CN110042164A - Lung cancer diagnosis and treatment lncRNA marker - Google Patents
Lung cancer diagnosis and treatment lncRNA marker Download PDFInfo
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Abstract
The invention discloses diagnosis and treatment lncRNA marker, the lncRNA marker is RP11-284F21.10.Present invention firstly discovers that RP11-284F21.10 expresses in adenocarcinoma of lung significant up-regulation, and the clinical diagnosis that can be applied to adenocarcinoma of lung using RP11-284F21.10 as potential molecular target is further demonstrated by large sample QPCR experiment.The present invention passes through CCK8 simultaneously the experiment proves that RP11-284F21.10 influences the proliferation of lung adenocarcinoma cell.
Description
Technical field
The invention belongs to biomedicine fields, are related to lung cancer diagnosis and treatment lncRNA marker, and specific marker is RP11-
284F21.10。
Background technique
Lung cancer is disease incidence and the highest cancer of lethality (FERLAY J, SOERJOMATARAM I, DIKSHIT in the world
R,et al.Cancer incidence and mortality worldwide:sources,methods and major
patterns in GLOBOCAN 2012[J].Int J Cancer.2015,136(5):E359-386.).Lung cancer contains four
Kind Main Subtype and a variety of secondary or extremely rare hypotype (BRAMBILLA E, TRAVIS W D, COLBY T V, et
al.The new World Health Organization classification of lung tumours[J].Eur
Respir J.2001,18 (6): 1059-1068.), each hypotype has specific biology and feature clinically.Pass through
Clinical pathological characteristic they can be divided into small cell carcinoma (Small cell lung cancer, SCLC) and non-small cell carcinoma
(Non-small cell lung cancer,NSCLC)(SUN S,SCHILLER J H,GAZDAR A F.Lung cancer
in never smokers--a different disease[J].Nat Rev Cancer.2007,7(10):778-790.)。
Wherein, non-small cell carcinoma is one of the most common type type in lung cancer, occupies 80% of lung cancer or more.It, can in non-small cell carcinoma
It is divided into three kinds of hypotypes, adenocarcinoma of lung, lung squamous cancer and large cell carcinoma.And adenocarcinoma of lung and lung squamous cancer are most common in non-small cell carcinoma
Type (YILDIZ O, BUYUKTAS D, EKIZ E, et al.Facial Nerve Palsy:An Unusual
Presenting Feature of Small Cell Lung Cancer[J].Case Reports in
Oncology.2011,4 (1): 35-38.), they have different pathogenesis and diagnostic mode.The two in contrast, lung gland
Cancer is easier to occur the periphery in lung and more common from nonsmoker, and lung squamous cancer be then easier to occur lung center and with
The smoking history of patient is highly relevant.
Lung cancer was once considered as solely belonging to the disease of smoker.However, the statistical analysis to the whole world is found, Nan Xingzhong
In 15% patients with lung cancer and women 53% patients with lung cancer it is unrelated with tobacco (PARKIN D M, BRAY F, FERLAY J,
et al.Global cancer statistics,2002[J].CA Cancer J Clin.2005,55(2):74-108.)。
Studies have found that lung cancer is the seventh-largest common cancer in non-smokers, uterine cancer, cancer of pancreas and prostate cancer are come
Before.In current patients with lung cancer, it is only less than 16% patient and is just diagnosed in the initial period of lung cancer, greatly mentioned
The high difficulty and cost for the treatment of.Therefore, it is very necessary to find the biomarker that can be identified and diagnose in lung cancer early stage.
This can greatly improve the efficiency for the treatment of, reduce the social cost for the treatment of.
Long-chain non-coding RNA (long non-coding RNAs, 1ncRNAs) is sent out with the development of cDNA chip technology
Existing, their conservative is poor, and expression quantity is low, therefore is once considered as transcription noise, and the loophole of genome transcribes (LIU
J.Control of protein synthesis and mRNA degradation by microRNAs[J].Curr Opin
Cell Biol.2008,20(2):214-221.).With the development of sequencing technologies, they are gradually recognized by people, and are become
Generation star RNA.Currently, application study of the lncRNA in adenocarcinoma of lung early diagnosis and prognosis prediction is also fewer, more
Lung cancer correlation 1ncRNAs need further to excavate.
Summary of the invention
In order to make up for the deficiencies of the prior art, one of the objects of the present invention is to provide one kind and adenocarcinoma of lung occurrence and development phase
The lncRNA biomarker of pass and its application in adenocarcinoma of lung diagnosing and treating.
The second object of the present invention is to provide a kind of method of the drug candidate of screening treatment adenocarcinoma of lung.
To achieve the goals above, the present invention adopts the following technical scheme:
The first aspect of the present invention provides a kind of detection reagent, and the reagent can detecte the water of RP11-284F21.10
It is flat.
Further, the reagent is selected from:
The probe of specific recognition RP11-284F21.10；Or
The primer of specific amplification RP11-284F21.10.
Further, the primer sequence of the specific amplification RP11-284F21.10 is as shown in NO.1~2 SEQ ID.
The second aspect of the present invention provides a kind of product, and the product includes reagent described in first aspect present invention.
Further, the product includes kit, chip, nucleic acid film item.
The third aspect of the present invention provides a kind of composition, and the composition includes a effective amount of RP11-284F21.10
Inhibitor.The inhibitor is selected from: as target sequence and being able to suppress RP11- using RP11-284F21.10 or its transcript
The disturbing molecule of 284F21.10 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 core
The construction of acid.
Further, the inhibitor is siRNA.
Further, the sequence of the siRNA is as shown in NO.7~12 SEQ ID.
Preferably, the sequence of the siRNA is as shown in NO.7~8 SEQ ID.
The fourth aspect of the present invention provides a kind of method of the drug candidate of screening treatment adenocarcinoma of lung, the method packet
It includes:
The system expressed or containing RP11-284F21.10 gene is handled with substance to be screened；With
Detect the expression of RP11-284F21.10 gene in the system；
Wherein, if the substance to be screened can reduce the level of RP11-284F21.10 gene, show this wait sieve
Selecting substance is the drug candidate for treating adenocarcinoma of lung.
The system is selected from: cell system, subcellular system, solution system, organizational framework, organ systems or animal body
System.
The candidate substances include but is not limited to: setting for RP11-284F21.10 gene or its upstream or downstream gene
Disturbing molecule, nucleic acid inhibitor, small molecule compound of meter etc..
The fifth aspect of the present invention provides any one of following application:
A. application of the reagent described in first aspect present invention in the tool of preparation diagnosis adenocarcinoma of lung；
B. application of the product described in second aspect of the present invention in the tool of preparation diagnosis adenocarcinoma of lung；
Application of the c.RP11-284F21.10 in the computation model of building diagnosis adenocarcinoma of lung；
Application of the d.RP11-284F21.10 in the drug of preparation treatment adenocarcinoma of lung；
E. application of the composition described in third aspect present invention in the drug of preparation treatment adenocarcinoma of lung；
Application of the f.RP11-284F21.10 in the drug candidate of screening treatment adenocarcinoma of lung.
Detailed description of the invention
Fig. 1 is the expression figure using QPCR detection RP11-284F21.10 gene in pulmonary adenocarcinoma；
Fig. 2 is to detect siRNA to the silencing efficiency figure of RP11-284F21.10；
Fig. 3 is the influence diagram that CCK8 method detection RP11-284F21.10 is proliferated lung adenocarcinoma cell.
Specific embodiment
The present invention after extensive and in-depth study, passes through high-flux sequence and bioinformatic analysis method, detection
LncRNA has found wherein there is obvious differential expression in the expression of tumor tissues and cancer beside organism in adenocarcinoma of lung sample
LncRNA inquires into its relationship between the occurrence and development of adenocarcinoma of lung, to find more for the diagnosis of adenocarcinoma of lung and targeted therapy
Good approaches and methods.By screening, present invention firstly discovers that RP11-284F21.10 conspicuousness up-regulation in adenocarcinoma of lung, simultaneously
According to the relationship between RP11-284F21.10 and adenocarcinoma of lung, determined by designing the siRNA for RP11-284F21.10
Its correlation between adenocarcinoma of lung proliferation.New tumor markers and treatment are provided for the early diagnosis and treatment of adenocarcinoma of lung
Target spot.
Term " level of expression " or " expression " refer generally to the amount of biomarker in biological sample." expression " one
As refer to that information is converted in cell the process of structure for existing and running.Therefore, as used in this article, " expression ", which can refer to, turns
Polynucleotides are recorded into, translate into polypeptide, or even polynucleotides and/or peptide modified (such as posttranslational modification of polypeptide).Turn
The polynucleotides of record, the polypeptide of translation or polynucleotides and/or the piece of peptide modified (such as posttranslational modification of polypeptide)
Section also should be regarded as expression, and no matter they are derived from the transcript generated by alternative splicing or the transcript by degradation, or
Person is derived from the post translational processing (such as passing through proteolysis) of polypeptide." gene of expression " includes being transcribed into polynucleotides (such as
MRNA), then translate into the gene of polypeptide, be also transcribed into RNA but do not translate into polypeptide gene (such as transhipment and ribosomes
RNA,miRNA,lncRNA,circRNA).In specific embodiment of the invention, " gene of expression ", which refers to, to be transcribed into
RNA but the gene for not translating into polypeptide.
Increased expression ", " increased expression ", " increased level ", " raised expression ", " raised expression water
It is flat " or " raised level " refer to relative to the control such as individual without disease or illness (such as cancer), internal contrast (example
Type of such as running one's home biomarker), or in patient group/group sample biomarker median expression level, it is a
The increased expression or increased level of biomarker in body.
" expression of reduction ", " expression of reduction ", " level of reduction ", " reduced expression ", " reduced expression water
It is flat " or " reduced level " refer to relative to control such as individual or internal contrast without disease or illness (such as cancer)
(such as type biomarker of running one's home), or in patient group/group sample biomarker median expression level,
The reduced expression or reduced level of biomarker in individual.In some embodiments, reduced expression is seldom
Expression or not.
RP11-284F21.10
The gene of transcription RP11-284F21.10 is located on No. 1 chromosome of people, the RP11-284F21.10 in the present invention
Including wild type, saltant type or its segment.One skilled in the art will appreciate that when carrying out sequencing analysis, it can be by primitive sequencer knot
Fruit compares onto the reference genome of people, therefore the RP11-284F21.10 in the selection result may include different transcription
This, as long as the RP11-284F21.10 on reference genome can be compared.In an embodiment of the present invention, a kind of representative
Property transcription RP11-284F21.10 gene nucleotide sequence as shown in ENST00000605886.1.
The present invention can use the expression of any method known in the art measurement gene.Those skilled in the art
It should be appreciated that the means of measurement gene expression are not importances of the invention.Biological marker can be detected on transcriptional level
The expression of object.
Some detections of lncRNA level or quantitative approach are known in the art and are suitable for provided herein
Method is to measure the level of biomarker.Illustrative method includes but is not limited to RNA trace (northern blots), core
The method of ribonuclease T. protection test and based on PCR.When biomarker is lncRNA molecule, lncRNA sequence or its segment
It can be used for preparing the probe of at least partial complementarity.Then using the method for such as based on PCR, RNA blotting (Northern
Blotting) or test strips detect any suitable measuring methods such as (dipstick assay), can be in probe in detecting sample
LncRNA sequence.
The measuring method can change according to the type of required lncRNA information.Illustrative method includes but unlimited
In the method (for example, qRT-PCR) of RNA trace (Northern blots) and based on PCR.The methods of qRT-PCR can also be right
The amount of lncRNA carries out accurate quantitative analysis in sample.
Any suitable measurement platform is used equally for determining the presence of lncRNA in sample.For example, measurement can be in test paper
Item (dipstick), film (membrane), chip (chip), disk (disk), test-strips (test strip), filter
(filter), microballoon (microsphere), slide glass (slide), porous plate (multiwell plate) or optical fiber (optical
Fiber form).Measurement system can have the solid support for being attached with nucleic acid corresponding with lncRNA thereon.It is described solid
Phase support may include for example plastics, silicon wafer, metal, resin, glass, film, particle, sediment (precipitate), gel,
Polymer, thin slice (sheet), ball, polysaccharide, capillary, film (film), plate or slide glass.After the measurement component can be prepared
It is packaged together as the kit for detecting lncRNA.
Nucleic acid can be labeled if necessary to make labeled lncRNA group.In general, sample can use
Method well-known in the art is marked.In certain embodiments, the sample is fluorescently labeled substance markers.It is exemplary
Fluorescent dye include but is not limited to xanthene (xanthene) dyestuff, fluorescein(e) dye, rhodamine, fluorescein isothiocynate
(FITC), 6- Fluoresceincarboxylic acid (FAM), 6- carboxyl -2', 4', 7', 4,7- chlordene fluorescein (HEX), 6- carboxyl -4', 5'- bis-
Chloro- 2', 7'- dimethoxyfluorescein (JOE or J), N, N, N', N'- tetramethyl -6- carboxyrhodamine (TAMRA or T), 6- carboxylic
Base-X- rhodamine (ROX or R), 5- carboxyrhodamine 6G (R6G5 or G5), 6- carboxyrhodamine 6G (R6G6 or G6) and rhodamine
110；Cyanine dye, such as Cy3, Cy5 and Cy7 dyestuff；Alexa dyestuff, such as Alexa-fluor-555；Cumarin, diethylamino
Butylcoumariii, umbelliferone；Benzimide dyestuff, such as Hoechst 33258；Phenanthridines dyestuff, such as texas Red (Texas
red)；Second ingot dyestuff；Acridine dye；Carbazole dye；Phenoxazine dye；Porphyrin dye；Polymethin dyes, BODIPY dyestuff, quinoline
Quinoline dyestuff, pyrene (pyrene), fluorescein chlorotriazine base (fluorescein chlorotriazinyl), R110, Eosin, JOE,
R6G, tetramethylrhodamine, lissamine, ROX and naphthofluorescein.
Nucleic acid may be present in the particular addressable position on solid support；Each position corresponds to biomarker
LncRNA sequence at least part.
In certain embodiments, 1) lncRNA measurement is the following steps are included: obtain one or more biomarkers
Surface-combination probe；2) make lncRNA group miscellaneous under conditions of being enough to provide specific binding with surface-combination probe
It hands over；3) nucleic acid being not associated in hybridization step is removed；With the lncRNA of 4) detection hybridization.
Hybridization can carry out under suitable hybridization conditions, and the stringency of the condition can optionally change.Typical item
Part is the complementary lncRNA on a solid surface between complementary binding members, i.e., in surface-combination probe and sample
Between be enough to generate probe/target compound.
In certain embodiments, using stringent hybridization conditions.Including temperature, salinity, polynucleotides concentration, hybridization
The selection of felicity condition including the stringency of time and wash conditions depends on experimental design, including sample source, capturing agent
Type, expected complementary degree etc., and can be used as routine experiment means come by those of ordinary skill in the art it is true
It is fixed.
After lncRNA hybridization procedures, the polynucleotides that surface is combined are washed to remove unbonded nucleic acid.It can be with
It is washed using any convenient washing scheme.In certain embodiments, wash conditions are stringent.Then it can use
Standard technique detection target lncRNA hybridizes with probe.
Kit, chip, nucleic acid film item
The present invention provides a kind of kit, the kit can be used for detecting the expression of RP11-284F21.10.
As preferred embodiment, the kit includes the one kind specifically bound with the lncRNA of biomarker
Or a variety of probes or primer.
As highly preferred embodiment, the kit also includes washing solution.
As highly preferred embodiment, the kit also includes the reagent for carrying out cross experiment, lncRNA separation
Or tools for purification, detection instrument and the positive and negative control.
As further preferred embodiment, the kit also includes the specification using the kit.The examination
Agent box can customize for use at home, clinical use or research use.
The kit includes the specific primer pair for expanding RP11-284F21.10；Standard DNA template；PCR is anti-
Answer liquid.
Chip in the present invention includes: solid phase carrier；And the oligonucleotides being orderly fixed on the solid phase carrier is visited
Needle, the oligonucleotide probe some or all of specifically correspond to shown in RP11-284F21.10 sequence.
The various common used materials in genetic chip field, such as, but not limited to nylon can be used in heretofore described solid phase carrier
Film, slide or silicon wafer, unmodified slide, plastic sheet through active group (such as aldehyde radical, amino) modification etc..
The conventionally fabricated side of biochip known in the art can be used in the preparation of the RP11-284F21.10 chip
Method.For example, 5 ' ends of probe are gone here and there containing amido modified poly- dT if solid phase carrier is using modification slide or silicon wafer, it can
Oligonucleotide probe is configured to solution, then point sample instrument is used on modification slide or silicon wafer, to be arranged in its point scheduled
Sequence or array, are then fixed by standing overnight, so that it may obtain lncRNA chip of the invention.
In the present invention, nucleic acid film item includes substrate and the specific recognition RP11- that is fixed in the substrate
284F21.10 oligonucleotide probe；The substrate can be any substrate suitable for immobilized oligonucleotide probe, such as nylon
Film, nitrocellulose filter, polypropylene screen, sheet glass, silica gel chip, miniature magnetic bead etc..
It includes RP11- that gene detecting kit or genetic chip or nucleic acid film item, which can be used for detecting, in the present invention
The expression of multiple genes (for example, multiple genes relevant to adenocarcinoma of lung) including 284F21.10 gene, by adenocarcinoma of lung
Multiple markers are detected simultaneously, are greatly improved the accuracy rate of adenocarcinoma of lung diagnosis.
In the present invention, as those of skill in the art know, it can be implemented in various ways marker water and realize
Flat the step of getting up with certain possibility or risk association.Preferably, mathematically composite marker object and one or more other
The measurement concentration of marker, and combined value is associated with basic diagnosis problem.Any suitable existing skill can be passed through
Art mathematical method combines the measurement of marker levels.
Preferably, the mathematical algorithm applied in marker combination is a kind of logarithmic function.Preferably, using such mathematics
Algorithm or such logarithmic function the result is that single value.According to basic diagnosis problem, can easily by such value with it is for example a
Body associates about the risk of adenocarcinoma of lung or with the other intentional diagnostic uses for helping to assess patients with lung adenocarcinoma.With a kind of excellent
The mode of choosing, such logarithmic function obtain as follows: individual segregation a) being entered group, such as normal person, has adenocarcinoma of lung risk
Individual, patient with adenocarcinoma of lung etc., b) marker of the significant difference between these groups is identified by univariate analysis,
C) logarithmic regressions analysis is to assess the independent difference value that can be used for assessing these differences and organize of marker, and d) constructs logarithmic function
Carry out composition independency difference value.In such analysis, marker is no longer independent, but represents a marker group
It closes.
Logarithmic function for marker combination to be got up with disease association is preferably developed using by applied statistical method
With the algorithm of acquisition.For example, suitable statistical method is discriminant analysis (DA) (i.e. linear, secondary, regular DA), Kernel method
(i.e. SVM), nonparametric technique (i.e. k- nearest neighbor classifiers), PLS (partial least square), method (the i.e. logic based on tree
Recurrence, CART, random forest method, boosting/bagging method), generalized linear model (i.e. logarithm regression), the side based on principal component
Method (i.e. SIMCA), broad sense Additive Model, the method based on fuzzy logic, the method based on artificial neural network and genetic algorithms.Skillfully
Technical staff merges in the suitable statistical method of selection to assess marker group of the invention thus to obtain suitable mathematical algorithm
Aspect will not be problematic.In one embodiment, for obtaining the statistical method of mathematical algorithm used in assessment adenocarcinoma of lung
Selected from DA (i.e. linear, secondary, rule based judgment analysis), Kernel method (i.e. SVM), nonparametric technique (i.e. k- nearest-neighbors point
Class device), PLS (partial least square), the method (i.e. logistic regression, CART, random forest method, propelled method) based on tree,
Or generalized linear model (i.e. logarithm regression).
Inhibitor and drug
Discovery based on inventor, the present invention provides the inhibitor of RP11-284F21.10 a kind of, the inhibitor
Property has no importance for the present invention, as long as it inhibits the functional expression of RP11-284F21.10 gene, citing comes
It says, inhibitor of the invention can be using RP11-284F21.10 gene as target sequence and be able to suppress RP11-284F21.10
The disturbing molecule of gene, comprising: shRNA (children purpura nephritis), siRNA (siRNA), dsRNA, Microrna, antisense nucleic acid,
Or it can express or be formed the construction of the shRNA, siRNA, dsRNA, Microrna, antisense nucleic acid.These inhibitor are made
To can be used for treating adenocarcinoma of lung for lowering the useful substance of RP11-284F21.10.
As a kind of preferred embodiment of the invention, the inhibitor of the RP11-284F21.10 is a kind of RP11-
284F21.10 the siRNA molecule of specificity.As used herein, " siRNA " refers to a kind of short-movie section double-strand
RNA molecule, can be using the lncRNA of homologous complementary sequence as the target specific lncRNA of degradation, this process is exactly RNA dry
Disturb (RNA interference) process.SiRNA can be prepared into the form of double-strandednucleic acid, it contain a positive-sense strand and
One antisense strand, this two chains only form double-strand under conditions of hybridization.One double-stranded RNA compound can be by being separated from each other
It is prepared by positive-sense strand and antisense strand.Therefore, for example, complementary positive-sense strand and antisense strand are chemical synthesis, and can be led to thereafter
Anneal is crossed, the double-stranded RNA compound of synthesis is generated.
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, in a specific embodiment of the present invention, the sequence of the siRNA such as SEQ ID
Shown in NO.7~8, is further tested in cellular level, as a result prove effectively inhibit in cell the siRNA
The expression of RP11-284F21.10 gene and the proliferation of lung adenocarcinoma cell.It will be appreciated by the appropriately skilled person that choosing
It selects the optimal subsequent experiment of siRNA progress of effect and is not meant to that other siRNA cannot play similar effect, carry out
The purpose of siRNA interference experiment be in order to prove the expression of RP11-284F21.10 really with the proliferation of lung adenocarcinoma cell and
Invasion are related to migration, in order to reduce cost, it will usually representative siRNA be selected to be tested.
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.
In the present invention, " drug ", " pharmaceutical composition " can be general.As in selectable embodiment, medicine group
Close the inhibitor and pharmaceutically acceptable carrier that object includes RP11-284F21.10 gene.Pharmaceutically acceptable carrier packet
It includes (but being not limited to): diluent, excipient such as lactose, sodium chloride, glucose, urea, starch, water etc., filler such as starch,
Sucrose etc.；Adhesive such as simple syrup, glucose solution, starch solution, cellulose derivative, alginates, gelatin and polyethylene pyrrole
Pyrrolidone；Wetting agent such as glycerol；Disintegrating agent such as dried starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and carbon
Sour hydrogen sodium；Sorbefacient quaternary ammonium compound, lauryl sodium sulfate etc.；Surfactant such as polyoxyethylene sorbitan rouge
Fat acid esters, lauryl sodium sulfate, glyceryl monostearate, hexadecanol etc.；Humectant such as glycerol, starch etc.；Absorption carrier
Such as starch, lactose, bentonite, silica gel, kaolin and soap clay；Lubricant such as talcum powder, calcium stearate and magnesium, poly- second two
Alcohol, boric acid powder etc..
In the present invention, pharmaceutical composition can be used different additives and be prepared, for example, buffer, stabilizer,
Bacteriostatic agent, isotonic agent, chelating agent, pH controlling agent and surfactant.
Pharmaceutical composition Orally-administrable of the present invention, parenteral administration are given by sucking spray delivery, part
Medicine, adenocarcinoma of lung administration, nasal administration, cheek administration, vagina administration are administered by the storage medicine device of implantation.It is preferred that oral administration or note
Penetrate administration.Pharmaceutical composition of the invention contains any commonly employed nontoxic pharmaceutical acceptable carrier, auxiliary material or excipient.In certain feelings
Under condition, medicinal acid, alkali or buffer can be used to adjust the pH of preparation to improve the steady of prepared compound or its form of administration
It is qualitative.Terms used herein parenteral route includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, breastbone
It is interior, bring up in, in damage location and intracranial injection or infusion techniques.As long as destination organization can be reached, pharmaceutical composition of the present invention
Object can give receptor by any approach.
Pharmaceutical composition of the present invention can be administered orally in the form of any peroral dosage form, including but not limited to capsule, piece
Agent, emulsion and water slurry, dispersing agent and solution.For oral tablet, common carrier includes lactose and cornstarch.It is general to go back
Lubricant such as magnesium stearate is added.In order to be administered with capsules per os, applicable diluent includes lactose and anhydrous corn
Starch.When water slurry and/or lotion is administered orally, active component can be suspended or dissolved in oily phase, and and emulsifier
And/or suspending agent merges.If necessary, some sweeteners and/or corrigent and/or colorant can be added.Where appropriate, can
The dosage unit preparations packet micro-capsule that will be used to be administered orally.For example, by the way that particulate matter is coated or is wrapped in polymer, wax etc.
It buries, the preparation can also be prepared and extended or maintained release.Pharmaceutical composition of the invention can be used for reducing endogenic
RP11-284F21.10 is overexpressed, by reducing the expression of RP11-284F21.10, so that treatment is because of RP11-284F21.10 table
The adenocarcinoma of lung up to caused by raising.
In the present invention, the compound of RP11-284F21.10 expression can will be inhibited as naked RNA together with delivery of agents
It is applied as nucleic acid (such as recombinant plasmid or viral vectors) to subject, the nucleic acid includes to inhibit RP11-284F21.10 expression
Sequence.Delivery of agents can be lipophilic agent, polycation, liposome etc..
In the present invention, term " effective quantity " refers to that its dosage is enough to treat disease, to be suitable for any therapeutic treatment
Reasonable interests/risk-ratio.The effective dose level of composition can according to the type of subject, the severity of disease,
The age of subject and gender, pharmaceutical activity, the sensibility to drug, administration time, administration route, excretion rate, treatment time,
With composition associated in drug and medical field other known facts determine.Pharmaceutical composition of the invention can be used alone
Or it is administered in combination with other therapeutic agents, and can be sequentially or simultaneously administered with conventional therapeutic agent.It can be used one or more
Composition is applied in dosage form.Consider all above-mentioned factors, minimum of the maximum efficiency without causing side effect can shown
Lower application composition is most important, which can be readily determined by those skilled in the art.
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.
Statistical analysis
In a specific embodiment of the present invention, experiment be all to be completed according to being at least repeated 3 times, result data be all with
The mode of mean+SD indicates, using SPSS18.0 statistical software come for statistical analysis, difference between the two
It is different to be examined using t, it is believed that there is statistical significance as P < 0.05.
Below with reference to specific embodiment further illustrate the present invention, the embodiment of the present invention for explaining only the invention,
It is not intended to limit protection scope of the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1 screens gene marker relevant to adenocarcinoma of lung
1, sample collection
35 pulmonary adenocarcinomas and corresponding cancer beside organism are collected, all patients do not receive other before surgery and control
It treats, therefrom optional 4 pulmonary adenocarcinomas and corresponding cancer beside organism carry out high-flux sequence.
2, the preparation and quantitative analysis of RNA sample
The extraction of tissue RNA is carried out using the tissue RNA extracts kit of QIAGEN, concrete operations by specification carries out.
The RNA of said extracted is subjected to agarose gel electrophoresis, using Nanodrop2000 to the concentration of mentioned RNA and pure
Degree is detected, and agarose gel electrophoresis detects RNA integrality, and Agilent2100 measures RIN value.Single requirement for construction data base RNA is total
5 μ g are measured, concentration >=200ng/ μ L, OD260/280 is between 1.8~2.2.
3, construction cDNA library and sequencing
The building and sequencing of cDNA library are completed by Hua Da gene, and steps are as follows:
1) rRNA is removed
The rRNA in total serum IgE is removed using Ribo-Zero kit；
2) fragmentation RNA
To complete RNA sequence, interrupted at random using metal ion, by RNA random fracture at the small of 200bp or so
Segment.
3) reversion synthesis cDNA
The building that cDNA library is carried out using the TruseqTM RNA sample Prep Kit of Illumina, in reverse transcription
Under the action of enzyme, using random primer, one chain cDNA of synthesis is inverted by template of lncRNA, when carrying out the synthesis of two chains, dNTPs examination
DTTP is replaced with dUTP in agent, making base in the second chain of cDNA includes A/U/C/G.
4) adaptor is connected
End Repair Mix is added to mend the cohesive end of double-strand cDNA at flat end, then adds one in 3 ' ends
A base, for connecting the connector of Y-shaped.
5) bis- chain of UNG enzymic digestion cDNA
The second chain of cDNA is digested with UNG enzyme, to make in library only comprising the first chain of cDNA.
6) Illumina X-Ten microarray dataset is used, 2*150bp sequencing is carried out.
4, high-throughput transcript profile sequencing data analysis
Deletion is not easy the lncRNA detected, and (i.e. the read count value of the lncRNA is big for 0 sample number in case
It is greater than always in normal for 0 sample number in the read count value of the 20% or lncRNA of total case sample size
The 20% of normal sample size) after, Differential expression analysis, differential expression lncRNA are carried out using the DESeq2 of R-3.3.3 tool
Screening criteria: FDR < 0.05, abs (log2FC)>2。
5, result
The results show that compared with cancer beside organism, on expression of the RP11-284F21.10 in pulmonary adenocarcinoma is significant
It adjusts.
The differential expression of 2 QPCR sequence verification RP11-284F21.10 gene of embodiment
1,35 samples of collection are carried out with the verifying of RP11-284F21.10 gene differential expression.
2, RNA is extracted
RNA sample is extracted using the tissue RNA extracts kit of QIAGEN, concrete operations are detailed in specification.
3、QPCR
1) reverse transcription reaction
It is anti-that lncRNA is carried out using FastQ μ ant the first chain of cDNA synthetic agent box (article No.: KR106) of Tiangeng company
Transcription, the first reaction of removal genomic DNA, are added 5 × gDNA B μ ffer, 2.0 μ l, 1 μ g of total serum IgE adds Rnase in test tube
Free ddH2O makes total volume to 10 μ l, 42 DEG C of heating 3min. in water-bath
By 10 × Fast RT B μ, 2.0 μ l, RT Enzyme Mix of ffer, 1.0 μ l, FQ-RT Primer Mix, 2.0 μ
L, RNase Free ddH25.0 μ l of O is added in above-mentioned test tube after mixing and is mixed together totally 20 μ l, 42 DEG C of heating in water-bath
15min, 95 DEG C of heating 3min.
2) design of primers
Drawn according to the coded sequence of RP11-284F21.10 gene and GAPDH gene design QPCR amplification in Genebank
Object is synthesized by Bo Maide biotech firm.Specific primer sequence is as follows:
RP11-284F21.10 gene:
Forward primer is 5 '-TTAGCCATAGCAACATAG-3 ' (SEQ ID NO.1)；
Reverse primer is 5 '-CAGATGATGACAGTAAGAT-3 ' (SEQ ID NO.2).
GAPDH gene:
Forward primer is 5 '-AATCCCATCACCATCTTCCAG-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-GAGCCCCAGCCTTCTCCAT-3 ' (SEQ ID NO.4).
3) QPCR amplification is examined
It with SuperReal PreMix Plus (SYBR Green) (article No.: FP205), is expanded, experimental implementation is by production
Product specification carries out.
Using 20 μ l reaction systems: 2 × SuperReal PreMix Plus 10 μ l, each 0.6 μ of forward and reverse primer (10 μM)
L, 5 × ROX Reference Dye△2 μ l, 2 μ l of DNA profiling, 4.8 μ l of sterile purified water.3 parallel pipes are arranged in each sample,
All amplified reactions are repeated three times the above reliability to guarantee result.
Amplification program are as follows: 95 ° of 15min, (95 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 32s) × 40 circulations, 95 DEG C of 15s, 60 DEG C
60s, 95 DEG C of 15s).
4) screening of cDNA template concentrations
After each sample cDNA is mixed, 10 times of gradients (10,100,1000,10000,100000 times) are carried out as template
Dilution, sample respectively takes 2 μ l to make template after dilution, is expanded respectively with target gene primer and reference gene primer, while
60-95 DEG C of progress melt curve analysis analysis carries out the screening of template concentrations according to amplification efficiency height and the unimodal principle of solubility curve.
According to solubility curve, it can be seen that when 10 times of dilutions of carry out of cDNA, the amplification efficiency of PCR is higher, and dissolution is bent
Line is unimodal relatively good.
5) sample RealTime PCR is detected
2 μ l will be taken to make template after cDNA10 times of each sample dilution, respectively with target gene primer and reference gene primer into
Row amplification.Simultaneously in 60-95 DEG C of progresss solubility curve analysis, purpose band is determined by melt curve analysis analysis and electrophoresis, 2-ΔΔCT
Method carries out relative quantification.
4, result
QPCR result is as shown in Figure 1, compared with cancer beside organism, on RP11-284F21.10 is expressed in pulmonary adenocarcinoma
It adjusts, difference has statistical significance (P < 0.05), prompts that RP11-284F21.10 is with higher in the diagnosis of adenocarcinoma of lung answers
With value.
The silencing of 3 RP11-284F21.10 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.
2, the design of siRNA
For the sequence design siRNA of RP11-284F21.10 gene, the siRNA sequence of design is as follows:
The sequence of negative control siRNA-NC:
Positive-sense strand: 5 '-UUCUCCGAACGUGUCACGU-3 ' (SEQ ID NO.5),
Antisense strand: 5 '-ACGUGACACGUUCGGAGAA-3 ' (SEQ ID NO.6)；
SiRNA1:
Positive-sense strand: 5 '-AUUGUGUAGGCUCAUGUUGCU-3 ' (SEQ ID NO.7),
Antisense strand: 5 '-CAACAUGAGCCUACACAAUAU-3 ' (SEQ ID NO.8)；
SiRNA2:
Positive-sense strand: 5 '-AUUGCAAUUACUUCUACAGGC-3 ' (SEQ ID NO.9),
Antisense strand: 5 '-CUGUAGAAGUAAUUGCAAUCA-3 ' (SEQ ID NO.10)；
SiRNA3:
Positive-sense strand is 5 '-AUUUCAGAUUCUAUACGAGAG-3 ' (SEQ ID NO.11),
Antisense strand is 5 '-CUCGUAUAGAAUCUGAAAUGU-3 ' (SEQ ID NO.12)
3, it transfects
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.
Experiment is divided into three groups: control group (A549), negative control group (siRNA-NC) and experimental group (siRNA1-3),
The sequence of middle negative control group siRNA-NC and RP11-284F21.10 gene is without homology, and concentration is the hole 20nM/, simultaneously respectively
It is transfected.Transfected using the Lipofectamine3000 kit of Invitrogen company, specific transfection method according to
The instruction of specification carries out.
4, QPCR detects the transcriptional level of RP11-284F21.10 gene
1) extraction of cell total rna
The total serum IgE in cell is extracted using QIAGEN cell RNA extracts kit, specific steps are detailed in specification.
2) reverse transcription step is the same as embodiment 2.
3) QPCR amplification step is the same as embodiment 2.
5, result
As a result as Fig. 2 shows that compared with control group A 549 and siRNA-NC group, experimental group (siRNA1~3) can be reduced
The level of RP11-284F21.10, wherein the effect of siRNA1 is the most significant, therefore siRNA1 is selected to carry out subsequent experimental.
The influence that 4 RP11-284F21.10 of embodiment is proliferated lung adenocarcinoma cell
1, human umbilical vein endothelial cell inoculation is cultured in 6 orifice plates, and is reached 85%-90% to cell density, is used liposome
3000 transfection siRNA1.More renew culture medium after serum free medium culture 4-6h.
2, it is cultivated for 24 hours after transfecting siRNA1, interference group cell and cellular control unit is digested, transfection is inoculated in 96 orifice plates
100 μ l (1 × 10 of A549 cell suspension and each control group afterwards4A/hole), in transfection 12h, for 24 hours, examined after 48h, 72h
It surveys.
3,10 μ l CCK8 solution are added to every hole.
4, culture plate is placed in incubator and cultivates 1-4h.
5, the absorbance at 450nm is measured using microplate reader, is horizontal axis by the longitudinal axis, time of absorbance value, drawn thin
Intracellular growth activity curve.
6, result
As a result as shown in figure 3, compared with the control group, with the increase of growth time, siRNA1 transfection group cell activity is bright
Aobvious to reduce, difference tool is statistically significant (P < 0.05), prompts the proliferation phase of RP11-284F21.10 and lung adenocarcinoma cell
Close, can as possible potential target apply in the treatment 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
<110>Beijing Yang Shen biology information technology Co., Ltd
<120>lung cancer diagnosis and treatment lncRNA marker
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ttagccatag caacatag 18
<210> 2
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
cagatgatga cagtaagat 19
<210> 3
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
aatcccatca ccatcttcca g 21
<210> 4
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gagccccagc cttctccat 19
<210> 5
<211> 19
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 5
uucuccgaac gugucacgu 19
<210> 6
<211> 19
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 6
acgugacacg uucggagaa 19
<210> 7
<211> 21
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 7
auuguguagg cucauguugc u 21
<210> 8
<211> 21
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 8
caacaugagc cuacacaaua u 21
<210> 9
<211> 21
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 9
auugcaauua cuucuacagg c 21
<210> 10
<211> 21
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 10
cuguagaagu aauugcaauc a 21
<210> 11
<211> 21
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 11
auuucagauu cuauacgaga g 21
<210> 12
<211> 21
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 12
cucguauaga aucugaaaug u 21
Claims (10)
1. a kind of detection reagent, which is characterized in that the reagent can detecte the level of RP11-284F21.10.
2. reagent according to claim 1, which is characterized in that the reagent is selected from:
The probe of specific recognition RP11-284F21.10；Or
The primer of specific amplification RP11-284F21.10.
3. application according to claim 2, which is characterized in that the primer sequence of the specific amplification RP11-284F21.10
Column are as shown in NO.1~2 SEQ ID.
4. a kind of product, which is characterized in that the product includes the described in any item reagents of claim 1-3.
5. product according to claim 4, which is characterized in that the product includes kit, chip, nucleic acid film item.
6. a kind of composition, which is characterized in that the composition includes the inhibitor of a effective amount of RP11-284F21.10.
7. composition according to claim 6, which is characterized in that the inhibitor is siRNA.
8. composition according to claim 7, which is characterized in that the sequence of siRNA is as shown in NO.7~12 SEQ ID.
9. a kind of method of the drug candidate of screening treatment adenocarcinoma of lung, which is characterized in that the described method includes:
The system expressed or containing RP11-284F21.10 gene is handled with substance to be screened；With
Detect the expression of RP11-284F21.10 gene in the system；
Wherein, if the substance to be screened can reduce the level of RP11-284F21.10 gene, show the object to be screened
Matter is the drug candidate for treating adenocarcinoma of lung.
10. any one of following application:
A. application of the described in any item reagents of claim 1-3 in the tool of preparation diagnosis adenocarcinoma of lung；
B. application of the product described in claim 4 or 5 in the tool of preparation diagnosis adenocarcinoma of lung；
Application of the c.RP11-284F21.10 in the computation model of building diagnosis adenocarcinoma of lung；
Application of the d.RP11-284F21.10 in the drug of preparation treatment adenocarcinoma of lung；
E. application of the described in any item compositions of claim 6-8 in the drug of preparation treatment adenocarcinoma of lung；
Application of the f.RP11-284F21.10 in the drug candidate of screening treatment adenocarcinoma of lung.
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| CN106729756A (en) * | 2017-02-28 | 2017-05-31 | 北京泱深生物信息技术有限公司 | Application of the biomarker as target in adenocarcinoma of lung diagnosis and treatment |
| CN107058499A (en) * | 2017-01-25 | 2017-08-18 | 河北医科大学第四医院（河北省肿瘤医院） | A kind of molecular marker for adenocarcinoma of lung diagnosis and treatment |
| CN109609648A (en) * | 2019-01-31 | 2019-04-12 | 泰山医学院 | LncRNA marker relevant to liver cancer and its detection primer and application |
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| CN107058499A (en) * | 2017-01-25 | 2017-08-18 | 河北医科大学第四医院（河北省肿瘤医院） | A kind of molecular marker for adenocarcinoma of lung diagnosis and treatment |
| CN106729756A (en) * | 2017-02-28 | 2017-05-31 | 北京泱深生物信息技术有限公司 | Application of the biomarker as target in adenocarcinoma of lung diagnosis and treatment |
| CN109609648A (en) * | 2019-01-31 | 2019-04-12 | 泰山医学院 | LncRNA marker relevant to liver cancer and its detection primer and application |
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Cited By (2)
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| CN111826442A (en) * | 2020-06-23 | 2020-10-27 | 温州医科大学 | Target PLEKHN1 for preventing lung cancer and application thereof |
| CN111826442B (en) * | 2020-06-23 | 2021-09-03 | 温州医科大学 | Target PLEKHN1 for preventing lung cancer and application thereof |
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