019); Anti-Cyclin A2 antibody [E23.1] (ab38);

MiR-518a-1-5p sequence is provided, synthesizes miR-518a-1-5p mimics by Shanghai Ji Ma company;

Experimental implementation:

The cultivation of cell and transfection are with reference to embodiment 7, and experiment is divided into blank group (not transfection) and transfection miR-518a-1-5p mimics group, and 48h harvesting after transfection, take actin as internal reference, step conventionally.Extract after total protein, use health to measure protein concentration for century BCA protein quantification test kit (article No. is CW0014), concrete operation step is shown in description.With 50 μ g/ hole loadings, row SDS-PAGE, utilize half-dried transferring film instrument by protein delivery on pvdf membrane, with the PEST buffer blind 2h containing 5% defatted milk powder, an anti antibody 4 DEG C of corresponding protein is closed and is spent the night, TEST solution washes film 15min × 3 time, add the HRP labelling of dilution two resist, and incubated at room 2h, TBST solution washes film 15min × 3 time, ECL liquid develops the color, and x-ray takes the photograph sheet exposure.The gray value of Image J software to band is adopted to analyze.

Experimental result show: in transfection miR-518a-1-5p mimics group FOXM1, MID1, MKI67 protein expression level comparatively blank group obviously lower, difference has statistical significance.

The mensuration of embodiment 10 miR-518a-1-5p on cell migration impact

Scratch experiment

A549 cell DMEM (containing 10%FBS) culture medium, 37 DEG C, 5%CO ₂, cultivate under saturated humidity condition, will the A549 cell of exponential phase be in by 1.5 × 10 ⁵individual/mL is seeded on 6 orifice plates, 37 DEG C, 5%CO ₂, cultivate 24h under saturated humidity condition.MiR-518a-1-5p sequence is issued Shanghai Ji Ma company, by its synthesis miR-518a-1-5p mimics and anti-miR-518a-1-5p.According to Lipofectamin ^tM2000 description transfection methods, transfection 4 groups of cells, be matched group (not transfection), transfection miR-518a-1-5pmimics group, transfection anti-miR-518a-1-5p group, transfection miR-518a-1-5pmimics+anti-miR-518a-1-5p respectively, often organize sample transfection concentrations 50nM; With aseptic yellow rifle head cut with aseptic 1 × PBS washed cell plate in each hole after transfection 24h, in this, as starting point, every 12h Taking Pictures recording.

After found that scratch experiment 24h, each group presents significant difference, obviously can observe transfection miR-518a-1-5p mimics group cell migration rates and be slower than matched group; And the transfer ability of transfection miR-518a-1-5pmimics+anti-miR-518a-1-5p group cell is obviously strengthened, we draw the migration of raising miR-518a-1-5p and can suppress lung adenocarcinoma cell thus.

Although describe the present invention with reference to various preferred embodiment, it will be appreciated by those skilled in the art that and can carry out various change, and available equivalents substitutes its assembly and do not deviate from elemental range of the present invention.In addition, many changes can be carried out and not deviate from its elemental range to make particular case or material be suitable for instruction of the present invention.

Therefore, the present invention is not intended to be defined in disclosed herein for carrying out particular of the present invention; On the contrary, this invention is intended to comprise all embodiments dropped in Claims scope.

Claims

1.mir-518a-1 and/or the application of its ripe miRNA in preparation prevention, diagnosis and/or treatment adenocarcinoma of lung transfering reagent.

2. application according to claim 1, it is characterized in that, described prevention, Diagnosis of pulmonary adenocarcinoma transfering reagent comprises based on high-flux sequence method and/or based on quantifying PCR method and/or the expression transcribing or detect based on immunologic detection method the target gene of its ripe miRNA regulation and control in adenocarcinoma of lung sample detecting mir-518a-1 and/or its ripe miRNA in adenocarcinoma of lung sample based on probing procedure, preferred employing northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, based on transcribing of mir-518a-1 and/or its ripe miRNA in the Flow cytometry adenocarcinoma of lung sample of microsphere, ELISA and/or colloidal gold strip is adopted to detect the expression of the target gene of its ripe miRNA regulation and control in adenocarcinoma of lung sample.

3. application according to claim 2, is characterized in that, the described primer comprising specific amplification mir-518a-1 and/or its ripe miRNA based on quantifying PCR method; Described comprises the probe with the nucleic acid array hybridizing of mir-518a-1 and/or its ripe miRNA based on probing procedure; Described immunologic detection method comprises the antibody that miRNA regulate gene expression protein-specific ripe with it is combined.

4. application according to claim 1, is characterized in that, described treatment adenocarcinoma of lung transfering reagent comprises the reagent of activity that transcribing and/or promoting its ripe miRNA raising mir-518a-1 and/or its ripe miRNA.

5. application according to claim 4, it is characterized in that, adopt the activity transcribing and/or promote its ripe miRNA raising mir-518a-1 and/or its ripe miRNA based on the microRNA gain-of-function technology of RNA and/or gene specific miR Mimics technology.

6. treat an adenocarcinoma of lung diversion medicaments compositions, it is characterized in that, described pharmaceutical composition comprises:

Receptible carrier on (b) pharmaceutics.

7. pharmaceutical composition according to claim 6, it is characterized in that, adopt the activity transcribing and/or promote its ripe miRNA raising mir-518a-1 and/or its ripe miRNA based on the microRNA gain-of-function technology of RNA and/or gene specific miR Mimics technology.

8. an adenocarcinoma of lung transfer diagnostic reagent, it is characterized in that, described adenocarcinoma of lung transfer diagnostic reagent can detect the expression that transcribing of mir-518a-1 and/or its ripe miRNA in adenocarcinoma of lung sample or immunologic detection method detect the target gene of its ripe miRNA regulation and control in adenocarcinoma of lung sample.

9. adenocarcinoma of lung transfer diagnostic reagent according to claim 8, it is characterized in that, described adenocarcinoma of lung transfer diagnostic reagent is based on high-flux sequence method and/or based on quantifying PCR method and/or the expression transcribing or detect based on immunization method the target gene of its ripe miRNA regulation and control in adenocarcinoma of lung sample detecting mir-518a-1 and/or its ripe miRNA in adenocarcinoma of lung sample based on probing procedure, preferred employing northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, based on transcribing of mir-518a-1 and/or its ripe miRNA in the Flow cytometry adenocarcinoma of lung sample of microsphere, ELISA and/or colloidal gold strip is adopted to detect the expression of the target gene of its ripe miRNA regulation and control in adenocarcinoma of lung sample.

10. adenocarcinoma of lung transfer diagnostic reagent according to claim 9, is characterized in that, the described primer comprising specific amplification mir-518a-1 and/or its ripe miRNA for quantifying PCR method; Described comprises the probe with the nucleic acid array hybridizing of mir-518a-1 and/or its ripe miRNA based on probing procedure; Described immunologic detection method comprises the antibody that miRNA regulate gene expression protein-specific ripe with it is combined.