CN105343896B - The new diagnosis and treatment target spot of nasopharyngeal carcinoma and its application - Google Patents

Abstract

The present invention relates to the new diagnosis and treatment target spot of nasopharyngeal carcinoma and its application, more particularly relates to VPS11 genes and its regulate and control purposes of the miRNA in diagnosis and treatment nasopharyngeal carcinoma.Invention analyzes tissues of nasopharyngeal carcinoma and cancer beside organism by high-flux sequence, obtain its mRNA and miRNA transcription group informations, go out to choose VPS11 genes through Analysis and Screening and miR 632 carries out quantitative fluorescent PCR verification and target checking, as a result it shows, VPS11 genes and miR 632 and nasopharyngeal carcinoma are closely related, and VPS11 is the target gene of miR 632 in nasopharyngeal carcinoma.The present invention provides new target spot for clinical diagnosis and prevention detection nasopharyngeal carcinoma, has good application value.

Description

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

Technical field

The present invention relates to molecular biology fields, are specifically related to new diagnosis and treatment target spot and its application of nasopharyngeal carcinoma, more specifically Be related to VPS11 genes and its regulate and control purposes of the miRNA in diagnosis and treatment nasopharyngeal carcinoma.

Background technology

Nasopharyngeal carcinoma (Nasopharyngeal carcinoma, NPC) be the common head-neck malignant tumor of south China it One, wherein male's incidence is approximately twice of women, with 40-50 Sui for the disease high-incidence season.Nasopharyngeal carcinoma is fallen ill (to be lost with inherent cause Pass neurological susceptibility), ebv infection, environmental factor, many factors such as eating habit it is related, early diagnosis, early treatment are to save Patient vitals and the most effective means improved the quality of living.Regrettably, nasopharyngeal carcinoma onset is hidden, and there is strong transfer to incline To.In addition, poorly differentiated squamous cell carcinoma is the most commonly seen histological type of nasopharyngeal carcinoma, and still, five year survival rate after radiation alone Only 50% or so, the main reason for being recurred after treatment or transfer prognosis be very poor, become treatment of nasopharyngeal carcinoma failure.Therefore, it identifies With identification and the relevant tumor marker of nasopharyngeal carcinoma, strive for that early detection, selection therapeutic regimen, prediction prognosis, monitoring are multiple Hair is shifted to nasopharyngeal carcinoma diagnosis and treatment with important clinical meaning.

MiRNA is the endogenic non-coding RNA with adjusting function of one kind found in eucaryote.Usually by Rna plymerase ii (Pol II) transcription generates.There are mainly two types of the typical effect modes of miRNA and said target mrna.In most of feelings Under condition, the not fully complementary pairings of 3 ' UTR of single-stranded miRNA and said target mrna in compound block the translation of target gene, to adjust Save gene expression.This mode mainly influences protein expression level, has no effect on the stability of mRNA.Another mode of action with SiRNA is similar, and when miRNA and mRNA complete complementaries match clock synchronization, Ago2 albumen directly results in its degradation by cutting mRNA, realizes Gene silencing.By taking the RNAi that siRNA is participated in as an example：SiRNA can be combined with RISC, identified mRNA targets as template, passed through alkali Base complementary pairing principle, mRNA are combined with the antisense strand in siRNA, displace positive-sense strand.Double-strand mRNA Dicer enzymes, ATP and The siRNA of 22nt or so is generated under unwindase collective effect, siRNA continues to form complex with RISC, with siRNA complementations MRNA is combined, and mRNA is made to be cracked by RNA enzyme.This process is also referred to as posttranscriptional gene silencing (PTGS).

In short, being presently believed to pairing degree of the miRNA in which way with target gene effect and miRNA and target gene It is related.When miRNA matches incomplete with target gene, miRNA is just to inhibit the expression of target gene to play a role；MiRNA with When the pairing of target gene section sequence is complete, it is possible to target gene be caused to be broken in complementary region and lead to gene silencing.In addition, MiRNAs sometimes also leads to the DNA methylation of histidine modification and promoter region, to influence the expression of target gene.Except this Outside, it has recently found that quickly taking off the new machine that polyadenylation (accelerated deadenylation) is miRNA inhibition of gene expression System.Find that miR-125b and let-7 can promote mRNA poly A tails (polyA tail) in mammalian cell Removal.Replace poly A tail with 3 ' histone stem-loop structures, can not only eliminate shadows of the miR-125b to mRNA contents It rings, effect synthesize to protein can also be reduced, it is seen that miRNA can pass through reduction translation efficiency and polyadenylation mRNA Concentration carrys out inhibition of gene expression.

Invention analyzes tissues of nasopharyngeal carcinoma and cancer beside organism by high-flux sequence, obtains itself mRNA and miRNA transcript profile letter Breath goes out to choose VPS11 genes through Analysis and Screening and miR-632 carries out quantitative fluorescent PCR verification and target checking, the results show that VPS11 genes and miR-632 and nasopharyngeal carcinoma are closely related, and VPS11 is the target gene of miR-632 in nasopharyngeal carcinoma.The present invention is Clinical diagnosis and prevention detection nasopharyngeal carcinoma provide new target spot, have good application value.

Invention content

The purpose of the present invention is to provide VPS11 genes and/or albumen in preparing treatment or diagnosis of nasopharyngeal carcinoma reagent Using.

The purpose of the present invention is to provide the preparations of regulation and control VPS11 genes and/or protein expression to prepare treatment nasopharyngeal carcinoma Application in reagent.

Further, the treatment nasopharyngeal carcinoma reagent refers to the reagent for the expression that can promote VPS11 genes.Those skilled in the art It is known to promote the expression of gene that one kind in following methods and/or several usually may be used：Regulate and control VPS11 by DNA level Gene：Including but not limited to increase the copy number of VPS11 genes, transfect the over-express vector of the gene containing VPS11；By transcribing water Heibei provincial opera control VPS11 genes：The including but not limited to startup of expression, the activation regulation and control VPS11 gene expressions of activation VPS11 genes Son, the transcription factor for inhibiting negative regulation VPS11 gene expressions, the inhibition using RNA perturbation techniques to inhibition VPS11 gene expressions Son is interfered；Regulate and control VPS11 genes by post-transcriptional level：Including but not limited to inhibit to promote the degradation of VPS11 gene mRNAs MicroRNA transcriptional expressions, import promote VPS11 gene expressions microRNA；Pass through level modulation VPS11 bases after translation Cause：Including but not limited to import promote VPS11 gene coded proteins molecule, inhibit negative regulation VPS11 gene expressions albumen, Promote the factor of VPS11 gene expressions and the expression of albumen.

Preferably, the carrier for promoting VPS11 gene expressions is contained in the treatment nasopharyngeal carcinoma reagent.

The purpose of the present invention is to provide the preparations of detection VPS11 genes and/or albumen to prepare diagnosis of nasopharyngeal carcinoma reagent In application.

Further, the diagnosis of nasopharyngeal carcinoma reagent includes detecting nasopharyngeal carcinoma with fluorescence quantifying PCR method, method for gene chip The expression of middle VPS11 genes.

The product for VPS11 genes in fluorescence quantifying PCR method detection nasopharyngeal carcinoma contains a pair of of specificity and expands Increase the primer of VPS11 genes；The genetic chip includes the probe with the nucleic acid array hybridizing of VPS11 genes.

Further, the diagnostic preparation of the nasopharyngeal carcinoma includes the expression that VPS11 albumen is detected with immunization method.It is preferred that institute It is western blot and/or ELISA and the inspection of/colloidal gold to state VPS11 protein expressions in immunologic detection method detection nasopharyngeal carcinoma Survey method.

Further, the ELISA method of the detection VPS11 albumen is to use ELISA detection kit.In the kit Commercially available VPS11 monoclonal antibodies can be used in antibody.Further, the kit includes：It is coated with VPS11 monoclonal antibodies Solid phase carrier, ELIAS secondary antibody, the substrate of enzyme, protein standard substance, negative controls, dilution, cleaning solution, enzyme reaction terminate liquid Deng.

Further, the colloidal gold method of the detection VPS11 albumen is using detection kit, and city can be used in the antibody The VPS11 monoclonal antibodies sold.Further, the gold-immunochromatographyreagent reagent for assay box uses colloidal gold immunochromatographimethod technology or colloid Golden percolation.Further, detection zone (T) specking on the gold-immunochromatographyreagent reagent for assay box nitrocellulose filter has anti-VPS11 Monoclonal antibody, quality control region (C) specking have Immunoglobulin IgG.

The purpose of the present invention is to provide a kind of PCR kit for fluorescence quantitative of detection nasopharyngeal carcinoma, which is characterized in that described Kit detects gene VPS11, and using special sense primer and downstream primer, upstream primer sequence is SEQ ID NO.4, under Trip primer sequence is SEQ ID NO.5.

Further, which is suitable for presently, there are all types fluorescence quantitative gene extender in the market, clever Sensitivity is high, it is quantitative quick and precisely, stability it is good, have a good application prospect.

Further, above-mentioned PCR kit for fluorescence quantitative component includes：Specific primer, internal control primer, quantitative fluorescent PCR Reaction solution.Wherein the specific primer includes sense primer and downstream primer, and upstream primer sequence is SEQ ID NO.4, Downstream primer sequence is SEQ ID NO.5.The internal control primer is β-actin internal control primers.

The purpose of the present invention is to provide the regulation and control miRNA of VPS11 genes and/or albumen prepare prevent, diagnosis and/or Treat the application in nasopharyngeal carcinoma reagent.

The purpose of the present invention is to provide the regulation and control miRNA preparations of detection VPS11 genes and/or albumen to prepare diagnosis nose Application in pharynx cancer reagent.

The purpose of the present invention is to provide the regulation and control miRNA reagents for adjusting VPS11 genes and/or albumen to prepare treatment nose Application in pharynx cancer reagent.

The regulation and control miRNA is mir-632 and/or miR-632.The sequence of mir-632 is shown in sequence table SEQ ID NO 1； The ripe miRNA of mir-632 is that miR-632 sequences are shown in sequence table SEQ ID NO 2.

Further, the prevention, diagnosis of nasopharyngeal carcinoma reagent include based on high-flux sequence method and/or based on quantitative PCR method and/or the transcription of mir-632 and/or miR-632 is detected in nasopharyngeal carcinoma sample based on probing procedure or based on exempting from Epidemic disease detection method detects the expression for the target gene that miR-632 regulates and controls in nasopharyngeal carcinoma sample, it is preferred to use northern hybridizes Method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE methods, in situ hybridization, bead-based flow-cytometry inspection Survey the transcription of mir-632 and/or miR-632 in nasopharyngeal carcinoma sample；Nasopharyngeal carcinoma is detected using ELISA and/or colloidal gold strip The expression for the target gene that miR-632 regulates and controls in sample.It is preferred that the target gene of the 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, preferred miR- The target gene of 632 regulation and control is VPS11.

Preferably, it is described based on quantifying PCR method include specific amplification mir-632 and/or miR-632 primer, Further preferably, specific amplification miR-632 primer sequences are SEQ ID NO 3；Described includes based on probing procedure With the probe of the nucleic acid array hybridizing of mir-632 and/or miR-632；The immunologic detection method includes regulating and controlling base with miR-632 The antibody combined by expression protein-specific.

Further, the reagent of the treatment nasopharyngeal carcinoma is the transcription and/or blocking for lowering mir-632 and/or miR-632 The active reagent of miR-632.

Preferably, using antisense oligonucleotides, antagomiRs, miRNA sponge, miRNA Erasers, Target The method of Masking and/or multiple target point antisense oligonucleotides lowers the transcription and/or blocking of mir-632 and/or miR-632 The activity of miR-632.

The present invention also aims to provide a kind of pharmaceutical composition for treating nasopharyngeal carcinoma, including：

(a) promote the preparation of VPS11 gene expressions；

(b) receptible carrier in pharmacy.

It is transcription and/or the blocking miR- for inhibiting mir-632 and/or miR-632 to promote the preparation of VPS11 gene expressions 632 active preparations.

Preferably, using antisense oligonucleotides, antagomiRs, miRNA sponge, miRNA Erasers, Target The method of Masking and/or multiple target point antisense oligonucleotides lowers the transcription and/or blocking of mir-632 and/or miR-632 The activity of miR-632.

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

Preferably, the nasopharyngeal carcinoma diagnosis reagent be based on high-flux sequence method and/or based on quantifying PCR method and/or The transcription of mir-632 and/or miR-632 in adenocarcinoma of lung sample is detected based on probing procedure or is based on immunization method detection sample The expression for the target gene that miR-632 regulates and controls in this, it is preferred to use northern hybridizing methods, miRNA chip of expression spectrum, core Mir-632 and/or miR- in enzyme protection analytical technology, RAKE methods, in situ hybridization, bead-based flow-cytometry detection sample 632 transcription；Using the expression of the target genes regulated and controled of miR-632 in ELISA and/or colloidal gold strip detection sample.

It is furthermore preferred that described includes drawing for specific amplification mir-632 and/or miR-632 for quantifying PCR method Object；Described based on probing procedure includes probe with the nucleic acid array hybridizing of mir-632 and/or miR-632；It is described to exempt from Epidemic disease detection method includes the antibody combined with miR-632 controlling genes expression protein-specific.Definition：

The method of the expression of detection miRNA includes mainly based on high throughput sequencing technologies, is based on nucleotide at this stage The miRNA detection methods of hybridization and based on PCR.MiRNA detection methods based on probe hybridization technique are a kind of direct Detection Methods, Sample rna need not be expanded in advance, including northern hybridizing methods, miRNA chip of expression spectrum, ribozyme protection analysis skill The technologies such as art, RAKE methods, in situ hybridization, bead-based flow-cytometry.

(1) Northern hybridizes

Also known as Northern blot is most classical detection eucaryote RNA sizes, estimates the experimental method of its abundance.Base Present principles are as follows：MiRNA samples are fixed on carrier (such as silicon chip, microballoon or film) first, then miscellaneous with the probe by label It hands over, signal detection is carried out after washing extra hybridization probe；It can also be first fixed on carrier and target miRNA sequence complementation Then DNA probe hybridizes with the sample miRNA by label, then carries out signal detection.The method of signal label includes isotope Label, fluorescent marker and nano gold mark etc..

(2) miRNA chip of expression spectrum

Principle is equally using the target molecule on label probe detection solid support.Pass through miRNA in design chips Gene and internalcontrol sequence, can Accurate Analysis go out the expression of corresponding miRNA in sample.Genetic chip has the excellent of high throughput Point can once detect whole expression of hundreds of genes in same sample.The liquid-phase chip that Luminex companies develop (Liquid chip) is also known as multifunctional suspending dot matrix (Multi analyte suspension array, MASA), be it is new Generation biochip technology.Liquid-phase chip system is made of many spherulas for main matrix, is fixed with not on each spherula Same probe molecule, in order to distinguish different probes, each is used for the sphere matrix of label probe all with there are one unique Color numbers constitute liquid-phase chip system as soon as these spherulas are suspended in a liquid-phase system.The system can be to same Multiple and different molecules in one trace sample are carried out at the same time quick qualitative and quantitative analysis, and this detection technique is referred to as FMAP (Flexible multianalyte profiling) technology.Molecule hybridization carries out in aaerosol solution, detection speed pole Soon.

(3) ribozyme protection analytical technology (RPA)

The detection of miRNA can also use ribozyme to protect analytical technology, and the probe marked and RNA samples to be measured are mixed Close, hybridize after thermal denaturation, non-hybridized RNA and the single-chain nucleic acid enzymic digestion of extra probe, after heat inactivation nuclease purifying by Probe, colour developing is separated by electrophoresis finally by denaturation PAGE in the RNA molecule of protection.This new method based on solution hybridization is simple Quickly, high sensitivity, but be also only used for analyzing known miRNA.

(4) RAKE methods

RAKE methods (RNA primed array based Klenow emzyme) are the bases in miRNA microarray The Klenow segments of DNA polymerase i, the method for making miRNA hybridize with fixed DNA probe are utilized on plinth.RAKE can be sensitive MiRNA is specifically detected, suitable for largely quickly screening all known miRNA.It can be in specific cell and tumour Detect miRNA express spectra situations.Moreover, RAKE methods can also be from the tissue of the paraffin embedding secured by formalin It isolates miRNA and analyzes it, to analyze the door that miRNA opens hope from archive sample.

(5) in situ hybridization (in situ hybridization)

Hybridization in situ technique can intuitively understand miRNA expression ways, be a kind of easier of observation miRNA spatial and temporal expressions Method, normal mark mode include digoxin, biotin, fluorescent marker etc..In situ hybridization (Locked on the basis of locked nucleic acid Nucleic Acid (LNA) based in situ hybridization (LNA-ISH)) it is the more probe side of current application Formula.

(6) bead-based flow-cytometry (bead-based flow cytometry)

It is a kind of liquid-phase chip technology, FCM analysis is organically combined with chip technology, had concurrently by this method The features such as flux is big, detection speed is fast, high sensitivity and specificity are good.

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

Fluoroscopic examination PCR instrument can draw dynamic changing curve to the cumulative speed of extension increasing sequence during entire PCR.Anti- Answer the initial concentration of target sequence in mixed system bigger, it is desirable that the PCR cycle number for obtaining amplified production specific output is (general to use Specific threshold recurring number Ct is expressed) it is fewer.Since miRNA length is only 22nt, it is such that traditional qRT-PCR is not suitable for amplification Short segment.There are several real time quantitative PCR methods for miRNA, such as tailing method, neck ring method now.Neck ring method is a kind of Ideal miRNA detects qRT-PCR methods：Special loop-stem structure primer is designed first, using miRNA to be measured as template reverse transcription The first chains of cDNA are synthesized, which is stem Loop primer, and stem loop structure, which is opened, substantially increases the length of cDNA, then Real-time quantitative PCR amplification is carried out by template design primer of the cDNA of synthesis.QRT-PCR has specificity height, sensitivity good, fast A variety of advantages such as fast simple.

(8) PCR sequencing PCR

MiRNA known to major part is to be sequenced to find and identify by cDNA clone.The method needs first to build miRNA CDNA library, then carry out PCR amplification, amplified production is then cloned on expression vector and is sequenced.Takada develops one kind and changes Into amplification PCR cloning PCR (miRNA amplification profiling, mRAP), mRAP methods first connect at the 3 ' ends of miRNA Connector, then with the reverse transcription primer reverse transcription with connector complementation.Because specific reverse transcriptase has end deoxynucleotide Transferase active, some nucleotide (mainly deoxycytidylic acid) can be connected to 3 ' ends of the cDNA chains that reverse transcription goes out.When 5 ' End connector is added a pair of of general primer and can be realized and expand the PCR of cDNA with after poly (C) cohesive end annealing of cDNA chains Increase.Due to mRAP High sensitivities, the expression quantity of miRNA in clone and a small amount of tissue of sequencing technologies detection can be directly used.Label Sequence PCR cloning PCR is a kind of to have developed detection efficiency on the basis of serial analysis of gene expression (SAGE) technology higher MiRAGE (miRNA SAGE) PCR cloning PCR, the method can detect multiple by generating big sub-series by single sequencing reaction MiRNA, hence it is evident that improve detection efficiency.

High-flux sequence (High-throughput sequencing) is also known as next-generation sequencing technologies (next Generation sequencing) it is the change for tradition being sequenced revolution, once to hundreds of thousands to millions of DNA Molecule carries out sequencing, greatly improves sequencing efficiency.This kind of large scale sequencing technology greatly improves multiple species and loses The solution reading rate of communication breath, to obtain the sequence information of all miRNA, decryption miRNA collection of illustrative plates provides guarantee.It is high-throughput simultaneously Sequencing to carry out the analysis of careful overall picture to the transcript profile and genome of species, so the depth that is otherwise known as It is sequenced (deep sequencing).The representative of high-flux sequence platform is 454 sequenator (Roch of Roche Holding Ag (Roche) GSFLX sequencer), the Solexa genome analysis instrument (Illumina Genome Analyzer) of Illumina companies and The SOLiD sequenators (ABI SOLiD sequencer) of ABI.

Immunologic detection method is carried out to determinand quantitative or qualitative using a kind of antibody or Multiple Antibodies as analytical reagent The detection method of analysis.Its basic principle is the interaction between antibody and antigen.To improve the quick of antigen and antibody test Perception, the substance that will easily be shown in known antibodies or antigenic mark, by detecting marker, reflection whether there is or not antigen-antibody reaction, To measure micro antigen or antibody indirectly.Common marker has enzyme, fluorescein, radioactive isotope, colloidal gold and electricity Sub- dense matter etc..Show that the specific reaction that object is carried out is known as immunolabelling technique on this antigen or antibody label (immunolabelling technique).Immunoassay technology most widely used at present mainly has：Enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA), colloidal gold immunity chromatography etc..

Enzyme-linked immunosorbent assay principle is to combine antigen or antibody with substrate (enzyme), it is made to keep immune response and enzyme Activity.The antigen or antibody of label and the ligand binding being coated on solid phase carrier, then it is allowed to and corresponding colorless substrate It acts on and display color, according to the range estimation of colour developing depth degree or measures OD values with microplate reader and judge result.

Colloidal gold strip is generally made of sample pad, gold-labelled pad, chromatographic film, four part of water absorption pad.Chromatographic material has nitre Change tunica fibrosa (NC), polyester film, nylon membrane and pvdf membrane etc., the film that different requirements may be selected, wherein NC films are needed according to experiment It is the most commonly used, it can determine the need for activating according to experiment concrete condition before or handle, be not necessarily to processing in most cases, i.e., It can be used directly.By gold mark protein solution even application in gold-labelled pad, dry at room temperature spare.NC films can capture a certain amount of Coating (antibody) and secondary antibody as detection line and nature controlling line.Finally sample pad, gold-labelled pad, NC films and blotting paper are fixed successively In PVC board, test strips.

The acquired technology of microRNA functions based on RNA be the precursor substance that is synthesized by exogenous supplement miRNAs come Increase the level of miRNAs.For example, can the artificial synthesized bob folder sample RNA (short consistent with endogenous miRNA sequence Hairpin RNA, shRNA), promoter is done by polymerase II or III, with virus for carrier transfectional cell, by Dicer enzyme modifications It is loaded into RISC afterwards to play a role, is equivalent to the level for increasing pre-miRNA, function and effect are stable and lasting.

This technique avoids the nonspecific actions of miRNA and gene for gene specific miR Mimics technologies.This people The specific oligonucleotide chain of work synthesis combined with 3 ' UTR complementations of target gene, is adjusted after capable of playing transcription identical with miRNA Section acts on.

The carrier permitted in the pharmacy of the pharmaceutical composition of the present invention is the load usually utilized in preparation Body, which includes lactose (lactose), dextrose (dextrose), sucrose (sucrose), sorbierite (sorbitol), sweet Reveal alcohol (mannitol), starch, acacia gum, calcium phosphate, alginates (alginate), gel (gelatin), calcium silicates, Microcrystalline cellulose, polyvinylpyrrolidone (polyvinylpyrrolidone), cellulose (cellulose), water, syrup, first Base cellulose (methyl cellulose), methyl hydroxybenzoate (methyl hydroxybenzoate), propyl hydroxy benzene Propyl formate (propyl hydroxybenzoate), talcum, magnesium stearate (stearic acid magnesium) and mineral Oily (mineral oil) etc., but it is not limited to this.

The pharmaceutical composition of the present invention can also include lubricant, wetting agent, sweetener, perfume (or spice) other than mentioned component Taste agent, emulsifier, suspending agent, preservative etc..The suitable carrier and preparation permitted in pharmacy is recorded in Lei Mingdengshi in detail Pharmacy pandect.

The pharmaceutical composition of the present invention can by it is oral or it is non-oral be administered, when as non-oral administration, can lead to Cross intravenous injection, intranasal injection, local injection, intraventricular injection, spinal cavity injection is subcutaneously injected, intraperitoneal injection, percutaneously The modes such as administration are administered.

The present invention pharmaceutical composition suitable dosage according to preparation ways, administering mode, patient year The factor of age, weight, gender, morbid state, food, administration time, administration route, drainage rate and draw property etc and can be with Carry out a variety of prescriptions, in general, skilled practitioner can be easily determined by and prescription to it is desirable treatment or prevention effectively to Pharmaceutical quantities.

The pharmaceutical composition of the present invention can be easy to implement according to general technical staff of the technical field of the invention Method, carried out using receptible carrier in pharmacy and/or excipient it is formulation, so as in the form of unit dose It prepares or interior prepares in the multicapacity container.At this point, dosage form be solution in oiliness or aqueous medium, suspension or Emulsion form can also be either extract, powder agent, granule, tablet or capsule form, can also include dispersion Agent or stabilizer.

Description of the drawings

Fig. 1 RT-PCR detect tissues of nasopharyngeal carcinoma miR-632 expressions

Fig. 2 RT-PCR detect tissues of nasopharyngeal carcinoma VPS11 gene expression doses

Specific implementation mode

Present invention will be further explained below with reference to specific examples, is only used for explaining the present invention, and should not be understood as to this The limitation of invention.It will be understood by those skilled in the art that：Without departing from the principle and spirit of the present invention may be used To carry out a variety of change, modification, replacement and modification to these embodiments, the scope of the present invention is limited by claim and its equivalent It is fixed.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition or according to the item proposed by manufacturer Part examinations.

The collection of 1 sample of embodiment and Total RNAs extraction

Tissue is all from the sample that hospital collects in December, -2013 in March, 2011, and experiment group selection disease inspection is diagnosed as nasopharynx 7 patients of cancer, control group are the cancer beside organism with sample.

Experimental method：

1 extracting method

1) 80mg tissue blocks are taken, 800 μ l Lysis/Binding buffer solutions are added, tissue block are carried out using homogenizer even Slurry.Sample will be placed in and keep low-temperature condition on ice during homogenate.

2) it adds in 1/10 volume Homogenate Additive to the above-mentioned tissue sample being homogenized, puts on ice Set 10min.

3) be added with the water-saturated phenols of Lysis/Binding buffer solution equivalent volumes, shake 45s, 10,000 × g room temperatures from Heart 5min.

4) it carefully takes out in supernatant to new test tube, the absolute ethyl alcohol of 1.25 times of volumes of addition, after mixing, moves into purification column In, 10,000 × g centrifuges 15s, outwells the liquid in collecting pipe.Since the maximum volume of pillar only has 700 μ l, again This multiple step operation, until all supernatants all filter completion.

5) 700 μ l miRNA eluents 1, room temperature are added into centrifugation pillar, 10,000 × g centrifuges 15s, outwells collection Liquid uses new collecting pipe instead.

6) the step for being added in centrifugal column with 500 μ l eluents 2/3 again, 10,000 × g, centrifuging 10s, repeat is primary.

7) 1min, 10,000 × g are centrifuged, extra liquid is discarded.

8) aforesaid liquid is transferred to new centrifuge tube, adds DEPC processing 30s, 10, the 000 × g of 100 μ l, 95 DEG C of preheatings, Centrifugation.

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

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

2 extraction standards

Measure the ratio of RNA concentration and 260nm/280nm：The purity requirement of total serum IgE is that OD260/OD280 values should be 1.8 To between 2.2；The detection of RNA integralities：The integrality of RNA is detected with 1% agarose gel electrophoresis；

According to the requirement of sequencing company, 3 μ g of total amount or more are sequenced in tiny RNA, and concentration is more than 300ng/ μ l.

Embodiment 2 is sequenced and data analysis

Foundation and the sequencing of upper machine of sequencing library are carried out by sequencing company, used sequenator is Illumina companies HiSeq2000 sequenators.

The data results provided according to company：Group carries out statistical analysis by 7 nasopharyngeal carcinoma groups and cancer, and P values are less than 0.05, and tumor group and the difference of control group by cancer at least will be more than the differential expression miRNA of 2 times or more, to differential expression MiRNA people is to select the apparent VPS11 genes of downward expression and the apparent has-mir-632 of up-regulated expression in filtering to enter us Research range, by software prediction has-mir-632 prediction target gene be VPS11.

1 sample of expression of VPS11 genes and miR-632 are adopted in embodiment 3Real-time PCR detection tissues of nasopharyngeal carcinoma Collection：

63 tissues of nasopharyngeal carcinoma and cancer beside organism are all from hospital's (acquisition time in December, -2013 in March, 2011), 63 Nasopharyngeal carcinoma determines pathologic feature and histological grade through two Pathologis.

2RNA is extracted

2.1miRNA extraction：

The processing for removing Rnase of related experiment article：

1. by bubble, 120 DEG C of high pressure 20min is invaded with DEPC flushings before the application of all glasswares, 180 DEG C of high temperature dry 2 Hour or more.

2. (such as by plastic ware：EP pipes/pipette tips) need before use with 0.1%DEPC water enchroachment (invasion)s bubble overnight, after drain liquid

Body, 120 DEG C of high pressure 20min, oven is dried spare.

(1) it is taken out from liquid nitrogen and freezes tumor tissues, weighed, be put into centrifuge tube, by 50-100mg tissues/mlTrizol Amount be added Trizol, 10% of tissue volume no more than Trizol volumes, fully be homogenized about 1-2min；

(2) after Trizol is added in tissue, 15-30 DEG C of incubation 5min makes it fully crack；

(3) miRNA that 1/10 volume is added is homogenized additive, is uniformly mixed under whirlpool number, places 10 minutes on ice；

(4) chloroform of same volume, whirlpool 30-60s mixings are added into lysate；

(5) room temperature maximum (top) speed (10000g) centrifuges 5 minutes, and the organic phase separation of water phase, interphase is made to be precipitated；Interphase is such as Fruit is not precipitated, and centrifuges again；

(6) it carefully draws in upper strata aqueous phase to new collecting pipe, records water phase volume；

(7) absolute ethyl alcohol of 1/3 volume, whirlpool or reverse several lower mixings are added into collecting pipe；

(8) filtration core filtering is added in lysate/alcohol mixeding liquid, filtration core is put into new collecting pipe, each sample With a filtration core；

(9) mixed liquor in upper step is moved into filtration core with pipette, it once can 700 μ l of receiving volume.More than 700 μ l after It is continuous to be filtered again with same filtration core；

(10) 10000g, which centrifuges 15s, makes liquid pass through filter core；

(11) filtrate is collected, if lysate/ethyl alcohol volume is more than 700 μ l, with new collecting pipe when continuing filtering, until All lysates/alcohol mixeding liquid filtering finishes, and collects filtered fluid, records volume；

(12) one step up in the room temperature absolute ethyl alcohol of 2/3 volume is added in the filtered fluid that is collected into；

(13) filtered fluid/alcohol mixeding liquid is added in second filtration core and is filtered, discard filtered fluid, each sample is with one Filtration core is put into the collecting pipe provided by a filtration core；

(14) with pipette by upper step mixed liquor move into filtration core, once can 700 μ l of receiving volume when.More than 700 μ L continues to be filtered again with same filtration core；

(15) 10000g, which centrifuges 15s, makes liquid pass through filter core；

(16) liquid filtered out is discarded, stays filtration core for eluting in next step；

(17) the miRNA washing lotions 1 (ethyl alcohol is added in working solution) of 700 μ l are added into filtration core, centrifuges 5-10s, discards The liquid eluted, collecting pipe are continuing with；

(18) the miRNA washing lotions 2/3 (ethyl alcohol is added in working solution) of 500 μ l are added into filtration core, centrifuges 5-10s, abandons Remove the liquid eluted；

(19) previous step is repeated；

(20) filtration core is put into new collecting pipe (being provided in kit).100 μ l95 DEG C are added to filtration core center The eluent of preheating or water without nuclease, maximum (top) speed centrifuge 20-30s and collect RNA lysates.

2.2mRNA extraction

Referring to embodiment 1.

3 reverse transcriptions synthesize cDNA

3.1 miRNA reverse transcriptions

The preparation of RT systems：5×miScript HiSpec Buffer 4μl；10×Nucleics Mix 2μl； miScript Reverse Transcriptase Mix 2μl；RNA 1μg；Nuclease-free H₂O filling-in is to 20 μ l. After 37 DEG C of heat preservation 60min keep reverse transcription reaction complete in 9700 type PCR instruments of ABI, 95 DEG C of 5min terminate reaction.80 μ l are added Nuclease-free H₂O is diluted to 100 μ l and is stored in -20 DEG C of refrigerators.

3.2mRNA reverse transcription

UsingIII Reverse Transcriptase (invitrogen, article No. 18080-044) into Row cDNA reverse transcriptions, experimental implementation are carried out by product description, and concrete operations are as follows：

Using Reverse Transcriptase kit, converse record is carried out to l μ g total serum IgEs with RT Buffer and synthesizes cDNA.Using 25 μ l Reaction system, each sample take 1 μ g total serum IgEs as template ribonucleic acid, following components are separately added into PCR pipe：

5 × RT Buffer, 5 μ l, 10mmol/l dNTP, 1.25 μ l, 0.1mmol/l DTT 2.5 μ l, 30 μm of mol/l Aqua sterilisa is added to 25 μ l of total system in 2 μ l, 200U/ μ l MMLV of OligodT 1.25 μ l, 1 μ g of template ribonucleic acid.42 DEG C are incubated 1 Hour, 72 DEG C 10 minutes, of short duration centrifugation.It is spare that -20 DEG C of refrigerators are put in cDNA preservations.

4 quantitative fluorescent PCRs

The RT-PCR systems of 4.1 miRNA：

3 parallel tube reactions are arranged in the detection of expression of miRNAs every time, using snRNA U6 as internal reference.

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

The RT-PCR systems of 4.2 mRNA：

3 parallel tube reactions are arranged in the detection of expression of mRNAs every time, using β-actin as internal reference.

Reaction system：Use PowerGreen PCR Master Mix (invitrogen, article No. 4367659) into Row amplification, experimental implementation are carried out by product description.Amplification program is：95 ° of 10min, (95 DEG C of 15sec, 60 DEG C of 60sec) × 45 A cycle.

5 statistical analysis

Real-time quantitative PCR amplification curve inflection point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tube Rate is close, and the limit is flat and present without raising up, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production is molten Solution curve is all unimodal, illustrates that amplified production only has one, is specific amplification；Divided using OriginPro8.1 softwares Analysis.Compare between statistical method mean and is examined using t, P<0.05 (significant difference) and P<0.01 (difference highly significant), which is set to, system Meter learns meaning, analyzes miR-632 expressions in cancerous tissue and cancer beside organism, as a result shows the expression of miR-632 in cancerous tissue It is apparently higher than cancer beside organism, the former is nearly 4 times (being specifically shown in Fig. 1) of the latter；The expression of VPS11 is significantly lower than in tissues of nasopharyngeal carcinoma Cancer beside organism, the former is the about one third (being specifically shown in Fig. 2) of the latter.

Embodiment 4miR-632 is verified with VPS11 regulation relationships

Material prepares：

It is thin that human nasopharyngeal carcinoma cell line CNE is purchased from Cell Bank of Chinese Academy of Sciences/Shanghai Inst. of Life Science, CAS Born of the same parents resource center.

Design of primers：

VPS11 amplimers：

Forward primer：5’-CTCCTCAACTGCTATACC-3’SEQ ID NO 4

Reverse primer：5’-GACTTCACTCTCACTCTT-3’SEQ ID NO 5

Amplified production length 78bp.

1. cell culture

Human nasopharyngeal epithelioma 1 is used containing dual anti-1640 culture mediums of RPMI of 10% fetal calf serum and 1% mycillin, 37 DEG C, 5%CO₂, secondary culture under conditions of saturated humidity, tested for RNA extractings or in next step when cell growth state is good.

2.miRNA is transiently transfected

It is transiently transfected, is operated according to Lipofectamin using cationic-liposome method^TM2000 reagent specifications into Row.For 24 hours by the good 5-8F and 6-10B cell inoculations to 12 orifice plates of growth conditions before transfection, cell count about 2 × 10⁴, often To the transfection same day, cell fusion degree is that 50-60% is to be tested for rule culture.20nM/40nM/80nM miRNA mimic are added Enter into 100u1Opti-MEM culture mediums, soft mixing；Separately diluted with 100u1Opti-MEM culture mediums 2u1Lipofectamin^TM2000 liposomes, soft mixing are incubated at room temperature 5min；Mix Opti-MEM- liposomes and Opti- MEM-miRNAs is incubated at room temperature 20min, to form transfection composite；Then said mixture is added in cell culture medium, gently Light mixing replaces complete medium after cultivating 6h.Wherein, nonspecific miR sequences are as negative control.It is carried after culture 48h It takes cell total rna or carries out next step experiment.

3. experimental result：

MiR-632mimics is transfected into human nasopharyngeal carcinoma cell line, cell total rna is extracted after 48h, blank control is not It is transferred to the human nasopharyngeal carcinoma cell line cell of miRNA, nonspecific miRNA sequence is as negative control.Quantitative PCR detection is predicted The horizontal variation of the mRNA of target gene VPS11.As a result show that experimental group VPS11 gene expression doses are lowered about compared with the control 35%, the experimental results showed that VPS11 is the target gene of miR-632, in human nasopharyngeal epithelioma 1 the up-regulation of miR-632 cause VPS11 The downward of gene.

Although having referred to various preferred embodiments describes the present invention, it will be appreciated by those skilled in the art that can carry out each Kind variation, and available equivalents substitute base region of its component without departing from the present invention.Come in addition, many changes can be carried out Specific condition or material is set to be suitable for the teachings of the present invention without departing from its base region.

Therefore, the present invention is not intended to be defined in the particular embodiment disclosed herein for carrying out the present invention；On the contrary, This invention is intended to all embodiments including falling within the scope of the appended claims.

Claims (6)

1. The application of 1.VPS11 genes and/or VPS11 albumen in preparing diagnosis of nasopharyngeal carcinoma reagent.
2. 2. application according to claim 1, which is characterized in that it includes fluorescence quantifying PCR method, method for gene chip to use Detect the expression of VPS11 genes in sample；Or the expression using VPS11 albumen in immunization method detection sample.
3. 3. application according to claim 2, which is characterized in that use the reagent of fluorescence quantifying PCR method diagnosis of nasopharyngeal carcinoma Including a pair of of specific primer, upstream primer sequence is SEQ ID NO.4, and downstream primer sequence is SEQ ID NO.5.
4. Applications of the miRNA of 4.VPS11 gene regulations in preparing diagnosis of nasopharyngeal carcinoma reagent, which is characterized in that the miRNA of regulation and control For mir-632 and/or miR-632.
5. 5. application according to claim 4, which is characterized in that it includes high-flux sequence method and/or quantitative PCR side to use The transcription of mir-632 and/or miR-632 in method and/or probing procedure detection sample, or detected using immunologic detection method The expression for the target gene that miR-632 regulates and controls in sample.
6. 6. application according to claim 4, which is characterized in that using northern hybridizing methods, miRNA express spectra cores Piece, ribozyme protection analytical technology, RAKE methods, in situ hybridization, bead-based flow-cytometry detection sample in mir-632 and/ Or the transcription of miR-632；Using the expression of the target genes regulated and controled of miR-632 in ELISA and/or colloidal gold strip detection sample Situation.