CN106702002A

CN106702002A - Biomarker for lung adenocarcinoma diagnosis and treatment

Info

Publication number: CN106702002A
Application number: CN201710111869.7A
Authority: CN
Inventors: 杨承刚; 宋宏涛
Original assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2017-02-28
Filing date: 2017-02-28
Publication date: 2017-05-24

Abstract

The invention discloses a biomarker for lung adenocarcinoma diagnosis and treatment. The expression of marker FIBIN in a lung adenocarcinoma tissue is down-regulated, and the early diagnosis of the lung adenocarcinoma can be performed by detecting the expression level of the FIBIN. The invention also proves that the up-regulation of the expression of the FIBIN can effectively inhibit the proliferation and invasion of a lung adenocarcinoma cell through the experiment, thereby prompting that the FIBIN can be used for treating the lung adenocarcinoma or the lung adenocarcinoma metastasis. The invention further discloses a method for screening a potential substance for treating the lung adenocarcinoma, namely, the potential substance can up-regulate or increase the expression level or the expression product activity of the FIBIN.

Description

A kind of biomarker for adenocarcinoma of lung diagnosis and treatment

Technical field

The invention belongs to biomedicine field, it is related to a kind of biomarker for adenocarcinoma of lung diagnosis and treatment, more particularly to Biomarker is FIBIN.

Background technology

Lung cancer is one of the incidence of disease and death rate highest tumour in global range.It is many country its incidence of disease still in Peak.The WHO recent statistics display whole world has 1,590,000 patients to die from lung cancer every year.The issue in 2016 of tumour Register of China Data display：2015, China newly send out cases of lung cancer about 73.3 ten thousand (male 50.93 ten thousand, women 22.4 ten thousand), the same period, China's lung Cancer death toll is that 61.02 ten thousand (male 43.24 ten thousand, and women 17.78 is ten thousand).Lung cancer oneself turn into China's illness rate and death rate highest Tumour, especially female lung cancer illness rate is significantly raised in recent years.At present, surgical operation, chemotherapy, radiotherapy is still the master of lung cancer therapy Want method.With the development of the multidisciplinary MDT complex treatments based on operation, and targeted therapy and immunization therapy development, The treatment of lung cancer achieves rapid progress.However, these do not change the present situation of lung cancer high mortality fundamentally, especially The patients with lung cancer totality prognosis for lacking target gene is still very poor, and survival rate is still hovered 15% or so within 5 years.Therefore, how to carry The therapeutic effect of these patients high is clinical key subjects in the urgent need to address.

Lung cancer can be divided into ED-SCLC (small lung cancer, SCLC) and non-small cell lung by histological type Cancer (non-small lung cancer, NSCLC), wherein NSCLC is divided into squamous carcinoma, gland cancer, large cell carcinoma, carcinoma sarcomatodes etc. again. According to driving gene existence, and it is squamous carcinoma and non-squama non-small cell NSCLC points with deepening continuously for studying lung cancer Lung cancer, rather than squama non-small cell lung cancer refers to just mainly adenocarcinoma of lung.Current data shows that 70% pulmonary adenocarcinoma can be detected To driving gene, and the ratio in lung squamous cancer only has 20%-30%, and both driving gene groups and dissmilarity.Many institutes Known, the patient for driving gene with the presence of targeting can substantially benefit from targeted therapy.It is understood that in molecular gene water Flat lung squamous cancer and adenocarcinoma of lung are " two kinds of diseases ".

With the development of molecular biology and bioinformatics technique, quasi- medical model advocate for lung cancer diagnosis and control Treatment opens brand-new thinking.Accurate medical science is the medical model based on molecular bioinformatics, and its key is using effective Biomarker, carries out specific individualized treatment.Therefore, adenocarcinoma of lung new effective Molecular biomarkers and signal are found Path, further elucidates the generation development mechanism of adenocarcinoma of lung, and the individuation to current adenocarcinoma of lung is precisely treated with important meaning Justice.

The content of the invention

In order to make up the deficiencies in the prior art, it is an object of the invention to provide a kind of gene that can be used for adenocarcinoma of lung diagnosis and treatment Biomarker, gene biological mark provided by the present invention has sensitivity and specificity higher for human lung adenocarcinoma, The detection and treatment of adenocarcinoma of lung can be used for as new biomarkers.

To achieve these goals, the present invention is adopted the following technical scheme that：

The invention provides the application of FIBIN genes or its albumen in the product for preparing diagnosis adenocarcinoma of lung.

Further, the product includes chip, preparation or kit.

Wherein, the chip includes genetic chip, protein-chip；The genetic chip includes solid phase carrier and fixation In the oligonucleotide probe of solid phase carrier, the oligonucleotide probe is included for detecting being directed to for FIBIN gene transcription levels The oligonucleotide probe of FIBIN genes；The protein-chip includes solid phase carrier and is fixed on the FIBIN eggs of solid phase carrier White specific antibody；The kit includes gene detecting kit and protein immunization detection kit；The genetic test Kit includes the reagent for detecting FIBIN gene transcription levels；The protein immunization detection kit includes FIBIN albumen Specific antibody

The invention provides a kind of chip, preparation or kit, it includes detecting FIBIN genes or the reagent of its albumen, When the significantly lower timing of FIBIN expression in sample, patient is with adenocarcinoma of lung or there is the risk for suffering from adenocarcinoma of lung.

Further, the reagent bag is selected from：The probe or specificity of specific recognition FIBIN expand the primer of FIBIN； Or the antibody or part of specific binding FIBIN albumen.

Further, the chip can be used for detect including including FIBIN genes multiple genes (for example, with adenocarcinoma of lung phase Multiple genes of pass) expression；The kit can be used for detect including including FIBIN genes multiple genes (for example, The multiple genes related to adenocarcinoma of lung) expression.

The answering in the product for preparing diagnosis adenocarcinoma of lung the invention provides chip recited above, preparation or kit With.

The invention provides purposes of the above-mentioned FIBIN in the potential material of screening treatment human lung adenocarcinoma.

The invention provides a kind of method for screening the treatment potential material of adenocarcinoma of lung, the potential material can promote, The expression of FIBIN genes or its albumen or activity.

The answering in the pharmaceutical composition for preparing prevention or treatment adenocarcinoma of lung the invention provides FIBIN genes or its albumen With.

Further, described pharmaceutical composition includes the accelerator of FIBIN genes, FIBIN albumen and/or its expression product.

The invention provides a kind of pharmaceutical composition for preventing or treating adenocarcinoma of lung, described pharmaceutical composition includes controlling Treat the accelerator of the FIBIN genes, FIBIN albumen and/or its expression product of effective dose.

Further, described pharmaceutical composition also includes and other medicine classes of the accelerator compatibility and pharmaceutically acceptable Carrier and/or auxiliary material.

Pharmaceutical composition of the invention can also can be with the drug combination of other treatment adenocarcinoma of lung, other therapeutic compound It is administered simultaneously with main active component, or even is administered simultaneously in same composition.

Pharmaceutical composition of the invention can also be with single composition or the dosage shape different from main active component Formula individually gives other therapeutic compounds.The Fractional of main component can be administered simultaneously with other therapeutic compounds, And other dosage can be administered alone.Over the course for the treatment of, can be according to the order of severity of symptom, the frequency of recurrence and treatment side The physiologic response of case, adjusts the dosage of pharmaceutical composition of the present invention.

The advantages of the present invention：

Present invention firstly discovers that the differential expression of FIBIN is to the generation development of adenocarcinoma of lung related, by detecting subject The expression of FIBIN in sample, it can be determined that whether subject suffers from adenocarcinoma of lung or the risk with adenocarcinoma of lung, so as to instruct to face Bed doctor provides prevention scheme or therapeutic scheme to subject.

Gene FIBIN as potential molecular target, for the clinical diagnosis and treatment of adenocarcinoma of lung provide new possibility, together When provide theoretical foundation for the study mechanism of adenocarcinoma of lung, for the personalized treatment of patients with lung adenocarcinoma provides new approach.

Brief description of the drawings

Fig. 1 is the expression figure in pulmonary adenocarcinoma using QPCR detection FIBIN genes；

Fig. 2 is the transfected condition figure in lung adenocarcinoma cell using QPCR detections FIBIN；

Fig. 3 is to detect the influence figure that FIBIN gene pairs lung adenocarcinoma cell is bred with CCK-8 methods；

Fig. 4 is with the influence figure of flow cytomery FIBIN gene pairs Apoptosis of Lung Adenocarcinoma Cell；

Fig. 5 is to detect the influence figure that FIBIN is migrated and attacked to lung adenocarcinoma cell using Transwell cells；Wherein scheme A It is influence figure that FIBIN is migrated to lung adenocarcinoma cell；Figure B is the influence figure that FIBIN is attacked to lung adenocarcinoma cell.

Specific embodiment

By in-depth study extensively, by a large amount of screenings, FIBIN is presented special to the present invention during adenocarcinoma of lung is found that first Different in nature low expression.It is demonstrated experimentally that by the special activity for expressing flat or expression product for improving FIBIN, can effectively suppress The growth and invasion and attack of lung adenocarcinoma cell, the effect of adenocarcinoma of lung is suppressed so as to reach

Biomarker

Term " biomarker " is its expression in tissue or cell and normal or healthy cell or tissue Expression compares any gene for changing.

It would be recognized by those skilled in the art that practicality of the invention is not limited to marker gene of the invention The gene expression of any specific variants is quantified.Used as nonrestrictive example, marker gene can have SEQ ID NO.1 Or the nucleotide sequence or amino acid sequence that SEQ ID NO.2 are specified.In some embodiments, it has and listed sequence The all sequences listed as described above at least 90% of at least 85% same or analogous cDNA sequence, 91%, 92%, 93%, 94%, 95%th, 96%, 97%, 98% or at least 99% same or analogous cDNA sequence.

The present invention can determine gene expression using any method known in the art.Those skilled in the art should manage Solution, the means for determining gene expression are not importances of the invention.The table of biomarker can be detected on transcriptional level Up to level.

In some embodiments, the expression of biomarker is detected on transcriptional level.Using nucleic acid hybridization skill Various methods that art carries out specific DNA and RNA measurements are well known by persons skilled in the art.Certain methods are related to electrophoretic separation (for example, Southern traces and Northern traces for detecting RNA for detecting DNA), but can also be unfavorable With the measurement (for example, by Dot blot) that DNA and RNA is carried out in the case of electrophoretic separation.Genomic DNA is (for example, come from People) Southern traces can be used to screen RFLP (RFLP), to detect influence polypeptide of the present invention The presence of inherited disorder.The RNA of form of ownership can be detected.

Chip

Described genetic chip of the invention includes：Solid phase carrier；And the widow on the solid phase carrier is fixed in order Nucleotide probe, described oligonucleotide probe specifically corresponds to the part or all of sequence shown in FIBIN.

Specifically, suitable probe can be designed according to gene of the present invention, is fixed on solid phase carrier, formed " oligonucleotide arrays ".Described " oligonucleotide arrays " refer to addressable point (i.e. with distinctive, addressablely The position that location is characterized) array, a coupled characteristic oligonucleotides is contained in each addressable point.According to need Will, oligonucleotide arrays can be divided into multiple Asia battle arrays.

In the present invention, the solid phase carrier is including plastic products, microparticle, membrane carrier etc..The plastic products can lead to Cross non-covalent or physical absorption mechanism to be combined with antibody or proteantigen, the most frequently used plastic products are made for polystyrene Small test tube, globule and micro-reaction plate；The microparticle is the microballoon or particle aggregated into by high polymer monomer, and its diameter is generally Micron, due to the functional group that can be combined with protein, easily forming chemical coupling with antibody (antigen), binding capacity is big；Institute Stating membrane carrier includes nitrocellulose filter, glass fibre element film and miillpore filter etc. nylon membrane.

Term " probe " refers to the molecule that can be combined with the particular sequence of another molecule or subsequence or other parts.Unless another Point out, term " probe " is often referred to be matched and another polynucleotides (often referred to as " target polynucleotide ") by complementary base With reference to polynucleotide probes.Lack sufficient sequence complementarity according to the stringency of hybridization conditions, probe energy and with the probe Target polynucleotide is combined.Probe can make direct or indirect mark, and its scope includes primer.Crossing system, including, but do not limit In：Solution, solid phase, mixed phase or in situ hybridization determination method.

Term " complementation " or " complementarity " are used to refer to polynucleotides (that is, the nucleosides for passing through basepairing rule correlation The sequence of acid).For example, sequence " 5'-A-G-T-3' " is complementary with sequence " 3'-T-C-A-5' ".Complementarity can be " part ", The base of only few of which nucleic acid is matched according to basepairing rule.Or, can also exist between nucleic acid " complete " or It is " total " complementary.Complementarity between nucleic acid chains has significant impact for the hybridization efficiency and intensity between nucleic acid chains. It is even more important in the detection method of this combination between amplified reaction and dependence nucleic acid.

Term " stringency " is used to refer to the condition carried out residing for nucleic acid hybridization：Temperature, ionic strength and other compounds The presence of (such as organic solvent).Under " low stringency condition ", nucleotide sequence of interest will with its exact complementary sequence, have The sequence of single base mispairing, closely related sequence (for example, the sequence with 90% or more high homology) and only portion Divide sequence (for example, the sequence with the 50-90% homologys) hybridization of homology.It is of interest under " medium stringent conditions " Nucleotide sequence by only with its exact complementary sequence, the sequence with single base mispairing and closely related sequence (for example, 90% or more high homology) hybridization.Under " stringency high ", nucleotide sequence of interest will only and its exact complementary sequence (depending on the condition of such as temperature) has the sequence hybridization of single base mispairing.In other words, under stringency high, High-temperature can be risen to exclude and the sequence hybridization with single base mispairing.

In the present invention for FIBIN genes oligonucleotide probe can be DNA, RNA, DNA-RNA chimera, PNA or Other derivatives.The length of the probe is not limited, as long as completing specific hybrid and purpose nucleotide sequence specificity knot Close, any length can.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the probe Length can grow to 60,80,100,150,300 base-pairs or longer, or even whole gene.Due to different probe lengths pair Hybridization efficiency, signal specificity have different influences, and the length of the probe is typically at least 14 base-pairs, most it is long it is general not More than 30 base-pairs, complementary length is optimal with 15-25 base-pair with purpose nucleotide sequence.The probe self-complementary Sequence is most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.

Heretofore described solid phase carrier can be using the various common used materials in genetic chip field, such as but not limited to nylon Film, the slide modified through active group (such as aldehyde radical, amino) or silicon chip, unmodified slide, plastic sheet etc..

Preparing for described FIBIN chips can be using the common manufacturing method of biochip known in the art.For example, such as Fruit solid phase carrier uses modification slide or silicon chip, and 5 ' ends of probe are gone here and there containing amido modified poly- dT, can be by oligonucleotides Probe is configured to solution, then uses point sample instrument by its point on modification slide or silicon chip, is arranged in predetermined sequence or array, Then fixed by standing overnight, so that it may obtain genetic chip of the invention.

Kit

The invention provides a kind of kit, the kit can be used to detect the expression of FIBIN.

Preferably, also containing the label for labeled RNA sample in described preparation or kit, and with the mark The corresponding substrate of note thing.Additionally, be may also include in described kit required for extracting RNA, PCR, hybridization, colour developing etc. Various reagents, including but not limited to：Extract, amplification liquid, hybridization solution, enzyme, comparison liquid, nitrite ion, washing lotion etc..Additionally, described Kit in also include operation instructions and/or chip image analysis software.

Drug screening

The invention provides purposes of the FIBIN in human lung adenocarcinoma diagnosis and treatment medicine is screened.I.e.：Processed with candidate substances and expressed The system of FIBIN；With the expression or activity for detecting FIBIN in the system；If the candidate substances can promote the expression of FIBIN Or activity, then show that the candidate substances are the potential materials for suppressing adenocarcinoma of lung.The system of described expression FIBIN for example can be Cell (or cell culture) system, described cell can be the cell of endogenous expression FIBIN；Or can be recombination expression The cell of FIBIN.The system of described expression FIBIN can also be subcellular fraction system, solution system, organizational framework, organ body System or animal system (such as animal model, the preferably animal model of non-human mammal, such as mouse, rabbit, sheep, monkey) etc..

The accelerator of FIBIN and pharmaceutical composition

Based on discovery of the invention, the invention provides a kind of pharmaceutical composition, described pharmaceutical composition includes FIBIN's Accelerator.

The accelerator of described FIBIN refers to any to improve FIBIN genes or expression product stability, raise FIBIN Expression, increase gene FIBIN effective acting time or promote FIBIN genes transcription material, these materials can use In the present invention, as the useful material of the expression for raising FIBIN genes, so as to can be used for preventing or treat adenocarcinoma of lung.

Used as a kind of preferred embodiment of the invention, the accelerator of described FIBIN is that a kind of expression containing FIBIN is carried Body.Described expression vector generally also contains promoter, replication orgin and/or marker gene etc..

The expression vector that method well-known to those having ordinary skill in the art can be used for needed for building the present invention.These methods include Recombinant DNA technology in vi, DNA synthetic technologys, In vivo recombination technology etc..Described expression vector preferably includes one or more Selected marker, to provide the phenotypic character of the host cell for selecting conversion, such as kalamycin, gentamicin, tide Mycin, amicillin resistance.

In the present invention, be various carriers known in the art, such as commercially available carrier, including plasmid, clay, bacteriophage, Virus etc..Expression vector can be gathered to the importing in host cell using electroporation, calcium phosphate method, liposome method, DEAE Portugals The known methods such as the combination of sugared method, microinjection, virus infection, liposome transfection and cell-membrane permeable peptide.

Term " host cell " includes prokaryotic and eukaryotic.The example of conventional prokaryotic host cell includes large intestine Bacillus, hay bacillus etc..Conventional eukaryotic host cell includes yeast cells, insect cell and mammalian cell.It is preferred that The host cell is eukaryotic, such as Chinese hamster ovary celI, COS cells.

Pharmaceutical composition

Pharmaceutical composition in the present invention includes the accelerator of FIBIN, and/or other medicine classes with the accelerator compatibility And pharmaceutically acceptable carrier and/or auxiliary material.

Pharmaceutically acceptable carrier can be that one kind can also be various, and the carrier includes but is not limited to diluent such as Lactose, sodium chloride, glucose, urea, starch, water etc.；Adhesive such as starch, pregelatinized starch, dextrin, maltodextrin, sugarcane Sugar, Arabic gum, gelatin, methylcellulose, carboxymethylcellulose calcium, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyethylene Than pyrrolidone, alginic acid and alginate, xanthans, hydroxypropyl cellulose and hydroxypropyl methyl cellulose etc.；Surfactant Such as polyoxyethylene sorbitan fatty acid ester, lauryl sodium sulfate, glyceryl monostearate, hexadecanol；Humectant Such as glycerine, starch；Absorption carrier such as starch, lactose, bentonite, silica gel, kaolin and soap clay etc.；Lubricant such as stearic acid Zinc, glycerin monostearate, polyethylene glycol, talcum powder, calcium stearate and magnesium, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, Sodium stearyl fumarate, polyoxyl 40 stearate, single bay sucrose acid ester, sldium lauryl sulfate, magnesium laurylsulfate, 12 Alkylsurfuric acid magnesium etc.；Filler such as mannitol (granular or powdery), xylitol, sorbierite, maltose, erythrose, microcrystalline cellulose Element, polymerization sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, carbon Sour calcium and sodium acid carbonate etc.；Disintegrant such as cross-linked ethylene pyrrolidones, sodium carboxymethyl starch, low-substituted hydroxypropyl ylmethyl, crosslinking Sodium carboxymethylcellulose, soybean polyoses etc..

Pharmaceutical composition in the present invention can also include stabilizer, bactericide, buffer, isotonic agent, chelating agent, pH controls The additive such as preparation and surfactant.

Term " effective dose " can produce function or activity to people and/or animal and can be connect by people and/or animal The amount received." pharmaceutically acceptable carrier " refers to the carrier for Therapeutic Administration, including various excipient and diluent.The art Language refers to such some medicament carriers：Themselves it is not necessary active component, and does not have undue toxicity after administration.Properly Carrier be well known to those of ordinary skill in the art.Pharmaceutically acceptable carrier can contain liquid in the composition, such as Water, salt solution, buffer solution.In addition, there is likely to be complementary material in these carriers, such as filler, lubricant, glidant, Wetting agent or emulsifying agent, pH buffer substance etc..Lipofectamine can also be contained in described carrier.

In the present invention, can using various methods well known in the art by described accelerator or its open gene or Its pharmaceutical composition delivers medicine to mammal.Including but not limited to：Hypodermic injection, intramuscular injection, for percutaneous administration of, administer locally to, Implantation, sustained release give；Preferably, the administering mode is that non-bowel gives.

Preferably, can be carried out using the means of gene therapy.Such as, can be directly by the accelerator of FIBIN by such as noting The method such as penetrate and deliver medicine to subject；Or, the ceneme (ratio that the promotion that can will carry FIBIN by certain approach is adjusted Such as expression vector or virus) it is delivered on target spot, concrete condition need to be depending on the type of described accelerator, and these are these Known to art personnel.

The effective dose of the accelerator of FIBIN of the present invention can be with the pattern of administration and the serious journey of disease to be treated Degree etc. and change.The selection of preferred effective dose can be determined (for example by those of ordinary skill in the art according to various factors By clinical test).Described factor is included but is not limited to：The pharmacokinetic parameter of the accelerator of described FIBIN is for example Bioavailability, metabolism, half-life period etc.；The order of severity of the disease to be treated of patient, the body weight of patient, the immune shape of patient Condition, approach of administration etc..For example, by an urgent demand for the treatment of situation, dosage separate several times can be daily given, or by dosage Reduce pari passu.

Term " sample " is used with its broadest sense.In a kind of implication, it is intended that including originating what is obtained from any Sample or culture, and biological and environmental samples.Biological specimen is available from animal (including people) and covers liquid, solid, group Knit and gas.Biological specimen includes blood product, blood plasma, serum etc..However, such sample should not be construed as limitation being applicable In sample type of the invention.

The present invention is further detailed explanation with reference to the accompanying drawings and examples.Following examples are merely to illustrate this Invention rather than limitation the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment, generally according to conventional strip Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) described in condition, or according to the condition proposed by manufacturer.

Embodiment 1 screens the gene marker related to adenocarcinoma of lung

1st, sample collection

It is each to collect 8 adenocarcinoma of lung cancer beside organisms and pulmonary adenocarcinoma sample.Adenocarcinoma of lung tumor tissues specimen sampling position is Vital tumor areas, positioned at tumor mass China and foreign countries 1/3 and normal structure junction, exclude tumor center substantially necrosis, calcification portion Divide and tumour periphery normal lung tissue；Ai Pang normal lung tissues sample takes from the position of more than borderline tumor 5cm, visually observes Without significant change.The acquirement of above-mentioned all samples is by the agreement of the committee of organizational ethics.

2nd, the preparation of RNA sample (is utilizedMiRNA kit are operated)

Liquid nitrogen is imported in mortar, the tissue for taking above-mentioned acquisition is put into mortar, is shredded in liquid nitrogen and grind into powder, Put into after shredding in liquid nitrogen and be ground to it is powdered, in then continuing at glass homogenizer；Tissue homogenate adds in glass homogenizer Enter Trizol reagents, in tissue abrasion on ice.Tissue homogenate after homogenate is transferred in the EP pipes without RNase, is stood at room temperature 5min.Extracted according to the specification in kit and separate RNA.It is specific as follows：

1) separation of RNA：

0.2m1 chloroforms are added in EP pipes, EP lids are covered tightly, 15s is acutely vibrated manually, it is fully mixed.At room temperature Hatching 5min.Then 15min is centrifuged with 14000g at 4 DEG C.Sample is divided into three layers after centrifugation, and RNA is present in upper strata aqueous phase.

2) RNA precipitate

The water that will be separate is mutually taken during 450 μ l move to the new EP pipes without RNase, according to 1:1 ratio adds 450 μ l different Propyl alcohol, hatches 10min at room temperature after mixing of turning upside down, 4 DEG C of 14000g are centrifuged 10min.

3) RNA wash-outs

It is careful after centrifugation to remove supernatant, add the ethanol of 1ml 75% (destroy the enzyme treatment, matching while using and in precooling on ice) punching RNA is washed, subsequent 4 DEG C of 7500g are centrifuged 5min.

4) RNA is redissolved

The careful supernatant removed after washing, opens EP pipe lids in superclean bench, and RNA samples are placed at room temperature 5-10min, dries.Add without RNase treatment water 20-50 μ l, water-bath 10min in 55-60 DEG C of water bath.

5) quality analysis of RNA sample

Spectrophotometer is detected：

NanoDrop1000 spectrophotometers detect RNA sample, the sample requirement of RNA-seq sequencings：OD260/OD280 is 1.8-2.2。

Agarose gel electrophoresis is detected：

The RNA of said extracted is entered into row agarose gel electrophoresis, Agilent Technologies 2100Bioanalyzer detects RNA sample quality, and observation 28S rRNA and 18S rRNA master tapes are obvious, complete without degraded, RNA Sex index is qualified, concentration reaches requirement, can be used for the gene expression profile of chip and screening experiment.

3rd, reverse transcription and mark

With Low RNA Input Linear Amplification Kit by mRNA reverse transcriptions into cDNA, while using Cy3 Difference labelling experiment group and control group.

4th, hybridize

Genetic chip using Aglient companies people's full-length genome chip of expression spectrum, the step of by chip operation instructions Carry out.

5th, data analysis

Chip results are analyzed using Agilent GeneSpring softwares, screening expression quantity has significant difference (standard is that the gene differs more than 2 times, and p with the expression quantity by cancer in cancer<0.05) gene.

6th, result

Result shows that expression quantity of the FIBIN in pulmonary adenocarcinoma is substantially less than the expression quantity in cancer beside organism.

The differential expression of the QPCR sequence verification FIBIN genes of embodiment 2

1st, large sample QPCR checkings are carried out to FIBIN gene differential expressions.Selected according to the sample collection mode in embodiment 1 Select adenocarcinoma of lung cancer beside organism and each 50 of pulmonary adenocarcinoma.

2nd, RNA extraction steps are as described in Example 1.

3rd, reverse transcription

1) reaction system：

The μ l of RNA templates 1, the μ l of random primer 1, distilled water adds to 12 μ l, mixes, slow-speed of revolution centrifugation, 65 DEG C of 5min, then It is placed on cooled on ice.

Continuation adds following ingredients in 12 μ l reaction solutions：

The μ l of 5 × reaction buffer 4, μ l, the AMV reverse transcriptions of 1 μ l, 10mM dNTP mixed liquors of RNase inhibitor (20U/ μ l) 2 The μ l of enzyme (200U/ μ l) 1；Fully mix and carry out centrifugal treating；

2) reverse transcription reaction condition

25 DEG C of 5min, 42 DEG C of 60min, 70 DEG C of 5min.

3) polymerase chain reaction

Design of primers：

It is biological by Bo Maide according to FIBIN genes in Genebank and the sequences Design QPCR amplimers of GAPDH genes Company synthesizes.Specific primer sequence is as follows：

FIBIN genes：

Forward primer is 5 '-TCTACCCAGAGATGTCCAA-3 ' (SEQ ID NO.3)；

Reverse primer is 5 '-GGTCATCGTTCTCCTCATAG-3 ' (SEQ ID NO.4).

GAPDH genes：

Forward primer is 5 '-AATCCCATCACCATCTTCCAG-3 ' (SEQ ID NO.5)；

Reverse primer is 5 '-GAGCCCCAGCCTTCTCCAT-3 ' (SEQ ID NO.6).

Prepare PCR reaction systems：

The μ l of 2 × qPCR mixed liquors 12.5, the μ l of gene primer 2.0, reverse transcription product 2.5 μ l, ddH₂O 8.0μl。

PCR reaction conditions：95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) × 40 circulations, 60 DEG C of 5min extensions.75 DEG C to 95 DEG C, heated up 1 DEG C per 20s, draw solubility curve.Using SYBR Green as fluorescent marker, in Light Cycler The enterprising performing PCR reaction of quantitative real time PCR Instrument, is analyzed by melt curve analysis and electrophoresis determines purpose band, and Δ Δ CT methods carry out phase To quantitative.

5th, statistical method

Experiment is all completed according to being repeated 3 times, and result data is represented in the way of mean+SD, Statistical analysis is carried out using SPSS18.0 statistical softwares, cancer is checked with the paired comparisons of cancer beside organism using t, it is believed that work as P< There is statistical significance when 0.05.

6th, result

Result is as shown in figure 1, compared with adenocarcinoma of lung cancer beside organism, FIBIN expresses downward, difference tool in pulmonary adenocarcinoma Statistically significant (P<0.05) it is, consistent with chip detection result.

The FIBIN overexpression of embodiment 3

1st, cell culture

Human A459 lung cancer cell line, with the RPMI1640 culture mediums containing 10% hyclone and 1%P/S 37 DEG C, 5% CO₂, cultivate in the incubator that relative humidity is 90%.Change within 2-3 days liquid 1 time, use the 0.25% trypsase routine containing EDTA Had digestive transfer culture.

Cell in blake bottle is digested and be seeded in 6 orifice plates with pancreatin, it is ensured that cell number is 2-8 × 10⁵Individual/ Hole, adds cell culture medium.Overnight, second day observation of cell density, cell density can be transfected for more than 70%.

2nd, the structure of gene overexpression carrier

CDNA sequence (as shown in SEQ ID NO.1) according to FIBIN synthesizes special pcr amplification primer thing, primer sequence It is as follows：

Forward primer：5’-CCGAAGCTTGCCACCATGGTGTTTCTGAAGTTC-3’(SEQ ID NO.7)

Reverse primer：5’-CGGCTCGAGTACTTTAAGATAATCATTTTTCAGCC-3’(SEQ ID NO.8)

Two restriction enzyme sites of HindIII and XhoI are added respectively in 5 ' end primers and 3 ' end primers.With adenocarcinoma of lung , used as amplification template, above-mentioned cDNA sequence is through restriction enzyme HindIII for the cDNA that patient tissue is extracted and reverse transcription is obtained Be inserted into after XhoI double digestions in the eukaryotic expression vector pcDNA3.1 through HindIII and XhoI double digestions, connection is obtained The recombinant vector pcDNA3.1-1 for obtaining is used for subsequent experimental.

3rd, transfect

Lung adenocarcinoma cell is divided into 3 groups, respectively control group (A549), blank control group (transfection pcDNA3.1-NC), reality Test group (transfection pcDNA3.1-1).The transfection of carrier, specific transfection method finger to specifications are carried out using liposome 2000 Showing is carried out.The transfection concentrations of pcDNA3.1 empty carriers and pcDNA3.1-1 are 0.5 μ g/ml.

4th, QPCR detects the transcriptional level of FIBIN genes

The extraction of 4.1 cell total rnas

1) cell culture fluid in 6 orifice plates is outwelled, is rinsed twice with PBS, each hole adds 1ml Trizol reagents, room temperature Place 5min.

2) 0.2m1 chloroforms are added, 15s, 4 DEG C, 12000g centrifugations 15min is acutely shaken.

3) water is mutually transferred in new pipe, adds 4.5m1 isopropanols, and room temperature places 10min；4 DEG C, 10000g centrifugations 10min.

4) liquid is outwelled, EP tube walls is washed with 75% ethanol of lml.4 DEG C, 7500g is centrifuged 5min.

5) 75% ethanol after cleaning is outwelled, room temperature hangs 5-10min.

6) DEPC water of the 25 μ l without RNase, -70 DEG C of preservations are added.

4.2 reverse transcription steps are with embodiment 2.

4.3 QPCR amplification steps are with embodiment 2.

5th, statistical method

Experiment is all completed according to being repeated 3 times, and result data is represented in the way of mean+SD, Statistical analysis is carried out using SPSS18.0 statistical softwares, the difference between FIBIN gene overexpressions group and control group uses t Inspection, it is believed that work as P<There is statistical significance when 0.05.

6th, result

Result such as Fig. 2 shows, with non-transfection group compared with empty plasmid group is transfected, overexpression in transfection FIBIN groups FIBIN, difference has statistical significance (P<0.05).

The influence of the FIBIN gene pairs lung adenocarcinoma cell of embodiment 4 propagation

Influenceed using CCK-8 experiment detection FIBIN gene pairs lung adenocarcinoma cells multiplication capacities.

1st, cell culture and transfection procedure are with embodiment 3.

2nd, cell was taken out in second day, basis of microscopic observation cell growth status, 1ml/ holes add the pancreatin containing EDTA, enter Row cell dissociation, removes pancreatin after the completion of waiting to digest, adding cell culture medium to mix makes cell suspend, and then carries out cytometer Number.

3rd, concentration of cell suspension is diluted to 15000/ml, afterwards toward being inoculated with 96 orifice plates, cell is added per hole The μ l of suspension 200, cell is controlled at 3000 or so, is inoculated with 8 multiple holes.PcDNA3.1-1 experimental groups and pcDNA3.1-NC are set Control group.4 piece of 96 orifice plate is spread altogether is respectively used to 24h, 48h, 72h, 96h4 detection time point.

4th, after 24h, first piece of 96 orifice plate is taken out, adds the CCK-8 of 10 μ l to detect liquid in every hole, 96 orifice plates are continued to put Enter and 4h or so is incubated in cell culture incubator, absorbance and record data of each hole at 450nm wavelength are detected with ELIASA.

5th, the operation in 4 is respectively repeated steps after 48h, 72h, 96h, the absorbance at each time point is finally counted, Make growth curve chart.

6th, statistical analysis

Experiment is all completed according to being repeated 3 times, and statistical analysis is carried out using SPSS18.0 statistical softwares, both it Between difference using t check, it is believed that work as P<There is statistical significance when 0.05.

7th, result

Result is as shown in figure 3, compared with the control, after pcDNA3.1-1 is transfected, the propagation of cell is substantially subject to experimental group Suppress, difference has a statistical significance (P<0.05) explanation FIBIN has the effect for suppressing cell propagation.

The influence of the FIBIN gene pairs Apoptosis of Lung Adenocarcinoma Cell of embodiment 5

Use the influence of flow cytomery FIBIN gene pairs Apoptosis.

1st, cell culture step is with embodiment 3.

2nd, cell transfecting step is with embodiment 3.

3rd, step

1) by 10 × sample-loading buffers of 3m1 27m1 distilled water dilutings.

2) collection of cellular samples and with the PBS of precooling.

3) 1 × sample-loading buffers of lml, 300g centrifugation 10min is added to suction out buffer solution in cell.

4) 1 × sample-loading buffers of lml are added again, and cell concentration in cell suspension is adjusted to 1 × 10⁶Individual/ml.

5) cell suspension is taken out into 100 μ l, in addition EP pipes.

6) by the Annexin V FITC addition EP pipes of 5 μ l, the liquid in EP pipes is mixed, lucifuge is incubated at room temperature 10min。

7) to 5 μ l PI dye liquors are added in EP pipes, lucifuge 5min at room temperature.

8) PBS solution of 500 μ l is added in EP pipes, is gently mixed, flow cytometer is detected in 1h.

3rd, statistical method

Experiment is all completed according to being repeated 3 times, and result data is represented in the way of mean+SD, Statistical analysis is carried out using SPSS13.0 statistical softwares, the t inspections of difference use between the two, it is believed that work as P<When 0.05 With statistical significance.

4th, result：

As shown in figure 4, experimental group is compared with control group, apoptosis rate is without obvious change (P for result<0.05), the knot Fruit illustrates that FIBIN differential expressions are on the apoptosis of lung adenocarcinoma cell without obvious influence.

The cell migration of embodiment 6 and Matrigel

1st, prepared by Transwell cells

Matrigel is ice bath melted under aseptic condition, and 20 times of dilutions are carried out with PBS, is layered on the volume in 50 μ l/ holes On the polycarbonate membrane of Transwell cells.37 DEG C of placement 4h, take out after Matrigel glue aggregates into gel, gently suction out Upper strata separates out liquid.The serum-free medium containing BSA of 50 μ l is added in per hole carries out hydration process, 37 DEG C of placements to basilar memebrane 30min。

2nd, cell suspension is configured

Cell removes serum starvation treatment 12-24h, and digestion process is carried out to cell, is centrifuged after terminating digestion, in removal Layer nutrient solution.Sedimentation cell is cleaned with PBS, adds the serum free medium containing BSA to carry out it resuspended.Adjustment is thin The density of born of the same parents is to 5 × l0⁵Individual/ml.

3rd, cell inoculation

The μ l of obtained cell suspension 200 (migration experiment is 100 μ l, and Matrigel is 200 μ l) are added to Transwell cells In.Room adds 1640 culture mediums of the 500 μ l containing FBS under 24 orifice plates.Cell is put into cell culture incubator and cultivates 24h.

4th, dye

Cell is dyeed after culture terminates using DAPI.Small ventricular cell is first rinsed 2 times with PBS, DAPI working solutions are put into Middle room temperature dyes 5-20min.Rinsed 2 times with PBS, be put into fluorescence microscopy Microscopic observation and count.

5th, result

Result compared with control group, the migration of experimental group and is invaded as shown in figure 5, after lung adenocarcinoma cell transfected plasmids Attack ability to be decreased obviously, as a result illustrate that FIBIN can suppress the migration and invasion and attack of adenocarcinoma of lung.

The explanation of above-described embodiment is only intended to understand the method for the present invention and its core concept.It should be pointed out that for this For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improvement can also be carried out to the present invention And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.

SEQUENCE LISTING

<110>Beijing Yang Shen biology information technologies Co., Ltd

<120>A kind of biomarker for adenocarcinoma of lung diagnosis and treatment

<160> 8

<170> PatentIn version 3.5

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<211> 636

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ggggactatg aggagaacga tgaccccgag aagtgccagc tgctcttcag ggtgagtgac 180

cacaggcgct gctcccaggg ggaggggagc caggttggca gcctgctgag cctcaccctg 240

cgggaggagt tcaccgtgct gggccgccag gtggaggatg ctgggcgcgt gctggagggc 300

atcagcaaaa gcatctccta cgacctagac ggggaagaga gctatggcaa gtacctgcgg 360

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Gln Gly Tyr Phe Asp Gly Pro Leu Tyr Pro Glu Met Ser Asn Gly Thr

20 25 30

Leu His His Tyr Phe Val Pro Asp Gly Asp Tyr Glu Glu Asn Asp Asp

35 40 45

Pro Glu Lys Cys Gln Leu Leu Phe Arg Val Ser Asp His Arg Arg Cys

50 55 60

Ser Gln Gly Glu Gly Ser Gln Val Gly Ser Leu Leu Ser Leu Thr Leu

65 70 75 80

Arg Glu Glu Phe Thr Val Leu Gly Arg Gln Val Glu Asp Ala Gly Arg

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Val Leu Glu Gly Ile Ser Lys Ser Ile Ser Tyr Asp Leu Asp Gly Glu

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Glu Ser Tyr Gly Lys Tyr Leu Arg Arg Glu Ser His Gln Ile Gly Asp

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Ala Tyr Ser Asn Ser Asp Lys Ser Leu Thr Glu Leu Glu Ser Lys Phe

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Lys Gln Gly Gln Glu Gln Asp Ser Arg Gln Glu Ser Arg Leu Asn Glu

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Asp Phe Leu Gly Met Leu Val His Thr Arg Ser Leu Leu Lys Glu Thr

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Leu Asp Ile Ser Val Gly Leu Arg Asp Lys Tyr Glu Leu Leu Ala Leu

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Claims

The application of 1.FIBIN genes or its albumen in the product for preparing diagnosis adenocarcinoma of lung.
2. application according to claim 1, it is characterised in that the product includes chip, preparation or kit.
3. a kind of chip, preparation or kit, it is characterised in that the reagent including detection FIBIN genes or its albumen.
4. chip according to claim 3, preparation or kit, it is characterised in that the reagent bag is selected from：Specificity is known The probe or specificity of other FIBIN expand the primer of FIBIN；Or the antibody or part of specific binding FIBIN albumen.
5. application of the chip, preparation or kit described in claim 3 or 4 in the product for preparing diagnosis adenocarcinoma of lung.
The purposes of 6.FIBIN genes or its albumen in the potential material of screening treatment human lung adenocarcinoma.
7. it is a kind of to screen the method for treating the potential material of adenocarcinoma of lung, it is characterised in that the potential material can promote, FIBIN The expression of gene or its albumen or activity.
The application of 8.FIBIN genes or its albumen in the pharmaceutical composition for preparing prevention or treatment adenocarcinoma of lung.
9. application according to claim 7, it is characterised in that described pharmaceutical composition includes FIBIN genes, FIBIN eggs The accelerator of white and/or its expression product.
10. a kind of pharmaceutical composition for preventing or treating adenocarcinoma of lung, it is characterised in that described pharmaceutical composition includes treatment The accelerator of the FIBIN genes, FIBIN albumen and/or its expression product of effective dose.