CN109371136A - One kind lncRNA relevant to adenocarcinoma of lung and its application - Google Patents

Abstract

The invention discloses a kind of applications of gene relevant to adenocarcinoma of lung, the gene is LINC02014, present invention firstly discovers that the differential expression of LINC02014 is to the occurrence and development of adenocarcinoma of lung related, and LINC02014 is demonstrated in patients with lung adenocarcinoma by QPCR and is significantly raised, the index for prompting the gene to can be used as diagnosis adenocarcinoma of lung is applied to clinic.The invention also discloses the expression by reducing LINC02014, can effectively inhibit the proliferation of lung adenocarcinoma cell, reduce the migration and invasion rate of lung adenocarcinoma cell, and LINC02014 is prompted to can be used as drug target for treating adenocarcinoma of lung and its transfer.

Description

One kind lncRNA relevant to adenocarcinoma of lung and its application

Technical field

The invention belongs to biomedicine fields, are related to one kind lncRNA relevant to adenocarcinoma of lung and its application, specifically relate to And application of the LINC02014 in adenocarcinoma of lung diagnosing and treating.

Background technique

From the 1980s, lung cancer gradually become in global range disease incidence and the highest cancer of lethality it One, and the trend risen year by year is presented in disease incidence and lethality, with the investigation statistics data according to American Cancer Society in 2016 It has been shown that, median lethal rate of the lung cancer in male and female patient are respectively 27% and 26%, occupy malignant tumour lethality it It is first.In China, the disease incidence and case fatality rate of lung cancer are risen year by year, and are had become maximum to population health and life threat pernicious Tumour.In recent years, with the research application to deepen continuously with a large amount of targeted drugs that lung cancer mutated gene is studied, the treatment of lung cancer Gradually change to the individualized treatment that gene is guiding.In the past, people focus on what lung cancer individualized treatment was studied On encoding gene, and with the continuous deepening of research it is found that long-chain non-coding RNA (long non-coding RNA, LncRNA very important important function) is played during malignant tumour occurrence and development, while its significant tumor tissues is special Anisotropic and its applied to diagnosing tumor a possibility that, also makes it have become hot spot new in tumor research.With sequencing technologies It continues to develop, more and more researchers identify novel lung cancer according to gene function using high-throughput genome screening technique. Attempt to develop more accurate diagnostic tool on the basis of carrying out effective parting to lung cancer, to preferably control lung cancer It treats.

By the regulation of gene, gene of eucaryote cell group is made of the pathophysiological process of disease the DNA sequence dna of bulky complex, By being transcribed into rna expression and transmitting biological heredity information.Mankind's genome sequencing as a result, it has been found that, the only base less than 2% Because sequence participates in protein coding, and most non-coding sequences are transcribed into the non-volume of long-chain that length is greater than 200 bases Code RNA, participates in the regulation of gene expression, promotes or inhibit generation, the development of kinds of tumors.All the time, molecular biology is ground The focus to make internal disorder or usurp concentrates on the mRNA (messenger RNA, mRNA) of coding protein, and largely without encoding histone function Non-coding RNA (non-coding RNA, ncRNA) be considered as " determined garbage sequence " of abiology function and by long-standing neglect. NcRNA does not have the characteristics such as typical atg start codon, open reading frame, promoter conserved region and terminator codon.According to NcRNA length is different, and ncRNA is divided into small non-coding RNA (small non-coding RNA, snRNA) and long-chain non-coding RNA, wherein lncRNA accounts for 80%.LncRNA is transcribed by RNA polymerase II, participates in epigenetic, alternative splicing, nuclear import etc. Process, transcription and functional disturbance can be used as tumor suppressor or carcinogenic factor functions.Therefore, lncRNA is furtherd investigate Biological function and its new thinking can be provided for clinical diagnosis and treatment with disease relationship.

Summary of the invention

In order to make up for the deficiencies of the prior art, an object of the present invention is to provide a kind of related to adenocarcinoma of lung occurrence and development Molecular marker.

The second object of the present invention is to provide the diagnostic products and means of a kind of adenocarcinoma of lung, pass through detection molecules marker Whether expression suffers from adenocarcinoma of lung judging patient or suffers from the risk of adenocarcinoma of lung.

The third object of the present invention provides a kind of pharmaceutical composition for treating adenocarcinoma of lung or method, passes through the downward of specificity Molecular marker treats adenocarcinoma of lung.

To achieve the goals above, the present invention adopts the following technical scheme:

The present invention provides the reagents of detection LINC02014 gene expression in the product of preparation diagnosis early stage adenocarcinoma of lung Using.

Further, the reagent is selected from:

The probe of specific recognition LINC02014；Or

The primer of specific amplification LINC02014.

Preferably, the primer sequence of the specific amplification LINC02014 gene such as SEQ ID NO.2 and SEQ ID Shown in NO.3.

The present invention provides a kind of product for diagnosing adenocarcinoma of lung, the product includes hybridizing skill by sequencing technologies, nucleic acid The expression of LINC02014 gene in art, nucleic acid amplification technologies detection sample.Wherein, the product includes but is not limited to Chip, preparation or kit.

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

The present invention provides application of the LINC02014 gene in the pharmaceutical composition of preparation prevention or treatment adenocarcinoma of lung.

Further, described pharmaceutical composition includes the lower adjustment of LINC02014.The lower adjustment is selected from: with LINC02014 Or its transcript is target sequence and the disturbing molecule for being able to suppress LINC02014 gene expression or genetic transcription, comprising: shRNA (children purpura nephritis), siRNA (siRNA), dsRNA, Microrna, antisense nucleic acid, or can express or be formed the shRNA, SiRNA, dsRNA, Microrna, antisense nucleic acid construction.

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

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

The present invention provides application of the LINC02014 gene in the potential substance of screening prevention or treatment adenocarcinoma of lung.

The present invention provides a kind of screening prevention or the methods of the potential substance for the treatment of adenocarcinoma of lung, which comprises

The system expressed or containing LINC02014 gene is handled with candidate substances；With

Detect the expression of LINC02014 gene in the system；

Wherein, if the candidate substances can reduce the expression or activity of LINC02014 gene, (preferably significantly reduce, it is such as low 20% or more, preferably low 50% or more；More preferably low 80% or more) then shows that the candidate substances are prevention or treatment lung gland The potential substance of cancer.The system is selected from: cell system, subcellular system, solution system, organizational framework, organ systems or dynamic Objects system.

The potential substance includes but is not limited to: designing for LINC02014 gene or its upstream or downstream gene Disturbing molecule, nucleic acid inhibitor, small molecule compound etc..

Detailed description of the invention

Fig. 1 is the expression figure using QPCR detection LINC02014 gene in pulmonary adenocarcinoma；

Fig. 2 is the transfected condition figure using QPCR detection LINC02014 in lung adenocarcinoma cell；

Fig. 3 is the influence diagram with CCK-8 method detection LINC02014 gene pairs lung adenocarcinoma cell proliferation；

Fig. 4 is the influence diagram with flow cytomery LINC02014 gene pairs Apoptosis of Lung Adenocarcinoma Cell；

Fig. 5-1 is the influence diagram migrated using the cell Transwell detection LINC02014 to lung adenocarcinoma cell；

Fig. 5-2 is the influence diagram invaded using the cell Transwell detection LINC02014 to lung adenocarcinoma cell.

Specific embodiment

The present invention is after extensive and in-depth study, most wide using current covering database by high throughput method LncRNA chip, detecting lncRNA in adenocarcinoma of lung sample, in the expression of tumor tissues and cancer beside organism, discovery wherein has obvious The lncRNA segment of differential expression inquires into its relationship between the generation of adenocarcinoma of lung, thus for adenocarcinoma of lung early detection and Targeted therapy finds better approaches and methods.By screening, present invention firstly discovers that LINC02014 conspicuousness in adenocarcinoma of lung Up-regulation.It is demonstrated experimentally that siRNA interferes silencing LINC02014, the proliferation and invasion of lung adenocarcinoma cell can be effectively inhibited, is The personalized treatment of adenocarcinoma of lung provides new way.

Molecular marker

" molecular marker " is its expression in tissue or cell and normal or healthy cell or tissue expression Level is compared to any gene to change.

It would be recognized by those skilled in the art that practicability of the invention is not limited to marker gene of the invention The gene expression of any specific variants is quantified.As unrestricted example, marker gene can have SEQ ID NO.1 Specified nucleotide sequence.The present invention can use any method known in the art measurement gene expression.Art technology Personnel should be appreciated that the means of measurement gene expression are not importances of the invention.Biology can be detected on transcriptional level The expression of marker.

LINC02014 gene

LINC02014 is located on No. 3 chromosomes of people, NC_000003.12 (130088863..130094304, Complement), nucleotide sequence is as shown in SEQ ID NO.1.LINC02014 in the present invention includes wild type, saltant type Or its segment.

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

Alternatively, when nucleic acid or its segment and another nucleic acid (or its complementary strand), a chain or its complementary series are 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 are cross selections.Typically, when one section of sequence at least about 14 nucleotide exists 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.As described herein, the length of cognate pair ratio can be longer sequence section, lead in certain embodiments It is often at least about 20 nucleotide, more frequently at least about 24 nucleotide, typically at least about 28 nucleotide, more Typically at least about 32 nucleotide, and preferably at least about 36 or more nucleotide.

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

Detection technique

LncRNA of the invention is 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 dye Expect terminator sequencing.Those skilled in the art it will be recognized that due to RNA in cell less stable and in an experiment It is more vulnerable to nuclease attack, therefore usually by RNA reverse transcription at DNA before sequencing.

The present invention simultaneously can expand nucleic acid (for example, ncRNA) before detection or with detection.Nucleic acid amplification technologies Exemplary, non-limitative example include but is not limited to: polymerase chain reaction (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 nucleic acid sequence It expands (NASBA).Those skilled in the art will be it will be recognized that certain amplification techniques (for example, PCR) needs will before amplification RNA reverse transcription is at DNA (for example, RT-PCR), and other amplification techniques then direct cloning RNA (for example, TMA and NASBA).

The polymerase chain reaction of commonly referred to as PCR uses denaturation, the annealing and primer extend of primer pair and opposite strand Multiple circulations, exponentially increase target nucleic acid sequence copy number；The amplification of the transcriptive intermediate of TMA is (substantial constant Temperature, multiple copies of target nucleic acid sequence are autocatalytically synthesized under conditions of ionic strength and pH, wherein target sequence is more A RNA copy autocatalytically generates other copy；The ligase chain reaction of LCR uses miscellaneous with the adjacent area of target nucleic acid The two groups of complementary DNA oligonucleotides handed over；Other amplification methods include for example: the commonly referred to as expansion based on nucleic acid sequence of NASBA Increase；Use the amplification of rna replicon enzyme (commonly referred to as Q β replicase) amplification probe molecule 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 present invention provides the product of the expression of LINC02014 gene in detection, the product includes (but unlimited In) preparation, chip or kit.Wherein chip includes: solid phase carrier；And orderly it is fixed on the few core on the solid phase carrier Thuja acid probe, the oligonucleotide probe some or all of specifically correspond to shown in LINC02014 sequence.

The solid phase carrier includes inorganic carrier and organic carrier, the inorganic carrier include but is not limited to have silicon carrier, Glass carrier, ceramic monolith etc.；The organic carrier includes polypropylene film, nylon membrane etc..

Term " probe " refers to can be with the molecule in conjunction with the particular sequence of another molecule or subsequence or other parts.Unless another It points out, term " probe " is often referred to match and another polynucleotides (often referred to as " target polynucleotide ") by complementary base In conjunction with polynucleotide probes.Lack sufficient sequence complementarity according to the stringency of hybridization conditions, probe energy and with the probe Target polynucleotide combines.Probe can make direct or indirect label, and range includes primer.Crossing system, including, but it is unlimited In: solution phase, solid phase, mixed phase or in situ hybridization measuring method.

Exemplary probe in the present invention includes PCR primer and gene specific DNA oligonucleotide probe, such as fixed In micro probe array, the quantitative nucleic acid enzyme protection on microarray substrate examine probe, the probe that is connect with molecular barcode and The probe being fixed on pearl.

These probes have the base sequence with the specific base sequence complementary of target gene.Here, so-called " complementation ", As long as hybridization, can not be complete complementary.These polynucleotides have usually relative to the specific base sequence 80% or more, preferably 90% or more, more preferable 95% or more, particularly preferred 100% homology.These probes can be DNA, It is also possible to RNA, furthermore it is possible to pass through PNA (Polyamide nucleic in part of it or whole nucleotides Acid, peptide nucleic acid), LNA (registered trademark, locked nucleic acid, Bridged Nucleic Acid, Cross-linked core Acid), ENA (registered trademark, 2 '-O, 4 '-C-Ethylene-bridged nucleic acids), GNA (Glycerol Nucleic acid, glycerol nucleic acid), the artificial replacement nucleic acid such as TNA (Threose nucleic acid, threose nucleic acid) obtains Polynucleotides.

The present invention provides a kind of kit, the kit can be used for detecting the expression of LINC02014.Preferably, institute Also containing the marker for labeled RNA sample, and substrate corresponding with the marker in the preparation or kit stated. In addition, may also include in the kit for various reagents needed for extracting RNA, PCR, hybridization, colour developing etc., including but not It is limited to: extract, amplification liquid, hybridization solution, enzyme, comparison liquid, developing solution, washing lotion etc..In addition, further including making in the kit Software is analyzed with specification and/or chip image.

Lower adjustment and pharmaceutical composition

Discovery based on the present inventor, the present invention provides a kind of purposes of the lower adjustment of LINC02014, are used to prepare suppression The pharmaceutical composition of adenocarcinoma of lung processed.As used herein, the lower adjustment of the LINC02014 include but is not limited to inhibitor, it is short of money Anti-agent, retarding agent, blocking agent, nucleic acid inhibitor etc..

The lower adjustment of the LINC02014 refers to any expression for lowering LINC02014 gene or inhibits The substance of the transcription of LINC02014 gene, these substances can be used for preventing as lowering the useful substance of LINC02014 Or treatment adenocarcinoma of lung.

As a kind of preferred embodiment of the invention, the lower adjustment of the LINC02014 is a kind of LINC02014 specificity SiRNA molecule.As used herein, " siRNA " refers to a kind of short-movie section double stranded rna molecule, can be with same The mRNA of source complementary series is the target specific mRNA of degradation, this process is exactly RNA interference (RNA interference) Process.SiRNA can be prepared into the form of double-strandednucleic acid, it contains a positive-sense strand and an antisense strand, this two chains Only double-strand is formed under conditions of hybridization.One double-stranded RNA compound can be made by the positive-sense strand that is separated from each other and antisense strand It is standby.Therefore, for example, complementary positive-sense strand and antisense strand are chemical synthesis, and can generate synthesis by anneal thereafter Double-stranded RNA compound.

When screening effective siRNA sequence, the present inventor is by largely comparing analysis, to find out optimal effective Segment.The present inventor's design has synthesized a variety of siRNA sequences, and they are transfected lung adenocarcinoma cell system by transfection reagent respectively It is verified, selects the optimal siRNA of interference effect, further tested in cellular level, as a result prove to exist for the siRNA The proliferation of the expression of LINC02014 gene and lung adenocarcinoma cell in cell can effectively be inhibited.

Nucleic acid inhibitor of the invention such as siRNA 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 transfection reagent quilt appropriate It is transported into the cell, or multiple technologies known in the art also can be used and be transported into the cell.

Pharmaceutical composition

The present invention also provides a kind of compositions, it contains lower adjustment and the medicine of a effective amount of LINC02014 Acceptable carrier on.The composition can be used for inhibiting adenocarcinoma of lung.The lower adjustment of any LINC02014 above-mentioned Preparation for composition.

As used herein, described " effective quantity " refer to people and/or animal can be generated function or it is active 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 medicament carriers some in this way: themselves not being necessary active constituent, and does not have after applying Excessive toxicity.Suitable carrier is well known to those of ordinary skill in the art.Pharmaceutically acceptable load in the composition Body can contain liquid, such as water, salt water, buffer.In addition, there is likely to be complementary substance in these carriers, as filler, Lubricant, glidant, wetting agent or emulsifier, pH buffer substance etc..Cell can also be contained in the carrier, and (host is thin Born of the same parents) transfection reagent.

The present invention can using with a variety of methods well known in the art by the lower adjustment or its encoding gene or its Pharmaceutical composition delivers medicine to mammal.Including but not limited to: subcutaneous injection, intramuscular injection, for percutaneous administration of, administer locally to, plant Enter, be sustained and give；Preferably, the administration mode is that non-bowel is given.

Preferably, the means that gene therapy can be used carry out.For example, directly the lower adjustment of LINC02014 can be passed through all As the methods of injection delivers medicine to subject；Alternatively, the expression list of the lower adjustment of LINC02014 can will be carried by certain approach Position (such as expression vector or virus etc. or siRNA or shRNA) is delivered on target spot, and is allowed to the LINC02014 of expression activity Lower adjustment, concrete condition need to be depending on the types of the lower adjustment, these are well-known to those skilled in the art.

Pharmaceutical composition of the invention can further include one or more anticancer agents.In specific embodiments, Pharmaceutical composition includes at least one compound for inhibiting LINC02014 gene expression and at least one chemotherapeutics.For this hair The chemotherapeutics of bright method, including but not limited to, DNA- alkylating agent, antitumor antibiotics agent, antimetabolite, tubulin stabilization Agent, tubulin destabilizing agent, hormone antagonist, topoisomerase enzyme inhibitor, kinases inhibitor, HMG-COA inhibitor, CDK inhibitor, cyclin inhibitors, caspase inhibitors, metal protease inhibitors, antisense nucleic acid, three chains Virus, bacterium and the exotoxin reagent of helical dna, aptamer and molecular modification.

Pharmaceutical acceptable carrier may include but be not limited to: virus, liposome, nano particle or polymer and any combination thereof.Phase The delivering carrier of pass may include but be not limited to: liposome, biocompatible polymer (including natural polymer and synthesized polymer Object), 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.

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 constituent, or even is administered simultaneously in same composition.

Pharmaceutical composition of the invention can also be with individual composition or the dosage shape different from main active constituent 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.It over the course for the treatment of, can be according to the 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 present invention provides a kind of screening prevention or the methods for the treatment of adenocarcinoma of lung drug, it may be assumed that

In experimental group, untested compound is added into cell culture system, and measures the expression of LINC02014； In control group, it is added without untested compound into same cultivating system, and measures the expression of LINC02014；Wherein, If the expression of LINC02014 is less than control group in experimental group, illustrate that the candidate compound is the downward of LINC02014 Agent.

In the present invention, the method further include: the candidate compound obtained to previous step further tests its suppression The effect of adenocarcinoma of lung processed illustrates the compound for prevention or controls if test compound has significant inhibitory effect to adenocarcinoma of lung Treat the potential substance of adenocarcinoma of lung.

In the present invention, term " sample " is with the use of its broadest sense.In a kind of meaning, it is intended that including from it is any come The sample or culture that source obtains, and biology and environmental samples.Biological sample available from animal (including people) and cover liquid, Solid, tissue and gas.Biological sample includes blood product, blood plasma, serum etc..However, such sample should not be construed as Limitation is suitable for the invention sample type.

The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.

Embodiment 1 screens gene marker relevant to adenocarcinoma of lung

1, sample collection

Respectively collect 5 adenocarcinoma of lung cancer beside organisms and pulmonary adenocarcinoma sample.Adenocarcinoma of lung tumor tissues specimen sampling position is Vital tumor areas is located at tumor mass China and foreign countries 1/3 and normal tissue junction, excludes the obvious necrosis of tumor center, calcification portion Point and tumour periphery normal lung tissue；Ai Pang normal lung tissue sample is derived from the position of borderline tumor 5cm or more, visually observes Without significant change.The acquirement of above-mentioned all samples passes through the agreement of the committee of organizational ethics.

2, the preparation of RNA sample (utilizesMiRNA kit is operated)

Liquid nitrogen is imported in mortar, the tissue of above-mentioned acquisition is taken to be put into mortar, and simultaneously grind into powder is shredded in liquid nitrogen, It is put into liquid nitrogen after shredding and is ground to powdered, then continued in glass homogenizer；Tissue homogenate adds in glass homogenizer Enter Trizol reagent, on ice tissue abrasion.Tissue homogenate after homogenate is transferred in the EP pipe of no RNA enzyme, is stood at room temperature 5min.Separation RNA is extracted according to the specification in kit.

It is specific as follows:

1) separation of RNA:

0.2m1 chloroform is added in EP pipe, covers tightly EP pipe lid, acutely vibrates 15s manually, mixes well it.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 EP pipe of new no RNA enzyme the water phase separated, it is different that 450 μ l are added according to the ratio of 1:1 Propyl alcohol, hatches 10min at room temperature after mixing of turning upside down, 4 DEG C of 14000g are centrifuged 10min.

3) RNA is eluted

Supernatant is carefully removed after centrifugation, and 75% ethyl alcohol of 1ml (destroy the enzyme treatment, matching while using are simultaneously pre-chilled on ice) punching is added RNA is washed, subsequent 4 DEG C of 7500g are centrifuged 5min.

4) RNA is redissolved

The supernatant after washing is carefully removed, opens EP pipe pipe lid in superclean bench, places RNA sample at room temperature 5-10min dries.It is added and handles water 20-50 μ l, water-bath 10min in 55-60 DEG C of water bath without RNA enzyme.

5) quality analysis of RNA sample

Spectrophotometer detection:

NanoDrop1000 spectrophotometer detects RNA sample, the sample requirement of RNA-seq sequencing, and: OD260/OD280 is 1.8-2.2。

Agarose gel electrophoresis detection:

The RNA of said extracted is subjected to agarose gel electrophoresis, Agilent Technologies 2100Bioanalyzer detects RNA sample quality, and observation 28S rRNA and 18S rRNA master tape is obvious, complete without degradation, RNA Sex index is qualified, concentration reaches requirement, can be used for the lncRNA express spectra and screening experiment of chip.

3, reverse transcription and label

With Low RNA Input Linear Amplification Kit by mRNA reverse transcription at cDNA, while using Cy3 Labelling experiment group and control group respectively.

4, hybridize

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

5, data are analyzed

Chip results are analyzed using Agilent GeneSpring software, screening expression quantity has significant difference The lncRNA of (p < 0.05).

6, result

The results show that expression quantity of the LINC02014 in pulmonary adenocarcinoma is significantly higher than the expression quantity in cancer beside organism.

The differential expression of 2 QPCR sequence verification LINC02014 gene of embodiment

1, large sample QPCR verifying is carried out to LINC02014 gene differential expression.According to the sample collection side in embodiment 1 Formula selects adenocarcinoma of lung cancer beside organism and pulmonary adenocarcinoma each 35.

2, RNA extraction step is as described in Example 1.

3, reverse transcription

1) reaction system:

1 μ l of RNA template, 1 μ l of random primer, distilled water add to 12 μ l, mix, then slow-speed of revolution centrifugation, 65 DEG C of 5min are put It cools down on ice.

Following ingredients are added in 12 μ l reaction solutions in continuation:

5 × reaction buffer, 4 μ l, 1 μ l, 10mM dNTP mixed liquor of RNase inhibitor (20U/ μ l), 2 μ l, AMV reverse transcription 1 μ l of enzyme (200U/ μ l)；It mixes well and carries 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 amplimer is designed according to the coded sequence of LINC02014 gene and GAPDH gene in Genebank, by winning The synthesis of Mai De biotech firm.Specific primer sequence is as follows:

LINC02014 gene:

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

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

GAPDH gene:

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

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

Prepare PCR reaction system:

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

PCR reaction condition: 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, every 20s heats up 1 DEG C, draws solubility curve.Using SYBR Green as fluorescent marker, in Light Cycler PCR reaction is carried out on fluorescence quantitative PCR instrument, determines that purpose band, Δ Δ CT method carry out phase by melt curve analysis analysis and electrophoresis To quantitative.

5, statistical method

Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD, Using SPSS18.0 statistical software come for statistical analysis, the paired comparisons of cancer and cancer beside organism are examined using t, it is believed that when P < There is statistical significance when 0.05.

6, result

As a result as shown in Figure 1, compared with adenocarcinoma of lung cancer beside organism, LINC02014 expresses up-regulation in pulmonary adenocarcinoma, poor It is different that there is statistical significance (P < 0.05), it is consistent with chip test result.

The silencing of 3 LINC02014 gene of embodiment

1, cell culture

Human A459 lung cancer cell line, with the RPMI1640 culture medium containing 10% fetal calf serum and 1%P/S in 37 DEG C, 5% CO₂, relative humidity be 90% incubator in cultivate.It changes within 2-3 days liquid 1 time, trypsase of the use 0.25% containing EDTA is conventional Had digestive transfer culture.

Cell in culture bottle is digested with pancreatin and is seeded in 6 orifice plates, guarantee cell number is 2-8 × 10⁵A/ Cell culture medium is added in hole.Overnight, second day observation cell density, cell density can be transfected for 70% or more.

2, the design of siRNA

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 '-UAAAAAGCCCCUUUCAUUCGG-3 ' (SEQ ID NO.8)

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

SiRNA-2:

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

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

SiRNA-3:

Positive-sense strand is 5 '-UCUUUGAAGACAAAUGGUGGA-3 ' (SEQ ID NO.12)

Antisense strand is 5 '-CACCAUUUGUCUUCAAAGAGA-3 ' (SEQ ID NO.13)

3, it transfects

Experiment is divided into three groups: control group (A549), negative control group (siRNA-NC) and experimental group (siRNA-1, SiRNA-2, siRNA-3), wherein without homology, concentration is the sequence of negative control group siRNA-NC and LINC02014 gene The hole 20nM/, while being transfected respectively.

4, QPCR detects the transcriptional level of LINC02014 gene

The extraction of 4.1 cell total rnas

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

2) 0.2m1 chloroform is added, acutely shakes 15s, 4 DEG C, 12000g is centrifuged 15min.

3) water phase is transferred in new pipe, and 4.5m1 isopropanol is added, is placed at room temperature for 10min；4 DEG C, 10000g is centrifuged 10min.

4) liquid is outwelled, washes EP tube wall with 75% ethyl alcohol of l ml.4 DEG C, 7500g, it is centrifuged 5min.

5) 75% ethyl alcohol after outwelling cleaning, room temperature hang 5-10min.

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

4.2 reverse transcription steps are the same as embodiment 2.

4.3QPCR amplification step is the same as embodiment 2.

5, statistical method

Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD, Using SPSS18.0 statistical software come for statistical analysis, the difference between LINC02014 Gene Experiments group and control group is adopted It is examined with t, it is believed that there is statistical significance as P < 0.05.

6, result

As a result such as Fig. 2 is shown, with non-transfection group compared with transfecting siRNA-NC group, the table of the LINC02014 in experimental group It is significantly reduced up to level, and the silencing efficiency for transfecting siRNA-1 is best, difference has statistical significance (P < 0.05).

The influence of 4 LINC02014 gene pairs lung adenocarcinoma cell of embodiment proliferation

Testing detection LINC02014 gene pairs lung adenocarcinoma cell proliferative capacity using CCK-8 influences.

1, cell culture and transfection procedure are with embodiment 3, and 6h changes liquid after transfection, place cell incubator and stay overnight.

2, cell to be taken out in second day, microscopically observation cell growth status, the pancreatin containing EDTA is added in the hole 1ml/, into Row cell dissociation removes pancreatin after the completion of digesting, and cell culture medium mixing, which is added, makes cell suspend, and then carries out cytometer Number.

3, concentration of cell suspension is diluted to 15000/ml, is inoculated with later into 96 orifice plates, cell is added in every hole 200 μ l of suspension, cell are controlled at 3000 or so, are inoculated with 8 multiple holes.SiRNA-1 experimental group and siRNA-NC control group are set. Altogether spread 4 piece of 96 orifice plate be respectively used to for 24 hours, 4 detection time points of 48h, 72h, 96h.

4, after for 24 hours, first piece of 96 orifice plate is taken out, the CCK-8 detection liquid of 10 μ l is added in every hole, 96 orifice plates are continued to put Enter and be incubated for 4h or so in cell incubator, detect absorbance value of each hole at 450nm wavelength with microplate reader and records data.

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

6, statistical analysis

Experiment is completed according to being repeated 3 times, using SPSS18.0 statistical software come for statistical analysis, the two it Between difference using t examine, it is believed that as P < 0.05 have statistical significance.

7, result

As a result as shown in figure 3, compared with the control, for experimental group after transfecting siRNA-1, the proliferation of cell obviously receives suppression System, difference have the function of that statistical significance (P < 0.05) illustrates that LINC02014 has and promotes cell Proliferation.

The influence of 5 LINC02014 gene pairs Apoptosis of Lung Adenocarcinoma Cell of embodiment

Use the influence of flow cytomery LINC02014 gene pairs Apoptosis.

1, cell culture step is the same as embodiment 3.

2, cell transfecting step is the same as embodiment 3.

3, step

1) 10 × sample-loading buffer of 3m1 27m1 distilled water is diluted.

2) collection of cellular samples and with pre-cooling PBS clean.

3) 1 × sample-loading buffer of lml is added in cell, 300g is centrifuged 10min, and buffer is sucked out.

4) 1 × sample-loading buffer of lml is added again, cell concentration in cell suspension is adjusted to 1 × 10⁶A/ml.

5) cell suspension is taken out into 100 μ l, be added in EP pipe.

6) the Annexin V FITC of 5 μ l is added in EP pipe, mixes the liquid in EP pipe, is protected from light incubation at room temperature 10min。

7) 5 μ l PI dye liquors are added into EP pipe, are protected from light 5min at room temperature.

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

3, statistical method

Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD, Using SPSS13.0 statistical software come t inspection for statistical analysis, that difference between the two uses, it is believed that as P < 0.05 With statistical significance.

4, result:

As a result as shown in figure 4, experimental group compared with the control group, apoptosis rate has significant change (P < 0.05), the knot Fruit explanation, LINC02014 inhibit the apoptosis of cell.

6 cell migration of embodiment and Matrigel

1, prepared by the cell Transwell

Matrigel is ice bath melted under aseptic condition, carries out 20 times of dilutions with PBS, is layered on the volume in 50 holes μ l/ On the polycarbonate membrane of the cell Transwell.37 DEG C of placement 4h, take out after Matrigel glue aggregates into gel, are gently sucked out Liquid is precipitated in upper layer.The serum-free medium containing BSA that 50 μ l are added in every hole carries out hydration process, 37 DEG C of placements to basilar memebrane 30min。

2, 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 culture solution.Sedimentation cell is cleaned with PBS, the serum free medium containing BSA is added, it is resuspended.Adjustment is thin The density of born of the same parents is to 5 × l0⁵A/ml.

3, cell inoculation

200 μ l of cell suspension (migration experiment is 100 μ l, and Matrigel is 200 μ l) is taken to be added to the cell Transwell In.1640 culture mediums of the 500 μ l containing FBS are added in room under 24 orifice plates.Cell is put into cell incubator and is cultivated for 24 hours.

4, it dyes

Cell is dyed after culture using DAPI.Small ventricular cell is first rinsed 2 times with PBS, DAPI working solution is put into Middle room temperature dyes 5-20min.It is rinsed 2 times, be put into fluorescence microscopy under the microscope and counted with PBS.

5, result

As a result as shown in figure 5, lung adenocarcinoma cell transfect RNA interfering after, compared with the control group, the migration of experimental group and Invasive ability decreased significantly, and as a result illustrate that LINC02014 can promote the migration and invasion of adenocarcinoma of lung.

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

Sequence table

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Claims (10)

1. detecting application of the reagent of LINC02014 gene expression in the product of preparation diagnosis early stage adenocarcinoma of lung.

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

The probe of specific recognition LINC02014；Or

The primer of specific amplification LINC02014.

3. it is a kind of diagnose adenocarcinoma of lung product, which is characterized in that the product include by sequencing technologies, nucleic acid hybridization technique, Nucleic acid amplification technologies detect the expression of LINC02014 gene in sample.

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

Application of the 5.LINC02014 gene in the pharmaceutical composition of preparation prevention or treatment adenocarcinoma of lung.

6. application according to claim 5, which is characterized in that described pharmaceutical composition includes the lower adjustment of LINC02014.

7. application according to claim 6, which is characterized in that the lower adjustment is siRNA.

8. application according to claim 6 or 7, which is characterized in that described pharmaceutical composition further includes and the lower adjustment Its other medicine class and pharmaceutically acceptable carrier and/or auxiliary material of compatibility.

Application of the 9.LINC02014 gene in the potential substance of screening prevention or treatment adenocarcinoma of lung.

10. a kind of method of the potential substance of screening prevention or treatment adenocarcinoma of lung, which is characterized in that the described method includes:

The system expressed or containing LINC02014 gene is handled with candidate substances；With

Detect the expression of LINC02014 gene in the system；

Wherein, if the candidate substances can reduce the expression or activity of LINC02014 gene, show that the candidate substances are preventions Or the potential substance for the treatment of adenocarcinoma of lung.