CN105343896A - New diagnosis and treatment target of nasopharynx cancer and application of new diagnosis and treatment target - Google Patents

Abstract

The invention relates to a new diagnosis and treatment target of nasopharynx cancer and application of the new diagnosis and treatment target, and more particularly relates to a VPS11 gene and application that the VPS11 regulates and controls miRNA in treatment of nasopharynx cancer. Nasopharynx cancer tissues and para-carcinoma tissues are analyzed through high-throughput sequencing, mRNA and miRNA transcriptome information is obtained, after analysis, VPS11 gene and miR-363 are screened out and selected to perform fluorescent quantitation PCR verification and target verification, a result shows that the VPS11 gene and miR-363 are closely related to nasopharynx cancer, and VPS11 is a target gene of miR-632 in nasopharynx cancer. Through the invention, a new target is provided for clinical diagnosis and prevention detection of nasopharynx cancer, and has very good application value.

Description

The new diagnosis and treatment target spot of nasopharyngeal carcinoma and application thereof

Technical field

The present invention relates to biology field, be specifically related to new diagnosis and treatment target spot and the application thereof of nasopharyngeal carcinoma, relate to VPS11 gene and the purposes of regulation and control miRNA in diagnosis and treatment nasopharyngeal carcinoma thereof more specifically.

Background technology

Nasopharyngeal carcinoma (Nasopharyngealcarcinoma, NPC) is one of common head-neck malignant tumor of south China, and wherein male's sickness rate is about the twice of women, is this disease high-incidence season with 40-50 year.Nasopharyngeal carcinoma morbidity is relevant with the many factors such as inherited genetic factors (genetic predisposition), ebv infection, environmental factors, dietary habit, the most effective means that early diagnosis, early treatment are redemption patient vitals and improve the quality of living.Regrettably, nasopharyngeal carcinoma onset is hidden, and has strong metastasis tendency.In addition, PDSCC is the most common histological type of nasopharyngeal carcinoma, but after radiation alone, five year survival rate is only about 50%, after treatment recurrence or transfer prognosis very poor, become the main cause for the treatment of of nasopharyngeal carcinoma failure.Therefore, identify the tumor marker relevant to nasopharyngeal carcinoma with qualification, strive for that early discovery, selection therapeutic regimen, predict prognosis, monitoring recurrence or transfer have important clinical meaning to nasopharyngeal carcinoma diagnosis and treatment.

MiRNA is the endogenic non-coding RNA with adjusting function of a class found in eukaryote.Usually generation is transcribed by rna plymerase ii (PolII).The typical effect mode of miRNA and said target mrna mainly contains two kinds.In most of the cases, the incomplete complementary pairing of 3 ' UTR of the strand miRNA in complex and said target mrna, blocks the translation of target gene, thus regulator gene is expressed.This mode major effect protein expression level, does not affect the stability of mRNA.Another kind of model of action is similar with siRNA, and when miRNA and mRNA complete complementary matches, Ago2 albumen passes through to cut mRNA and directly causes it to degrade, and realizes gene silencing.Participate in for siRNA RNAi: siRNA can be combined with RISC, and as template identification mRNA target, by base pair complementarity principle, the antisense strand in mRNA and siRNA combines, and displaces positive-sense strand.Double-strand mRNA produces the siRNA of about 22nt under Dicer enzyme, ATP and unwindase combined effect, and siRNA continues to form complex with RISC, is combined, makes mRNA by RNA enzymatic lysis with the mRNA of siRNA complementation.This process is also referred to as PTGS (PTGS).

In a word, currently think that miRNA is relevant with the pairing degree of genes of interest effect and miRNA and genes of interest in which way.When miRNA and genes of interest match incomplete, miRNA is just to suppress the expression of genes of interest to play a role; When miRNA and genes of interest section sequence are matched complete, genes of interest just may be caused to cause gene silencing in the fracture of complementary district.In addition, miRNAs sometimes also causes the DNA methylation of histidine modification and promoter region, thus affects the expression of target gene.In addition, recently find that de-polyadenylation (accelerateddeadenylation) is the new mechanism of miRNA inhibition of gene expression fast.In mammalian cell, find that miR-125b and let-7 can promote the removal of mRNA poly A tail (polyAtail).Poly A tail is replaced with 3 ' histone stem-ring structure, not only can eliminate the impact of miR-125b on mRNA content, can also reduce the effect to protein synthesis, visible miRNA carrys out inhibition of gene expression by the concentration reducing translation efficiency and polyadenylation mRNA.

Invent and analyze tissues of nasopharyngeal carcinoma and cancer beside organism by high-flux sequence, obtain itself mRNA and miRNA transcript profile information, filter out by analysis and choose VPS11 gene and miR-363 and carry out quantitative fluorescent PCR checking and target checking, result shows, VPS11 gene and miR-363 and nasopharyngeal carcinoma closely related, in nasopharyngeal carcinoma, VPS11 is the target gene of miR-632.The present invention is that clinical diagnosis and prevention detection nasopharyngeal carcinoma provide new target spot, has good using value.

Summary of the invention

The object of the present invention is to provide VPS11 gene and/or the application of albumen in preparation treatment or diagnosis of nasopharyngeal carcinoma reagent.

The object of the present invention is to provide the application of the preparation of regulation and control VPS11 gene and/or protein expression in preparation treatment nasopharyngeal carcinoma reagent.

Further, described treatment nasopharyngeal carcinoma reagent refers to the reagent of the expression that can promote VPS11 gene.Those skilled in the art know and promote that the expression of gene can adopt the one in following method and/or several usually: by DNA level regulation and control VPS11 gene: include but not limited to increase the copy number of VPS11 gene, transfection containing the over-express vector of VPS11 gene; By transcriptional level control VPS11 gene: include but not limited to the expression of activation VPS11 gene, activate the promoter of regulation and control VPS11 gene expression, suppress the transcription factor of negative regulation VPS11 gene expression, adopt suppression of RNA perturbation technique to suppression VPS11 gene expression to disturb; By post-transcriptional level regulation and control VPS11 gene: include but not limited to suppress the microRNA transcriptional expression of promotion VPS11 gene mRNA degraded, import the microRNA promoting VPS11 gene expression; By translating rear level modulation VPS11 gene: include but not limited to import the molecule promoting VPS11 gene coded protein, the albumen suppressing negative regulation VPS11 gene expression, promote the factor of VPS11 gene expression and the expression of albumen.

Preferably, containing the carrier promoting VPS11 gene expression in described treatment nasopharyngeal carcinoma reagent.

The preparation detecting VPS11 gene and/or albumen is the object of the present invention is to provide to prepare the application in diagnosis of nasopharyngeal carcinoma reagent.

Further, described diagnosis of nasopharyngeal carcinoma reagent comprises the expression detecting VPS11 gene in nasopharyngeal carcinoma with fluorescence quantifying PCR method, method for gene chip.

Described contains the primer of a pair specific amplification VPS11 gene for the product of VPS11 gene in fluorescence quantifying PCR method detection nasopharyngeal carcinoma; Described gene chip comprises the probe with the nucleic acid array hybridizing of VPS11 gene.

Further, the diagnostic preparation of described nasopharyngeal carcinoma comprises the expression detecting VPS11 albumen with immunization method.In preferred described immunologic detection method detection nasopharyngeal carcinoma, VPS11 protein expression is westernblot and/or ELISA and/gold colloidal detection method.

Further, the ELISA method of described detection VPS11 albumen is for using ELISA detection kit.Antibody in described test kit can adopt commercially available VPS11 monoclonal antibody.Further, described test kit comprises: wrap by the solid phase carrier of VPS11 monoclonal antibody, ELIAS secondary antibody, the substrate of enzyme, protein standard substance, negative controls, diluent, cleaning mixture, enzyme reaction stop buffer etc.

Further, the colloidal gold method of described detection VPS11 albumen is for using detection kit, and described antibody can adopt commercially available VPS11 monoclonal antibody.Further, described gold-immunochromatographyreagent reagent for assay box adopts colloidal gold immunochromatographimethod technology or gold colloidal percolation.Further, detection zone (T) specking on described gold-immunochromatographyreagent reagent for assay box nitrocellulose filter has anti-VPS11 monoclonal antibody, quality control region (C) specking has immunoglobulin IgG.

The object of the present invention is to provide a kind of PCR kit for fluorescence quantitative detecting nasopharyngeal carcinoma, it is characterized in that, described test kit detects gene VPS11, adopts special forward primer and downstream primer, forward primer sequence is SEQIDNO.4, and downstream primer sequence is SEQIDNO.5.

Further, this PCR kit is suitable for all types fluorescence quantitative gene extender deposited at present commercially, highly sensitive, quantitatively quick and precisely, good stability, has a good application prospect.

Further, above-mentioned PCR kit for fluorescence quantitative component comprises: Auele Specific Primer, internal reference primer, fluorescence quantitative PCR reaction solution.Wherein said Auele Specific Primer comprises forward primer and downstream primer, and forward primer sequence is SEQIDNO.4, and downstream primer sequence is SEQIDNO.5.Described internal reference primer is β-actin internal reference primer.

The object of the present invention is to provide the application of the regulation and control miRNA of VPS11 gene and/or albumen in preparation prevention, diagnosis and/or treatment nasopharyngeal carcinoma reagent.

The regulation and control miRNA preparation detecting VPS11 gene and/or albumen is the object of the present invention is to provide to prepare the application in diagnosis of nasopharyngeal carcinoma reagent.

The object of the present invention is to provide and regulate the application of the regulation and control miRNA reagent of VPS11 gene and/or albumen in preparation treatment nasopharyngeal carcinoma reagent.

Described regulation and control miRNA is mir-632 and/or miR-632.The sequence of mir-632 is shown in sequence table SEQ IDNO1; The ripe miRNA of mir-632 is that miR-632 sequence is shown in sequence table SEQ IDNO2.

Further, described prevention, diagnosis of nasopharyngeal carcinoma reagent comprise based on high-flux sequence method and/or based on quantifying PCR method and/or the expression transcribing or detect based on immunologic detection method the target gene of miR-632 regulation and control in nasopharyngeal carcinoma sample detecting mir-632 and/or miR-632 in nasopharyngeal carcinoma sample based on probing procedure, preferably adopt northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, transcribing based on mir-632 and/or miR-632 in the Flow cytometry nasopharyngeal carcinoma sample of microsphere; ELISA and/or colloidal gold strip is adopted to detect the expression of the target gene of miR-632 regulation and control in nasopharyngeal carcinoma sample.The target gene of preferred described miR-632 regulation and control is ENPP4, PDZD2, ATP1A2, TMEM9B, ARHGAP11A, MSLN, CR1, CRY2, MAPK14, CTNS, ADRA2A, SWAP70, TMEM184B, SNED1, KCNJ16, PLAC8, PGK1, POU2AF1, SMPD3, RBM38, VPS11, PLSCR4, SGTA, ST6GAL1, SMPD1, C10orf81, ITIH5, LIMD2, JHDM1D, EIF3J, MS4A1, CD22, SPAG6, CD40LG, MRPL19, the target gene of preferred miR-632 regulation and control is VPS11.

Preferably, the described primer comprising specific amplification mir-632 and/or miR-632 based on quantifying PCR method, further preferably, specific amplification miR-632 primer sequence is SEQIDNO3; Described comprises the probe with the nucleic acid array hybridizing of mir-632 and/or miR-632 based on probing procedure; Described immunologic detection method comprises the antibody be combined with miR-632 regulate gene expression protein-specific.

Further, the reagent of described treatment nasopharyngeal carcinoma is the reagent transcribing and/or block the activity of miR-632 lowering mir-632 and/or miR-632.

Preferably, adopt antisense oligonucleotide, antagomiRs, miRNA sponge, miRNAErasers, TargetMasking and/or Mutiple Targets antisense oligonucleotide method lower the activity transcribing and/or block miR-632 of mir-632 and/or miR-632.

The present invention also aims to provide a kind of pharmaceutical composition for the treatment of nasopharyngeal carcinoma, comprise:

A () promotes the preparation of VPS11 gene expression;

Receptible carrier on (b) pharmaceutics.

Promote that the preparation of VPS11 gene expression is the preparation transcribing and/or block miR-632 activity suppressing mir-632 and/or miR-632.

Preferably, adopt antisense oligonucleotide, antagomiRs, miRNA sponge, miRNAErasers, TargetMasking and/or Mutiple Targets antisense oligonucleotide method lower the activity transcribing and/or block miR-632 of mir-632 and/or miR-632.

The present invention also aims to provide a kind of nasopharyngeal carcinoma diagnosis reagent, it is characterized in that, described nasopharyngeal carcinoma diagnosis reagent can detect the expression that transcribing of mir-632 and/or miR-632 in sample or immunologic detection method detect the target gene of miR-632 regulation and control in sample.

Preferably, described nasopharyngeal carcinoma diagnosis reagent, based on high-flux sequence method and/or based on quantifying PCR method and/or the expression transcribing or detect based on immunization method the target gene of miR-632 regulation and control in sample detecting mir-632 and/or miR-632 in adenocarcinoma of lung sample based on probing procedure, preferably adopts northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, transcribing based on mir-632 and/or miR-632 in the Flow cytometry sample of microsphere; ELISA and/or colloidal gold strip is adopted to detect the expression of the target gene of miR-632 regulation and control in sample.

Preferred, the described primer comprising specific amplification mir-632 and/or miR-632 for quantifying PCR method; Described comprises the probe with the nucleic acid array hybridizing of mir-632 and/or miR-632 based on probing procedure; Described immunologic detection method comprises the antibody be combined with miR-632 regulate gene expression protein-specific.Definition:

Present stage, the method for expression that detects miRNA mainly comprised based on high throughput sequencing technologies, miRNA detection method based on nucleotide hybridization and PCR-based.MiRNA detection method based on probe hybridization technology is a kind of direct Detection Method; do not need to increase in advance to sample rna, comprise northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, the technology such as flow cytometry based on microsphere.

(1) Northern hybridization

Also known as the detection eukaryote RNA size that RNA engram technology is the most classical, estimate the experimental technique of its abundance.Ultimate principle is as follows: first at the upper fixing miRNA sample of carrier (as silicon chip, microsphere or film etc.), then with the probe hybridization through labelling, carry out signal detection after washing unnecessary hybridization probe; Also can first fix the DNA probe with the complementation of target miRNA sequence on carrier, then hybridize with the sample miRNA through labelling, then carry out signal detection.The method of signal labelling comprises isotopic labeling, fluorescent labeling and nano gold mark etc.

(2) miRNA chip of expression spectrum

Principle is the target molecule on usage flag probe in detecting solid support equally.By miR-96 gene in design chips and internal reference sequence, Accurate Analysis the expression of corresponding miRNA in sample can be gone out.Gene chip has high-throughout advantage, once can detect whole expression of a hundreds of gene in same sample.The liquid-phase chip (Liquidchip) of Luminex company development, also known as multifunctional suspending dot matrix (Multianalytesuspensionarray, MASA), is the biochip technology of new generation.Liquid-phase chip system is that main matrix is formed by many spherulas, often kind of spherula is fixed with different probe molecules, in order to distinguish different probes, each sphere matrix for label probe is all with a unique color numbers, these spherulas are suspended in a liquid-phase system, just constitute liquid-phase chip system.This system can carry out qualitative and quantitative analysis fast to the multiple different moleculars in same trace sample simultaneously, and this detection technique is called as FMAP (Flexiblemultianalyteprofiling) technology.Molecular hybridization carries out in aaerosol solution, and detection speed is exceedingly fast.

(3) ribozyme protection analytical technology (RPA)

The detection of miRNA can also adopt ribozyme to protect analytical technology; the probe that labelling is good and RNA sample to be measured mixing; hybridize after thermal denaturation; the RNA of not hybridizing and unnecessary probe single-chain nucleic acid enzymic digestion; the shielded RNA molecule of purification after heat inactivation nuclease; finally by degeneration PAGE electrophoretic separation probe, colour developing.This new method based on solution hybridization is simple and quick, highly sensitive, but also can only be used for analyzing known miRNA.

(4) RAKE method

RAKE method (RNAprimedarraybasedKlenowemzyme) is the Klenow fragment utilizing DNA polymerase i on the basis of miRNAmicroarray, makes the method that miRNA is hybridized with fixing DNA probe.RAKE sensitivity can detect miRNA specifically, is applicable to screen all miRNA that oneself knows fast in a large number.MiRNA express spectra situation can be detected in specific cell and tumor.Moreover, RAKE method can also be isolated miRNA and analyze it from the paraffin-embedded tissue secured by formalin, for analyzing the door that miRNA opens hope from file specimen.

(5) in situ hybridization (insituhybridization)

Hybridization in situ technique can intuitively understand miRNA expression way, and be a kind of easier method of observation miRNA spatial and temporal expression, normal mark mode comprises digoxin, biotin, fluorescent labeling etc.In situ hybridization (LockedNucleicAcid (LNA) basedinsituhybridization (LNA-ISH)) on locked nucleic acid basis is the more probe mode of current application.

(6) based on the flow cytometry (bead-basedflowcytometry) of microsphere

Be a kind of liquid-phase chip technology, FCM analysis organically combines with chip technology by the method, has that flux is large, detection speed is fast concurrently, a feature such as highly sensitive and specificity is good.

(7) Real-Time Fluorescent Quantitative PCR Technique (Real-timePCR, RT-PCR)

Fluoroscopic examination PCR instrument can draw dynamic changing curve to the cumulative speed of extension increasing sequence in whole PCR process.In reaction mixture, the initial concentration of target sequence is larger, requires that the PCR period (generally expressing with specific threshold period Ct) obtaining amplified production specific output is fewer.Because miRNA length is only 22nt, traditional qRT-PCR is not suitable for increasing so short fragment.There is several real time quantitative PCR method for miRNA now, as tailing method, neck ring method etc.Neck ring method is that a kind of desirable miRNA detects qRT-PCR method: first design special loop-stem structure primer, with miRNA to be measured for template reverse transcription synthesis cDNA first chain, this cDNA one end is stem Loop primer, stem circulus is opened and substantially increases the length of cDNA, carries out real-time quantitative PCR amplification for template design primer subsequently with the cDNA of synthesis.QRT-PCR has that specificity is high, sensitivity good, the multiple advantage such as simple fast.

(8) sequencing

The known miRNA of major part is found by cDNA cloning and sequencing and identifies.This method needs the cDNA library first building miRNA, then carries out pcr amplification, and amplified production is cloned into subsequently on expression vector and checks order.Takada develops a kind of amplification cloning (miRNAamplificationprofiling, mRAP) of improvement, and mRAP method first connects joint, then with the reverse transcription primer reverse transcription with joint complementation at 3 ' of miRNA end.Because specific reverse transcription has terminal deoxynucleotidyl transferase activity, some nucleotide (mainly dCMP 1beta-Deoxyribofuranosylcytosine-5'-phosphate) can be connected to 3 ' end of the cDNA chain that reverse transcription goes out.After poly (C) sticky end of 5 ' end connector and cDNA chain is annealed, add the pcr amplification that a pair general primer can realize cDNA.Due to mRAP High sensitivity, the expression of miRNA in can directly organizing on a small quantity by Cloning and sequencing technology for detection.Sequence label cloning is that one has developed the higher miRAGE of detection efficiency (miRNASAGE) cloning on the basis in serial analysis of gene expression (SAGE) technology, this method is by generating large sub-series, multiple miRNA can be detected by single sequencing reaction, significantly improve detection efficiency.

High-flux sequence (High-throughputsequencing) is the change to tradition order-checking revolution also known as sequencing technologies of future generation (nextgenerationsequencing), once to millions of DNA moleculars, sequencing is carried out to hundreds of thousands, greatly improve order-checking efficiency.This kind of large scale sequencing technology greatly improves the solution reading rate of multiple species hereditary information, and for obtaining the sequence information of all miRNA, deciphering miRNA collection of illustrative plates provides guarantee.Simultaneously high-flux sequence makes the analysis transcript profile of species and genome being carried out to careful overall picture become possibility, so degree of depth order-checking (deepsequencing) that is otherwise known as.The representative of high-flux sequence platform is 454 sequenators (RochGSFLXsequencer) of Roche Holding Ag (Roche), the Solexa gene element analyzer (IlluminaGenomeAnalyzer) of Illumina company and the SOLiD sequenator (ABISOLiDsequencer) of ABI.

Immunologic detection method is using a kind of antibody or Multiple Antibodies as analytical reagent, carries out quantitatively or the detection method of qualitative analysis determinand.Its ultimate principle is the interaction between antibody and antigen.For improving the sensitivity of antigen and antibody test, by the material of easily display in known antibodies or antigenic mark, by certification mark thing, reflect with or without antigen antibody reaction, thus indirectly measure antigen or the antibody of trace.Conventional label has enzyme, fluorescein, radiosiotope, gold colloidal and electron dense substances etc.The specific reaction that on this antigen or antibody labeling, display object is carried out is called immunolabelling technique (immunolabellingtechnique).Immunoassay technology most widely used at present mainly contains: elisa (enzyme-linkedimmunosorbentassay, ELISA), colloidal gold immunity chromatography etc.

Elisa principle is combined antigen or antibody and substrate (enzyme), makes it keep the activity of immunoreation and enzyme.The antigen of labelling or antibody and the ligand binding that is coated on solid phase carrier, then make it and corresponding colorless substrate effect and Show Color, measure OD value result of determination according to the range estimation of colour developing depth degree or by microplate reader.

Colloidal gold strip is generally made up of sample pad, gold mark pad, chromatographic film, adsorptive pads four part.Chromatographic material has nitrocellulose membrane (NC), polyester film, nylon membrane and pvdf membrane etc., need to select the different film required according to test, wherein NC film is the most conventional, can determine whether to need activation or process according to test concrete condition before using, in most cases without the need to process, can directly use.Gold is marked protein solution even application on gold mark pad, dry for subsequent use under room temperature.NC film can catch a certain amount of bag by (antibody) and two anti-as detection line and nature controlling line.Finally sample pad, gold mark pad, NC film and absorbent paper are fixed on PVC board successively, test strips.

Namely microRNA gain-of-function technology based on RNA raises the level of miRNAs by the precursor substance of exogenous supplementary miRNAs synthesis.Such as, sample RNA (shorthairpinRNA can be pressed from both sides by the synthetic bob consistent with endogenous miRNA sequence, shRNA), promoter is done by polymerase II or III, take virus as vector-transfected cell, being loaded into RISC after Dicer enzyme modification plays a role, and is equivalent to raise the level of pre-miRNA, and action effect is stable and lasting.

Gene specific miRMimics technology this technique avoids the nonspecific action of miRNA and gene.This synthetic with the specific oligonucleotide chain that combines of target gene 3 ' UTR complementation, can play and identical with miRNA transcribe rear regulating action.

Being included in the carrier that the pharmaceutics of pharmaceutical composition of the present invention is permitted is the carrier usually utilized when preparation, this carrier comprises lactose (lactose), dextrose (dextrose), sucrose (sucrose), sorbitol (sorbitol), mannitol (mannitol), starch, acacia gum, calcium phosphate, alginate (alginate), gel (gelatin), calcium silicates, microcrystalline Cellulose, polyvinylpyrrolidone (polyvinylpyrrolidone), cellulose (cellulose), water, syrup, methylcellulose (methylcellulose), methyl hydroxybenzoate (methylhydroxybenzoate), propyl hydroxy propyl benzoate (propylhydroxybenzoate), Talcum, magnesium stearate (stearicacidmagnesium) and mineral oil (mineraloil) etc., but it is not limited thereto.

Pharmaceutical composition of the present invention can also comprise lubricant, wetting agent, sweeting agent, flavouring agent, emulsifying agent, suspending agent, antiseptic etc. except mentioned component.The carrier be applicable to that pharmaceutics is permitted and preparation are recorded in Lei Mingdengshi pharmacy pandect in detail.

Pharmaceutical composition of the present invention by oral or parenterally carry out administration, during as non-oral administration, by intravenous injection, intranasal injection, local injection, intracerebral ventricle injection, spinal cavity is injected, subcutaneous injection, lumbar injection, the modes such as percutaneous dosing carry out administration.

The dosage be applicable to of pharmaceutical composition of the present invention can carry out multiple prescription according to the factor of age of preparation ways, administering mode, patient, body weight, sex, morbid state, food, administration time, route of administration, drainage rate and being quick on the draw property and so on, usually, skilled doctor can easily determine and prescription to desired treatment or prevent effective dosage.

The method that pharmaceutical composition of the present invention can easily be implemented according to general technical staff of the technical field of the invention, to utilize on pharmaceutics receptible carrier and/or excipient formulation to carry out, thus can with the preparation of unit dose form or in be contained in multicapacity container and prepare.Now, dosage form is solution, suspension or emulsion form in oiliness or aqueous medium, or also can be extractum, powder agent, granule, tablet or capsule form, can also comprise dispersant or stabilizing agent.

Accompanying drawing explanation

Fig. 1 RT-PCR detects tissues of nasopharyngeal carcinoma miR-632 expression

Fig. 2 RT-PCR detects tissues of nasopharyngeal carcinoma VPS11 gene expression dose

Detailed description of the invention

Below in conjunction with specific embodiment, setting forth the present invention further, only for explaining the present invention, and can not limitation of the present invention be interpreted as.Those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.The experimental technique of unreceipted actual conditions in the following example, the usually conveniently conditioned disjunction condition examinations of advising according to manufacturer.

The collection of embodiment 1 sample and Total RNAs extraction

Tissue is all from the sample that hospital's in March, 2011 in December ,-2013 is collected, and experimental group selects sick inspection to be diagnosed as 7 routine patients of nasopharyngeal carcinoma, and matched group is the cancer beside organism of same sample.

Experimental technique:

1 extracting method

1) get 80mg piece of tissue, add 800 μ lLysis/Binding buffer, use homogenizer to carry out homogenate to piece of tissue.In the process of homogenate, sample will be placed in and keep low temperature state on ice.

2) add 1/10 volume HomogenateAdditive again in the tissue sample of above-mentioned homogenate, place 10min on ice.

3) water-saturated phenol with Lysis/Binding buffer equivalent volumes is added, concussion 45s, the centrifugal 5min of 10,000 × g room temperature.

4) the careful supernatant that takes out is in new test tube, adds the dehydrated alcohol of 1.25 times of volumes, after mixing, moves in purification column, and 10,000 × g, centrifugal 15s, outwell the liquid in collecting pipe.Maximum volume due to pillar only has 700 μ l, therefore repeats this step operation, until all supernatants have all filtered.

5) in centrifugal pillar, add 700 μ lmiRNA eluents 1, room temperature, 10,000 × g, centrifugal 15s, outwell collection liquid, use new collecting pipe instead.

6) add in centrifugal column with 500 μ l eluents 2/3 again, 10,000 × g, centrifugal 10s, repeat this step once.

7) centrifugal 1min, 10,000 × g, discard unnecessary liquid.

8) aforesaid liquid is transferred to new centrifuge tube, add the DEPC process 30s of 100 μ l95 DEG C preheatings, 10,000 × g, centrifugal.

9) nanodrop is used to measure the ratio of RNA concentration and 260nm/280nm.

10) RNA obtained is stored in-80 DEG C of refrigerators.

2 extract standard

Measure the ratio of RNA concentration and 260nm/280nm: the purity requirement of total serum IgE is that OD260/OD280 value should between 1.8 to 2.2; The detection of RNA integrity: the integrity detecting RNA with 1% agarose gel electrophoresis;

According to the requirement of order-checking company, tiny RNA order-checking total amount 3 more than μ g, concentration is at 300ng/ more than μ l.

Embodiment 2 checks order and data analysis

Foundation and the order-checking of upper machine of sequencing library is carried out, the HiSeq2000 sequenator that the sequenator used is Illumina company by order-checking company.

Data results according to company provides: 7 routine nasopharyngeal carcinoma groups and the other group of cancer carry out statistical analysis, P value is less than 0.05, and the difference of the other matched group of tumor group and cancer at least will differential expression miRNA more than more than 2 times, be select in filtration to lower to express the research range that obvious VPS11 gene and the obvious has-mir-632 of up-regulated expression enter us to differential expression miRNA people, the prediction target gene through software prediction has-mir-632 is VPS11.

Embodiment 3Real-timePCR detects the expression of VPS11 gene and miR-632 in tissues of nasopharyngeal carcinoma

1 sample collecting:

63 routine tissues of nasopharyngeal carcinomas and cancer beside organism are all from hospital (acquisition time in March, 2011 in December ,-2013), and 63 routine nasopharyngeal carcinoma are through two Pathologis determination pathologic features and histological grade.

2RNA extracts

2.1miRNA extracts:

The process of removing Rnase of related experiment article:

1. invade bubble, 120 DEG C of high pressure 20min by all rinsing with DEPC before all glass drying oven application, 180 DEG C of high temperature dry more than 2 hours.

2. need before being used by plastic ware (as: EP pipe/rifle head) to spend the night with 0.1%DEPC water enchroachment (invasion) bubble, rear control dry liquids, 120 DEG C of high pressure 20min, baking box is dried for subsequent use.

(1) from liquid nitrogen, take out frozen tumor tissues, weigh, put into centrifuge tube, add Trizol by the amount of 50-100mg tissue/mlTrizol, tissue volume can not more than 10% of Trizol volume, and abundant homogenate is about 1-2min;

(2) after tissue adds Trizol, hatch 5min for 15-30 DEG C, make its abundant cracking;

(3) add the miRNA homogenate additive of 1/10 volume, mix homogeneously under whirlpool number, place 10 minutes on ice;

(4) in lysate, add the chloroform of same volume, whirlpool 30-60s mixes;

(5) centrifugal 5 minutes of room temperature maximum (top) speed (10000g), makes aqueous phase organic facies be separated, and mesophase spherule is separated out; If mesophase spherule is not separated out, recentrifuge;

(6) careful upper strata aqueous phase of drawing is in new collecting pipe, record aqueous phase volume;

(7) in collecting pipe, the dehydrated alcohol of 1/3 volume is added, whirlpool or put upside down several lower mixing;

(8) lysate/alcohol mixeding liquid is added filtration core to filter, filtration core puts into new collecting pipe, each sample filtration core;

(9) with pipet, the mixed liquor in upper step is moved into filtration core, once can receiving volume 700 μ l.Continue again to filter with same filtration core more than 700 μ l;

(10) the centrifugal 15s of 10000g makes liquid pass through filter element;

(11) collect filtrate, if lysate/ethanol contend is more than 700 μ l, with new collecting pipe when continuing to filter, until all lysates/alcohol mixeding liquid filters complete, collect filtrate, recording volume;

(12) the room temperature dehydrated alcohol of 2/3 volume is added in the filtrate collected in previous step;

(13) added in second filtration core by filtrate/alcohol mixeding liquid and filter, discard filtrate, each sample filtration core, puts into the collecting pipe provided by filtration core;

(14) with pipet by upper step mixed liquor move into filtration core, once can receiving volume 700 μ l time.Continue again to filter with same filtration core more than 700 μ l;

(15) the centrifugal 15s of 10000g makes liquid pass through filter element;

(16) discard the liquid filtered out, stay filtration core for next step eluting;

(17) in filtration core, add the miRNA washing liquid 1 (adding ethanol in working solution) of 700 μ l, centrifugal 5-10s, discards the liquid eluted, and collecting pipe continues to use;

(18) in filtration core, add the miRNA washing liquid 2/3 (adding ethanol in working solution) of 500 μ l, centrifugal 5-10s, discards the liquid eluted;

(19) previous step is repeated;

(20) filtration core is put into new collecting pipe (test kit provides).Add the eluent of 100 μ l95 DEG C preheatings to filtration core center or do not contain the water of nuclease, the centrifugal 20-30s of maximum (top) speed collects RNA lysate.

2.2mRNA extract

See embodiment 1.

3 reverse transcription synthesis cDNA

3.1miRNA reverse transcription

The preparation of RT system: 5 × miScriptHiSpecBuffer4 μ l; 10 × NucleicsMix2 μ l; MiScriptReverseTranscriptaseMix2 μ l; RNA1 μ g; Nuclease-freeH ₂o fills to 20 μ l.After in ABI9700 type PCR instrument, 37 DEG C of insulation 60min make reverse transcription reaction completely, 95 DEG C of 5min cessation reactions.Add 80 μ lNuclease-freeH ₂o is diluted to 100 μ l and is stored in-20 DEG C of refrigerators.

3.2mRNA reverse transcription

Adopt iIIReverseTranscriptase (invitrogen, article No. 18080-044) carries out cDNA reverse transcription, and experimental implementation is undertaken by product description, and concrete operations are as follows:

Use Reverse Transcriptase kit, with RT Buffer, converse record synthesis cDNA is carried out to l μ g total serum IgE.Adopt 25 μ l reaction systems, each sample gets 1 μ g total serum IgE as template ribonucleic acid, in PCR pipe, add following component respectively:

5 × RT Buffer 5 μ l, 10mmol/ldNTP1.25 μ l, 0.1mmol/lDTT2.5 μ l, 30 μm of mol/lOligodT2 μ l, 200U/ μ lMMLV1.25 μ l, template ribonucleic acid 1 μ g, add aquesterilisa to total system 25 μ l.Hatch 1 hour for 42 DEG C, 72 DEG C 10 minutes, of short duration centrifugal.It is for subsequent use that-20 DEG C of refrigerators are put in cDNA preservation.

4 quantitative fluorescent PCRs

The RT-PCR system of 4.1miRNA:

The detection of expression of miRNAs arranges 3 parallel pipe reactions, using snRNAU6 as internal reference at every turn.

Amplification program: 95 DEG C of 10min; 40 circulations (95 DEG C of 10s, 60 DEG C of 30s).

The RT-PCR system of 4.2mRNA:

The detection of expression of mRNAs arranges 3 parallel pipe reactions, using β-actin as internal reference at every turn.

Reaction system: use Power greenPCRMasterMix (invitrogen, article No. 4367659) increases, and experimental implementation is undertaken by product description.Amplification program is: 95 ° of 10min, (95 DEG C of 15sec, 60 DEG C of 60sec) × 45 circulations.

5 statistical analysis

Real-time quantitative PCR amplification curve flex point is clear, and the overall collimation of amplification curve is good, shows that the amplification efficiency of each reaction tube is close, and the limit is flat and without raising up now, exponent phase slope is comparatively large, illustrates that amplification efficiency is higher; Sample amplified production solubility curve is all unimodal, illustrates that amplified production only has one, is specific amplification; OriginPro8.1 software is adopted to analyze.Compare between statistical method mean and adopt t inspection, P<0.05 (significant difference) and P<0.01 (difference highly significant) is decided to be statistical significance, analyze miR-632 expression in cancerous tissue and cancer beside organism, in result display cancerous tissue, the expression of miR-632 is apparently higher than cancer beside organism, and the former is nearly 4 times (specifically seeing Fig. 1) of the latter; In tissues of nasopharyngeal carcinoma, the expression of VPS11 is starkly lower than cancer beside organism, and the former is about 1/3rd (specifically the seeing Fig. 2) of the latter.

Embodiment 4miR-632 and VPS11 regulation relationship are verified

Material prepares:

Human nasopharyngeal carcinoma cell line CNE is purchased from Chinese Academy of Sciences's cell bank/Shanghai Inst. of Life Science, CAS cellular resources center.

Design of primers:

VPS11 amplimer:

Forward primer: 5 '-CTCCTCAACTGCTATACC-3 ' SEQIDNO4

Reverse primer: 5 '-GACTTCACTCTCACTCTT-3 ' SEQIDNO5

Amplified production length 78bp.

1. cell culture

Human nasopharyngeal epithelioma 1 adopts containing 10% hyclone and the dual anti-RPMI1640 culture medium of 1% mycillin, at 37 DEG C, and 5%CO ₂, saturated humidity condition under Secondary Culture, when cell growth state is good be used for RNA extracting or next step experiment.

2.miRNA transient transfection

Adopt cationic-liposome method to carry out transient transfection, operate according to Lipofectamin ^tM2000 reagent description are carried out.Before transfection, 5-8F and 6-10B cell good for growth conditions is inoculated in 12 orifice plates by 24h, cell counting about 2 × 10 ⁴, cellar culture was to transfection same day, and cell fusion degree is that 50-60% tests.20nM/40nM/80nMmiRNAmimic is joined in 100u1Opti-MEM culture medium, softly mix; Another 100u1Opti-MEM culture medium dilution 2u1Lipofectamin ^tM2000 liposomees, softly mix, incubated at room 5min; Mixing Opti-MEM-liposome and Opti-MEM-miRNAs, incubated at room 20min, to form transfection composite; Then said mixture is added in cell culture medium, mixes gently, after cultivating 6h, change complete medium.Wherein, nonspecific miR sequence is as negative control.Extract cell total rna after cultivating 48h or carry out next step experiment.

3. experimental result:

By miR-632mimics transfection in human nasopharyngeal carcinoma cell line, extract cell total rna after 48h, blank is the human nasopharyngeal carcinoma cell line cell not proceeding to miRNA, and nonspecific miRNA sequence is as negative control.The level change of the mRNA of quantitative PCR detection prediction target gene VPS11.Result shows experimental group VPS11 gene expression dose compared with the control and lowers about 35%, and experimental result shows that VPS11 is the target gene of miR-632, and in human nasopharyngeal epithelioma 1, the rise of miR-632 causes the downward of VPS11 gene.

Although describe the present invention with reference to various preferred embodiment, it will be appreciated by those skilled in the art that and can carry out various change, and available equivalents substitutes its assembly and do not deviate from elemental range of the present invention.In addition, many changes can be carried out and not deviate from its elemental range to make particular case or material be suitable for instruction of the present invention.

Therefore, the present invention is not intended to be defined in disclosed herein for carrying out particular of the present invention; On the contrary, this invention is intended to comprise all embodiments dropped in Claims scope.

Claims (10)

1.VPS11 gene and/or the application of VPS11 albumen in preparation treatment or diagnosis of nasopharyngeal carcinoma reagent.

2. application according to claim 1, is characterized in that, treatment nasopharyngeal carcinoma reagent refers to the reagent of the expression that can promote VPS11 gene, preferably, containing the carrier promoting VPS11 gene expression in treatment nasopharyngeal carcinoma reagent.

3. application according to claim 1, is characterized in that, the expression that diagnosis of nasopharyngeal carcinoma reagent comprises fluorescence quantifying PCR method, method for gene chip detects VPS11 gene in nasopharyngeal carcinoma; Or the expression of VPS11 albumen is detected with immunization method.

4. application according to claim 3, is characterized in that, fluorescence quantifying PCR method diagnosis of nasopharyngeal carcinoma comprises a pair Auele Specific Primer, and forward primer sequence is SEQIDNO.4, and downstream primer sequence is SEQIDNO.5.

The application of regulation and control miRNA in preparation diagnosis and/or treatment nasopharyngeal carcinoma reagent of 5.VPS11 gene and/or albumen, it is characterized in that, described regulation and control miRNA is mir-632 and/or miR-632.

6. application according to claim 5, it is characterized in that, diagnosis of nasopharyngeal carcinoma reagent comprises based on high-flux sequence method and/or based on quantifying PCR method and/or the expression transcribing or detect based on immunologic detection method the target gene of miR-632 regulation and control in nasopharyngeal carcinoma sample detecting mir-632 and/or miR-632 in nasopharyngeal carcinoma sample based on probing procedure, preferred employing northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, based on transcribing of mir-632 and/or miR-632 in the Flow cytometry nasopharyngeal carcinoma sample of microsphere, ELISA and/or colloidal gold strip is adopted to detect the expression of the target gene of miR-632 regulation and control in nasopharyngeal carcinoma sample.

7. application according to claim 5, is characterized in that, treatment nasopharyngeal carcinoma reagent is the reagent transcribing and/or block the activity of miR-632 lowering mir-632 and/or miR-632.

8. application according to claim 7, it is characterized in that, adopt antisense oligonucleotide, antagomiRs, miRNA sponge, miRNAErasers, TargetMasking and/or Mutiple Targets antisense oligonucleotide method lower the activity transcribing and/or block miR-632 of mir-632 and/or miR-632.

9. treat a medicine for nasopharyngeal compositions, pharmaceutical composition comprises:

A () promotes the preparation of VPS11 gene expression;

Receptible carrier on (b) pharmaceutics.

10. pharmaceutical composition according to claim 9, its feature exists, and promotes that the preparation of VPS11 gene expression is the preparation transcribing and/or block miR-632 activity suppressing mir-632 and/or miR-632.