CN104857513B

CN104857513B - Nasopharyngeal carcinoma diagnosis and treatment marker and its application

Info

Publication number: CN104857513B
Application number: CN201510290983.1A
Authority: CN
Inventors: 杨承刚; 宋宏涛
Original assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2015-06-01
Filing date: 2015-06-01
Publication date: 2018-07-17
Anticipated expiration: 2035-06-01
Also published as: CN104857513A

Abstract

The present invention relates to nasopharyngeal carcinoma diagnosis and treatment marker and its applications, more particularly relate to the new application of mir 326 and/or miR 326 in diagnosis and treatment nasopharyngeal carcinoma.Invention analyzes tissues of nasopharyngeal carcinoma by high-flux sequence, obtain its miRNA expression data, and then carry out bioinformatic analysis, it chooses miR 326 and carries out molecular biology verification, as a result it shows, the specificity overexpressions in tissues of nasopharyngeal carcinoma of miR 326 can be used for clinical diagnosis and prevent detection, have good actual application value.

Description

Nasopharyngeal carcinoma diagnosis and treatment marker and its application

Technical field

The present invention relates to molecular biology fields, are specifically related to nasopharyngeal carcinoma diagnosis and treatment marker and its application, more specifically It is related to the new application of mir-326 and/or miR-326 in diagnosis and treatment nasopharyngeal carcinoma.

Background technology

MiRNA is usually transcribed by rna plymerase ii (Pol II) and is generated.Pol II form hairpin structure after being incorporated in Near neck ring DNA sequence dna.The transcript of generation is cut through modification 5 ' cap sequences of addition and 3 ' end polyadenylic acid tail structures It cuts, the product of generation is known as primary miRNA (pri-miRNA), which may be up to thousands of or hundreds of nucleotide, may include Multiple miRNA ring structures.

Single pri-miRNA may contain one to six miRNA precursors.These hairpin structures are each by the core of about 70nt or so Thuja acid forms.Each hairpin structure is accompanied by partial sequence in favor of effective shear treatment.Double stranded hairpin RNA in pri-miRNA Structure is called nucleoprotein (the DiGeorge Syndrome Critical Region 8) identification of DGCR8, and DGCR8 is same Drosha enzymes form microprocessor (microprocessor) complex together.In the complex, DGCR8 organizes Drosha albumen RNase III structural domains make its at about 11 nucleotide of hairpin structure cut pri-miRNA, make its discharge hair fastener knot Structure.The hairpin structure of release be before miRNA (pre-miRNA), pre-miRNA 3 ' there are two hanging nucleotide, Pre-miRNA 5 ' is phosphoric acid group, and 3 ' be hydroxy groups.

In endochylema, pre-miRNA hairpin structures are through RNase III Dicer cutting process.This endogenous ribose core 3 ' interactions of the sour enzyme (endoribonuclease) with hairpin structure and the completion cutting on the 3 ' of ring and 5 ' arms, generate length The miRNA of about 22nt not perfect matchings：MiRNA* duplex structures.

There are mainly two types of the typical effect modes of miRNA and said target mrna.In most cases, single-stranded in compound The not fully complementary pairings of 3 ' UTR of miRNA and said target mrna, block the translation of target gene, to adjust gene expression.This mode It is main to influence protein expression level, have no effect on the stability of mRNA.Recently, research queries Translational repression theory, It was found that repressed said target mrna s and miRNAs, which is gathered in jointly in endochylema, is referred to as P corpusculums (processing bodies, P- Bodies region), this region also concentrate the enzyme of many participation mRNA degradations.P corpusculums are probably as untranslated MRNA carries out the container of temporary reversible storage, reduces the expression of some specific P corpusculums constitutive proteins and can mitigate miRNA mediations Gene expression inhibition effect.P corpusculums are the certain areas in endochylema, it includes the protein of process after participating in a variety of transcriptions, Such as：MRNA degrades (mRNA degradation), nonsense mediates mRNA declines (nonsense-mediated mRNA Decay, NMD), the gene silencing (RNA-mediated gene silencing) that Transcription inhibition and RNA are mediated.

Another mode of action is similar with siRNA, and when miRNA and mRNA complete complementaries match clock synchronization, Ago2 albumen is by cutting It cuts mRNA and directly results in its degradation, realize gene silencing.By taking the RNAi that siRNA is participated in as an example：SiRNA can be combined with RISC, be made MRNA targets are identified for template, and by base pair complementarity principle, mRNA is combined with the antisense strand in siRNA, displaces justice Chain.Double-strand mRNA generates the siRNA of 22nt or so under Dicer enzymes, ATP and unwindase collective effect, and siRNA continues same RISC Complex is formed, is combined with the mRNA of siRNA complementations, mRNA is made to be cracked by RNA enzyme.It is heavy that this process is also referred to as posttranscriptional gene Silent (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.

Nasopharyngeal carcinoma (Nasopharyngeal carcinoma, NPC) is that one kind betiding mucous membrane of nasopharynx, is had higher pernicious The malignant tumour of degree and extremely strong transfer ability.Nasopharyngeal carcinoma is multiple-factor inheritance disease, and (heredity is easily with inherent cause for morbidity Perception), ebv infection, environmental factor, many factors such as eating habit it is related, early diagnosis, early treatment are to save patient Life and the most effective means improved the quality of living.Regrettably, nasopharyngeal carcinoma onset is hidden, and has strong metastasis tendency.According to Statistics, about 75% patient just arrived late period when medical, regional nodes and/or DISTANT METASTASES IN occur, is recurred after treatment Or the main reason for shifting then poor prognosis, becoming treatment of nasopharyngeal carcinoma failure.Therefore, the tumor marker of screening for nasopharyngeal cancer, strives Take early detection, selection therapeutic regimen, prediction prognosis, monitoring recurrence or transfer that there is important clinic to nasopharyngeal carcinoma diagnosis and treatment Meaning.

The present invention analyzes tissues of nasopharyngeal carcinoma by high-flux sequence, obtains its miRNA expression data, and then carry out biological letter Credit analysis is ceased, standby miRNA is chosen and carries out molecular biology verification, the results show that miRNA provided by the invention and nasopharyngeal carcinoma are close Cut phase is closed, and be can be used for clinical diagnosis and is prevented detection, has good actual application value.

Invention content

The purpose of the present invention is to provide mir-326 and/or its maturation miRNA to prepare prevention, diagnose and/or treat lung Application in gland cancer transfering reagent.The sequence of mir-326 is shown in sequence table SEQ ID NO 1.The ripe miRNA of mir-326 is Its sequence of miR-326 is shown in sequence table SEQ ID NO 2.

Further, the prevention, diagnosis adenocarcinoma of lung transfering reagent include based on high-flux sequence method and/or based on fixed It measures PCR method and/or the transcription of mir-326 and/or miR-326 in adenocarcinoma of lung sample is detected based on probing procedure or is based on Immunologic detection method detects the expression for the target gene that miR-326 regulates and controls in adenocarcinoma of lung sample, it is preferred to use northern is miscellaneous Friendship method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE methods, in situ hybridization, bead-based flow-cytometry Detect the transcription of mir-326 and/or miR-326 in adenocarcinoma of lung sample；Lung gland is detected using ELISA and/or colloidal gold strip The expression for the target gene that miR-326 regulates and controls in cancer sample.

Preferably, it is described based on quantifying PCR method include specific amplification mir-326 and/or miR-326 primer； Described based on probing procedure includes probe with the nucleic acid array hybridizing of mir-326 and/or miR-326；It is described immune Detection method includes the antibody combined with miR-326 controlling genes expression protein-specific.

Further, the treatment adenocarcinoma of lung transfering reagent includes that inhibitor and/or inhibitor combination lower mir-326 And/or miR-326 transcription and/or block miR-326 activity.

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-326 and/or miR-326 The activity of miR-326.

The present invention also aims to provide a kind of inhibition adenocarcinoma of lung transfering reagent, which is characterized in that the inhibition lung gland Metastasis of cancer reagent includes：

(a) inhibitor and/or inhibitor combination, the inhibitor and/or inhibitor combination lower mir-326 And/or miR-326 transcription and/or block miR-326 activity；

(b) receptible carrier in pharmacy.

The present invention also aims to provide a kind of adenocarcinoma of lung transfer diagnostic reagent, which is characterized in that the adenocarcinoma of lung turns The transcription of mir-326 and/or miR-326 or immunologic detection method detection lung gland in adenocarcinoma of lung sample can be detected by moving diagnostic reagent The expression for the target gene that miR-326 regulates and controls in cancer sample.

Preferably, the adenocarcinoma of lung transfer diagnostic reagent is based on high-flux sequence method and/or is based on quantifying PCR method And/or the transcription of mir-326 and/or miR-326 in adenocarcinoma of lung sample is detected based on probing procedure or is based on immunization method Detect the expression for the target gene that miR-326 regulates and controls in adenocarcinoma of lung sample, it is preferred to use northern hybridizing methods, miRNA Chip of expression spectrum, ribozyme protection analytical technology, RAKE methods, in situ hybridization, bead-based flow-cytometry detect adenocarcinoma of lung sample The transcription of mir-326 and/or miR-326 in this；Using miR- in ELISA and/or colloidal gold strip detection adenocarcinoma of lung sample The expression of the target gene of 326 regulation and control.

It is furthermore preferred that described includes drawing for specific amplification mir-326 and/or miR-326 for quantifying PCR method Object；Described based on probing procedure includes probe with the nucleic acid array hybridizing of mir-326 and/or miR-326；It is described to exempt from Epidemic disease detection method includes the antibody combined with miR-326 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 inhibitor and/or inhibitor combination lower the transcription of mir-326 and/or miR-326 and/or block miR- 326 activity, which belongs to, makes miRNA functions lose, multiple keys during theoretically can miRNA being synthesized and be functioned Step is intervened：1) it blocks the processing of pri-miRNA or pre-miRNA and goes out nuclear process；2) by being tied with pre-miRNA It closes, it is prevented to be processed and enter induction silencing complex (RISC)；3) it by being combined with the ripe miRNA in RISC, plays Competitive inhibition.But at present mainly by importing exogenous miRNA antagonists to block its effect to target gene.Specifically Interference method includes：Antisense oligonucleotides (antisense miRNA oligonucleotides, AMOs), antagomiRs, MiRNA sponges (miRNA Sponge), miRNA Erasers, Target Masking and multiple target point antisense oligonucleotides (multiple target AMU, MTg AMU) etc..

Antisense OligodeoxynucleotideTechnique Technique is artificial synthesized single-stranded with ripe miRNA complementations i.e. according to basepairing rule Backward nucleotide chain plays the effect of competitive inhibitor.In order to prevent by the degradation of nuclease and phosphodiesterase etc., need pair The antisense RNA chain carries out different chemical modifications, often takes the mode of multiple technologies mixing modification to obtain in practical application Obtain stability most preferably.The antisense oligonucleotides utilized as active ingredient in the present invention has and the SEQ ID in sequence table The sequence of 1- the 8th, the 7th nucleotide sequence complementation of 2- the 8th or 2- in the nucleotide sequence of NO 2.Most preferably, originally The antisense oligonucleotides utilized as active ingredient in invention has mutual with the entire nucleotide sequence of sequence table SEQ ID NO 2 The sequence of benefit.

AntagomiRs technologies are to carry out cholesterol modification and 4 D2EHDTPA sites to 3 ' ends of antisense oligonucleotides chain Modification, 5 ' ends carry out 2 D2EHDTPA site modifications, and full chain is modified using 2 '-OMe-AMOs.The technological merit is, thio Phosphoric acid modifies the melting temperature for reducing antagomiR dimers, to ensure that its stability.

The antisense miRNA oligonucleotide fragments of miRNA Sponge technologies, that is, multiple repetitions, the kind of the sequence and target miRNA Subsequence partial complementarity inserts it into 3 ' UTR of the Green fluorescent protein fusion vector that oneself builds, by cytomegalovirus Promoter guides, and plays the effect of more binding domain competitive inhibitors.In order to and the more stable combinations of target miRNA, AGO2's Share zone (9-12 nucleotide) devises 4 unpaired base-pairs.It is demonstrated experimentally that miRNA ponge have and The similar function and effect of AMOs.The technology is often used to inhibit the function of some miRNA family.

MiRNA Erasers technologies.It is similar to miRNA Sponge technologies, but only by antisense miRNA widow's core of 2 repetitions Acid fragments are inserted into 3 ' UTR of reporter gene, and pass through gland under the control of U6 promoters with target miRNA complete complementaries Viral vectors transfectional cell.Since it is not seed sequence specificity, so being used only for inhibiting the function of some miRNA. The acting duration of miRNA Erasers technologies is also shorter than miRNA Spong technologies.

MiR-Masking technologies.1 kind of miRNA enlightening often has multiple said target mrna binding sites, while regulating and controlling multiple proteins Expression.Therefore, inhibit the expression of certain a kind of miRNA, target mRNA can not only be regulated and controled, it is also possible to will produce and not need " target external effect (off-target effect) ".And the appearance of miR-Masking technologies preferably solves the problems, such as this. It is 1 section of antisense base sequences combined with said target mrna, with some 3 ' end UTR of said target mrna of high compatibility and miRNA Complementation combines, to specifically block the interaction of miRNA and the said target mrna, simultaneously to the horizontal or other function of the miRNA It does not have an impact.

Multiple target point Antisense OligodeoxynucleotideTechnique Technique, which refers to, is designed into multiple antisense oligonucleotides acid fragments with 1 nucleotide chain On, the activity of multiple miRNA is made it have while inhibiting, and then adjust a variety of target proteins.The technology is suitable for while adjusting Act on multiple miRNA of Mr. Yu mRNA.

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-326 expressions

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 Beijing Friendship Hospital's (in December, -2011 in January, 2010), and experiment group selection disease inspection is diagnosed as nasopharynx 9 patients of cancer, control group choose the nasopharyngeal tissue of 5 normal persons of the same period.

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：Tumor group (9) and Normal group (5) carry out statistics credit Analysis, P values are less than 0.05, and tumor group and the difference of Normal group at least will be more than the differential expression miRNA of 2 times or more, It is to select the apparent has-mir-326 of up-regulated expression in filtering to enter our research range to differential expression miRNA people.

The relationship of embodiment 3 electronically validating miR-326 and nasopharyngeal carcinoma

Limitation research type is " expression profiling by array ", " non-coding RNA The data set that profiling by array " meet following standard will be included in our research：1. selected data collection must be same When include full-length genome mRNA transcript profiles data and miRNA expression data；2. these data come from nasopharyngeal carcinoma case group and The nasopharyngeal tissue of control group；3. this research considers normalized or raw data set；Through screening 5 sets of miRNA data sets (table 1) is included in our research.

The selection of 1 data of table

T- is carried out after carrying out background correction and standardization by 5 sets of miRNA initial data of transcript profile Data Analysis Software pair Test obtains P values, calculates effect quantity, then utilizes Fisher to examine and merges P values, merges effect quantity using random-effect model, Differential expression miRNA is screened, setting F.D.R. values ＜ 0.05 filters out the miRNA of 27 differential expressions, wherein expression altogether The miRNA of up-regulation has 12, and the miRNA that expression is lowered has 15.MiR-326 expression is apparent in the gene of up-regulation.

The expression of miR-326 in embodiment 4Real-time PCR detection tissues of nasopharyngeal carcinoma

1 sample collection：

78 nasopharyngeal carcinoma tumor tissues and 21 normal nasopharyngeal tissues are all from Beijing Friendship Hospital's (acquisition time 2009 In December, -2011 in June), 78 tumor tissues determine pathologic feature and histological grade through two Pathologis, 21 Control group Pathologic specimen all turns out to be normal nasopharyngeal tissue.

2miRNA is extracted：

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, 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/ml Trizol is added in the amount of Trizol, and 10% of tissue volume no more than Trizol volumes is fully 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.

3 miRNA reverse transcriptions

The preparation of RT systems：

Component	Concentration	Volume (μ l)
			Total RNA	-	1μg

miScript HiSpec Buffer	5×	4
			Nucleics Mix	10×	2
miScript Reverse Transcriptase Mix	-	2
			Nuclease-free H₂O	-	Up to 20
Total Volume	-	20

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 Nuclease-free H are added₂O is diluted to 100 μ l and is stored in -20 DEG C of refrigerators, is used for subsequent experimental.

4 quantitative fluorescent PCRs

The preparation of RT-PCR systems：

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

PCR programs：

95℃10min；40 cycles (95 DEG C of 10s, 60 DEG C of 30s).Utilize melting curve detection product special after circulation terminates It is anisotropic：97 DEG C are to slowly warm up to from 60 DEG C, 5 fluorescence signals of every DEG C of acquisition.

5 statistical analysis

It is analyzed using OriginPro8.1 softwares.Compare between statistical method mean and is examined using t, P<0.05 (difference Significantly) and P<0.01 (difference highly significant) is set to statistically significant, miR-326 tables in analysis tumor tissues and normal structure Up to level, as a result show that the expression of miR-326 in tumor tissues is apparently higher than positive tissue, the former is 4.3 times of the latter, is specifically shown in Fig. 1.

A kind of 5 nasopharyngeal carcinoma detection kit of embodiment

Tiny RNA extracts kit：mirVana^TMmiRNA Isolation Kit

MiRNA reverse transcriptions

The preparation of RT systems：

Component	Concentration	Volume (μ l)
			Total RNA	-	1μg
miScript HiSpec Buffer	5×	4
			Nucleics Mix	10×	2
miScript Reverse Transcriptase Mix	-	2
			Nuclease-free H₂O	-	Up to 20
Total Volume	-	20

Quantitative fluorescent PCR

The preparation of RT-PCR systems：

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

Applications of the 1.mir-326 and/or miR-326 in preparing diagnosis of nasopharyngeal carcinoma reagent.
2. application according to claim 1, which is characterized in that the diagnosis of nasopharyngeal carcinoma reagent includes being based on high-flux sequence Method and/or detect mir-326 and/or miR-326 in sample based on quantifying PCR method and/or based on probing procedure Transcription or the expression that the target gene that miR-326 regulates and controls in sample is detected based on immunologic detection method.
3. according to application according to claim 2, which is characterized in that using northern hybridizing methods, miRNA express spectras Mir-326 in chip, ribozyme protection analytical technology, RAKE methods, in situ hybridization, bead-based flow-cytometry detection sample And/or the transcription of miR-326；Using the target genes regulated and controled of miR-326 in ELISA and/or colloidal gold strip detection sample Expression.
4. application according to claim 2, which is characterized in that described based on quantifying PCR method includes specific amplification The primer of mir-326 and/or miR-326；Described based on probing procedure includes core with mir-326 and/or miR-326 The probe of acid sequence hybridization；The immunologic detection method includes resisting with what miR-326 controlling genes expression protein-specific was combined Body.
5. application according to claim 2, which is characterized in that detect turn of miR-326 in sample based on quantifying PCR method It records, the primer used in quantitative PCR is SEQ ID NO 3 in sequence table.