CN105821131B - Osteosarcoma miRNA marker - Google Patents
Osteosarcoma miRNA marker Download PDFInfo
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- CN105821131B CN105821131B CN201610272993.7A CN201610272993A CN105821131B CN 105821131 B CN105821131 B CN 105821131B CN 201610272993 A CN201610272993 A CN 201610272993A CN 105821131 B CN105821131 B CN 105821131B
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Abstract
The present invention provides a kind of miRNA markers, which is miRNA550-a1.The present invention goes out satisfactory 2 sets of osteosarcoma miNRA data sets and 4 sets of osteosarcoma mRNA data sets using NCBI GEO data retrieval, analyzes above-mentioned data set, screens a kind of and closely related miRNA of osteosarcoma --- miRNA550-a1.MiRNA550-a1 can be used for judging whether osteosarcoma occurs to shift or anticipation osteosarcoma shifts risk.Inspection proves that miRNA550-a1 can effectively distinguish osteosarcoma transfer sample and non-diverting sample.On this basis, miRNA550-a1 can be also used for the drug that preparation inhibits bone and flesh tumor metastasis or prevention bone and flesh tumor metastasis.The present invention provides new thinking for the molecular mechanism research of osteosarcoma, while providing new diagnostic method for the clinical bone and flesh tumor metastasis of diagnosis on a molecular scale, and provide new drug target for bone and flesh tumor metastasis treatment.
Description
Technical field
The present invention relates to biomedicine field, it is related to a kind of osteosarcoma miRNA marker and its application, and in particular to a kind of
MiRNA550-a1 marker relevant to bone and flesh tumor metastasis and its application.
Background technique
Osteosarcoma (Osteosarcoma, OS) is that a kind of primary malignancy bone of most common non-hematopoietic origin is swollen
Tumor readily occurs in the stage that bone mushrooms out, and most of patient age is between 12-25 years old, and second high incidence age is 50
Year old or more.The typical happening part of OS is the metaphysis of bone, and Lung metastases most easily occur.Once Lung metastases, Patients with Osteosarcoma occurs
Survival rate be only 20%.Therefore, osteosarcoma is diagnosed early, it is shifted and is predicted and is intervened for improving bone and flesh
The prognosis of tumor patient has very important significance.
MicroRNA (miRNA) is a kind of non-coding RNA molecule being widely present in animals and plants and virus, and length is about
21-22nt, the negative regulation target gene in such a way that miRNA is sheared and inhibits protein translation.It is estimated that in organism, there are about 1/3
Gene by miRNA regulation.The study found that the generation of miRNA and tumour, development and transfer etc. are closely related.Due to miRNA
Structure has stability, is not easy to be degraded by endogenous RNA degrading enzyme (RNase), therefore can be used as the diagnosis marker of disease.
The expression of detection miRNA can provide reference frame for the clinical diagnosis of cancer.The unconventionality expression of miRNA is straight
The abnormal expression for resulting in and occurring and shift relevant gene to cancer is connect, and then induces the generation of cancer.In following clinic
In treatment, miRNA acts not only as the correlation marker of osteosarcoma early diagnosis and cancer progression, is also used as osteosarcoma
The target site for the treatment of.It finds and is applied to clinic to miRNA relevant with osteosarcoma occurrence and development and its target gene is identified for miRNA
Treatment provides basis.
With the reduction of sequencing cost, we can be studied between miRNA and mRNA by the sequence information of miRNA
Relationship.There is a large amount of miRNA information in present NCBI GEO database, the present invention is by transcribing the osteosarcoma delivered
Group data are analyzed, and are filtered out miRNA related with bone and flesh tumor metastasis, are verified it in clinic with quantitative fluorescent PCR (qPCR)
Expression in sample, and its mechanism to bone and flesh tumor metastasis is studied in osteosarcoma cell line, it is mentioned for osteosarcoma diagnosing and treating
For foundation.
Summary of the invention
The purpose of the present invention is to provide a kind of miRNA markers that can be used for judging bone and flesh tumor metastasis.
To achieve the goals above, present invention employs following technical solutions:
The present invention provides one kind to shift risk for assessing osteosarcoma, diagnoses whether osteosarcoma occurs transfer, judges bone
Sarcoma shifts the miRNA whether recurred, and the miRNA marker is miRNA550-a1.The miRNA550-a1 is selected from following
At least one of group: initial miRNA, miRNA550-a1 precursor miRNA of miRNA550-a1, maturation miRNA550-a1；It is described
The initial miRNA of miRNA550-a1 can be sheared in people's cell and be expressed as mature miRNA550-a1；The miRNA550-a1
Precursor miRNA can be sheared in people's cell and be expressed as mature miRNA550-a1.
It should be known that miRNA550-a1 of the invention includes the functional equivalent of composing type nucleic acid molecules, i.e. variant,
The identical function of complete miRNA550-a1 nucleic acid molecules is shown, although they are by the missing of nucleotide residue, displacement or insert
Enter and is mutated.
Those skilled in the art are known, in order to guarantee the stability of miRNA, can increase in the one or both ends of miRNA and protect
Shield property base, such as TT, can also modify miRNA base, but not influence the function of miRNA.Therefore, those skilled in the art
Member is known, under conditions of not influencing miRNA550-a1 function, carries out base modification to miRNA550-a1 or increases at both ends
The sequence for adding base to obtain is also contained within protection scope of the present invention.
In some specific embodiments of the invention, the miRNA550-a1 is mature miRNA550-a1.
Although being mature miRNA550-a1, those skilled in the art used in some specific embodiments
It is contemplated that initial miRNA (pi-miRNA550-a1), precursor miRNA (pre-miRNA550-a1) can obtain and maturation
The same technical effect of miRNA550-a1, because cell has initial miRNA (pi-miRNA550-a1), precursor further
MiRNA (pre-miRNA550-a1) is processed as the ability of mature miRNA550-a1.
MiRNA550-a1 nucleic acid molecules of the invention can exist in single-stranded or double-stranded form.Mature miRNA550-
A1 is mainly in single stranded form, and miRNA550-a1 precursor is part from complementation, to form duplex structure.Nucleic acid of the invention
Molecule can be the form of RNA, DNA, PNA, LNA.
Further, above-mentioned anticipation osteosarcoma shifts risk, judges whether osteosarcoma occurs transfer, judges that bone and flesh tumor metastasis is multiple
The tool of hair includes but is not limited to chip, kit.The tool includes expressing water for miRNA550-a1 in sample to be tested
Flat reagent.The reagent can be the primer or probe for miRNA550-a1.
The present invention also provides application of the above-mentioned miRNA550-a1 in high-flux sequence platform.Pass through high-flux sequence
The expression that can know miRNA550-a1 in sample osteosarcoma tissue to be detected, by the result of sample to be tested with osteosarcoma cancer
Side tissue is compared, and is easily determined sample to be tested with the presence or absence of the risk of transfer or is easily determined whether sample to be tested has occurred and that
Transfer or judge whether bone and flesh tumor metastasis recurs.Therefore, miRNA550-a1 expression is obtained by high-flux sequence
Application with osteosarcoma correlation is also contained within protection scope of the present invention.
The present invention provides one kind to shift risk for prejudging osteosarcoma, diagnoses whether osteosarcoma occurs transfer, judges bone
Sarcoma shifts the chip whether recurred, and the chip includes solid phase carrier；And it is fixed on the few nucleosides on the solid phase carrier
Acid probe, the oligonucleotide probe include specifically corresponding to some or all of miRNA550-a1 sequence.Widow's core
Thuja acid probe may also include in the prior art it has been reported that can be used for judging osteosarcoma whether occur shift or judge
Osteosarcoma shifts risk or judges the oligonucleotide probe of miRNA that whether bone and flesh tumor metastasis recurs.By the inspection of a variety of miRNA
Probing needle, which is placed, is also contained in this by detecting the case where a variety of miRNA index joints judge bone and flesh tumor metastasis on the same chip
Within the protection scope of invention.
Further, the solid phase carrier includes the various common used materials in the adoptable genetic chip field of solid phase carrier, example
Such as, but not limited to, nylon membrane, the slide or silicon wafer, unmodified slide, plastics modified through active group (such as aldehyde radical, amino)
Piece etc..
The conventional manufacturing method of biochip known in the art can be used in the preparation of the miRNA chip.
The present invention also provides one kind to shift risk for prejudging osteosarcoma, diagnose whether osteosarcoma occurs transfer, judgement
The kit whether bone and flesh tumor metastasis recurs, the kit include for detecting miRNA550- in subject's osteosarcoma tissue
The reagent of the expression of a1.Compared with the expression of the miRNA550-a1 in osteosarcoma cancer beside organism, if passing through kit
The expression of miRNA550-a1 significantly increases in detection osteosarcoma tissue, then judges that the osteosarcoma of the subject shifts risk
It is very high or occurred shift or shift again.
Further, the reagent includes the primer and/or probe for miRNA550-a1.The reagent further includes being directed to
In the prior art it has been reported that can be used for judging whether osteosarcoma shifts, perhaps judge osteosarcoma shift risk or
Judge the primer and/or probe of the miRNA whether bone and flesh tumor metastasis recurs.The detection primer of a variety of miRNA and/or probe are put
Set in same reagent box by detect the case where a variety of miRNA indexs joint judges bone and flesh tumor metastasis be also contained in it is of the invention
Within protection scope.
MiRNA550-a1 of the invention can be natural or artificial synthesized, or use can express
The carrier transfection cell of the DNA fragmentation of miRNA550-a1 obtains.The carrier includes viral vectors, eukaryotic vector.
Viral vectors can be any carrier appropriate, including but not limited to retroviral vector, adenovirus vector, gland
Viral related viral vectors, herpesviral (such as herpes simplex virus, vaccinia virus and Epstein-Barr virus) carrier, alphavirus vectors.
Carrier for expression of eukaryon can be any expression vector appropriate, including but not limited to pCMV-Myc expression vector,
PcDNA3.0 expression vector, pcDNA3.1 expression vector, pEGFP expression vector, pEF Bos expression vector, pTet expression vector,
PTRE expression vector or modified carrier on the basis of known expression vector, such as pBin438, pCAMBIA1301
Deng.
The DNA fragmentation that miRNA550-a1 can be expressed can obtain in the following way: from ncbi database
(http://www.ncbi.nlm.nih.gov/nuccore) finds the position in the genome miRNA550-a1 and specific sequence
Column information determines the position of the initial miRNA of miRNA550-a1 according to genome sequence, miRNA initial in miRNA550-a1
Meter specific primer is installed, amplification can be obtained the DNA fragmentation of expression miRNA550-a1.
The advantages of the present invention:
Present invention firstly discovers that miRNA550-a1 expression is to the occurrence and development of osteosarcoma related, by detection by
The expression of examination person miRNA550-a1, it can be determined that whether subject suffers from osteosarcoma or judge that subject whether there is
The risk of bone and flesh tumor metastasis perhaps osteosarcoma transfer and relapse prevention scheme or is controlled so that clinician be instructed to provide to subject
Treatment scheme.
Detailed description of the invention
Fig. 1 qPCR detects miRNA550-a1 in the cancerous tissue of the Patients with Osteosarcoma shifted and the expression of cancer beside organism
Situation；
Fig. 2 qPCR detects expression of the miRNA550-a1 in osteosarcoma cell line；
The inhibiting effect that Fig. 3 anti-miRNA-550a1 invades miRNA550-a1；
Influence of Fig. 4 anti-miRNA-550a1 to MG-63 cell invasion.
Specific embodiment
Carry out the technical solution that the present invention is furture elucidated below by specific embodiment.
The screening of embodiment 1 and the closely related miRNA of bone and flesh tumor metastasis
1, the data retrieval of osteosarcoma miRNA
Construct term (" osteosarcoma " [MeSH Terms] OR " osteosarcoma " [All Fields]) AND
(" gse " [Filter] AND " Homo sapiens " [Organism]), according to preset screening sample strategy, in NCBI
GEO (Gene Expression Omnibus) database retrieval, has obtained 2 sets of osteosarcoma miNRA data sets and 4 sets of osteosarcoma
MRNA data set.
Preset screening sample strategy: limitation research type be " expression profiling by array ",
" non-coding RNA profiling by array ", the data set for meeting following standard will be included in our research: 1.
Selected data collection the mRNA transcript profile data including full-length genome and miRNA must express data simultaneously；2. these data come from
In the cell of the biopsy or culture of osteosarcoma case group and control group；3. this research considers normalized or original number
According to collection.
2, osteosarcoma mRNA and miRNA expression Data Integration analysis
The miRNA and mRNA of 2.1 screening differential expressions
By transcript profile Data Analysis Software to 2 sets of osteosarcoma miNRA initial data and 4 sets of osteosarcoma mRNA initial data
Progress t-test obtains P value after carrying out background correction and standardization, calculates effect quantity, is then examined using Fisher and merge P value,
Effect quantity is merged using random-effect model, screens differential expression miRNA and mRNA, intersection is found, filters out 15 differences altogether
The miRNA of expression, wherein expression up-regulation gene 5, expression lower gene 10.
The identification of the miRNA target gene of 2.2 osteosarcoma differential expressions
MiRTarBase database (http://mirtarbase.mbc.nctu.edu.tw) downloads mankind miRNA target base
Because of information, while the miR-96 gene in cancer sample with differential expression is screened, 453 miRNA- target genes are obtained and close
System pair.
The biological information network of 2.3 osteosarcoma differential expression miRNA and differential expression target gene composition
The biology being made of using Cytoscape software building osteosarcoma differential expression miRNA and differential expression target gene
Information network figure.
The functional annotation of the target gene of 2.4 osteosarcoma differential expressions
The enrichment of GO function is carried out by gene of the DAVID to differential expression and KEGG access is enriched with.The enrichment of KEGG access
The target gene of 136 differential expressions can sift out in the library KEGG as the result is shown, concentrate on cell cycle, Apoptosis, cell
The signal paths such as communication, transcriptional control.
The screening of 2.5 osteosarcoma differential expression miRNA
Based on the expression Data Integration analysis of osteosarcoma disease mRNA, miRNA as a result, we have obtained differential expression
miRNA——miRNA550-a1。
The miRNA550-a1 of 2 qPCR of embodiment verifying differential expression
1, the confluence analysis of the miRNA data set obtained for NCBI GEO database retrieval is as a result, selection miRNA550-
A1 carries out qPCR verifying.
It verifies the selection osteosarcoma tissue shifted and the osteosarcoma tissue not shifted each 5 is verified.On
State the operation Operated Specimens that sample tissue is all from Patients with Osteosarcoma.All samples obtained obtain the committee, organizational ethics
Agree to.Whether the clinical data of tissue samples includes: gender, age, metastases situation, pathological grading, recurrence and other are given birth to
Change Testing index etc..
2, osteosarcoma cancerous tissue and cancer beside organism's RNA extraction process
Liquid nitrogen is added into tissue, is fully ground into powder, Trizol is added, stands 5min；The chlorine of about 1/5 volume is added
It is imitative, it turns upside down and mixes well, stand 5-10min at room temperature；4 DEG C, carefully draw supernatant after 12000rpm high speed centrifugation 15min
Liquid moves into new 1.5ml centrifuge tube, and -20 DEG C of isometric isopropanols are added, are sufficiently mixed by inversion, are placed in 10min on ice；4℃,
After 12000rpm high speed centrifugation 15min, supernatant is abandoned, is added 70% ethyl alcohol (4 DEG C preservation) of 2/5 volume into precipitating, 4 DEG C,
12000rpm centrifugation 5min；Supernatant is abandoned, the processed water of appropriate DEPC is added after precipitating room temperature is dried and sufficiently dissolves；
Nanodrop2000 ultraviolet specrophotometer measures RNA purity and concentration.RNA quality judging standard: the OD260/ of RNA sample
OD280 ratio is between 1.7-2.2；Total serum IgE electrophorogram has clearly 28S, 18S band；After 70 DEG C of water-baths keep the temperature 1 hour
Map no significant difference before electrophorogram and water-bath heat preservation.
3, reverse transcription synthesizes cDNA
Using Reverse Transcriptase kit, converse record is carried out to l μ g total serum IgE with RT Buffer and synthesizes cDNA.Using 25 μ l
Reaction system, each sample take 1 μ g total serum IgE as template ribonucleic acid, following components are separately added into PCR pipe: DEPC water, 5 × inverse
Transcription buffer, 10mmol/L dNTP, 0.1mmol/L DTT, 30 μm of mol/L Oligo dT, 200U/ μ l MMLVRT, template
RNA.42 DEG C of incubation 1h, 72 DEG C of 10min, of short duration centrifugation.
4, qPCR reacts
MRNA fluorescent quantitation upstream and downstream PCR primer, synthesis are designed using primer-design software Primer Premier 5.0
Primer sequence, is operated using SYBR Green PCR Master Mix kit, and specific steps by specification is operated, adopted
With 25 μ l reaction systems, 3 parallel pipes are arranged in each sample, and all amplified reactions repeat to be repeated three times above to guarantee result
Reliability.Prepare following reaction system: 12.5 μ l of SYBR Green Mix, 1 μ l of forward primer (5 μM/μ l), reverse primer
(5 μM/μ l) 1 μ l, template cDNA 2.0 μ l, no 8.5 μ l of enzyme water.Operations are carried out on ice.With SYBR Green I work
For fluorescent marker, PCR reaction is carried out on Light Cycler fluorescence quantitative PCR instrument, passes through melt curve analysis analysis and electrophoresis
Determine that purpose band, Δ Δ CT method carry out relative quantification.
5, result
As shown in Figure 1, compared with the osteosarcoma tissue not shifted, in the osteosarcoma tissue that has shifted
The expression of miRNA550-a1 significantly increases, consistent with miRNA data set confluence analysis result in embodiment 1.
Expression of 3 miRNA550-a1 of embodiment in osteosarcoma cell line
1, cell culture
Osteosarcoma cell line MG-63, U2-OS are cultivated in DMEM culture medium, by the normal osteoblast system of people
HFOB1.19 is cultivated in F12 and DMEM mixed culture medium (1:1) and 10% fetal calf serum, is placed in 37 DEG C, 5%CO2Incubator
In.
2、qPCR
2.1 cell total rnas extract
The extraction of cell total rna is carried out using the RNA extracts kit of QINGEN company, instruction carries out to specifications.
2.2 qPCR
Step is the same as embodiment 2.
3, result
As shown in Fig. 2, compared with the normal osteoblast system hFOB1.19 of people, in osteosarcoma cell line MG-63, U2-OS
The expression of miRNA550-a1 is significantly raised (P < 0.05).
The inhibiting effect that embodiment 4anti-miRNA-550a1 expresses miRNA550-a1
1, design synthesis is directed to the antisense oligonucleotides (anti-miRNA550-a1) of miRNA550-a1
Find miRNA550-a1's in ncbi database (http://www.ncbi.nlm.nih.gov/nuccore)
Sequence information synthesizes anti-miRNA550-a1 and random controls by TaKaRa design according to the sequence information of miRNA550-a1
Sequence.
2, cell culture
MG-63 cultural method is the same as embodiment 3.
3, cell transfecting
MG-63 cell is divided into two groups, respectively inhibition negative control group (anti-NC), miRNA550-a1 inhibition group
(anti-miRNA550-a1).By negative control group and constituents for suppressing not Zhuan Ran anti-NC and anti-miRNA550-a1, use
Transfection reagent Lipofectamine TM 2000 is transfected, and transfection method is referring to specification.Anti-NC and anti-
The working concentration of miRNA550-a1 is 5 μM.48h collects group of cells and is used for subsequent experimental after transfection.
4, qPCR is tested
Cell total rna extracts and PCR step is the same as embodiment 3.
The result shows that compared with inhibiting negative control group (anti-NC), miRNA550-a1 inhibition group (anti-
MiRNA550-a1 the level of miRNA550-a1) is remarkably decreased, and shows that anti-miRNA550-a1 can effectively inhibit
The expression of miRNA550-a1.
Embodiment 5 studies influence of the miRNA550-a1 to osteosarcoma cancer cell invasion ability
1, cell culture is the same as embodiment 4
2, Matrigel
The MG-63 cell of transfection 48h is digested and is counted using pancreatin, and 105 cells is taken to be placed in 1.5mL EP pipe, is added
Cell is resuspended in 200 μ l serum free mediums, is added in the cell transwell by paving matrigel, bottom chamber is added 10%
The DMEM culture medium of FBS puts 37 DEG C of people, 5%CO2Incubator culture is for 24 hours.The cell transwell is taken, wipes the thin of the inside with cotton swab
Born of the same parents, and the inside remaining cell is gently washed off with PBS.5 visuals field are taken to be counted at random under microscope after fixed dyeing.
3, result
Compared with inhibiting negative control group (anti-NC), miRNA-2116 inhibition group (anti-miRNA550-a1) is passed through
The Leukopenia of the cell the transwell basilar memebrane of matrigel 45% was spread.Show that anti-miRNA550-a1 can
The invasive ability of MG-63 cell is significantly inhibited, that is, shows that miRNA550-a1 is conducive to the migration and invasion of MG-63 cell.
The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.
Claims (10)
1.miRNA550-a1 is shifted risk in preparation anticipation osteosarcoma, diagnose whether osteosarcoma occurs transfer, judges osteosarcoma turn
Move the application in the tool whether recurred, which is characterized in that the miRNA550-a1 is selected from at least one of the following group:
Initial miRNA, miRNA550-a1 precursor miRNA of miRNA550-a1, maturation miRNA550-a1；The miRNA550-a1 is initial
MiRNA can be sheared in people's cell and be expressed as mature miRNA550-a1；The miRNA550-a1 precursor miRNA can be in people
It is sheared into the cell and is expressed as mature miRNA550-a1.
2. application according to claim 1, which is characterized in that the miRNA550-a1 is mature miRNA550-a1.
3. application according to claim 1, which is characterized in that be used for high-flux sequence platform, obtained by high-flux sequence
Know the expression of miRNA550-a1 described in sample to be tested osteosarcoma tissue, the miRNA550-a1 and bone and flesh are known in analysis
The correlation of tumor metastasis.
4. a kind of anticipation osteosarcoma shifts risk, diagnoses whether osteosarcoma occurs transfer, judges what whether bone and flesh tumor metastasis recurred
Chip, which is characterized in that the chip includes solid phase carrier；And it is fixed on the oligonucleotide probe on the solid phase carrier,
The oligonucleotide probe includes specifically corresponding to some or all of miRNA550-a1 described in claim 1 sequence.
5. a kind of anticipation osteosarcoma shifts risk, diagnoses whether osteosarcoma occurs transfer, judges what whether bone and flesh tumor metastasis recurred
Kit, which is characterized in that the kit includes described in claim 1 in subject's osteosarcoma tissue for detecting
The reagent of the expression of miRNA550-a1；With the expression water of miRNA550-a1 described in the osteosarcoma tissue that does not shift
It is flat to compare, if the expression of miRNA550-a1 described in osteosarcoma tissue significantly increases, judge the osteosarcoma of the subject
The risk shifted is high or has occurred to shift or shift to have recurred.
6. kit according to claim 5, which is characterized in that the reagent includes for the miRNA550-a1
Primer and/or probe.
7.miRNA550-a1 inhibitor inhibits the drug of bone and flesh tumor metastasis, invasion or prevention bone and flesh tumor metastasis, invasion in preparation
In application.
8. application according to claim 7, which is characterized in that the drug includes the miRNA550-a1 inhibitor.
9. application according to claim 8, which is characterized in that the miRNA550-a1 inhibitor is able to suppress
The expression of miRNA550-a1 or the function of being able to suppress miRNA550-a1.
10. application according to claim 9, which is characterized in that the miRNA550-a1 inhibitor is described
The antisense oligonucleotides of miRNA550-a1 or the miRNA550-a1 analogies.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103476947A (en) * | 2011-03-02 | 2013-12-25 | 格路福生物制药公司 | Enhanced biodistribution of oligomers |
| CN104774966A (en) * | 2015-05-01 | 2015-07-15 | 北京泱深生物信息技术有限公司 | Lung adenocarcinoma miRNA marker |
| CN104784704A (en) * | 2015-05-01 | 2015-07-22 | 北京泱深生物信息技术有限公司 | Composition related to lung adenocarcinoma metastasis and application thereof |
| CN105506156A (en) * | 2016-01-29 | 2016-04-20 | 北京泱深生物信息技术有限公司 | Molecular marker for diagnosing osteosarcoma |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103476947A (en) * | 2011-03-02 | 2013-12-25 | 格路福生物制药公司 | Enhanced biodistribution of oligomers |
| CN104774966A (en) * | 2015-05-01 | 2015-07-15 | 北京泱深生物信息技术有限公司 | Lung adenocarcinoma miRNA marker |
| CN104784704A (en) * | 2015-05-01 | 2015-07-22 | 北京泱深生物信息技术有限公司 | Composition related to lung adenocarcinoma metastasis and application thereof |
| CN105506156A (en) * | 2016-01-29 | 2016-04-20 | 北京泱深生物信息技术有限公司 | Molecular marker for diagnosing osteosarcoma |
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