CN107365859A - Molecular markers of the LncRNA as diagnosis and treatment osteosarcoma - Google Patents
Molecular markers of the LncRNA as diagnosis and treatment osteosarcoma Download PDFInfo
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Abstract
The present invention relates to a kind of detection and its application of the long-chain non-coding RNA for osteosarcoma examination, specifically utilize long-chain non-coding chip of expression spectrum technology, by variance analysis, the lncRNA of a notable low expression in osteosarcoma tissue is screened, is named as LINC01996.Compared with normal cancer beside organism, LINC01996 notable low expressions in osteosarcoma tissue, and further confirm that expression quantity of the LINC01996 in osteosarcoma tissue is significantly lower than normal cancer beside organism in large sample fluorescent quantitative PCR experiment.LINC01996 will further enrich the research of osteosarcoma pathogenesis, and also the early diagnosis for osteosarcoma and Prognosis scoveillance provide new tumor markers and therapy target.
Description
Technical field
The invention belongs to biomedical sector, is related to molecular markers of the LncRNA as diagnosis and treatment osteosarcoma.
Background technology
Osteosarcoma is derived from the malignant tumour of mesenchymal tissue, and its principal causative is characterized as that the tumour cell bred in vivo is straight
Connect to form prematurity bone or osteoid tissue.It is a kind of most common primary malignant tumor of human skeletal system.Typical bone and flesh
Knurl is a kind of rare (account for whole malignant tumours 0.2%) high carcinogenic malignant tumour, the about annual each million people of its incidence of disease
In have three.Osteosarcoma mainly appears on longer bone and least a portion of soft tissue.Its principal pathogenetic crowd concentrates on 10
~20 years old teenagers, have the incidence of disease high, the early stage rate of transform is high, cures the features such as survival rate is low.X-ray, tomography skill
Art, nuclear magnetic resonance, Angiography and dynamic scintigraphy technology etc. are widely used in the sick diagnosis, tumour occurs
Degree and type of surgery judge etc..But the diagnosis of osteosarcoma is carried out using above-mentioned clinical means, frequently result in patient's
The state of an illness is delayed, and misses optimal treatment period.Therefore it is urgent problem to be solved to find a kind of osteosarcoma early diagnosis marker.
The gene of encoding proteins matter about 2-3 ten thousand, only accounts for the 2% of human genome in human body, and remaining 98% is not compiled
The genomic DNA of code protein is considered as initially not have functional, is the rubbish in organism, commonly known as " rubbish
DNA”.But current research shows, these junk DNAs mostly it is transcribed generation non-coding RNA (non-coding RNA,
ncRNA).According to the difference of ripe transcript size, ncRNA can be divided into small molecule ncRNA (such as siRNA, miRNA, piRNA
Deng), moderate-length ncRNA (70-200nt) and long ncRNA (long ncRNA, LncRNA,>200nt).
At present, that ncRNA area researches are more is small molecule ncRNA, and the research to LncRNA is also in the starting stage.
Because interior sequences have excessive terminator codon, LncRNA can not be translated into protein, and they are typically with rna transcription
This form exercises its biological function, such as the cell differentiation in growth course, cell propagation, Apoptosis and steroid metabolism
Deng.
Nearest research finds that LncRNA and lung cancer, non-hodgkin lymphoma, skin T cell lymphoma and chronic lymphatic are thin
The tumor diseases such as born of the same parents' leukaemia are relevant.This show the canceration of LncRNA and cell have it is extremely close contact, LncRNA is thin
Played an important role in born of the same parents' propagation, differentiation and canceration.At present, the mankind lncRNAs genes cloned are more than 40,000
It is individual, but most lncRNA Unknown Function.
At present, the basic blank of research about LncRNA in terms of the pathogenesis of osteosarcoma, medical diagnosis on disease and treatment, because
This application is using bioinformatic analysis research and inquirement LncRNA in terms of the pathogenesis of osteosarcoma, medical diagnosis on disease and treatment
Effect, while provide experiment basis and research direction for follow-up further investigation.
The content of the invention
It is an object of the invention to provide a kind of long-chain non-coding RNA mark for diagnosis and treatment osteosarcoma.Profit of the invention
Prove expressions of the LINC01996 in osteosarcoma tissue significantly lower than the water in cancer beside organism with chip and QPCR experiments
It is flat, thus can be using LINC01996 as diagnosis and treatment osteosarcoma molecular marker.
In order to test above-mentioned purpose, present invention employs following technical scheme：
The invention provides the reagent of detection long-chain non-coding RNA expression to prepare osteosarcoma auxiliary diagnosis or prognosis prediction
Application in product.
The long-chain non-coding RNA of the present invention is named as LINC01996 in NCBI, its gene I/D：102724660,
LINC01996 transcript sequence is that Genbank accession number NR_145431.1 (has 1270bp length, corresponding DNA sequences
Row are as shown in SEQ ID NO.1).
Further, the reagent is using SYBR Green, TaqMan probe, molecular beacon, double cross probe or multiple
Close the pcr amplification primer thing used during probe in detecting LINC01996 expression quantity.
In specific embodiments of the present invention, the primer sequence is as shown in SEQ ID NO.2 and SEQ ID NO.3.
The invention provides a kind of product for being used for osteosarcoma auxiliary diagnosis or prognosis prediction, the product includes detection
The reagent of LINC01996 expressions.
Further, the reagent includes SYBR Green, TaqMan probe, molecular beacon, double cross probe or compound spy
Pin detects the pcr amplification primer thing used during LINC01996 expression quantity.
In specific embodiments of the present invention, the primer sequence is as shown in SEQ ID NO.2 and SEQ ID NO.3.
Further, foregoing product includes but is not limited to chip, kit, test paper or high-flux sequence platform；It is high
Flux microarray dataset is a kind of instrument of special diagnosis osteosarcoma, with the development of high throughput sequencing technologies, to people's
The structure of rna expression spectrum, which will turn into, very easily to work.Composed, held by the rna expression for contrasting Disease and normal population
The exception for easily analyzing which RNA is related to disease.Therefore, non-LINC01996 exception and bone is known in high-flux sequence
Sarcoma correlation falls within LINC01996 purposes, equally within protection scope of the present invention.
The kit include detection LINC01996 expression quantity reagent, the reagent include with LINC01996 or its
The nucleic acid that DNA sequence dna combines, the nucleic acid include SYBR Green, TaqMan probe, molecular beacon, double cross probe or multiple
Close the pcr amplification primer thing used during probe in detecting LINC01996 expression quantity.
The chip includes the reagent of detection LINC01996 expression quantity, and the reagent includes and LINC01996 or its DNA
The nucleic acid that sequence combines, the nucleic acid include the probe that can detect LINC01996 expression quantity.
The test paper includes the reagent of detection LINC01996 expression quantity, and the reagent includes and LINC01996 or its DNA
The nucleic acid that sequence combines, the nucleic acid include the probe that can detect LINC01996 expression quantity.
The invention provides a kind of pharmaceutical composition for being used to treat osteosarcoma, described pharmaceutical composition includes
LINC01996 activator.
Further, the activator is unrestricted, as long as LINC01996 expressions or raising can be increased
LINC01996 functional activities.
The activator include LINC01996 in itself, promoted type miRNA, promoted type transcription regulatory factor or promoted type target
To micromolecular compound.
The pharmaceutical composition of the present invention can be administered alone as medicine or be applied together with other medicines.Can be with this hair
The other medicines that bright pharmaceutical composition is applied together are unrestricted, as long as it does not damage the therapeutic or preventative medicine of the present invention
The effect of compositions.
The pharmaceutical composition of the present invention can be prepared into various formulations as needed.Including but not limited to, percutaneous, mucous membrane, nose,
Buccal, sublingual or the oral tablet used, solution, granule, patch, paste, capsule, aerosol or suppository.
The route of administration of the pharmaceutical composition of the present invention is unrestricted, as long as it can play desired therapeutic effect or prevention
Effect, include but is not limited to intravenously, intraperitoneal, intraocular, intra-arterial, intrapulmonary, orally, in vesicle, intramuscular, tracheae
It is interior, subcutaneous, local by pleura by skin, suction, intra-ventricle intra-articular by mucous membrane, skin, stomach, directly
Intestines, vagina, in skull, in urethra, in liver, in knurl.In some cases, can systematically be administered.It is office in some cases
Portion it is administered.
The dosage of the pharmaceutical composition of the present invention is unrestricted, as long as obtaining desired therapeutic effect or preventive effect i.e.
Can, appropriate determination can be carried out according to symptom, sex, age etc..Medicine composition or the prophylactic agent combination of the present invention
The dosage of thing can use therapeutic effect for example to disease or preventive effect to be determined as index.
Present invention also offers application of the foregoing activator in the medicine for preparing treatment osteosarcoma.
Present invention also offers a kind of method for diagnosing osteosarcoma, methods described comprises the following steps：
(1) sample of subject is obtained；
(2) expression of LINC01996 in Samples subjects is detected；
(3) LINC01996 measured expression is associated with the whether ill of subject.
(4) compared with the control, LINC01996 expression reduces, then the subject be judged with osteosarcoma or
Risk of the subject with osteosarcoma is high or Patients with Osteosarcoma is judged as prognosis mala.
Present invention also offers a kind for the treatment of method of osteosarcoma, methods described include increase LINC01996 expression quantity or
Person strengthens LINC01996 functional activity.
, can be by adding test to osteosarcoma cell present invention also offers a kind of screening technique of bone and flesh tumor medicine
The expression that some after medicine or after testing drug is applied to osteosarcoma animal pattern measures LINC01996 in period is surveyed
Determining tumour medicine improves the effect of tumor prognosis.More specifically, when LINC01996 expression is adding or applied test
When being raised after medicine or when recovering normal level, the medicine may be selected as the medicine for improving tumor prognosis.
In the context of the present invention, whether " diagnosis osteosarcoma " includes judging subject with osteosarcoma, judgement
Subject whether there is the risk with osteosarcoma, judge Patients with Osteosarcoma to the reactivity of drug therapy or judge bone and flesh
The prognosis situation of knurl patient.
" treatment " used herein is covered treatment-related in such as mankind of the mammal with relevant disease or illness
Disease or morbid state, and including：
(1) prevention disease or morbid state occur in mammal, especially when the mammal is susceptible in the disease
Diseased state, but when being not yet diagnosed with this morbid state；
(2) suppress disease or morbid state, that is, prevent its generation；Or
(3) disease or morbid state are alleviated, even if disease or morbid state disappear.
Term " treatment " is usually directed to the treatment mankind or animal (for example, being applied by animal doctor), wherein can reach some pre-
The therapeutic effect of phase, for example, suppressing the development (including reduce development speed, stop development) of illness, improving illness and healing
Illness.Also include the treatment as precautionary measures (such as prevention).Pair illness is not developed into also but develop into illness danger
The purposes of the patient of danger, is also included within term " treatment ".
The advantages of the present invention：
The present invention's is found that a kind of molecular marker for diagnosing osteosarcoma, can be in osteosarcoma using the molecular marker
The early stage of generation can be used as judging, there is provided the survival rate of patient.
The medicine of the activator including LINC01996 of the present invention can be used as the medicine of new osteosarcoma.
Brief description of the drawings
Fig. 1 shows the statistical chart using QPCR detection LINC01996 expressions in osteosarcoma tissue；
Fig. 2 shows the statistical chart that situation is overexpressed using QPCR detections LINC01996；
Fig. 3 shows that LINC01996 is overexpressed the statistical chart influenceed on human osteosarcoma cell proliferation.
Specific embodiment
The present invention is further detailed explanation with reference to the accompanying drawings and examples.Following examples are merely to illustrate this
Invention rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in embodiment, generally according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in, or according to the condition proposed by manufacturer.
Embodiment 1 screens the non-long-chain coding RNA of differential expression
1st, research object：
Choose the classifiable tumor sample of 3 Patients with Osteosarcoma that hospital orthopedics row operative treatment finely cuts off and 3 normal
Cancer beside organism, tumor specimen pathological diagnosis turns out to be osteosarcoma.Operation take sample be in charge of immediately loading cryopreservation tube it is quick-frozen,
It is in vitro to being respectively less than 30min between quick-frozen.
2nd, tissue RNA is always extracted
2.1 tissue homogenate
About 50mg tissue samples are taken to be put into the mortar after high-temperature sterilization is handled, the 1ml of addition Trizol solution, fully
Tissue abrasion is moved into liquid-transfering gun in 1.5ml centrifuge tube, is overturned under mixing 10, is stored at room temperature 10 minutes, answers nucleic acid-protein
Compound is kept completely separate.
2.2 separation phase
4 DEG C, 12000rpm centrifugation 5-10 minutes, take supernatant.Centrifuge obtained precipitation and include epicyte, polysaccharide, high score
Son, contain RNA in supernatant.Take the even dress solution of clarification to add 0.2ml chloroform, cover lid, acutely concussion 15 seconds, make it
Fully mixed Uniform, is stored at room temperature 5 minutes.4 DEG C again, 12000rpm centrifugation 10min, sample can be divided into three layers, and RNA is mainly on upper strata
In aqueous phase, upper strata aqueous phase is transferred in the new pipe of new pipe (about 500 μ L), is careful not to be drawn onto intermediate layer and lower floor's liquid, it is no
DNA and protein contamination can then be caused.
2.3 RNA precipitate
The isopropanol that 0.5ml is freezed in advance is added in new pipe, is positioned over after mixing in -20 DEG C 30 minutes, latter 4 DEG C,
12000rpm is centrifuged 10 minutes, abandons supernatant.RNA precipitate is often invisible before centrifugation, and glue is formed in pipe side and ttom of pipe after centrifugation
Precipitation.
2.4 RNA are washed
Supernatant carefully is outwelled, leaves and takes precipitation.1ml 75% ethanol (being prepared with DEPC water) is added, vortex oscillation mixes sample
Product washing precipitation RNA, can not such as be vortexed mixing, can use acutely reverse manually mix 2 minutes.Then 4 DEG C, 7000rpm centrifuges 5 points
Clock.
2.5 RNA are redissolved
Supernatant is carefully abandoned after centrifugation, can carefully draw supernatant with pipettor, is careful not to be drawn onto precipitation.Room temperature hangs 10 points
Clock or so dries RNA precipitate, is careful not to allow RNA precipitate to be completely dried in order to avoid reducing RNA dissolubility.Finally use in right amount
DEPC water dissolving precipitation, it is divided into -70 DEG C of aliquot until completely dissolved and preserves or carry out reverse transcription immediately.
3rd, RNA quality testings
The measure of total tissue RNA purity and concentration
Use the concentration and purity of NanoDropND-1000 types ultraviolet specrophotometer measure total tissue RNA, it is necessary first to
It is measured after carrying out background zeroing using DEPC water, operating procedure is as follows：
(1) 1 μ L DEPC water (zeroing) or RNA sample is added dropwise to the surface for measuring pedestal；
(2) drop can form fluid column between upper bottom base and be automatically performed measure automatically, RNA concentration and quality it is various
Parameter will automatically generate Parameter File in computer；
(3) after having determined one, the survey of next sample is carried out in wiped clean after the sample liquid of base surface again
It is fixed；
(4) measurement result：
Concentration calculates：
Readings is 1 40 μ g RNA/ml of expression under A260.(calculation formula is sample RNA concentration：The μ of A260* extension rates * 40
g/ml.Specific calculated example is as follows：
RNA is dissolved in 40 μ l DEPC water, takes 5 Μ l 1:100 are diluted in 495 μ l DEPC water, measure A260=
0.21, RNA concentration=0.21*100*40 μ g/ml=840 μ g/ml
5 μ l are taken to be used for after measuring, remaining sample RNA is 35 μ l, and remaining RNA total amounts are：35 μ l*840 μ g/ml=29.4 μ
g；
Purity detecting：
The A260/A280 ratios of RNA solution are a kind of common methods of RNA purity detectings, preferably pure RNA its
OD260/A280 ratio is 1.8-2.1, and purity is higher closer to 2.0
OD260/A280 ratio>2.1 or<1.8 are regarded as RNA pollutions.
4th, total tissue RNA integrity mensuration'
Through 1% denaturing formaldehyde agarose gel electrophoresis, observed under ultraviolet transmission light, detect RNA integrality.
5th, array experiment
Applicant selects the 12*135K of Arraystar companies of U.S. offer human LncRNA Microarray
(v2.0) chip, the chip data source almost contains all authoritative LncRNA databases, such as NCBI Refseq, UCSC
Known Gene6.0, Gencode v13, RNA db2.0, NRED, LincRNAs, while also to mRNA sequence research, mRNA numbers
According to being mainly derived from The collaborative consensus coding sequence (CCDS) project.
5.1 LncRNA chip hybridizations：
Key step is as follows：
(1) double-strand complementation cRNA is synthesized：
Using Invitrogen SuperScript ds-cDNA synthetic agent box, 5 μ g total serum IgEs are taken to add 100pmol's
Oligo dT primers (special primer), closed according to Invitrogen SuperScript ds-cDNA synthetic agent box handbook
Into；
(2) mark purifying double-strand complementation cDNA
Ds-cDNA is purified and marked in strict accordance with Nimblegen gene expression analyisis operation manuals,
In simple terms, first 4 μ g RNase is added in ds-cDNA and be incubated 10 minutes in 37 DEG C, then with phenol chloroform-isoamyl
The cleaning of alcohol mixed liquor is then precipitated with ice-cold ethanol；Ds-cDNA is marked according to Nimblegen gene expression
Analyisis operation manuals use Nimblegen One-Color DNA marker kits, first take 1 μ g ds-cDNA, add
1OD Cy3 primers are incubated 10 minutes in 98 DEG C, then add 100pmol triphosphoric acids DNA and archaeal dna polymerase carboxylic
Cardinal extremity large fragment (and be sufficiently mixed and be incubated 2 hours in 37 DEG C.The 0.5MEDTA reaction solutions for being eventually adding 0.1 volume terminate instead
Should, finally purified with isopropanol ethanol precipitation；
(3) labeling effciency quality testing：Enter line efficiency quality testing to ds-cDNA with NanoDropND-1000；
(4) chip hybridization：
Ds-cDNA the and Microarrays chips that 4 μ g are marked with to Cy3 fluorescent dyes put Nimblegen into
In hybridization buffer liquid, 42 DEG C of incubation 16-20 hours, the chip after hybridization is existed in hybridization cultivating chamber
Cleaned under ozone-free environment according to Nimblegen wash buffer kits manuals；
5.2 IMAQs and data analysis
Chip after cleaning is put into Axon genepix 4000B chip scanners, opens genepix6.0 softwares with 5 μm
The resolution ratio of pixel is scanned, and the scan image of acquisition is input to NimbleScan softwares with tiff format and carries out Grid Align
And data analysis.These data are needed by quantile standardization and the Average normalized analysis of sane multi-chip, so as to be marked
Chip expression data of standardization, then enter data into Agilent genespring GX softwares and analyzed, image it is quantitative and
Standardized data processing be fully completed after with tabular form output data, mRNA the and LncRNA data of acquisition, by folding times
Rate is screened, mRNA the and LncRNA data for filtering out differential expression remake into mRNA the and LncRNA data of differential expression,
The huge mRNA and LncRNA scatter diagrams of differential expression and volcano figure (mark, while application software Agilent
The cluster analysis chart of genespring GX softwares carries out cluster analysis to data.
13rd, result
MRNA and LncRNA data have been obtained after IMAQ and data analysis, have set the standard of differential expression：
Fold chang >=2.0, P value≤0.05.The mRNA and LncRNA of differential expression between two groups are obtained according to this standard screening.
The data that screening obtains show that osteosarcoma tissue is compared with normal structure, have 875 LncRNA differential expressions, wherein raising 569
It is individual, lower 306.
The difference expression gene that the checking of the large sample of embodiment 2 filters out
Based on the selection result of embodiment 1, according to P value size, LINC01996 is selected to be verified.
1st, sample is collected
Osteosarcoma tissue and each 50 of normal cancer beside organism are collected according to the method for embodiment 1.
2nd, verified on transcriptional level
Reagent：Reverse transcription reagent box (DDR037A) is purchased from precious bioengineering (Dalian) Co., Ltd.Real-time (the Real- of fluorescence
Time) the SYBR Premix Ex Taq used in quantitative PCR (polymerase chain reaction)TM(Tli RNaseH
Plus) kit produces for Japanese Takara companies.
2.1 extract tissue RNA
Step is the same as embodiment 1.
2.2 design of primers
According to LINC01996 transcript sequences, primer is designed by NCBI design of primers instrument (Primer BLAST),
Sense primer：5’-CTGGAGATAGAGTGAGAC-3’(SEQ ID NO.2)；Anti-sense primer：5’-
CCTTATTTGGTGCTTTCT-3’(SEQ ID NO.3)。
According to GAPDH (reference gene) primers, sense primer：5’-CTCTGGTAAAGTGGATATTGT-3’
(SEQ ID NO.4)；5’-GGTGGAATCATATTGGAACA-3’(SEQ ID NO.5).
2.3 reverse transcription reaction
With the total serum IgE (1 μ g) of extraction for template, following reaction system is added, is specially：Buffer
4 μ L,μ L, Random 6mers (100 μ of 1 μ L, Oligo dT Primer (50 μM) of RT Enzyme Mix 1
M) 1 μ L, with the ddH without RNase2O supplies reaction volume for 20 μ L.Above-mentioned mixed liquor is placed in 37 DEG C of 15min, 85 DEG C of 5s,
Obtain cDNA.The cDNA detects available for lncRNA Real-time PCR.
2.4 Real-time PCR
By Japanese Takara companiesPremix Ex TaqTMWhat (Tli RNaseH Plus) kit was recommended
Primer optimum concentration (10 μM), by LINC01996 primers with deionized water dissolving, and establishes following reaction system：SYBR
Premix Ex TaqTMUnder 1 μ L, PCR sense primers (10 μM) of (2 ×) 25 μ L, ROX Reference Dye (50 ×) 1 μ L, PCR
The μ L of primer (10 μM) 1 μ L, cDNA 4 are swum, sterilize ddH2O 18μL.With 95 DEG C of 20s pre-degenerations, it is denatured by 95 DEG C of 10s, 60 DEG C of 20s
Annealing, 70 DEG C of 10s extension processes circulate 40 times, obtain Ct values.As a result relative quantification method, formula 2 are used-△△ctCalculate.It is real
Test and be repeated 3 times.
3rd, result
As a result as Fig. 1 shows that compared with normal structure, LINC01996 expressions significantly reduce in osteosarcoma tissue, poor
It is different that there is statistical significance (P<0.05).
The LINC01996 of embodiment 3 is overexpressed
1st, plasmid construction
LINC01996 cDNA is obtained by the method for embodiment 2, extension increasing sequence is as shown in SEQ ID NO.1
LINC01996 cDNA sequence.Above-mentioned cDNA sequence is inserted into eukaryotic expression vector pcDNA3.1, connects acquisition
Recombinant vector pcDNA3.1-LINC01996 is used for subsequent experimental.
2nd, the culture and transfection of osteosarcoma cell
2.1 cell culture
By osteosarcoma cell line 143B with 10% hyclone is contained, each 100U/ml of penicillin and streptomycin RPMI1640 is in 37
DEG C, 5%CO2Saturated humidity case in cultivate.
2.2 cell transfecting
(1) day before transfection is by 0.5-2*105Individual tumour cell is suspended in the culture medium that 500 μ l are free of antibiotic, inoculation
To 24 well culture plates.
(2) transfection same day cell density should reach 80%-90%, prepare following compound A：1 μ g DNAs are diluted in nothing
In blood serum medium, gently mix；Compound B：Take 4 μ l Lipofectamine2000 to be diluted in serum free medium, mix
It is even.
(3) compound A and B are mixed, gently mixed, is incubated at room temperature.
(4) 100 μ l liposome compounds are added in tumour cell, gently mixes up and down, cell is put into 37 DEG C
Containing 5%CO2Incubator is incubated 5-7 hours.
(5) plus 1ml contains the growth medium of 2 times of normal serums and antibiotic concentration, and it is small to continue culture cell 18-24
When.
3rd, detection pcDNA3.1-LINC01996 overexpression situation is tested using QPCR
3.1 extraction cell total rnas are operated using conventional method.
3.2 reverse transcription
Step is the same as embodiment 2.
3.3 QPCR
Step is the same as embodiment 2.
3.4 result
As a result as shown in Fig. 2 pcDNA3.1-LINC01996 can be successfully overexpressed, difference has statistical significance (P<
0.05)。
Measure of the embodiment 4LINC01996 expression to human osteosarcoma cell proliferation ability
1st, step：
Osteosarcoma cell 143B after transfecting 24 hours is inoculated in 96 porocyte culture plates, per hole 2*103Individual cell/
The μ l of hole/200, cell are grouped as follows：
Experimental group 1 (control group)：Osteosarcoma cell transfects pcDNA3.1；
Experimental group 2：Osteosarcoma cell transfects pcDNA3.1-LINC01996.
By cell in 37 DEG C, 5%CO2After incubator is incubated 24 hours again, according to Brd U cell proliferation reagent boxes
The specification of (Chemicon International), measure cell proliferation rate.
2nd, statistical method
Experiment is all completed according to being repeated 3 times, and result data is represented in a manner of mean+SD,
Statistical analysis is carried out using SPSS13.0 statistical softwares, difference between the two is examined using t, it is believed that works as P<Have when 0.05
It is statistically significant.
3rd, result
As a result as shown in figure 3, there is statistical significance compared to experimental group 1, the cells proliferation slowed down of experimental group 2, difference
(P<0.05).It is above-mentioned test result indicates that, LINC01996 expression can suppress human osteosarcoma cell proliferation.
The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.It should be pointed out that for this
For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improvement can also be carried out to the present invention
And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.
Sequence table
<110>Bo Jian Bioisystech Co., Ltd of Guan County
<120>Molecular markers of the LncRNA as diagnosis and treatment osteosarcoma
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1270
<212> DNA
<213>People source (Homo sapiens)
<400> 1
gccgagacag gacaaatatt tgagctctgg agtttgagac cagcctgggc aacagagtga 60
gactctgtct ctacagaaag ttttaaaatt agccaggtgt agtgacacat gcttgtagtc 120
cccattactt ggaaggccaa ggcgggagga tggcttgagc ctgggaggtc aaggcatcag 180
tgagcgatgg ttgtgccact gcctggagat agagtgagac cctgtctcaa aaaagaaaga 240
aagaaagaaa gaaaaatccc taagacctga cctgcctact ccctagaaag caccaaataa 300
ggaaaagcac tccggaagaa tgaagctaag aaggctgtgc tctctgaggc atccgggcag 360
ccttcactgg agggacaagg tccagtggga ccttcttact caagatggct tcaattccaa 420
tcaccagaga gttttgagaa ccagtgggag gggttcctgg gcagcggggc tctgctccta 480
ccgggatttg tttattcagc tgatgttaat aaatgaagct gaaggtaaca agctgtcact 540
tagcatctcc aggctggacc agaactgcca ttggttaagt ttcagacttc agccccaaag 600
tacagtgtca ccaccttgga tgaaggatat taattagtgt catgagggag atcacagtct 660
cattcccctc cgtcactagc acgagactga aaaaaaaaaa gaaaatcaaa ttcagttaga 720
acagagacgt gctgagaagc tgggccagag accccgcggg gaacctgccg ttgtttgtca 780
aaaaacgagg ccagcgagga agaagccaac catgcctccc gctccactcg ctttggaacc 840
cacagaatgt tcaacagggc agcacctttc agttattaag acactcagat tccaccctgg 900
tgcagtgaag caaactggat taaccacgcg gcacgtggca gagccacagc gcaaaccacg 960
gctcctgctg ctgaggaaaa tacaggcacc tcggccactc tgggctgtgg gttcgacgtg 1020
agcagcctca gggcagagac ggattctgtt catgtctgtc ccctctgcca tcccaggtct 1080
caggggttct gttcacctga ccatggcaga gttgttttgc ccacctgtga aaaccactcc 1140
aaccatcatg gttaccattg cattgcatgt tggcaaattt ggggaacatc accttccagg 1200
ccacttacag cactcgaata agggctttga caaaacacaa gcgttattat gaatataata 1260
ataatcagcg 1270
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ctggagatag agtgagac 18
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ccttatttgg tgctttct 18
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
ctctggtaaa gtggatattg t 21
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ggtggaatca tattggaaca 20
Claims (10)
1. detect application of the reagent of long-chain non-coding RNA expression in osteosarcoma auxiliary diagnosis or prognosis prediction product is prepared；
The long-chain non-coding RNA is LINC01996, and the corresponding DNA sequence dna of long-chain non-coding RNA is as shown in SEQ ID NO.1.
2. application according to claim 1, it is characterised in that the reagent is visited using SYBR Green, TaqMan
Pin, molecular beacon, double cross probe or combined probe detect the pcr amplification primer used during the long-chain non-coding RNA expression quantity
Thing.
3. application according to claim 2, it is characterised in that the primer sequence such as SEQ ID NO.2 and SEQ ID
Shown in NO.3.
4. according to the application any one of claim 1-3, it is characterised in that the product include kit, chip or
Test paper.
5. a kind of product for being used for osteosarcoma auxiliary diagnosis or prognosis prediction, it is characterised in that the product includes test right
It is required that the reagent of the long-chain non-coding RNA expression any one of 1-4.
6. product according to claim 5, it is characterised in that the reagent includes SYBR Green, TaqMan probe, divided
Sub- beacon, double cross probe or combined probe detect the pcr amplification primer thing used during the long-chain non-coding RNA expression quantity.
7. product according to claim 6, it is characterised in that the primer sequence such as SEQ ID NO.2 and SEQ ID
Shown in NO.3.
8. a kind of pharmaceutical composition for being used to treat osteosarcoma, it is characterised in that described pharmaceutical composition includes claim 1-4
Any one of long-chain non-coding RNA activator.
9. pharmaceutical composition according to claim 8, it is characterised in that the activator includes increase long-chain non-coding
The activator of rna level, or improve the activator of the long-chain non-coding RNA functional activity.
10. application of the activator in the medicine for preparing treatment osteosarcoma described in claim 8 or 9.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710725189.4A CN107365859B (en) | 2017-08-22 | 2017-08-22 | Molecular markers of the LncRNA as diagnosis and treatment osteosarcoma |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710725189.4A CN107365859B (en) | 2017-08-22 | 2017-08-22 | Molecular markers of the LncRNA as diagnosis and treatment osteosarcoma |
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| CN107365859A true CN107365859A (en) | 2017-11-21 |
| CN107365859B CN107365859B (en) | 2018-09-18 |
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ID=60310823
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113817819A (en) * | 2021-10-22 | 2021-12-21 | 常州市第二人民医院 | Application of LINC01996 in diagnosis of allergic airway inflammation |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015118156A1 (en) * | 2014-02-07 | 2015-08-13 | Vib Vzw | Inhibition of neat1 for treatment of solid tumors |
-
2017
- 2017-08-22 CN CN201710725189.4A patent/CN107365859B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015118156A1 (en) * | 2014-02-07 | 2015-08-13 | Vib Vzw | Inhibition of neat1 for treatment of solid tumors |
| CN105960459A (en) * | 2014-02-07 | 2016-09-21 | 非营利性组织佛兰芒综合大学生物技术研究所 | Inhibition of NEAT1 for treatment of solid tumors |
Non-Patent Citations (4)
| Title |
|---|
| GENBANK: "NR_145431.1", 《NCBI》 * |
| Z.Q. PENG ET AL.: "Increased expression of the lncRNA BANCR and its prognostic significance in human osteosarcoma", 《GENETICS AND MOLECULAR RESEARCH》 * |
| 尤龙飞等: "lncRNA在骨肉瘤中的研究进展", 《医学临床研究》 * |
| 顾宗欣等: "长链非编码RNA在骨肉瘤中表达的研究进展", 《实用骨科杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113817819A (en) * | 2021-10-22 | 2021-12-21 | 常州市第二人民医院 | Application of LINC01996 in diagnosis of allergic airway inflammation |
| CN113817819B (en) * | 2021-10-22 | 2023-04-07 | 常州市第二人民医院 | Application of LINC01996 in diagnosis of allergic airway inflammation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107365859B (en) | 2018-09-18 |
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