CN108359731A - The application of AACS and its regulation and control lncRNA in bone and flesh tumor metastasis diagnosis and treatment - Google Patents
The application of AACS and its regulation and control lncRNA in bone and flesh tumor metastasis diagnosis and treatment Download PDFInfo
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Abstract
The present invention relates to the application of AACS and its regulation and control lncRNA in bone and flesh tumor metastasis diagnosis and treatment.It is existing research shows that AACS genes participate in aliphatic acid synthesis and neuronal cell development etc., the application is found that its new purposes, AACS and its regulation and control lncRNA differential expressions in bone and flesh tumor metastasis, after the expression for inhibiting them, the transfer of osteosarcoma cell is suppressed, and shows that the application has great importance in accurate medical field.
Description
Technical field
The present invention relates to molecular biology fields, are specifically related to AACS and its regulation and control lncRNA in bone and flesh tumor metastasis diagnosis and treatment
In application, more particularly relate to AACS and regulate and control its expression lncRNA-ENSG00000279233 in diagnosis and treatment bone and flesh tumor metastasis
In application.
Background technology
Existing studies have shown that AACS genes ginseng aliphatic acid synthesizes (Acetoacetyl-CoA synthetase gene is
abundant in rat adipose,and related with fatty acid synthesis in mature
Adipocytes, Biochem Biophys Res Commun.2005Sep 16；335(1):215-9.) and neuronal cell
Development (Acetoacetyl-CoA synthetase is essential for normal neuronal development,
Biochem Biophys Res Commun.2012Oct 19；427(2):398-403) etc. important metabolic activity does not have still
The relationship that researches show that it between osteosarcoma.
LncRNA is that a kind of transcript length is more than 200nt, lack effectively open reading frame, not coding protein
RNA molecule.It is catalyzed transcription synthesis by RNA polymerase II, is distributed mainly on nucleus, is present in cytoplasm on a small quantity.Research
It was found that lncRNA tends to be folded into thermodynamically stable two level more or more advanced structure functions, part lncRNA passes through
Montage becomes with the polyA tails and promoter structure similar to mRNA, and it can be dynamic in histoorgan atomization
Expression and different montage modes, therefore lncRNA has time and Region-specificity.LncRNA presses transcript location not at present
It is same to fall into 5 types:Positioned at the lncRNA of intergenic region；Natural antisense chain lncRNA；Include sub-district lncRNA；Positive-sense strand
lncRNA；Two-way lncRNA.MiRNA is a kind of short sequence of non-coding for not having Open reading frame, length for 18-25nt
RNA is widely present in eucaryote.It is hydroxyl that, which there are the ends phosphate group 3' at the ripe ends miRNA 5', this feature makes
It with most of oligonucleotides and the degradation fragment of function RNA be distinguished conservatives of the overwhelming majority miRNA with height,
Timing and tissue characteristics.
Once the expression number of the miRNA of high-flux sequence research metastatic bone sarcoma patients and Healthy People was used before applicant
According to discovery miR-4520-3p, miR-1299 (ZL2016100688631 and ZL related to bone and flesh tumor metastasis
2016100686566), in the application, inventor further to bone and flesh tumor metastasis patient, primary patient and normal population, from
MRNA to miRNA to lncRNA carries out various dimensions research.The present invention provides and the closely related AACS genes of bone and flesh tumor metastasis, tune
The lncRNA-ENSG00000279233 for controlling AACS gene expressions, good thinking is provided for osteosarcoma Mechanism Study, is bone and flesh
Tumor clinical diagnosis provides new molecular marker, has great importance.
Invention content
The purpose of the present invention is to provide detection AACS genes and the non-coding RNA preparation for regulating and controlling its expression to prepare diagnosis
Application in osteosarcoma transfering reagent, the non-coding RNA are lncRNA.
Further, lncRNA ENSG00000279233.
The sequence of ENSG00000279233 is shown in sequence table SEQ ID NO 1.
Further, diagnosis osteosarcoma transfering reagent includes based on high-flux sequence method and/or being based on quantifying PCR method
And/or it is examined based on AACS or ENSG00000279233 transcriptions in probing procedure detection sample or based on immunologic detection method
The expression of AACS albumen in test sample sheet.
Preferably, miscellaneous using northern hybridizing methods, chip of expression spectrum, ribozyme protection analytical technology, RAKE methods, original position
The transcription of AACS or ENSG00000279233 in friendship, bead-based flow-cytometry detection sample；Using ELISA and/or glue
Body gold test paper strip detects the expression of AACS albumen in sample.
Preferably, include the primer of specific amplification AACS or ENSG00000279233 based on quantifying PCR method；It is based on
Probing procedure includes the probe with the nucleic acid array hybridizing of AACS or ENSG00000279233；The immunologic detection method
Include the antibody combined with AACS protein-specifics.
The purpose of the present invention is to provide a kind of prevention osteosarcoma transfering reagent, including lower AACS or
The transcription of ENSG00000279233 and/or the active reagent for inhibiting AACS or ENSG00000279233.
The expression of the known suppressor of those skilled in the art or lncRNA usually may be used one kind in following methods and/or
It is several：Including but not limited to by the albumen of the inhibition of gene expression of the suppressor of activating genes of interest, activating genes of interest,
Inhibit destination gene expression, activation that the microRNA of target gene degradation, importing is promoted to promote purpose base using RNA perturbation techniques
Because of the molecule of coding protein degradation, inhibit the factor of promotion destination gene expression and the expression of albumen.
Inhibit AACS by activating the suppressor of AACS genes, activation to inhibit the albumen of AACS gene expressions, importing
The siRNA of gene expression, activation promote the microRNA of AACS mRNA degradations, import the molecule for promoting AACS protein degradations, suppression
System promotes the factor of AACS gene expressions and the expression of albumen；Or suppressor, the activation of activation ENSG00000279233 genes
The albumen of ENSG00000279233 gene expressions, the siRNA for importing inhibition ENSG00000279233 expression, activation is inhibited to promote
The microRNA of ENSG00000279233 degradations, inhibit to promote the factor of ENSG00000279233 gene expressions and the table of albumen
It reaches.
Further, the reagent also includes receptible carrier in pharmacy.
The purpose of the present invention is to provide above-mentioned prevention osteosarcoma transfering reagent prepare treatment osteosarcoma diversion medicaments or
Application in preparation.
The purpose of the present invention is to provide above-mentioned bone and flesh tumor metastasis diagnostic preparations in preparing bone and flesh tumor metastasis diagnostic tool
Application.
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 is RT-PCR detection each group AACS genes and ENSG00000279233 expressions
Fig. 2 is the expression of each group AACS genes or ENSG0000027923 after detection transfection
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
3 metastatic bone sarcoma patients, 3 primary Patients with Osteosarcoma and 3 Healthy Peoples.Transfer group chooses metastases
Lesion, metastasis site are lung, primary group of selection tumour primary lesion, and normal healthy controls choose mark of the amputation sample away from tumour distal end
This.All blood samples and pathological examination answer it is true and reliable, research ratify through Ethics Committee, patient's informed consent.
RNA extraction standards：RNA purity：OD260/280≤1.8,28S/18S≤1；RNA integralities：Zhi≤7.0 RIN.
RNA integrality detection methods：Agilent 2100 (RNA 6000Nano kit), (Ago-Gel is dense for agarose gel electrophoresis
Degree：1% agarose gel；Voltage：5V/cm；Time：20min).
LncRNA and mRNA sequencings require：
Sample requirements：≥200ng；Sample concentration：C≥20ng/μL；Sample purity：RIN≥7.0,28S/18S≥
1.0。
MiRNA sequencings require：
Sample requirements (single)：≥1μg；Sample concentration：15ng/μL≤c≤500ng/μL；Sample purity：OD260/
280=1.8~2.2；OD260/230≥2.0；28S:18S≥1.5；RIN≥8.0.
Embodiment 2 is sequenced and data analysis
Sequencing：
LncRNA and mRNA microarray datasets：Hiseq2500 125PE complete the chain specific transcriptional group sequencing of 9 people
(lncRNA), the output of each sample is not less than 10Gb data.
MRNA and lncRNA analyses：
1 tophat is compared onto reference gene group, and reference gene group comes from Ensembl V84；
2.cuffquant quantifies the expression quantity and normalization output of lncRNA and mRNA；
3.cuffdiff packets compare the differential expression of lncRNA and mRNA between two groups.
Screening criteria P<0.01, abs (log2 (fold change))>2；307 difference expression genes (106 are screened
Up-regulation, 201 lower)；50 differential expression lncRNA (27 up-regulations, 23 lower).After comprehensive analysis and artificial screening, AACS genes
And regulates and controls non-coding RNA-ENSG00000279233 that it is expressed and enter our research range.AACS genes are in transfer group height
Express (primary group of transfer group VS), but primary group between healthy control group but without difference, show its only with osteosarcoma turn
Phase shift is closed, and AACS genes are the nearby gene of long-chain non-coding RNA-ENSG00000279233, while data analysis is shown
It is also the target gene of lncRNA, the two coexpression related coefficient is up to 96%.
In 3 Real-time PCR detection osteosarcoma sample of embodiment AACS genes and regulate and control its expression lncRNA table
Up to situation
1 sample collection：
The peripheral blood of 14 metastatic bone sarcoma tumor patients, 15 primary Patients with Osteosarcoma and 20 normal healthy controls come
From hospital.
2 Total RNAs extractions：
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.
Leucocyte detaches
(1) 2m1 anticoagulation cirumferential bloods are taken (blood sampling time is no more than 3h)；
(2) isometric sterile PB S are added to be sufficiently mixed in peripheral blood, form cell suspension；
(3) 4m1 lymphocyte separation mediums are added in another centrifuge tube；
(4) draw 4m1 cell suspensions be gently added to along tube wall lymphocyte separation medium surface (pay attention to not with lymphocyte
Separating liquid mixes).Centrifuge 1500rpm 20min；
(5) boundary layer (tunica albuginea) is gently sucked out with suction pipe to enter in another centrifuge tube.Sterile cold PBS is washed 2 times, is washed for last 1 time
Washing can move into cell suspension in EP pipes, and supernatant is removed in centrifugation, for extracting RNA.
RNA is extracted
(1) first add lml Trizol in EP pipes, if freeze-stored cell is directly added into Trizol, be not required to thaw, piping and druming cracking
After be stored at room temperature 5-l0min；
(2) 0.2m1 chloroforms are added, acutely shakes 15s, is stored at room temperature 2-3min, 1 2000 leave heart 15min at 4 DEG C；
(3) the supernatant water 600ul that makes an appointment carefully is sucked out and moves into another centrifuge tube (being careful not to be extracted into albumin layer), is added 500：
1 isopropanol overturns mixing, is stored at room temperature 10min；
(4) 4 DEG C of 1 2000g centrifuge l 0min, abandon supernatant, bottom visible white substance；
(5) the rotation washing of 75% cold ethyl alcohol of lml is added, cleans isopropanol；
(6) 4 DEG C of 7500g centrifuge 5min, and dry in the air 5-l0min after removal ethyl alcohol, translucent, with 20u1DEPC water dissolutions
RNA.3u1RNA samples are taken, the electrophoresis in 1.5% Ago-Gel；Lu1RNA samples in UV spectrophotometer measuring concentration,
It is considered as RNA sample qualifications in 1.8-2.0 with A260/280.
3 reverse transcriptions
MRNA reverse transcriptions：
It takes 1 μ g total serum IgEs as template ribonucleic acid, usesIII Reverse Transcriptase
(invitrogen, article No. 18080-044) carries out cDNA reverse transcriptions, and experimental implementation is carried out by product description.The cDNA of acquisition
It is spare that -20 DEG C of refrigerators are put in preservation.
4 quantitative fluorescent PCRs
Design of primers：
ENSG00000279233
Sense primer：5’-TCTTTCCCTTTCCTCTACC-3’(SEQ ID NO 2)
Downstream primer：5’-AATACAGCAGCATCCTCT-3’(SEQ ID NO 3)
AACS genes (NM_001319839.1)
Sense primer：5’-GTCCGTTGAGTCATATTC-3’(SEQ ID NO 4)
Downstream primer：5’-CACAACCTCATCATACAC-3’(SEQ ID NO 5)
The preparation of the RT-PCR systems of mRNA and lncRNA：
| Reactive component | Concentration | Volume (μ l) |
| mix | 2× | 10 |
| Sense primer | 10uM | 0.5 |
| Downstream primer | 10uM | 0.5 |
| cDNA | - | 2 |
| Nuclease-free H2O | - | Filling-in is to 25 μ l |
3 parallel tube reactions are arranged in the detection of expression of mRNAs every time, using actin as internal reference.
Amplification program is：95 ° of 10min, 45 cycles (95 DEG C of 15s, 55 DEG C of 60s).
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；According to the relative quantification formula of qRT-PCR：2-
Δ Ct × 100% compares AACS genes and ENSG00000279233 in primary group of osteosarcoma, transfer group group and Normal group
In expression.As a result it shows：Relative to primary group of AACS gene and ENSG00000279233 in the high table of osteosarcoma transfer group
Reach, expression quantity respectively at 2.14 times and 2.53 times or so, the two primary group between control group differential expression it is not statistically significant
(being specifically shown in attached drawing 1).Result above demonstrates the result of the confluence analysis of high-throughput transcript profile expression data.
4 AACS genes of embodiment and ENSG00000279233 are verified with osteosarcoma transfer relationship
One, material prepares：
(1) Specimen origin
Human osteosarcoma cell line MG-63 is purchased from Shanghai cell research institute of the Chinese Academy of Sciences.
(2) main agents
LipofectamineTM2000Transfection Reagent(Invitrogen)。
(3) main solution
1, cell culture fluid
+ 10% standard fetal calf serum of DMEM culture mediums.
2, PBS (balanced salt solution)
8g NaCl, 0.25g KCl, 1.44g Na are dissolved in 800m1 distilled water2HPO4With 0.24g KH2PO4 HCl
The pH value of solution is adjusted to 7.4, water is added to be settled to 1L, high pressure sterilization, room temperature preservation.
3,0.25% tryptic digestive juice
0.25g trypsase is added in 100m1 deionized waters, filter filtration sterilization, and packing is spare.
4, siRNA structures and synthesis
According to 2.0 (http of Photographing On-line software siDirect version://design.rnai.jp/), according to AACS
The corresponding siRNA of sequence design in gene and ENSG00000279233.Be sent to after design Synesis Company synthesis, siRNA control by
Company provides.
Two, experimental method
1, cell passes on
(1) culture solution original in the culture bottle for covering with cell is discarded, 0.25% trypsin solution 1m1, covering is added
Cellular layer, bottleneck disinfection, capping；
(2) cellular change is observed under inverted microscope, over time, former adherent cell gradually tends to be round,
Cytoplasm bounces back, and space between cells increases, and discards pancreatin in also non-levitating, and the culture that 5ml contains 10% fetal calf serum is added
Liquid terminates digestion；
(3) cell count：Above-mentioned cell suspension 0.5mI is taken, is instilled in blood cell counting plate after appropriate dilution, by leucocyte
Counting method number quadrangle four big lattice inner cell sum, when counting, only count nucleus and the complete cell of cytoplasm, cell in heaps
It is calculated by a cell, by the total number of cells in 4 block plaids by following formula scales at the cell number in every milliliter of cell suspension：
Big lattice total number of cells/4 × 10 total number of cells/ml=44× extension rate；
(4) it according to cell counts, is further diluted to every milliliter with DMEM complete culture solutions and contains 3 × 105A cell
Concentration is sub-packed in culture bottle (every bottle of 8m1/), is positioned over 37 DEG C, 5%CO2It is cultivated in incubator.
Cell is grouped：Blank control group (transfection liposome group)；Negative control group (transfects nonspecific siRNA groups)；
Transfect AACS-siRNA groups；Transfect ENSG0000027923-siRNA groups；Simultaneously transfect AACS-siRNA and
ENSG0000027923-siRNA groups.
2. transiently transfecting
Transfection：According to LipofectamineTMThe step of 2000Transfection Reagent are provided carries out.
1. before transfection for 24 hours, the cell pancreatin of logarithmic growth phase is digested and is counted, dense with DMEM culture mediums adjustment cell
Degree is 1 × 105/ ml takes 2m1 to be inoculated in six orifice plates, is positioned over 37 DEG C, 5%CO2It cultivates in incubator, is merged up to 80% in cell
When for transfecting.With the DMEM medium cultures 3-4h without serum before transfection.
2. preparing transfection liquid：
A liquid：250u1 serum free mediums dilute 4.0ugDNA, mild mixing；
B liquid：250u1 serum free mediums dilute 10u1Lipofectamine, and mild mixing is placed at room temperature for 5min；
3. transfecting：A liquid is mixed with B liquid, and compound is directly added in every hole by incubation at room temperature 20min, is shaken
Culture plate, gently mixing.In CO2Liquid is changed after 37 DEG C of heat preservations 24-48h, 6h in incubator, the culture medium containing serum is added.
Using the front and back AACS genes of Real-time PCR methods detection transfection, the variation of ENSG0000027923 expression：It carries
Take the RNA of each group cell, measure RNA concentration and purity, carry out reverse transcription reaction, every group of DNA profiling be carried out at the same time AACS genes,
The Real-time PCR reactions of ENSG0000027923 and internal reference, in triplicate, specific steps are with reference to embodiment 3 for experiment.
3.Transwell migration experiments
Detect cell migration situation when, first use trypsin digestion cell, and the culture medium that serum-free is added prepare it is slender
Born of the same parents' suspension；By 1 × 105A cell inoculation is in interior cultures of the placement 600u1 containing 10%FBS in the small interiors Transwell, lower layer
Base is placed in 37 DEG C, 5%CO2Cell incubator in cultivate；The cell scraper for using cotton swab carefully not migrate small interior afterwards for 24 hours
Fall；Absolute methanol fixes 1-5min, and cell overturning is dried；Using 0.1% violet staining 20min；It is cleaned 2 times using PBS；
Cell is transferred in 24 orifice plates containing PBS；Cell migration situation is observed under inverted microscope, takes the 6-8 visual field at random, into
Row cell count.
4. experimental result
Real-time PCR detect transfection efficiency.With the method for double standard curves compare each group AACS genes or
The expression of ENSG0000027923.As a result (see Fig. 2) is shown：The expression base of liposome transfection group, nonspecific transfection group gene
This similar, no significant difference；Transfection AACS-siRNA group AACS gene expressions have dropped 71%, transfection
The ENSG0000027923 expression of ENSG0000027923-siRNA groups has dropped 65%, it is worth mentioning at this point that, transfect AACS-
The ENSG0000027923 expression of siRNA groups has dropped 7%, and transfection ENSG0000027923-siRNA group AACS gene expressions decline
9%, while transfecting in AACS-siRNA and ENSG0000027923-siRNA groups, AACS genes and ENSG0000027923's
Expression declines 79% and 70% respectively, with related well in expression between display AACS genes and ENSG0000027923
Property.
The number for transfecting AACS-siRNA groups and transfecting the MG63 cell migrations of ENSG0000027923-siRNA groups is aobvious
It writes and is less than blank control group (p<0.05), show to inhibit AACS genes and ENSG0000027923 expression that will inhibit osteosarcoma cell
Migration, transfection AACS-siRNA groups, transfection ENSG0000027923-siRNA groups and transfect simultaneously AACS-siRNA and
ENSG0000027923-siRNA group cell migration numbers are respectively 133,139 and 120, blank control group cell migration number
It it is 172, negative control group (transfection non-specific sequences) cell migration number is 190, with blank control group without statistics
Difference.
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 this
Particular embodiment for carrying out the present invention disclosed herein；On the contrary, it is intended to cover including falling in claims model
Enclose interior all embodiments.
Sequence table
<110>Beijing Yang Shen biology information technologies Co., Ltd
<120>The application of AACS and its regulation and control lncRNA in bone and flesh tumor metastasis diagnosis and treatment
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ctctctgtgg ggacactgat gctctccaag aaggtacttc ttgaatcagt gacccttatt 180
gtctttttct gatgagggtc taaggttttc cttcagtgaa tcagtgctgt cttatctgga 240
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tgtgggcttg cgtccagggc acccagctgg gctctgggct ctttctctcc ccagataagg 480
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gggaggctct ggccggcgta gtccttgccg ccagccttca cagcgggttc tctgagggtc 1140
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cctctctggt gggtgccgtg ggccacaggg tgcccagaac gcagtccccg tgcctctgtt 1320
tctgtgctgc ctccactcac cgtcagcctt cattcggagt aggtgcgcat gctgtgcaaa 1380
gcccttccac acacctgatc tcagttgctc tctgtgcaaa agtcagagag gctttccctg 1440
catttcctgt ttgaacagtg tcctggcctc catctttagc tttgacagtg tttaccatgg 1500
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aacagcagat gcttttggtg tctggtgagt gaatgggggg tggggagctg gtcctgtgac 1680
cctggtgagg cgggacaaac ttgtcttcct cacacccatc ttacttcctc ttatgaggaa 1740
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gctcacccag aataaagacc tggggacccc gcgagggtca tggccaagtg gaatggactc 1860
ctggcatttg agggcttccc gactgcagcc ctcaggcagc catggctgtc ccaagtccag 1920
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tggcttcttc ctttctcttt ccctttcctc taccttttcc cctctcccca gaggaaatgg 2100
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cgagacgttt gcatctcaca gcgggagggc cggcgacatc acatgaagtg acaggcaggc 2280
ccttggaagc cggtgcttag atccttaatt agttcacacg tcgactgaat tttcaagtga 2340
atgaatttta attacatctc aggttaaaaa aaaaaaaagg cgccagtgat cgaggactcg 2400
tcactgggct ctgttgctcc tgaagtttcc tagcccacaa cacaccaaca ctgccaaggg 2460
ctcttctgga ttcaaggtga aacacatgtg ccataaatct tggagctctg aatgtttgga 2520
aagggcccga ctgtgagaag aagtaacaca ccgtcccgtg cagatggctg gctctgagga 2580
ggagttcatg ggagcttggg gacactcttg cctctagttc taggaagctg ggccacttct 2640
gaagtaatgg caatatcaat aaagtaatgg tctttatcat agaataacgt gataaaatat 2700
atagagaagt aaaaaagtat aaataaaagt aaaatcatca taaaacatag tagctaggca 2760
cttctgaagc tgtgtgtgca ctgattcatt cacccagtga ctcacagcct tatagcctag 2820
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ucaaaaaaua uaaaaaugga c 21
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ccauuuuuau auuuuuugaa a 21
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Claims (10)
1. detecting AACS genes and/or regulating and controlling the non-coding RNA preparation of its expression in preparing diagnosis osteosarcoma transfering reagent
Using the non-coding RNA is ENSG00000279233.
2. application according to claim 1, which is characterized in that diagnosis osteosarcoma transfering reagent includes being based on high-flux sequence
Method and/or based on quantifying PCR method and/or based on probing procedure detection sample in AACS or ENSG00000279233
Transcription or the expression that AACS albumen in sample is detected based on immunologic detection method.
3. application according to claim 1, which is characterized in that using northern hybridizing methods, chip of expression spectrum, ribozyme
Protect analytical technology, RAKE methods, in situ hybridization, bead-based flow-cytometry detection sample in AACS or
The transcription of ENSG00000279233；Using the expression of AACS albumen in ELISA and/or colloidal gold strip detection sample.
4. application according to claim 3, which is characterized in that based on quantifying PCR method include specific amplification AACS or
The primer of ENSG00000279233；Include miscellaneous with the nucleic acid sequence of AACS or ENSG00000279233 based on probing procedure
The probe of friendship；The immunologic detection method includes the antibody combined with AACS protein-specifics.
5. application according to claim 4, which is characterized in that specific amplification AACS primer sequences are shown in sequence table SEQ ID
NO.4 and SEQ ID NO.5；Specific amplification ENSG00000279233 primer sequences are shown in sequence table SEQ ID NO.2 and SEQ
ID NO.3。
6. a kind of prevention osteosarcoma transfering reagent, including lower the transcription of AACS or ENSG00000279233 and/or inhibit AACS
Or the active reagent of ENSG00000279233.
7. prevention osteosarcoma transfering reagent according to claim 6, which is characterized in that by the inhibition for activating AACS genes
Gene, activation inhibit the albumen, the siRNA for importing inhibition AACS gene expressions, activation of AACS gene expressions to promote AACS mRNA
The microRNA of degradation, the factor and albumen for promoting the molecule of AACS protein degradations, inhibition to promote AACS gene expressions are imported
Expression, it is preferred that the siRNA sequence of AACS gene expressions is inhibited to see sequence table SEQ ID NO.6 and SEQ ID NO.7.
8. prevention osteosarcoma transfering reagent according to claim 5, which is characterized in that activation ENSG00000279233 bases
The suppressor of cause, activation inhibit the albumen of ENSG00000279233 gene expressions, import inhibition ENSG00000279233 tables
The microRNA of the siRNA, activation promotion ENSG00000279233 degradations that reach, inhibit to promote ENSG00000279233 gene tables
The expression of the factor and albumen that reach, it is preferred that the siRNA sequence of ENSG00000279233 expression is inhibited to see sequence table SEQ ID
NO.8 and SEQ ID NO.9.
9. the prevention osteosarcoma transfering reagent according to claim 6-8 any one, which is characterized in that also include pharmacy
Upper receptible carrier.
10. prevention osteosarcoma transfering reagent described in claim 6-9 any one prepare treatment osteosarcoma diversion medicaments or
Application in preparation.
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