CN105624313A - Molecular marker for diagnosing and treating adenocarcinoma of lungs - Google Patents
Molecular marker for diagnosing and treating adenocarcinoma of lungs Download PDFInfo
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- CN105624313A CN105624313A CN201610136775.0A CN201610136775A CN105624313A CN 105624313 A CN105624313 A CN 105624313A CN 201610136775 A CN201610136775 A CN 201610136775A CN 105624313 A CN105624313 A CN 105624313A
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Classifications
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention discloses a molecular marker for diagnosing and treating the adenocarcinoma of lungs. A gene UPK3B is lowly expressed in the tissue of the adenocarcinoma of lungs; by promoting the expression of the gene UPK3B, the death of cells of the adenocarcinoma of lungs can be promoted; the sensitivity and specificity of diagnosis and treatment of the adenocarcinoma of lung are greatly improved, and meanwhile new molecular target spots are provided for treatment of the adenocarcinoma of lungs.
Description
Technical field
The invention belongs to biomedicine field, it relates to a kind of molecular marked compound for adenocarcinoma of lung diagnosis and treatment, described molecular marked compound is UPK3B.
Background technology
Cancer is one of major disease affecting socio-economic development and living standards of the people, and along with increasing the weight of of global industry and environmental pollution, lung cancer has leapt to the first place of whole world cancer morbidity and mortality ratio, and annual whole world lung cancer death number is more than 1,000,000. Lung cancer atmosphere nonsmall-cell lung cancer and small cell lung cancer. Nonsmall-cell lung cancer accounts for 85%, and small cell lung cancer accounts for 15%. Nonsmall-cell lung cancer comprises gland cancer, squama cancer, large cell carcinoma and adenosquamous carcinoma etc., and wherein the ratio of gland cancer is the highest, accounts for the 50% of whole lung cancer. Adenocarcinoma of lung originates from the epithelial cell of less tunica mucosa bronchiorum juice mostly, and most of gland cancer is positioned at around lung tissue, swollen block spherical in shape, near pleura position. Around in type lung cancer, the sickness rate of adenocarcinoma of lung occupies first of each histological type of lung cancer, and its sickness rate is in obviously raising trend. In adenocarcinoma of lung patient, female patients is more, and age of onset is also less.
Adenocarcinoma of lung early clinic symptom is hidden, and not easily finds, most patient arrive middle and advanced stage when medical, loses best operation opportunity, even if operation, most of patients still can die from invasion and attack or the transfer that adenocarcinoma of lung recurs. Having report to show, after early stage adenocarcinoma of lung patient's art, 5 years survival rates are about 90%, I-II phase patient, 5 years survival rates lower than 70%, and patients with terminal is only 1%. Therefore, early find, early treatment is the key reducing adenocarcinoma of lung patient's high mortality, improving prognosis. Clinical upper adenocarcinoma of lung early diagnosis depends on X ray examination, bronchoscopy, radionuclide inspection, cytolgical examination, cut open chest detects, ECT inspection, mediastinoscopy etc. Utilize existing detection methods to distinguish tumour and the mesothelioma of pleura of adenocarcinoma of lung and other types of lung, there is bigger difficulty.
In recent years, along with molecular biological development, some new biomarkers occur, not only the pathological diagnosis of adenocarcinoma of lung are provided a great help, and for its Index for diagnosis, treatment plan selection provide more fully foundation, played unique effect to a certain extent. Find the molecular marker of adenocarcinoma of lung early diagnosis and relapse and metastasis further, it is the effective means improving tumour patient curative ratio and survival rate, and the Diagnosis and Treat of adenocarcinoma of lung is had important theory significance and clinical value.
Summary of the invention
In order to make up the deficiencies in the prior art, it is an object of the invention to provide a kind of molecular marked compound-UPK3B gene that can be used for adenocarcinoma of lung diagnosis and treatment. Compare the Diagnosis and Treat method of traditional adenocarcinoma of lung, it may also be useful to gene marker carrys out Diagnosis and Treat adenocarcinoma of lung and has susceptibility, specificity and non-invasive.
In order to realize above-mentioned purpose, the present invention adopts following technical scheme:
The present invention provides UPK3B gene and expression product diagnoses the application in the product of adenocarcinoma of lung in preparation.
Further, described product comprises: by the expression level of RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip detection UPK3B gene and expression product thereof to diagnose the product of adenocarcinoma of lung.
Further, described RT-PCR diagnoses the product of adenocarcinoma of lung at least to comprise the primer of one pair of specific amplified UPK3B gene; The product of described real-time quantitative PCR diagnosis adenocarcinoma of lung at least comprises the primer of one pair of specific amplified UPK3B gene; The product of described immunodetection diagnosis adenocarcinoma of lung comprises: the antibody being combined with UPK3B protein-specific; The product of described in situ hybridization diagnosis adenocarcinoma of lung comprises: with the probe of the nucleic acid array hybridizing of UPK3B gene; The product of described chip diagnosis adenocarcinoma of lung comprises: protein chip and gene chip; Wherein, protein chip comprises the antibody being combined with UPK3B protein-specific, and gene chip comprises the probe of the nucleic acid array hybridizing with UPK3B gene.
Such as, further, described gene chip can be used for detecting the expression level of the multiple genes (relevant to adenocarcinoma of lung multiple genes) comprising UPK3B gene. Described protein chip can be used for detecting the expression level of the multiple protein (such as relevant to adenocarcinoma of lung multiple protein) comprising UPK3B albumen. By detecting the mark of multiple adenocarcinoma of lung simultaneously, can greatly improve the accuracy rate of adenocarcinoma of lung diagnosis.
The present invention provides a kind of product diagnosing adenocarcinoma of lung, and described product can diagnose adenocarcinoma of lung by the expression level of UPK3B gene in detection lung tissue.
Further, described product comprises chip or test kit; Wherein, described chip comprises gene chip, protein chip; Described test kit comprises gene detecting kit, protein immunization detection kit.
Such as, further, described gene detecting kit can be used for detecting the expression level of the multiple genes (relevant to adenocarcinoma of lung multiple genes) comprising UPK3B gene. Described protein immunization detection kit can be used for detecting the expression level of the multiple protein (such as relevant to adenocarcinoma of lung multiple protein) comprising UPK3B albumen. Multiple marks of adenocarcinoma of lung are detected simultaneously, can greatly improve the accuracy rate of adenocarcinoma of lung diagnosis.
The present invention provides UPK3B gene and expression product treats the application in the pharmaceutical composition of adenocarcinoma of lung in preparation.
Further, described pharmaceutical composition comprises increases UPK3B genetic expression, strengthens UPK3B expressive function and/or strengthens the reagent of UPK3B expression product activity.
Further, described reagent comprises: the reagent of the nucleic acid containing energy encoding function UPK3B albumen, the activator of UPK3B albumen, the reagent containing UPK3B protein.
Wherein, the reagent of the described nucleic acid containing energy encoding function UPK3B albumen can be single-chain nucleic acid (such as mRNA) or the double-strandednucleic acid (such as DNA) of the UPK3B albumen translating into activity form under favourable condition, described nucleic acid can be connected on expression vector or recombinate in host cell, as long as active UPK3B albumen can be encoded into, the carrying mode of any a kind of UPK3B gene. Described UPK3B albumen activator refers to stimulates UPK3B protein-active, increase UPK3B protein-active, promote UPK3B protein-active, enhancing UPK3B albumen activates, make UPK3B protein-active sensitization or raise the reagent of UPK3B protein-active, such as the transcriptional activation agent of demethylation reagent, UPK3B promotor and/or enhanser specificity, the agonist (as activated antibody) etc. of UPK3B albumen.
Further, aforementioned pharmaceutical compositions also comprises pharmaceutically acceptable carrier, and described carrier can be one can also be multiple, and described carrier includes but not limited to that thinner is such as lactose, sodium-chlor, glucose, urea, starch, water etc.; Tackiness agent is such as starch, pregelatinized Starch, dextrin, Star Dri 5, sucrose, gum arabic, gelatin, methylcellulose gum, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyoxyethylene glycol, PVP, Lalgine and alginates, xanthan gum, hydroxypropylcellulose and Vltra tears etc.; Tensio-active agent is such as polyoxyethylene sorbitan aliphatic ester, sodium lauryl sulphate, glyceryl monostearate, cetyl alcohol etc.; Cause wet agent such as glycerine, starch etc.; Absorption carrier is such as starch, lactose, the de-soil of spot, silica gel, kaolin and soap clay etc.; Lubricant is such as Zinic stearas, glyceryl monostearate, polyoxyethylene glycol, talcum powder, calcium stearate and magnesium, polyoxyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyl 40 stearate, single bay sucrose acid ester, sodium laurylsulfate, lauryl alcohol magnesium sulfate, Stepanol MG etc.; Weighting agent is such as N.F,USP MANNITOL (granular or powdery), Xylitol, sorbyl alcohol, maltose, erythrose, Microcrystalline Cellulose, polymerization sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and sodium bicarbonate etc.; Disintegrating agent is such as cross-linked ethylene pyrrolidone, sodium starch glycolate, low-substituted hydroxypropyl ylmethyl, croscarmellose sodium, soybean polysaccharide etc.
Described pharmaceutical composition can use different additives to be prepared, such as stablizer, sterilant, buffer reagent, etc. penetration enhancer, sequestrant, pH control agent and tensio-active agent.
Stablizer comprises Human serum proteins, L-amino acid, sugar and derivatived cellulose. L-amino acid can also comprise any one in glycine, halfcystine and L-glutamic acid. Carbohydrate comprises monose, such as glucose, seminose, semi-lactosi, fructose etc.; Sugar alcohol, such as N.F,USP MANNITOL, Inositol nf12 99, Xylitol etc.; Disaccharides, such as sucrose, maltose, lactose etc.; Saccharan, such as dextran, hydroxypropylated starch, sulfuration chrondroitin, hyaluronic acid etc. and their derivative. Derivatived cellulose comprises methylcellulose gum, ethyl cellulose, Natvosol, hydroxypropylcellulose, HPMC and sodium cellulose glycolate.
Tensio-active agent comprises ion or nonionogenic tenside, such as polyoxyethylene alkyl ester, sorbitanic list acyl ester, glycerin fatty acid ester.
Additive buffer reagent can comprise boric acid, phosphoric acid, acetic acid, citric acid, L-glutamic acid and corresponding salt (their basic metal or alkaline rare earth metal salt, such as sodium salt, sylvite, calcium salt and magnesium salt). Repone K, sodium-chlor, sugar and glycerine is comprised Deng penetration enhancer. Sequestrant comprises sodium ethylene diamine tetracetate and citric acid.
Present invention also offers a kind of pharmaceutical composition treating adenocarcinoma of lung, described pharmaceutical composition comprises increases UPK3B genetic expression, strengthen UPK3B expressive function and/or the reagent of enhancing UPK3B expression product activity.
The medicine of the present invention may be used for disappearance or the deficiency of the UPK3B albumen of supplementary endogenous property, by improving the expression of UPK3B albumen, thus the adenocarcinoma of lung that treatment causes because of the minimizing of UPK3B albumen.
The carrier carrying gene of the present invention is various carrier known in the art, such as commercially available carrier, comprise plasmid, clay, phage, virus etc.
Further, in the present invention, the nucleic acid of UPK3B albumen or coding UPK3B albumen can be given by liposome, and the effect of described liposome is by drug targeting in specific tissue, and increases the transformation period of medicine. Liposome comprises emulsifying agent, pore forming material, liquid fatty substance, solid-state lipid, insoluble monolayer, phosphatide dispersion agent, tensio-active agent etc. Described liposome can also comprise can with target to cell in acceptor molecule be combined or other treatment or immunogenic composition.
The pharmaceutical composition of the present invention can be formulated into any administration type, such as, utilize in the intradermal injection of syringe or other device, subcutaneous injection, intravenous injection, peritoneal injection, intrapleural injection, intravesical injection, coronary artery or intra-tumoral injection, oral administration, rectal administration.
The mode that the drugs delivery tissue of the present invention or the mode of cell can be divided in external or body. Vitro formats comprise by the medicine containing UPK3B gene or containing UPK3B protein drugs delivery cell in, then by Transplanted cells or return be passed in body. In body, mode comprises directly by the medicine containing UPK3B gene or containing in the infusion of medicine in-vivo tissue of UPK3B protein.
The medicine of the present invention also can with the drug combination of other treatment adenocarcinoma of lung, other treatment compound can with main activeconstituents (such as, the nucleic acid of UPK3B albumen or encoding said proteins) administration simultaneously, even administration simultaneously in same composition. Other therapeutic compound can also be given separately with independent composition or the dosage form different from main activeconstituents. The part dosage of main component (such as the nucleic acid of UPK3B albumen or encoding said proteins) can with the administration simultaneously of other therapeutic compound, and other dosage can be individually dosed.
Over the course for the treatment of, according to the physiologic response of the frequency of the severity of symptom, recurrence and treatment plan, the dosage of pharmaceutical composition of the present invention can be adjusted.
In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell. The example of conventional prokaryotic host cell comprises intestinal bacteria, Bacillus subtilus etc. Conventional eukaryotic host cell comprises yeast cell, insect cell and mammalian cell. Goodly, this host cell is eukaryotic cell, such as Chinese hamster ovary celI, COS cell etc.
The present invention can be DNA, RNA, DNA-RNA mosaic, PNA or other derivative for the probe of UPK3B gene. The length of described probe does not limit, as long as completing specific hybrid and object nucleotide sequence specific binding, any length can. The length of described probe can be as short as 25,20,15,13 or 10 base length. Equally, the length of described probe can grow to 60,80,100,150,300 base pairs or longer, even whole gene. Owing to hybridization efficiency, signal specificity are had different impacts by different probe length, the length of described probe is at least 14 base pairs usually, the longest generally it is no more than 30 base pairs, best with 15-25 base pair with the length of object nucleotide sequence complementation. Described probe self-complementary sequences most preferably less than 4 base pairs, in order to avoid affecting hybridization efficiency.
The specific antibody of the albumen of UPK3B described in the present invention comprises monoclonal antibody, polyclonal antibody. The specific antibody of described UPK3B albumen comprises complete antibody molecule, any fragment of antibody or modification, such as, and chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc. As long as described fragment can retain and the binding ability of UPK3B albumen. It is well known to a person skilled in the art for detecting the preparation of the antibody of protein level, and the present invention can use any method to prepare described antibody, as described fragment by chemical method de novo synthesis or can utilize recombinant DNA technology to synthesize.
In the context of the present invention, " UPK3B gene " comprises the polynucleotide of any function equivalent of people's UPK3B gene and people's UPK3B gene. UPK3B gene comprises and has more than 70% homology with UPK3B gene (ID_80761) DNA sequence dna at present international common core sequence databank GeneBank, and coding identical function protein DNA sequence;
Preferably, the encoding sequence of UPK3B gene comprises any one DNA molecular following:
(1) DNA sequence dna shown in SEQ ID NO.1;
(2) the DNA sequence dna hybridization limited with (1) under strict conditions and coding identical function protein DNA sequence;
(3) DNA sequence dna limited with (1) or (2) has 70%, preferably, more than 90% homology, and coding identical function protein DNA molecule.
In specific embodiment of the invention scheme, the encoding sequence of described UPK3B gene is the DNA sequence dna shown in SEQIDNO.1.
In the context of the present invention, UPK3B gene expression product comprises people's UPK3B albumen and the partial peptide of people's UPK3B albumen. The partial peptide of described UPK3B albumen contains the functional domain relevant to adenocarcinoma of lung.
" UPK3B albumen " comprises any function equivalent of UPK3B albumen and UPK3B albumen. Described function equivalent comprises UPK3B albumen conservative property variant protein matter or its active fragments, or its reactive derivative, allelic variant, natural mutation, induced mutants, under high or low stringent condition can with the protein coded by the DNA of the DNA hybridization of people UPK3B.
Preferably, UPK3B albumen is the protein with following amino acid sequences:
(1) protein being made up of the aminoacid sequence shown in SEQ ID NO.2;
(2) aminoacid sequence shown in SEQIDNO.2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and had the protein derivative by the aminoacid sequence shown in SEQIDNO.2 of identical function with the aminoacid sequence shown in SEQIDNO.2. The amino acid whose number replacing, lack or adding is generally 1-50, it is preferred that 1-30, and more preferably 1-20,1-10 is individual best.
(3) with the aminoacid sequence shown in SEQIDNO.2, there is at least 80% homology (being also called sequence iden), preferably, with the homology of the aminoacid sequence at least about 90% to 95% shown in SEQIDNO.2, it it is often the polypeptide that the aminoacid sequence of 96%, 97%, 98%, 99% homology is formed.
In specific embodiment of the invention scheme, described UPK3B albumen is the protein with the aminoacid sequence shown in SEQIDNO.2.
Usually, in a protein, one or more amino acid whose modification can not affect the function of protein. Those skilled in the art can approve the amino acid changing single amino acids or little per-cent or be conservative modifications to indivedual interpolations of aminoacid sequence, disappearance, insertion, replacement, and wherein the change of protein produces the protein with similar function. Intimate amino acid whose conservative replacement table is provided to be well known in the art.
By adding the fusion rotein that the example of the protein of an amino acid or multiple Modification of amino acid residues is UPK3B albumen. Peptide or protein for merging with UPK3B albumen do not limit, as long as the fusion rotein of gained retains the biologic activity of UPK3B albumen.
The UPK3B albumen of the present invention also comprises the non-conservative modification to the aminoacid sequence shown in SEQIDNO.2, as long as the protein through modifying still can retain the biologic activity of UPK3B albumen. The amino acid number of sudden change normally 10 or less in this type of modifying protein, such as 6 or less, such as 3 or less.
In the context of the present invention, " treatment adenocarcinoma of lung " comprises generation and the recurrence of any symptom that can eliminate, alleviate, alleviate, reverse or prevent or postpone illness, namely comprises the therapeutic intervention to disease and Primary preventive intervention.
The advantage of the present invention and useful effect:
Late Cambrian of the present invention UPK3B gene expression dose is relevant to adenocarcinoma of lung, by the expression of UPK3B in detection experimenter's lung tissue, it can be determined that whether experimenter suffers from adenocarcinoma of lung, thus instruct clinicist to provide prevention scheme or treatment plan to experimenter.
Present invention finds the new molecular marked compound-UPK3B gene of a kind of adenocarcinoma of lung, utilize molecular marked compound to realize the Diagnosis and Treat of disease, compare traditional means, have more susceptibility, specificity, non-invasive.
Accompanying drawing explanation
Fig. 1 display utilizes QPCR to detect the expression of UPK3B gene in pulmonary adenocarcinoma;
Fig. 2 display utilizes QPCR to detect the expression of UPK3B gene in lung adenocarcinoma cell.
Concrete enforcement mode
Below in conjunction with drawings and Examples, the present invention is further detailed explanation. Following examples are only not used in for illustration of the present invention and limit the scope of the invention. The experimental technique of unreceipted concrete condition in embodiment, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.
Embodiment 1 screens the gene marker relevant to adenocarcinoma of lung
1, sample collection
Each collection 8 example adenocarcinoma of lung cancer beside organism and pulmonary adenocarcinoma samples. Above-mentioned sample is the excision sample of adenocarcinoma of lung patient, and the acquirement of above-mentioned all samples is all by the agreement of the council of organizational ethics.
2, the preparation of RNA sample (utilizesMiRNAkit operates)
The tissue of above-mentioned acquisition drops in liquid nitrogen after shredding and is ground to powder shape, according to the specification sheets extraction and isolation RNA in test kit. Specific as follows:
1) separation of RNA:
A. tissue homogenate or cell add RNA-ReagentII1ml;
B. room temperature places 3min, acutely shakes 15s after adding 0.2ml chloroform;
C. it is placed in and prevents 10min on ice;
D.12000g, 4 DEG C of centrifugal 15min;
E. the aqueous phase shifting 80% enters in new 2mlEP pipe, adds the dehydrated alcohol of 1/2 amount, jolting;
F. the aforesaid liquid being less than 700 �� l is transferred toRNAMinicolumn, the centrifugal 60s of 10000g room temperature after jolting.
2) RNA purifying:
A. toRNAMinicolumn adds 500 �� lRWCWashBuffer, the centrifugal 30s of 10000g;
B. add 500 �� lRWBWashBuffer, the centrifugal 30s of 10000g, after repeating twice, take maximum centrifugal completely dryRNAMinicolumn;
C. adding, to pillar, the DEPC water that 15 �� l are preheated to 70 DEG C, room temperature is centrifugal at full speed after placing 2min.
3, high-throughput transcript profile order-checking
1) RNA-seq reads section location
First the reading section of inferior quality is removed and obtain cleaning reading section, then TopHatv1.3.1 is utilized cleaning fragment to be mated with reference to genome (hg19) with UCSCH.sapiens, the index built in advance of H.sapiensUCSChg19 version is downloaded from TopHat homepage, and as with reference to genome, when utilizing TopHat to mate with genome, allow each to read section (defaulting to 20) and have multiple coupling site, maximum 2 mispairing. TopHat sets up possible shearing site storehouse according to exon region and GT-AG shear signal, the reading section not navigating to genome is navigated on genome according to these shearing site storehouses. We use the system default parameter of TopHat method.
2) transcript abundance assessment
The reading segment file matched is by Cufflinksv1.0.3 process, and RNA-seq segment number is carried out the relative abundance of standardized calculation transcript by Cufflinksv1.0.3. FPKM value refers to match in each 1,000,000 order-checking fragments the segment number of the exon region of specific gene 1kb length. The fiducial interval of FPKM estimated value is calculated by Bayes's inference method. The GTF comment file of the reference that Cufflinks uses downloads (Homo_sapiens.GRCh37.63.gtf) from Ensembl database.
3) detection of difference expression gene
The EnsemblGTF file of download and the source document that mated by TopHat are transferred to Cuffdiff, and Cuffdiff uses original matching files again to estimate the gene expression abundance of the transcript listed in GTF file, and checkout discrepancy is expressed. Only having q value < 0.01 in Cuffidff exports, test display is successfully more just considered as differential expression.
4, result
RNA-seq result shows, and the expression amount of UPK3B gene in pulmonary adenocarcinoma is significantly lower than the expression amount in cancer beside organism.
The differential expression of embodiment 2QPCR sequence verification UPK3B gene
1, UPK3B gene differential expression is carried out large sample QPCR checking. According to each 50 examples of the sample collection way selection adenocarcinoma of lung cancer beside organism in embodiment 1 and pulmonary adenocarcinoma.
2, the concrete operation steps of QPCR is as follows:
(1) RNA extracts
RNA extraction step is as described in Example 1.
(2) reverse transcription
A. configuring mixed solution: OligodT (50 ��Ms) 1 �� l in Microtube, dNTP mixed solution (10mM) 1 �� l, RNA template 5 �� g, adds ddH2O to 10 �� l, mixes;
B. in PCR instrument, sex change, annealing reaction is carried out according to following reaction conditions: after 65 DEG C of 5min, place immediately on ice;
C. in above-mentioned Microtube pipe, following inverse transcription reaction liquid is prepared:
Reaction solution 10 �� l after above-mentioned sex change, annealing,Buffer4 �� l, RNase inhibitor (40U/ �� l) 0.5 �� l,RTase (200U/ �� l) 1 �� l, ddH2O (RNase-free) 4.5 �� l, mixes;
D. in PCR instrument, reverse transcription reaction is carried out according to following reaction conditions:
(30 DEG C of 10min) �� 3, (42 DEG C of 45min) �� 4, (95 DEG C of 5min) �� 5, after process, be placed on ice;
E. PCR reaction solution is prepared on ice by following formula:
PremixExTaqII (TliRNaseHPlus) (2 ��) 12.5 �� l, forward primer (10 ��Ms) 1 �� l, reverse primer (10 ��Ms) 1 �� l, DNA profiling (< 100ng) 2.0 �� l, ddH2O8.5 �� l;
The cycling condition of F.PCR is as follows:
95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 45s) �� 40, choose ��-actin as internal reference. The primer sequence of PCR is as follows:
The primer sequence of UPK3B:
Forward primer: 5 '-CAGCATGATCGTCATTACC-3 ' (SEQIDNO.3),
Reverse primer: 5 '-GTAGAGGCTGCCAAGAAG-3 ' (SEQIDNO.4)
The primer sequence of ��-actin:
Forward primer: 5 '-CCTGGGCATGGAGTCCTGTG-3 ' (SEQIDNO.5)
Reverse primer: 5 '-TCCTTCTGCATCCTGTCG-3 ' (SEQIDNO.6)
Using SYBRGreen as fluorescent marker, in the enterprising performing PCR reaction of LightCycler fluorescence real-time quantitative PCR instrument, determining object band by melt curve analysis analysis and electrophoresis, �� �� CT method carries out relative quantification.
3, statistical method
Experiment all completes for 3 times according to repetition, result data are all represent in the way of mean+SD, employing SPSS13.0 statistical software carries out statistical study, and difference between the two adopts t inspection, it is believed that when P < has statistical significance when 0.05.
4, result
As shown in Figure 1, compared with adenocarcinoma of lung cancer beside organism, UPK3B gene down-regulated expression in pulmonary adenocarcinoma, difference has statistical significance (P < 0.05) to result, consistent with RNA-sep result.
The process LAN of embodiment 3UPK3B gene
1, cell cultures
Human A459 lung cancer cell line, with the RPMI1640 substratum containing 10% foetal calf serum and 1%P/S at 37 DEG C, 5%CO2, relative humidity be 90% incubator in cultivate. Within 2-3 days, change liquid 1 time, it may also be useful to 0.25% contains the conventional had digestive transfer culture of trypsinase of EDTA.
2, the process LAN of UPK3B gene
The structure of 2.1UPK3B expression vector
Encoding sequence (as shown in SEQIDNO.1) design of amplification primers according to UPK3B gene, primer sequence is as follows:
Forward primer: 5 '-CCGAAGCTTGCCACCATGGGGCTACCCTGG-3 ' (SEQIDNO.7)
Reverse primer: 5 '-CGGGCGGCCGCCTCCCGGCGCCCCATCTCC-3 ' (SEQIDNO.8)
From cDNA library (the clontech company becoming Human fetal spleen, article No.: the encoding sequence of the UPK3B gene of amplification total length in 638831), above-mentioned cDNA sequence is inserted in the eukaryotic expression vector pcDNA3.1 of restriction enzyme HindIII and NotI double digestion after restriction enzyme HindIII and NotI double digestion, connects the recombinant vectors pcDNA3.1-UPK3B obtained and is used for subsequent experimental.
2.2 transfection
Lung adenocarcinoma cell is divided into two groups, is respectively control group (transfection pcDNA3.1 empty carrier) and UPK3B process LAN group (transfection pcDNA3.1-UPK3B). Using liposome 2000 to carry out the transfection of carrier, concrete transfection method carries out according to the instruction of specification sheets. The working concentration of pcDNA3.1 empty carrier and pcDNA3.1-UPK3B is 0.5 �� g/ml.
2.3RT-PCR detection
Concrete steps are with embodiment 2.
3, statistical method
Experiment all completes for 3 times according to repetition, result data are all represent in the way of mean+SD, employing SPSS13.0 statistical software carries out statistical study, and difference between the two adopts t inspection, it is believed that when P < has statistical significance when 0.05.
4, result
As shown in Figure 2, compared with the cell of transfection pcDNA3.1 empty carrier, in the cell of transfection pcDNA3.1-UPK3B, the content of UPK3B significantly raises, and difference has statistical significance (P < 0.05).
The impact that embodiment 4UPK3B gene pairs lung adenocarcinoma cell withers and dies
Use the impact of flow cytomery UPK3B gene pairs apoptosis.
1, cell culture step is with embodiment 3.
2, cell transfecting step is with embodiment 3.
3, step
After cell transfecting 72h, it may also be useful to precooling PBS washed cell, then use 0.25% trypsin digestion cell, stop digestion, use PBS resuspended in the cell of centrifugal collection, be 1 �� 10 by cell quantification6Individual/ml, gets the 200 above-mentioned cell suspensions of �� l and is placed in Eppendorf pipe, add 10 �� lAnnexin-V-FITC and mix even, and dyeing 15min is hatched in room temperature dark place, and before upper machine, 5min adds 10mg/L iodate third ingot (PI) and dyes 5 �� l. The cell of untransfected siRNA is used for standard quantitative with Annexin-V-FITC and PI dyeing respectively. Two Colour Fluorescence cell cytometry is carried out, observing apoptosis cell percentages with FACS flow cytometer.
3, statistical method
Experiment all completes for 3 times according to repetition, result data are all represent in the way of mean+SD, employing SPSS13.0 statistical software carries out statistical study, the t inspection that difference between the two adopts, it is believed that when P < has statistical significance when 0.05.
4, result:
The apoptosis rate of transfection pcDNA3.1-UPK3B group is (27.32 �� 0.014) %, the apoptosis rate of transfection pcDNA3.1 empty carrier group is (8.98 �� 0.011) %, above-mentioned difference has statistical significance (P < 0.05), the above results shows, the process LAN of UPK3B gene promotes that withering of lung adenocarcinoma cell is died.
The explanation of above-described embodiment is method and the core concept thereof for understanding the present invention. , it is also possible to the present invention carries out some improvement and modification, it is noted that for the person of ordinary skill of the art, under the premise without departing from the principles of the invention these improve and modify also by the protection domain falling into the claims in the present invention.
Claims (10)
- The application in the product of preparation diagnosis adenocarcinoma of lung of 1.UPK3B gene and expression product thereof.
- 2. application according to claim 1, it is characterised in that, described product comprises: by the expression level of RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip detection UPK3B gene and expression product thereof to diagnose the product of adenocarcinoma of lung.
- 3. application according to claim 2, it is characterised in that, described RT-PCR diagnoses the product of adenocarcinoma of lung at least to comprise the primer of one pair of specific amplified UPK3B gene; The product of described real-time quantitative PCR diagnosis adenocarcinoma of lung at least comprises the primer of one pair of specific amplified UPK3B gene; The product of described immunodetection diagnosis adenocarcinoma of lung comprises: the antibody being combined with UPK3B protein-specific; The product of described in situ hybridization diagnosis adenocarcinoma of lung comprises: with the probe of the nucleic acid array hybridizing of UPK3B gene; The product of described chip diagnosis adenocarcinoma of lung comprises: protein chip and gene chip; Wherein, protein chip comprises the antibody being combined with UPK3B protein-specific, and gene chip comprises the probe of the nucleic acid array hybridizing with UPK3B gene.
- 4. diagnose the product of adenocarcinoma of lung for one kind, it is characterised in that, described product can diagnose adenocarcinoma of lung by the expression level of UPK3B gene in detection lung tissue.
- 5. product according to claim 4, it is characterised in that, described product comprises chip or test kit; Wherein, described chip comprises gene chip, protein chip; Described test kit comprises gene detecting kit, protein immunization detection kit.
- The application in the pharmaceutical composition of preparation treatment adenocarcinoma of lung of 6.UPK3B gene and expression product thereof.
- 7. application according to claim 6, it is characterised in that, described pharmaceutical composition comprises increases UPK3B genetic expression, strengthen UPK3B expressive function and/or the reagent of enhancing UPK3B expression product activity.
- 8. application according to claim 7, it is characterised in that, described reagent comprises: the reagent of the nucleic acid containing energy encoding function UPK3B albumen, the activator of UPK3B albumen, the reagent containing UPK3B protein.
- 9. application according to claim 7 or 8, it is characterised in that, described pharmaceutical composition also comprises pharmaceutically acceptable carrier.
- 10. treat the pharmaceutical composition of adenocarcinoma of lung for one kind, it is characterised in that, described pharmaceutical composition comprises increases UPK3B genetic expression, strengthen UPK3B expressive function and/or the reagent of enhancing UPK3B expression product activity.
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| CN107881234A (en) * | 2017-11-09 | 2018-04-06 | 南京卡迪睿伯生物技术有限公司 | One group of adenocarcinoma of lung related gene label and its application |
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