CN107058499A - A kind of molecular marker for adenocarcinoma of lung diagnosis and treatment - Google Patents

Abstract

The invention discloses a kind of molecular marker for adenocarcinoma of lung diagnosis and treatment, adenocarcinoma of lung is diagnosed and treated using molecular marker with specificity and sensitiveness.The high expression in patients with lung adenocarcinoma present invention finds molecular marker LOC101926969, the expression of the gene is disturbed with siRNA, the propagation of cell can be suppressed, the invasion and attack number and transport number of cancer cell is reduced, based on this, the present invention provides a kind of new approach for the diagnosis and treatment of adenocarcinoma of lung.

Description

A kind of molecular marker for adenocarcinoma of lung diagnosis and treatment

Technical field

The invention belongs to biomedicine field, it is related to a kind of molecular marker for adenocarcinoma of lung diagnosis and treatment, it is specific described Target is LOC101926969.

Background technology

Lung cancer is one of most common malignant tumour of the mankind, is also the main cause of cancer related mortality.Although lung cancer is examined Disconnected and treatment method and technology constantly update raising, but overall prognosis is still undesirable, and its death rate is still in first of cancer, The survival rate of 5 years is less than 15%.And non-small cell lung cancer accounts for more than the 85% of all lung cancer patients, in non-small cell lung cancer In, adenocarcinoma of lung is wherein topmost part again, and its maximally effective treatment method is that total pneumonectomy adds suitable putting of auxiliary Strategy is treated, even if being the patient of I phases, after lump excision completely, the patient for still having approximately more than 25% is recurred in a short time, sternly Ghost image rings harm human health and life.

In recent years as the research for lung cancer occurring development mechanism is goed deep into, some therapy targets, and new target are searched out Bring new breakthrough to the trial for the treatment of method for the diagnoses and treatment of lung cancer, but be due to it has now been found that can the property of medicine target spot The few and its abnormal distribution crowd's narrow range of quantity, therefore applicable crowd's narrow range for the treatment of method and also presence are controlled accordingly The problems such as treating resistance so that therapeutic effect faces the challenge.So from molecular level, screening newly related to non-small cell lung cancer Can property of medicine target spot and the new therapeutic strategy of development turn into the lasting focus of lung cancer research field.

With the appearance of genome microarray and full-length genome and transcript profile sequencing technologies, it is found that mankind's transcript profile has greatly Antisense gene, overlapping genes and the non-coding RNA expression (non-coding RNAs, ncRNAs) of amount.LncRNA is a class length More than the non-coding RNA of 200bp nucleotides, length can reach 100kb sometimes.LncRNA wide expressions in the mankind, and Played a significant role in multiple physiology courses of gene global regulation.Studied confirmation lncRNA and a variety of human diseases, Especially tumor disease is closely related.Obvious positive correlation, the i.e. hair in disease is presented in the generation of some lncRNA and disease Overexpression state is presented in corresponding lncRNA during hair tonic exhibition；Some lncRNA and disease show certain negative Close, i.e., corresponding lncRNA expression quantity substantially reduction compared with normal structure or normal person in the generation evolution of disease. But it is due to lncRNA not encoding proteins matter in itself, it is also considerably less that it plays the research specifically acted in biological process. The lncRNA related to adenocarcinoma of lung is found, and inquires into its mechanism in adenocarcinoma of lung pathogenic process, for the early diagnosis of disease And targeted therapy, and the scientific research of lncRNA genes is all significant.

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 molecule mark for adenocarcinoma of lung diagnosis and treatment Will thing.

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

The product of diagnosis early stage adenocarcinoma of lung is being prepared the invention provides the reagent of detection LOC101926969 gene expressions In application.

Further, the reagent is selected from：

Specific recognition LOC101926969 probe；Or

Specific amplification LOC101926969 primer.

Preferably, the primer sequence of described specific amplification LOC101926969 genes such as SEQ ID NO.2 and SEQ Shown in ID NO.3.

The invention provides a kind of product for diagnosing adenocarcinoma of lung, the product includes hybridizing skill by sequencing technologies, nucleic acid The expression of LOC101926969 genes in art, nucleic acid amplification technologies detection sample.Wherein, the product includes (but not limiting In) chip, preparation or kit.

Further, the nucleic acid amplification technologies are selected from PCR, reverse transcriptase polymerase chain reaction, transcription Jie Amplification, ligase chain reaction, strand displacement amplification and the amplification based on nucleotide sequence led.

The invention provides LOC101926969 genes answering in the pharmaceutical composition for preparing prevention or treatment adenocarcinoma of lung With.

Further, described pharmaceutical composition includes LOC101926969 lower adjustment.The lower adjustment is selected from：With LOC101926969 or its transcript are target sequence and can suppress the interference of LOC101926969 gene expressions or genetic transcription Molecule, including：ShRNA (children purpura nephritis), siRNA (siRNA), dsRNA, Microrna, antisensenucleic acids, or can express or Form the construction of the shRNA, siRNA, dsRNA, Microrna, antisensenucleic acids.

Further, described lower adjustment is siRNA.It is preferred that the siRNA sequence such as SEQ ID NO.8, SEQ ID Shown in NO.9.

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

The invention provides application of the LOC101926969 genes in the potential material of screening prevention or treatment adenocarcinoma of lung.

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

The system expressed or containing LOC101926969 genes is handled with candidate substances；With

Detect the expression of LOC101926969 genes in the system；

Wherein, if the candidate substances can reduce expression or the activity of LOC101926969 genes, (preferably significantly reduce, It is such as low by more than 20%, it is preferably low by more than 50%；More preferably low more than 80%), then it is prevention or treatment to show the candidate substances The potential material of adenocarcinoma of lung.The system is selected from：Cell system, subcellular fraction system, solution system, organizational framework, organ systems Or animal system.

The candidate substances include but is not limited to：Designed for LOC101926969 genes or its upstream or downstream gene Disturbing molecule, nucleic acid inhibitor, micromolecular compound etc..

The advantages of the present invention：

Present invention firstly discovers that LOC101926969 differential expression is to the generation development of adenocarcinoma of lung related, the base is pointed out Because being used as diagnosing the index of adenocarcinoma of lung.

The present invention is experimentally confirmed, the expression of siRNA silence LOC101926969 genes, can suppress lung adenocarcinoma cell Propagation, reduce migration and the invasion and attack rate of lung adenocarcinoma cell, point out LOC101926969 genes to be used to control as drug target Treat adenocarcinoma of lung and its transfer.

Brief description of the drawings

Fig. 1 is to detect expression figure of the LOC101926969 genes in pulmonary adenocarcinoma using QPCR；

Fig. 2 is to detect transfected condition figures of the LOC101926969 in lung adenocarcinoma cell using QPCR；

Fig. 3 is to detect the influence figure that LOC101926969 gene pairs lung adenocarcinoma cell is bred with CCK-8 methods；

Fig. 4 is the influence figure with flow cytomery LOC101926969 gene pairs Apoptosis of Lung Adenocarcinoma Cell；

Fig. 5 is to detect the influence figure that LOC101926969 is migrated and attacked to lung adenocarcinoma cell using Transwell cells； Wherein figure A is the influence figure that LOC101926969 is migrated to lung adenocarcinoma cell；It is that LOC101926969 is invaded lung adenocarcinoma cell to scheme B The influence figure attacked.

Specific embodiment

The present invention is most wide using current covering database by high throughput method by in-depth study extensively LncRNA has found wherein have substantially in the expression of tumor tissues and cancer beside organism in lncRNA chips, detection adenocarcinoma of lung sample The lncRNA fragments of differential expression, inquire into its relation between the generation of adenocarcinoma of lung, thus early detection for adenocarcinoma of lung and Targeted therapy finds more preferable approaches and methods.By screening, present invention firstly discovers that LOC101926969 shows in adenocarcinoma of lung The up-regulation of work property.It is demonstrated experimentally that siRNA disturb silence LOC101926969, can effectively suppress lung adenocarcinoma cell propagation and Invasion and attack, new way is provided for the accurate treatment of adenocarcinoma of lung.

Molecular marker

" molecular marker " is the expression of its expression in tissue or cell and normal or healthy cell or tissue Level compares any gene changed.

It would be recognized by those skilled in the art that the practicality of the present invention is not limited to the marker gene of the present invention The gene expression of any specific variants is quantified.As nonrestrictive example, marker gene can have SEQ ID NO.1 The nucleotide sequence specified.In some embodiments, it has and the same or analogous cDNA of listed sequence at least 85% All listed sequences at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% as described above of sequence 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 the importances of the present invention.The table of biomarker can be detected on transcriptional level Up to level.

LOC101926969 genes

LOC101926969 is located in No. 2 areas of the short arm of a chromosome 1 of people, a kind of representational people LOC101926969 genes Nucleotide sequence as shown in SEQ ID NO.1.LOC101926969 in the present invention includes wild type, saltant type or its piece Section.

It would be recognized by those skilled in the art that the practicality of the present invention is not limited to appointing to the target gene of the present 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 comparison 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 The acid sequence phase same sex, 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 (the phase same sex) therebetween.When hybridization has more than specific general loss When selective, there is cross selection.Typically, when one section of sequence at least about 14 nucleotides is present at least about When the 55% phase same sex, preferably at least about 65%, more preferably at least about 75% and the most preferably at least about 90% phase same sex, hair Raw selective cross.It is as described herein, cognate pair than length can be longer sequence section, 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 the importances of the present invention.The table of biomarker can be detected on transcriptional level Up to level.

Detection technique

The present invention lncRNA detected using multiple nucleic acids technology known to persons of ordinary skill in the art, these skills Art includes but is not limited to：Nucleic acid sequencing, nucleic acid hybridization and nucleic acid amplification technologies.

The exemplary, non-limitative example of Nucleic acid sequencing techniques includes but is not limited to chain terminator (Sanger) sequencing and contaminated Expect terminator sequencing.One of ordinary skill in the art it will be recognized that due to RNA in cell less stable and in an experiment Be more vulnerable to nuclease attack, thus before sequencing generally by RNA reverse transcriptions into DNA.

The present invention can simultaneously be expanded before detection or with detection to nucleic acid (for example, ncRNA).Nucleic acid amplification technologies Exemplary, non-limitative example include but is not limited to：PCR (PCR), reverse transcriptase polymerase chain reaction (RT- PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and based on nucleotide sequence Expand (NASBA).One of ordinary skill in the art will be it will be recognized that some amplification techniques (for example, PCR) needs will before amplification RNA reverse transcriptions are into DNA (for example, RT-PCR), and other amplification techniques then direct cloning RNA (for example, TMA and NASBA).

Commonly referred to as PCR PCR uses annealing and the primer extend of denaturation, primer pair and opposite strand Multiple circulations, exponentially increase target nucleic acid sequence copy number；The amplification of TMA transcriptive intermediate is (in substantial constant Temperature, ionic strength and pH under conditions of autocatalytically synthesize multiple copies of target nucleic acid sequence, wherein target sequence is more Individual RNA copies autocatalytically generate other copy；LCR ligase chain reaction uses miscellaneous with the adjacent area of target nucleic acid The two groups of complementary DNA oligonucleotides handed over；Other amplification methods are included for example：The commonly referred to as NASBA expansion based on nucleotide sequence Increase；Use rna replicon enzyme (the commonly referred to as Q β replicase) amplification of amplification probe molecule in itself；Amplification method based on transcription； And the sequence amplification of self―sustaining.

The nucleic acid of non-amplification or amplification can be detected by any conventional means in the present invention.

Chip, kit

The invention provides the product of the expression of LOC101926969 genes in detection, the product is included (but not It is limited to) preparation, chip or kit.Its chips 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 LOC101926969.

Term " probe " refers to the molecule that can be combined with the particular sequence or subsequence or other parts of another molecule.Unless another Point out, term " probe " is often referred to can be by complementary base pairing and another polynucleotides (often referred to as " target polynucleotide ") 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.

The invention provides a kind of kit, the kit can be used for detection LOC101926969 expression.It is preferred that, Also contain the label for labeled RNA sample, and the bottom corresponding with the label in described preparation or kit Thing.In addition, may also include in described kit for extracting the various reagents needed for RNA, PCR, hybridization, colour developing etc., including But it is not limited to：Extract, amplification liquid, hybridization solution, enzyme, comparison liquid, nitrite ion, washing lotion etc..In addition, also being wrapped in described kit Include operation instructions and/or chip image analysis software.

Lower adjustment and pharmaceutical composition

Discovery based on the present inventor, the invention provides a kind of purposes of the lower adjustment of LOC101926969, for making The standby pharmaceutical composition for suppressing adenocarcinoma of lung.As used herein, described LOC101926969 lower adjustment includes but is not limited to suppression Preparation, antagonist, retarding agent, blocking agent, nucleic acid inhibitor etc..

Described LOC101926969 lower adjustment refers to any expression for lowering LOC101926969 genes or suppression The material of the transcription of LOC101926969 genes, these materials can be used as the material useful for lowering LOC101926969 In prevention or treatment adenocarcinoma of lung.

As a kind of preferred embodiment of the present invention, the lower adjustment of the LOC101926969 is a kind of LOC101926969 special The siRNA molecule of the opposite sex.As used herein, described " siRNA " refers to a kind of short-movie section double stranded rna molecule, energy Enough specific mRNA that degraded by target of the mRNA of homologous complementary sequence, this process is exactly RNA interference (RNA Interference) process.SiRNA can be prepared into the form of double-strandednucleic acid, and it contains a positive-sense strand and one anti- Adopted chain, this two chains only form double-strand under conditions of hybridization.One double-stranded RNA compound can be by the positive-sense strand that is separated from each other Prepared with antisense strand.Therefore, for example, complementary positive-sense strand and antisense strand are chemical syntheses, can pass through annealing thereafter Hybridization, produces the double-stranded RNA compound of synthesis.

When screening effective siRNA sequence, the present inventor is analyzed by substantial amounts of compare, so as to find out optimal effective Fragment.The present inventor's design has synthesized a variety of siRNA sequences, and they are transfected into lung adenocarcinoma cell system by transfection reagent respectively Verified, select the optimal siRNA of interference effect, further tested in cellular level, as a result prove to exist for the siRNA Can effectively suppress the expression of LOC101926969 genes in cell, and lung adenocarcinoma cell propagation.

The nucleic acid inhibitor such as siRNA of the present invention can be with chemical synthesis, can also be 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 composition, it contains the described LOC101926969 of effective dose lower adjustment, with And pharmaceutically acceptable carrier.Described composition can be used for suppressing adenocarcinoma of lung.Under any foregoing LOC101926969 Adjustment is used equally for the preparation of composition.

As used herein, described " effective dose " refer to that people and/or animal can be produced function or activity and can by people and/ Or the amount that animal is received." pharmaceutically acceptable carrier " refers to the carrier for Therapeutic Administration, including various figurations Agent and diluent.The term refers to some such medicament carriers：Themselves it is not necessary active component, and does not have after administration Undue toxicity.Suitable carrier is well known to those of ordinary skill in the art.It is pharmaceutically acceptable in the composition to carry Body can contain liquid, such as water, salt solution, buffer solution.In addition, complementary material is there is likely to be in these carriers, such as filler, Lubricant, glidant, wetting agent or emulsifying agent, pH buffer substance etc..Cell can also be contained in described carrier, and (host is thin Born of the same parents) transfection reagent.

The present invention can use with a variety of 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；It is preferred that, the administering mode is that non-bowel is given.

It is preferred that, it can be carried out using the means of gene therapy.Such as, directly LOC101926969 lower adjustment can be passed through The methods such as injection deliver medicine to subject；Or, LOC101926969 lower adjustment can will be carried by certain approach Ceneme (such as expression vector or virus etc., or siRNA or shRNA) is delivered on target spot, and is allowed to expression activity Adjusted under LOC101926969, concrete condition need to be depending on the type of described lower adjustment, and these are those skilled in the art Known.

The pharmaceutical composition of the present invention can be further comprising one or more anticancers.In specific embodiments, Compound of the pharmaceutical composition comprising at least one suppression LOC101926969 gene expressions and at least one chemotherapeutics.For this The chemotherapeutics of the method for invention, includes but is not limited to, DNA- alkylating agents, antitumor antibiotics agent, antimetabolite, tubulin is steady Determine agent, tubulin destabilizing agent, hormone antagonist, topoisomerase enzyme inhibitor, kinases inhibitor, HMG-COA suppresses Agent, CDK inhibitor, cyclin inhibitors, caspase inhibitors, metal protease inhibitors, antisensenucleic acids, three Virus, bacterium and the exotoxin reagent of chain helical dna, aptamer, and molecular modification.

Pharmaceutical acceptable carrier may include but be not limited to：Virus, liposome, nano particle or polymer and its any combination.Phase The delivering supporting agent of pass may include but be not limited to：Liposome, biocompatible polymer (including natural polymer and synthesized polymer Thing), lipoprotein, polypeptide, polysaccharide, lipopolysaccharides, artificial viral envelope, inorganic (including metal) particle and bacterium or virus (example Such as baculoviral, adenovirus and retrovirus), bacteriophage, sticking grain or plasmid vector.

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

The pharmaceutical composition of the present 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, testing compound is added into cell culture system, and determine LOC101926969 expression water It is flat；In control group, testing compound is added without into same cultivating system, and determine LOC101926969 expression water It is flat；Wherein, if LOC101926969 expression is more than control group in experimental group, illustrate that the candidate compound is LOC101926969 lower adjustment.

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

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 is obtained, 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 Sample type of the limitation suitable for the present 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 method 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

Respectively 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, excludes tumor center substantially necrosis, calcification portion Divide and the peripheral normal lung tissue of tumour；Ai Pang normal lung tissues sample is derived from more than borderline tumor 5cm position, visually observes Without significant change.The acquirement of above-mentioned all samples passes through 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, takes the tissue of above-mentioned acquisition to be put into mortar, is shredded in liquid nitrogen and grind into powder, Put into after shredding in liquid nitrogen and be ground to powdered, then continued in 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 of no RNase, stood at room temperature 5min.Separation RNA is extracted according to the specification in kit.It is specific as follows：

1) RNA separation：

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 Hatch 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

450 μ l are taken to move in the new EP pipes without RNase the aqueous phase separated, according to 1:It is different that 1 ratio adds 450 μ l Propyl alcohol, hatches 10min at room temperature after mixing of turning upside down, 4 DEG C of 14000g centrifuge 10min.

3) RNA is eluted

It is careful after centrifugation to remove supernatant, add 1ml75% ethanol (destroy the enzyme treatment, matching while using and in precooling on ice) and rinse RNA, subsequent 4 DEG C of 7500g centrifuge 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 and handle water-bath 10min in water 20-50 μ l, 55-60 DEG C of water bath without RNase.

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 lncRNA express spectras and screening experiment of chip.

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 Kang Cheng biology-Human lncRNA Array, carries out the step of by chip operation instructions miscellaneous Hand over.

5th, data analysis

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

6th, result

As a result show, expression quantity of the LOC101926969 in pulmonary adenocarcinoma is significantly higher than the expression in cancer beside organism Amount.

The differential expression of embodiment 2QPCR sequence verification LOC101926969 genes

1st, large sample QPCR checkings are carried out to LOC101926969 gene differential expressions.Received according to the sample in embodiment 1 Mode set selects 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, Ran Houfang In 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：

QPCR amplimers are designed according to the coded sequence of LOC101926969 genes and GAPDH genes in Genebank, Synthesized by Bo Maide biotech firms.Specific primer sequence is as follows：

LOC101926969 genes：

Forward primer is 5 '-TTCTGAGTGTCGGATAAC-3 ' (SEQ ID NO.2)；

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

GAPDH genes：

Forward primer is 5 '-AATCCCATCACCATCTTCCAG-3 ' (SEQ ID NO.4)；

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

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, determines purpose band, Δ Δ CT methods carry out phase by melt curve analysis analysis and electrophoresis 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, Carry out statistical analysis using SPSS18.0 statistical softwares, the paired comparisons of cancer and cancer beside organism are examined using t, it is believed that work as P< There is statistical significance when 0.05.

6th, result

As a result as shown in figure 1, compared with adenocarcinoma of lung cancer beside organism, on LOC101926969 is expressed in pulmonary adenocarcinoma Adjust, difference has statistical significance (P<0.05) it is, consistent with chip testing result.

The silence of embodiment 3LOC101926969 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, relative humidity for 90% incubator in cultivate.Change within 2-3 days liquid 1 time, it is conventional using 0.25% trypsase containing EDTA Had digestive transfer culture.

Cell in blake bottle is digested and is 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 cell density, cell density can be transfected for more than 70%.

2nd, siRNA design

Negative control siRNA sequence (siRNA-NC)：

Positive-sense strand is 5 '-UUCUCCGAACGUGUCACGU-3 ' (SEQ ID NO.6)

Antisense strand is 5 '-ACGUGACACGUUCGGAGAA-3 ' (SEQ ID NO.7)

siRNA-1：

Positive-sense strand is 5 '-UACAGAAAAUAAGUCAUAGAA-3 ' (SEQ ID NO.8)

Antisense strand is 5 '-CUAUGACUUAUUUUCUGUAGU-3 ' (SEQ ID NO.9)

siRNA-2：

Positive-sense strand is 5 '-UUCUUCUUUGUACAGUUACCA-3 ' (SEQ ID NO.10)

Antisense strand is 5 '-GUAACUGUACAAAGAAGAAAU-3 ' (SEQ ID NO.11)

3rd, transfect

Experiment is divided into three groups：Control group (A549), negative control group (siRNA-NC) and experimental group (siRNA1, SiRNA2), wherein negative control group siRNA is with the sequence of LOC101926969 genes without homology, and concentration is 20nM/ holes, together When transfected respectively.

4th, QPCR detects the transcriptional level of LOC101926969 genes

The extraction of 4.1 cell total rnas

1) cell culture fluid in 6 orifice plates is outwelled, 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) aqueous phase is 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 are washed with lml 75% ethanol.4 DEG C, 7500g centrifuges 5min.

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

6) DEPC water of 25 μ 1 without RNase, -70 DEG C of preservations are added.

4.2 reverse transcription step be the same as Examples 2.

4.3 QPCR amplification steps be the same as Examples 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, the difference between LOC101926969 Gene Experiments group and control group are carried out using SPSS18.0 statistical softwares Examined using t, it is believed that work as P<There is statistical significance when 0.05.

6th, result

As a result such as Fig. 2 is shown, with non-transfection group compared with transfecting siRNA-NC groups, the LOC101926969's in experimental group Expression is significantly reduced, and difference has statistical significance (P<0.05).

The influence of embodiment 4LOC101926969 gene pairs lung adenocarcinoma cell propagation

Using CCK-8 experiment detection LOC101926969 gene pairs lung adenocarcinoma cells multiplication capacity influences.

1st, cell culture and transfection procedure be the same as Example 3,6h changes liquid after transfection, places cell culture incubator and stays overnight.

2nd, cell was taken out in second day, micro- Microscopic observation cell growth status, 1ml/ holes add the pancreatin containing EDTA, enter Row cell dissociation, waits to remove pancreatin after the completion of digesting, and adding cell culture medium and mixing 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 1 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, 10 μ 1 CCK-8 detection liquid is added 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 examine, it is believed that work as P<There is statistical significance when 0.05.

7th, result

As a result as shown in figure 3, compared with the control, experimental group is after transfection siRNA-1, and the propagation of cell substantially receives suppression System, difference has statistical significance (P<0.05) illustrate that LOC101926969 has the effect for promoting cell propagation.

The influence of embodiment 5LOC101926969 gene pairs Apoptosis of Lung Adenocarcinoma Cell

Use the influence of flow cytomery LOC101926969 gene pairs Apoptosis.

1st, cell culture step be the same as Example 3.

2nd, cell transfecting step be the same as Example 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) cell is added into 1 × sample-loading buffers of lml, 300g centrifugation 10min suction out buffer solution.

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 1, added in EP pipes.

6) 5 μ l Annexin V FITC are added in EP pipes, mixes the liquid in EP pipes, lucifuge is incubated at room temperature 10min。

7) 5 μ 1PI dye liquors are added into EP pipes, at room temperature lucifuge 5min.

8) 500 μ l PBS solution 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 of difference use between the two is examined, it is believed that work as P<When 0.05 With statistical significance.

4th, result：

As a result as shown in figure 4, experimental group is compared with control group, apoptosis rate has significant change (P<0.05), the knot Fruit illustrates that LOC101926969 suppresses the apoptosis of 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, are 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 that 50 μ l are 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 processing 12-24h, carries out digestion process to cell, is centrifuged after terminating digestion, in removal Layer nutrient solution.Sedimentation cell is cleaned with PBS, the serum free medium containing BSA is added and it is resuspended.Adjustment is thin The density of born of the same parents is to 5 × l0⁵Individual/ml.

3rd, cell is inoculated with

The μ 1 (migration experiment is 100 μ 1, and Matrigel is 200 μ 1) of cell suspension 200 is taken to be added to Transwell cells In.Room adds 1640 culture mediums of 500 μ 1 containing FBS under 24 orifice plates.Cell is put into cell culture incubator and cultivates 24h.

4th, dye

Cell is dyed 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

As a 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 LOC101926969 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 and its core concept of the present invention.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 improve 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>Hospital of Hebei Medical University the 4th

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

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 2720

<212> DNA

<213>People source

<400> 1

gtcacccagg ccggagtgca gtggcatgat cacagctcac tgaagcctcg acttcccagg 60

cttaagtgat cctcctacct cactcagcct cccgagtagt tgggaccaca ggcatgtgcc 120

accacatctg gctattttta gtattttttg tagagatggg gtttcgccat gctgctcagg 180

caggtctcaa actcctggcc tcaagccatc tgcttgtgtt ggcctcccaa agtgctggat 240

tacaggcgtg caccaccacc cccggcctaa ttgaattttt attttggaag aaccatatac 300

ctttatacat attctttcta tgacttattt tctgtagttg aagttcaagg gtgtttgaca 360

gtgtgatgtg gttattgtta acctgtatgt gggggcatac agatatgttg ttttactttt 420

tcccataagg ctaagatcat ctccagaaat attaattttt tagacagcgt gattatagtt 480

atttaatact catatttatc aaaatattat atgttaaaac caataaccat ctgaacttat 540

ttgaatatag tttttcctat tctgttctta gaaatttcag tcattaacaa tctttacact 600

tagaaagtgt tttaatttta ctttgatgac aggaatagtg tttttctcaa tgaaatatat 660

aatagcagct tataagtgat aaccgttttc attctgtttt taagagaaaa tactttggtc 720

ttttttacca tgatagttta gtgtgtgatg tcataaaaat gggctgtagc catttaaatt 780

cttgttcaag tgcagaaact aagagacttc agtttctatt aagccatgtc aactcctatt 840

aggagacctt tagtggttcc agttccaggg ccttcttgaa gaaattgtga tacttgtgta 900

agtatatcta gagttttctt tccagcattg gctaagtcac atagctgaga aatatgatac 960

atggagaagg ttgctgactg gatgaacttt tctacattga atactttaaa tctggattct 1020

agaataaaat atattgatgc atatttgagt tgtcaactgt ttatgtcttg ttcttttcag 1080

cttcaacatt gctttatggc atgtgtggtc gtttttcact ccattgttgt tgtttaccca 1140

gtttatgggg gttgtaatgt ttatcacact ccttggatga tttccgaaga tcaccccatc 1200

ctgagatctg ctgccttaca cagtggaggc tgttttctga gtgtcggata actctgttat 1260

taaataagtg catattgaga atactcactc cagatgctta gaggcatgtt gagaaggaca 1320

aagatagtgc ctcctgtcag gtcacttaca tcagaacttg caggaaacct gctttataca 1380

gcagttgaca ggtgtggaaa ttgaagctca tggaaatgaa agttaataca ctaaattttc 1440

aaggagttaa tgaaaactga catttctgct aaaaacagca tgttctccct gtgggatttt 1500

aaaggaaggt caatggtaac tgtacaaaga agaaatggac atagtctcat tgttagcagt 1560

ctgaaaatag ttgctagcac ctccatgccc acgccaatgt ctccagcctc cttcaagcac 1620

cttgctgtgt cttggaacca ttgggagccc tcacaggagg cccttcgttc ctgtatgcat 1680

gtggtgccac aagctgcttt gggcccggag gaatcctaca catctcagaa cacttcctca 1740

cctgaccctg tcacccatct ctccccttct catgtgctca gccccttctt tgactcttga 1800

attttgagtt tttacagatg tttgggagct cttactctga catgaattta taattgtaat 1860

ggagactcag acaacgttgt agatcacaga gtgaactatg ctttttagtg ttaaatgcaa 1920

tagcttaaga tagaatgttt tacttgttac ataaatgctg gttttctttc agatttaagg 1980

acatatactt tattttttta agagataggg tcttctatgt tgtccaggct ggctttgaac 2040

tcctgggatc aagtgatcct cctgcctcag ccttcgaagt agttgggact acaggcccac 2100

gccaccgtgc atggctggac acgtaaattt gaattgaatg gttaaacatc cagctagctg 2160

aaagcatggc agaccctaac agaaaagcta cagtgtgttt ttgcaactat gaagtgaatg 2220

gtttcctggg gaaaattgtg actttgtata actatttttg aaaccagaat aaattatatt 2280

tcacttgcat attcttaaat tattaaaatt ttcagaagtc agtgatacag aaatactatt 2340

ttgcaatgtt aatctgtttg agtctttgga gaaagtggtt tcattgtagg tacatgatgc 2400

actcttaata ttttaaacaa atagttcact cttccattta agggatatca gttccttgta 2460

taaaatgact ggatatgtat aaagcaatta tgttgtcatg tgcctttaac cagctttagt 2520

aattactata atctcatatt tatgatagtt ttgttaggtg acaggaccaa atgaaaatat 2580

tttatgtttt cccatcactt tagattttat cattgtgtaa attactgggt ttttagcatt 2640

tcctaatgtg aagttttaat catttttaag tatacatatt ttttttctgt accatttaaa 2700

taaaatattt ttataacttt 2720

<210> 2

<211> 18

<212> DNA

<213>Artificial sequence

<400> 2

ttctgagtgt cggataac 18

<210> 3

<211> 19

<212> DNA

<213>Artificial sequence

<400> 3

gcaagttctg atgtaagtg 19

<210> 4

<211> 21

<212> DNA

<213>Artificial sequence

<400> 4

aatcccatca ccatcttcca g 21

<210> 5

<211> 19

<212> DNA

<213>Artificial sequence

<400> 5

gagccccagc cttctccat 19

<210> 6

<211> 19

<212> RNA

<213>Artificial sequence

<400> 6

uucuccgaac gugucacgu 19

<210> 7

<211> 19

<212> RNA

<213>Artificial sequence

<400> 7

acgugacacg uucggagaa 19

<210> 8

<211> 21

<212> RNA

<213>Artificial sequence

<400> 8

uacagaaaau aagucauaga a 21

<210> 9

<211> 21

<212> RNA

<213>Artificial sequence

<400> 9

cuaugacuua uuuucuguag u 21

<210> 10

<211> 21

<212> RNA

<213>Artificial sequence

<400> 10

uucuucuuug uacaguuacc a 21

<210> 11

<211> 21

<212> RNA

<213>Artificial sequence

<400> 11

guaacuguac aaagaagaaa u 21

Claims (10)

1. detect application of the reagent of LOC101926969 gene expressions in the product for preparing diagnosis early stage adenocarcinoma of lung.

2. application according to claim 1, it is characterised in that the reagent is selected from：

Specific recognition LOC101926969 probe；Or

Specific amplification LOC101926969 primer.

3. it is a kind of diagnose adenocarcinoma of lung product, it is characterised in that the product include by sequencing technologies, nucleic acid hybridization technique, The expression of LOC101926969 genes in nucleic acid amplification technologies detection sample.

4. product according to claim 3, it is characterised in that the nucleic acid amplification technologies be selected from PCR, Reverse transcriptase polymerase chain reaction, the amplification of transcriptive intermediate, ligase chain reaction, strand displacement amplification and based on nucleotide sequence Amplification.

5.LOC101926969 application of the gene in the pharmaceutical composition for preparing prevention or treatment adenocarcinoma of lung.

6. application according to claim 5, it is characterised in that described pharmaceutical composition includes LOC101926969 downward Agent.

7. application according to claim 6, it is characterised in that described lower adjustment is siRNA.

8. the application according to claim 6 or 7, it is characterised in that described pharmaceutical composition also includes and the lower adjustment Other medicine classes and pharmaceutically acceptable carrier and/or auxiliary material of compatibility.

Application of the 9.LOC101926969 genes in the potential material of screening prevention or treatment adenocarcinoma of lung.

10. a kind of method for the potential material for screening prevention or treatment adenocarcinoma of lung, it is characterised in that methods described includes：

The system expressed or containing LOC101926969 genes is handled with candidate substances；With

Detect the expression of LOC101926969 genes in the system；

Wherein, if the candidate substances can reduce expression or the activity of LOC101926969 genes, show that the candidate substances are Prevention or the potential material for the treatment of adenocarcinoma of lung.