CN106729756A

CN106729756A - Application of the biomarker as target in adenocarcinoma of lung diagnosis and treatment

Info

Publication number: CN106729756A
Application number: CN201710112636.9A
Authority: CN
Inventors: 边洋; 李曙光
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: 2017-05-31
Anticipated expiration: 2037-02-28
Also published as: CN106729756B

Abstract

A kind of application the invention discloses biomarker as target in adenocarcinoma of lung diagnosis and treatment, specific biomarker is CLINT1.Worldwide adenocarcinoma of lung incidence continues to increase, it is necessary to effective biomarker is to diagnose and/or treat adenocarcinoma of lung.The present invention is experimentally confirmed, CLINT1 up-regulateds in patients with lung adenocarcinoma, and reducing the expression of CLINT1 genes can suppress propagation and the invasion and attack of lung adenocarcinoma cell.CLINT1 disclosed by the invention is pointed out to can be used for early diagnosing the mark of adenocarcinoma of lung；The potential drug target spot that the gene can also be treated as adenocarcinoma of lung simultaneously, for the precision medical treatment of adenocarcinoma of lung provides basis.

Description

Application of the biomarker as target in adenocarcinoma of lung diagnosis and treatment

Technical field

The invention belongs to biomedicine field, it is related to application of the biomarker as target in adenocarcinoma of lung diagnosis and treatment, has The biomarker of body is CLINT1.

Background technology

Lung cancer is one of the incidence of disease and death rate highest tumour, serious harm human health in global range.Lung cancer can To be divided into non-small cell lung cancer (non small cell lung cancer, NSCLC) and ED-SCLC (small cell Lung cancer, SCLC).Wherein NSCLC accounts for the 80%-85% of lung cancer sum, and its disease development is rapid, and transfer velocity is fast, Recurrence rate is higher.Most patients have been developed to middle and advanced stage when finding, poor prognosis, and survival rate is less than 15% within 5 years.Early stage receives to control The NSCLC Patients on Recurrence rates for the treatment of up to 40%, and more than 70% patient be local Invasion or distant metastasis when being found and cannot Operation.

At present, surgical operation, chemotherapy, radiotherapy is still the primary treatment regimen of lung cancer.In recent years, as multidisciplinary synthesis are treated The development of (multidisciplinary team, MDT), the treatment of lung cancer achieves significant progress.However, lung cancer life in 5 years Rate is deposited still to hover 15% or so.Accurate medical model is the trend and direction of current oncotherapy, and its key is using effective Biomarker, carry out specific individualized treatment.Therefore, the new effective molecular marker of lung cancer and potential treatment are found Target spot is just particularly important.

With the completion of the Human Genome Project, there is brand-new understanding in gene level to lung cancer.It is believed that dividing Sub- gene level lung squamous cancer and adenocarcinoma of lung are " two kinds of diseases ".Available data also points out to be more easy to find biology in adenocarcinoma of lung Mark, and may therefrom benefit.

Accurate medical model is advocated as the diagnosis and treatment of lung cancer open brand-new thinking.Accurate medical science is based on molecular biosciences The medical model of informatics, its key is, using effective biomarker, to distinguish target group, so as to carry out specific individuality Change treatment.Therefore, lung cancer new effective Molecular biomarkers and signal path are found, lung cancer is further elucidated and is sent out Exhibition mechanism, is that the individuation of lung cancer precisely treats the new theoretical foundation of offer as current study hotspot and direction.

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 related to adenocarcinoma of lung generation development Biomarker.

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

The invention provides a kind of purposes of CLINT1 genes, the drug regimen for preparing prevention or treatment adenocarcinoma of lung Thing.

Further, described pharmaceutical composition includes the lower adjustment of CLINT1.The lower adjustment is selected from：With CLINT1 or its turn This is recorded for target sequence and the disturbing molecule of CLINT1 gene expressions or genetic transcription can be suppressed, including：ShRNA (bobby pins RNA), siRNA (SiRNA), dsRNA, Microrna, antisensenucleic acids, or can express or form the shRNA, small interference RNA, dsRNA, Microrna, the construction of antisensenucleic acids；Or the protein bound binding molecule that specificity is encoded with CLINT1 (if suppressing the antibody or part of CLINT1 protein actives).

Further, described lower adjustment is selected from the group the siRNA of sequence：SEQ ID NO.8、SEQ ID NO.9.

The invention provides a kind of lower adjustment for preventing or treating the CLINT1 of adenocarcinoma of lung, the lower adjustment is selected from：

Nucleic acid inhibitor, protein inhibitor, proteolytic enzyme, protein binding molecule, it can be in albumen or gene level The expression of the albumen of upper downward CLINT1 genes or its coding or activity.

In the present invention, the lower adjustment of the CLINT1 can also be used to suppress invasion and attack and the propagation of lung adenocarcinoma cell.

The invention provides a kind of pharmaceutical composition for preventing or treating adenocarcinoma of lung, described pharmaceutical composition contains：

The lower adjustment of CLINT1 recited above；With

Pharmaceutically acceptable carrier.

The invention provides a kind of purposes of CLINT1 genes, the potential material for screening prevention or treatment adenocarcinoma of lung.

The invention provides a kind of method of the potential material for screening prevention or treatment adenocarcinoma of lung, methods described includes：

The system expressed or containing CLINT1 genes or its albumen for encoding is processed with candidate substances；With

Detect expression or the activity of the albumen of CLINT1 genes or its coding in the system；

Wherein, if the candidate substances can reduce expression or the activity of CLINT1 genes, (preferably significantly reduce, it is such as low More than 20%, it is preferably low by more than 50%；More preferably low more than 80%), then show that the candidate substances are to prevent or treat lung gland The potential material of cancer.The system is selected from：Cell system, subcellular fraction system, solution system, organizational framework, organ systems or dynamic Objects system.

The candidate substances are included but is not limited to：For CLINT1 genes or the albumen or its upstream or downstream base of its coding Disturbing molecule, nucleic acid inhibitor, binding molecule (such as antibody or part), the micromolecular compound of cause or protein design.

In the present invention, described method also includes：To obtain potential material carry out further cell experiment and/or Animal experiment, further selects and determines for prevention, alleviation or the useful material for the treatment of adenocarcinoma of lung with from candidate substances.

The invention provides a kind of purposes of CLINT1 genes, the product for preparing diagnosis adenocarcinoma of lung.Wherein, the product Product include but is not limited to chip, preparation or kit.

Further, the product includes the reagent of detection CLINT1 gene expressions.

Further, the reagent is selected from：

The probe of specific recognition CLINT1；Or

The primer of specific amplification CLINT1；Or

The antibody or part of the albumen of specific binding CLINT1 codings.

Preferably, the primer of described specific amplification CLINT1 genes is primer pair, sequence such as SEQ ID NO.3 and Shown in SEQ ID NO.4.

Brief description of the drawings

Fig. 1 is the expression figure in pulmonary adenocarcinoma using QPCR detection CLINT1 genes；

Fig. 2 is the transfected condition figure in lung adenocarcinoma cell using QPCR detections CLINT1；

Fig. 3 is to detect the influence figure that CLINT1 gene pairs lung adenocarcinoma cell is bred with CCK-8 methods；

Fig. 4 is with the influence figure of flow cytomery CLINT1 gene pairs Apoptosis of Lung Adenocarcinoma Cell；

Fig. 5 is to detect the influence figure that CLINT1 is migrated and attacked to lung adenocarcinoma cell using Transwell cells；Wherein scheme A is the influence figure that CLINT1 is migrated to lung adenocarcinoma cell；Figure B is the influence figure that CLINT1 is attacked to lung adenocarcinoma cell.

Specific embodiment

The present invention, by high throughput method, the most wide base of database is covered using current by in-depth study extensively Because of chip, gene finds wherein have substantially expression poor in the expression of tumor tissues and cancer beside organism in detection adenocarcinoma of lung sample Different genetic fragment, inquires into itself relation and the generation of adenocarcinoma of lung between, so as to be the early detection and targeted therapy of adenocarcinoma of lung Find more preferable approaches and methods.By screening, present invention firstly discovers that CLINT1 conspicuousnesses are raised in adenocarcinoma of lung.Experiment card It is bright, by reducing the expression of CLINT1, can effectively suppress the growth and invasion and attack of lung adenocarcinoma cell, point out detection The expression of CLINT1 genes can turn into one of auxiliary diagnostic index of adenocarcinoma of lung early diagnosis, disturb CLINT1 gene expressions Can turn into and prevent or treatment adenocarcinoma of lung or the new way of adenocarcinoma of lung transfer.

CLINT1 genes

CLINT1 is taken positioned at the area 3 of No. 5 chromosome long arms of people 3, a kind of nucleotides of representational people CLINT1 genes Sequence and amino acid sequence are as shown in SEQ ID NO.1 and SEQ ID NO.2.CLINT1 in the present invention includes wild type, dashes forward Modification or its fragment.

People CLINT1 nucleotides full length sequence of the invention or its fragment can generally use PCR TRAPs, recombination method or people The method of work synthesis is obtained.For PCR TRAPs, can be according to published relevant nucleotide sequence, and with commercially available cDNA storehouses Or the cDNA storehouses as prepared by conventional method well known by persons skilled in the art obtain relevant sequence as template, amplification.Work as sequence When row are more long, it is often necessary to carry out twice or multiple PCR is expanded, the fragment for then again amplifying each time is spliced by proper order Together.

It would be recognized by those skilled in the art that practicality of the invention is not limited to appoint target gene of the invention The gene expression of what specific variants is quantified.If when nucleic acid or its fragment and other nucleic acid (or its complementary strand) optimal comparisons When (have appropriate nucleotides inserted or missing), nucleotide base at least about 60%, usually at least about 70%, more Usually at least about 80%, it is preferably at least about 90% and more preferably at least about there is nucleosides in 95-98% nucleotide bases Acid sequence homogeny, then the two sequences are " substantially homologous " (or substantially similar).

Or, when nucleic acid or its fragment with another nucleic acid (or its complementary strand), a chain or its complementary series in selectivity When hybridizing under hybridization conditions, then there is substantially homologous or (homogeny) therebetween.When hybridization has more than specific general loss When selective, there is cross selection.Typically, exist at least about when at least about 14 the one of nucleotides section of sequences 55% homogeny, preferably at least about 65%, more preferably at least about 75% and most preferably at least about 90% homogeny when, hair Raw selective cross.It is as described herein, cognate pair than length can be sequence section more long, lead in certain embodiments Often it is at least about 20 nucleotides, more frequently at least about 24 nucleotides, typically at least about 28 nucleotides, more Typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides.

Therefore, polynucleotides of the invention and SEQ ID NO.1 preferably have at least 75%, more preferably at least 85%, more Preferably at least 90% homology.It is highly preferred that in the presence of at least 95%, more preferably at least 98% homology.

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.

Lower adjustment and pharmaceutical composition

Discovery based on the present inventor, the invention provides a kind of purposes of the lower adjustment of CLINT1, suppresses for preparing The pharmaceutical composition of adenocarcinoma of lung.As used herein, the lower adjustment of described CLINT1 include but is not limited to inhibitor, antagonist, Retarding agent, blocking agent, nucleic acid inhibitor etc..

The lower adjustment of described CLINT1 genes or albumen refers to any activity for reducing CLINT1 albumen, reduces The stability of CLINT1 genes or albumen, the expression for lowering CLINT1 albumen, reduction CLINT1 albumen effective acting times or suppression The material of the transcription and translation of CLINT1 genes processed, these materials are used equally to the present invention, used as useful for lowering CLINT1 Material, so as to can be used for preventing or treat adenocarcinoma of lung.For example, described inhibitor is：Nucleic acid inhibitor, protein inhibitor, Antibody, part, proteolytic enzyme, protein binding molecule, as long as it can lower CLINT1 albumen on albumen or gene level Or the expression of its encoding gene.

Used as a kind of selection mode of the invention, the lower adjustment of described CLINT1 is that a species specificity is combined with CLINT1 Antibody.Described antibody can be monoclonal antibody or polyclonal antibody.CLINT1 protein immune animals, such as rabbit can be used, Mouse, rat etc. produce polyclonal antibody；Various adjuvants can be used to strengthen immune response, including but not limited to Freund's adjuvant Deng.Similar, expression CLINT1 or its cell with antigenic fragment can be used to immune animal to produce antibody.Institute The antibody stated can also be monoclonal antibody, and such monoclonal antibody can be prepared using hybridoma technology.Antibody it is " special Property " refer to that antibody can be incorporated into CLINT1 gene outcomes or fragment.It is preferred that referring to that those can be with CLINT1 gene outcomes or fragment With reference to but nonrecognition and be incorporated into the antibody of other non related antigen molecules.Antibody of the invention can be by technology in the art Various technologies are prepared known to personnel.The present invention not only include complete monoclonal or polyclonal antibody, but also including With immunocompetent antibody fragment, such as Fab ' or (Fab)²Fragment；Heavy chain of antibody；Antibody light chain；Genetically engineered list Chain Fv molecules；Or chimeric antibody.The antibody of anti-CLINT1 albumen can be used in immunohistochemistry technology, in detection biopsy specimen CLINT1 protein contents, be also used as prevent liver cancer transfer and invasion and attack specific treatment agent.In blood sample or urine CLINT1 albumen direct measure can as the auxiliary diagnosis of tumour and more after observation index, also can be as tumour in early days The foundation of diagnosis.Antibody by ELISA, Western Blot engram analysis, or can be coupled with detection moiety, by chemistry The methods such as luminous, tagging are detected.

Used as a kind of preferred embodiment of the invention, the lower adjustment of the CLINT1 is a kind of specific small interference of CLINT1 RNA molecule.As used herein, described " siRNA " refers to a kind of short-movie section double stranded rna molecule, can be with homologous complementary The mRNA of sequence is the target specific mRNA of degraded, and this process is exactly RNA interference (RNA interference) processes.It is small RNA interfering can be prepared into the form of double-strandednucleic acid, and it contains a positive-sense strand and an antisense strand, and this two chains are only in hybridization Under conditions of form double-strand.One double-stranded RNA compound can be prepared by the positive-sense strand that is separated from each other and antisense strand.Therefore, For example, complementary positive-sense strand and antisense strand are chemical syntheses, and the double-strand of synthesis by anneal, can be produced thereafter RNA compounds.

When effective siRNA sequence is screened, the present inventor compares analysis by substantial amounts of, optimal effective so as to find out Fragment.The present inventor's design has synthesized various siRNA sequences, and they are transfected into lung adenocarcinoma cell system by transfection reagent respectively Verified, selected the optimal siRNA of interference effect, they are had the sequence shown in SEQ ID NO.8, SEQ ID NO.9 respectively Row, further test in cellular level, as a result prove that suppression efficiency is very high for test cell line.

Used as a kind of optional mode of the invention, the lower adjustment of described CLINT1 can also be a kind of " children purpura nephritis (Small hairpin RNA, shRNA) ", it is the non-coding small RNA molecular that can form hairpin structure, children purpura nephritis energy Enough by RNA interference channels come the expression of suppressor.As described above, shRNA can be expressed by double-stranded DNA template.Double-stranded DNA Template is inserted into a carrier, such as plasmid or viral vectors, and a promoter is then connected in vitro or in vivo carries out table Reach.ShRNA can be cut into siRNA molecule in the presence of DICER enzymes in eukaryotic, hence into RNAi approach. " shRNA expression vectors " refers to the plasmid that some this areas are conventionally used for building shRNA structures, exist on the usual plasmid " Every sequence " and MCS positioned at " intervening sequence " both sides or for replacing sequence so that people can by shRNA (or Analog) corresponding DNA sequence dna inserted by way of forward and reverse MCS or replace thereon for replacing sequence, RNA after DNA sequence dna transcription can form shRNA (ShortHairpin) structure.Described " shRNA expression vectors " is current Obtained through that can be bought by commercially available approach completely, such as some viral vectors.

Nucleic acid inhibitor of the invention such as siRNA can be with chemical synthesis, it is also possible to by a recombinant nucleic acid structure Expression cassette is prepared after being transcribed into single stranded RNA.The nucleic acid inhibitors such as siRNA, can be by using appropriate transfection reagent quilt It is transported to intracellular, or can be also transported to using multiple technologies known in the art intracellular.

Pharmaceutical composition

Present invention also offers a kind of pharmaceutical composition, it contains the lower adjustment of the described CLINT1 of effective dose, and Pharmaceutically acceptable carrier.Described composition can be used to suppress adenocarcinoma of lung.The lower adjustment of any foregoing CLINT1 For the preparation of composition.

As used herein, described " effective dose " refer to people and/or animal can be produced function or activity and can by people and/ Or the amount that animal is received.The effective dose of lower adjustment can become with order of severity of the pattern of administration and disease to be treated etc. Change.The selection of preferred effective dose can be determined (such as by clinic by those of ordinary skill in the art according to various factors Experiment).Described factor is included but is not limited to：The pharmacokinetic parameter of the lower adjustment of described CLINT1 genes is for example biological Utilization rate, metabolism, half-life period etc.；The order of severity of the disease to be treated of patient, the body weight of patient, the immune state of patient, Approach of administration etc..

" pharmaceutically acceptable carrier " refers to the carrier for Therapeutic Administration, including various excipient and dilution Agent.The term refers to such some medicament carriers：Themselves it is not necessary active component, and does not have undue poison after administration Property.Suitable carrier is well known to those of ordinary skill in the art.Pharmaceutically acceptable carrier can contain in the composition Liquid, 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..Examination can also be transfected containing cell (host cell) in described carrier Agent.

The present invention can use with various methods well known in the art by described lower adjustment or its encoding 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, plant Enter, be sustained and 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 lower adjustment of CLINT1 by such as noting The method such as penetrate and deliver medicine to subject；Or, can by certain approach by carry CLINT1 lower adjustment ceneme (such as Expression vector or virus etc., or siRNA or shRNA) it is delivered on target spot, and be allowed to be adjusted under the CLINT1 of expression activity, tool Body situation need to be depending on the type of described lower adjustment, and these are well-known to those skilled in the art.

Term " host cell " can be prokaryotic, such as bacterial cell；Or low eukaryotic, such as yeast cells； Or higher eucaryotic cells, such as mammalian cell.Representative example has：Escherichia coli, the bacterial cell of streptomyces；Fungi Cell such as yeast；Plant cell；The insect cell of fruit bat S2 or Sf9；Zooblast of CHO, COS or 293 cells etc..

Converting host cell with recombinant DNA can be carried out with routine techniques well known to those skilled in the art.When host is original When core biology is such as Escherichia coli, the competent cell that can absorb DNA can be harvested after exponential phase of growth, use CaCl₂Method treatment, institute With the step of it is generally well-known in the art.Another method is to use MgCl₂.If desired, conversion can also use the side of electroporation Method is carried out.When host is eucaryote, following DNA transfection methods are can select：Calcium phosphate precipitation, conventional mechanical methods are such as Microinjection, electroporation, liposome packaging etc..

Pharmaceutical composition in the present invention includes the lower adjustment of CLINT1, and/or other medicines with the lower adjustment compatibility Class 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.

Drug screening

The invention provides it is a kind of screen prevention or treatment adenocarcinoma of lung medicine method, i.e.,：

In experimental group, to adding testing compound in cell culture system, and the expression of CLINT1 is determined；Right According to group, to being added without testing compound in same cultivating system, and the expression of CLINT1 is determined；Wherein, such as fruit The expression of CLINT1 in group is tested more than control group, then illustrates the lower adjustment that the candidate compound is CLINT1.

In the present invention, described method also includes：Its suppression is further tested the candidate compound that previous step is obtained The effect of adenocarcinoma of lung processed, if test compound has significant inhibition to adenocarcinoma of lung, illustrates the compound for prevention or controls Treat the potential material of adenocarcinoma of lung.

Chip, kit

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 CLINT1.

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.

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.

In the present invention for CLINT1 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 for example be included but is not limited to using the various common used materials in genetic chip field Plastic products, microparticle, membrane carrier etc..The plastic products can be resisted by non-covalent or physical absorption mechanism and antibody or albumen Original is combined, small test tube, globule and micro-reaction plate that the most frequently used plastic products are made for polystyrene；The microparticle is The microballoon or particle aggregated into by high polymer monomer, its diameter is generally micron, due to the functional group that can be combined with protein, Chemical coupling easily is formed with antibody (antigen), binding capacity is big；The membrane carrier includes nitrocellulose filter, glass fibre element film And the miillpore filter such as nylon membrane.

Preparing for described CLINT1 chips can be using the common manufacturing method of biochip known in the art.For example, If solid phase carrier uses modification slide or silicon chip, 5 ' ends of probe are gone here and there containing amido modified poly- dT, can be by few nucleosides Acid 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 battle array Row, are then fixed, so that it may obtain genetic chip of the invention by standing overnight.

The invention provides a kind of kit, the kit can be used to detect the expression of CLINT1 genes or albumen.It is excellent Choosing, the label for labeled RNA sample is also contained in described preparation or kit, and it is corresponding with the label Substrate.Additionally, the various reagents required for extracting RNA, PCR, hybridization, colour developing etc. are may also include in described kit, Including but not limited to：Extract, amplification liquid, hybridization solution, enzyme, comparison liquid, nitrite ion, washing lotion etc..Additionally, in described kit Also include operation instructions and/or chip image analysis software.

The component of kit can be packed in the form of aqueous medium or in the form of lyophilized.Appropriate container in kit Typically at least include a kind of bottle, test tube, flask, PET bottle, syringe or other containers, wherein a kind of component can be placed, and And preferably, suitably decile can be carried out.When there is more than one component in kit, will generally also be included in kit Second, third or other additional containers, wherein being positioned separately additional component.However, the component of various combination can be wrapped It is contained in a bottle.Kit of the invention generally also will include a kind of container for accommodating reactant, sealing for Commercial distribution.This container may include the plastic containers of injection molding or blowing mould, wherein required bottle can be retained.

In the present invention, term " sample " is used with its broadest sense.In a kind of implication, it is intended that including from it is any come Sample or culture that source obtains, and biological and environmental samples.Biological specimen available from animal (including people) and cover liquid, Solid, tissue and gas.Biological specimen includes blood product, blood plasma, serum etc..However, such sample should not be construed as Limitation is applied to 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:ColdSpring 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 (utilizes E.Z.N.A.MiRNA 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 uses people's full-length genome chip of expression spectrum of Aglient companies.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 CLINT1 in pulmonary adenocarcinoma is significantly higher than the expression quantity in cancer beside organism.

The differential expression of embodiment 2QPCR sequence verification CLINT1 genes

1st, large sample QPCR checkings are carried out to CLINT1 gene differential expressions.According to the sample collection mode in embodiment 1 Selection 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：

Coded sequence according to CLINT1 genes and GAPDH genes in Genebank designs QPCR amplimers, is stepped by rich Moral biotech firm synthesizes.Specific primer sequence is as follows：

CLINT1 genes：

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

Reverse primer is 5 '-GGACCATATCTGTATTCT-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 as shown in figure 1, compared with adenocarcinoma of lung cancer beside organism, CLINT1 up-regulateds in pulmonary adenocarcinoma, difference With statistical significance (P<0.05) it is, consistent with chip detection result.

The silence of embodiment 3CLINT1 genes

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 design of siRNA

Negative control siRNA sequence (siRNA-NC)：

Positive-sense strand is 5 '-UUCUCCGAACGUGUCACGU-3 ' (SEQ ID NO.7)

Antisense strand is 5 '-ACGUGACACGUUCGGAGAA-3 ' (SEQ ID NO.8)

siRNA-1：

Positive-sense strand is 5 '-UUAUCACUGAAUGGAAAAGCA-3 ' (SEQ ID NO.9)

Antisense strand is 5 '-CUUUUCCAUUCAGUGAUAAAU-3 ' (SEQ ID NO.10)

siRNA-2：

Positive-sense strand is 5 '-UCCAAUUCUUUUUGUUGUCUU-3 ' (SEQ ID NO.11)

Antisense strand is 5 '-GACAACAAAAAGAAUUGGAGA-3 ' (SEQ ID NO.12)

siRNA-3：

Positive-sense strand is 5 '-AUACUUGUCUUUGUUCUUCUU-3 ' (SEQ ID NO.13)

Antisense strand is 5 '-GAAGAACAAAGACAAGUAUGU-3 ' (SEQ ID NO.14)

3rd, transfect

Experiment is divided into three groups：Control group (A549), negative control group (siRNA-NC) and experimental group (siRNA1, SiRNA2, siRNA-3), without homology, concentration is 20nM/ holes to the sequence of wherein negative control group siRNA and CLINT1 genes, Transfected respectively simultaneously.

4th, QPCR detects the transcriptional level of CLINT1 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 l ml.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.

4.3QPCR 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 CLINT1 Gene Experiments 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 siRNA-NC groups are transfected, the expression water of the CLINT1 in experimental group Flat to significantly reduce, difference has statistical significance (P<0.05).

The influence of embodiment 4CLINT1 gene pairs lung adenocarcinoma cell propagation

Influenceed using CCK-8 experiment detection CLINT1 gene pairs lung adenocarcinoma cells multiplication capacities.

1st, with embodiment 3,6h changes liquid after transfection for cell culture and transfection procedure, places cell culture incubator overnight.

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.SiRNA-1 experimental groups and siRNA-NC control groups are set. 4 piece of 96 orifice plate is spread altogether is respectively used to 4 detection time points of 24h, 48h, 72h, 96h.

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 siRNA-1 is transfected, the propagation of cell substantially receives suppression to experimental group System, difference has statistical significance (P<0.05) explanation CLINT1 has the effect for promoting cell propagation.

The influence of embodiment 5CLINT1 gene pairs Apoptosis of Lung Adenocarcinoma Cell

Use the influence of flow cytomery CLINT1 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 SPSS18.0 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 significantly raises (P to result<0.05), the result is said Bright, the overexpression of CLINT1 suppresses the apoptosis of lung adenocarcinoma cell.

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 as shown in figure 5, lung adenocarcinoma cell transfection RNA interfering after, compared with control group, the migration of experimental group and Invasive ability is decreased obviously, and as a result illustrates that CLINT1 can promote 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>Application of the biomarker as target in adenocarcinoma of lung diagnosis and treatment

<160> 14

<170> PatentIn version 3.5

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atgaacatgc tttggtcacg aatgttaaaa gacaacaaaa agaattggag aagagtttat 240

aagtcgttgc tgctcctagc ttacctcata aggaatggat cagagcgtgt tgttacaagt 300

gccagagaac acatttatga tttacgatcc ctggaaaatt accactttgt agatgagcat 360

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ttaggtgagc tgagtgataa aattggaagc acaattgatg acaccatcag caagttccgg 660

aggaaagata gagaagactc tccagaaaga tgcagcgaca gcgatgagga aaagaaagcg 720

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ccttccaaaa ccattgatct tggagcagca gcacattaca caggggacaa agcaagtcca 900

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Gly Ser Thr Ile Asp Asp Thr Ile Ser Lys Phe Arg Arg Lys Asp Arg

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Thr Gly Gly Ser Ala Asp Leu Phe Gly Gly Phe Ala Asp Phe Gly Ser

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Ala Ala Ala Ser Gly Ser Phe Pro Ser Gln Val Thr Ala Thr Ser Gly

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Pro Val Ala Ser Ser Gly Glu Phe Phe Gly Ser Ala Ser Gln Pro Ala

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Thr Val Gly Leu Gly Leu Pro Met Ser Arg Ser Gln Pro Leu Gln Asn

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Claims

The purposes of 1.CLINT1 genes, it is characterised in that the pharmaceutical composition for preparing prevention or treatment adenocarcinoma of lung.
2. purposes according to claim 1, it is characterised in that described pharmaceutical composition includes the lower adjustment of CLINT1.
3. purposes according to claim 2, it is characterised in that described lower adjustment is selected from the group the siRNA of sequence：SEQ ID NO.8、SEQ ID NO.9。
4. a kind of lower adjustment for preventing or treating the CLINT1 of adenocarcinoma of lung, it is characterised in that the lower adjustment is selected from：

Nucleic acid inhibitor, protein inhibitor, proteolytic enzyme, protein binding molecule, it can be upper and lower in albumen or gene level Adjust expression or the activity of the albumen of CLINT1 genes or its coding.
5. a kind of pharmaceutical composition for preventing or treating adenocarcinoma of lung, it is characterised in that described pharmaceutical composition contains：

The lower adjustment of the CLINT1 described in claim any one of 2-4；With

Pharmaceutically acceptable carrier.
6. a kind of purposes of CLINT1 genes, it is characterised in that the potential material for screening prevention or treatment adenocarcinoma of lung.
7. it is a kind of screen prevention or treatment adenocarcinoma of lung potential material method, it is characterised in that methods described includes：

The system expressed or containing CLINT1 genes or its albumen for encoding is processed with candidate substances；With

Detect expression or the activity of the albumen of CLINT1 genes or its coding in the system；

Wherein, if the candidate substances can reduce expression or the activity of CLINT1 genes, show the candidate substances be prevention or Treat the potential material of adenocarcinoma of lung.
8. a kind of purposes of CLINT1 genes, it is characterised in that the product for preparing diagnosis adenocarcinoma of lung.
9. purposes according to claim 8, it is characterised in that the product includes the examination of detection CLINT1 gene expressions Agent.
10. purposes according to claim 9, it is characterised in that the reagent is selected from：

The probe of specific recognition CLINT1；Or

The primer of specific amplification CLINT1；Or

The antibody or part of the albumen of specific binding CLINT1 codings.