CN107475386B - Long-chain non-coding RNA marker for diagnosis and treatment osteosarcoma - Google Patents
Long-chain non-coding RNA marker for diagnosis and treatment osteosarcoma Download PDFInfo
- Publication number
- CN107475386B CN107475386B CN201710723968.0A CN201710723968A CN107475386B CN 107475386 B CN107475386 B CN 107475386B CN 201710723968 A CN201710723968 A CN 201710723968A CN 107475386 B CN107475386 B CN 107475386B
- Authority
- CN
- China
- Prior art keywords
- osteosarcoma
- linp1
- long
- coding rna
- chain non
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
A kind of long-chain non-coding RNA of present invention offer and its application.The present invention research shows that expression of the LINP1 in osteosarcoma tissue and in cancer beside organism there are significant differences, think accordingly LINP1 can be used as diagnose osteosarcoma molecular marker.Low expression level is presented in osteosarcoma tissue according to LINP1, LINP1, effects of the research LINP1 in osteosarcoma, the results showed that the proliferation of osteosarcoma cell can obviously be inhibited by being overexpressed LINP1 are overexpressed in osteosarcoma cell.Present invention is disclosed important function of the LINP1 in osteosarcoma pathogenesis, and new molecular labeling and drug target are also provided for the clinical diagnosis of osteosarcoma, treatment and Prognosis scoveillance.
Description
Technical field
The invention belongs to biomedical sector, it is related to the long-chain non-coding RNA marker for diagnosis and treatment osteosarcoma.
Background technology
Osteosarcoma is derived from the malignant tumour of mesenchymal tissue, and principal causative is characterized as that the tumour cell being proliferated in vivo is straight
It connects 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
Tumor is a kind of rare (account for whole malignant tumours 0.2%) high carcinogenic malignant tumour, the about annual each million people of incidence
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 incidence high, and 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 diagnosis of the disease, 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 a kind of osteosarcoma early diagnosis marker is found to be a problem to be solved.
The gene of coding protein about 2-3 ten thousand, only accounts for the 2% of human genome in human body, remaining 98% is not compiled
The genomic DNA of code protein is considered as initially not having functional, is the rubbish in organism, commonly known as " rubbish
DNA”.But it is current studies have shown that these junk DNAs mostly 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).
Currently, ncRNA area researches it is more be small molecule ncRNA, and the research of LncRNA is still in infancy.
Since interior sequences are there are excessive terminator codon, LncRNA cannot be translated into protein, they are typically with rna transcription
This form exercises its biological function, such as cell differentiation, cell Proliferation, Apoptosis and the steroid metabolism in growth course
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 related.This show the canceration of LncRNA and cell have it is extremely close contact, LncRNA is thin
It plays an important role in born of the same parents' proliferation, differentiation and canceration.Currently, the mankind lncRNAs genes cloned are more than 40,000
It is a, but the Unknown Function of overwhelming majority lncRNA.
Currently, the basic blank of research in relation to 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 providing experiment basis and research direction for follow-up further investigation.
Invention content
The purpose of the present invention is to provide a kind of long-chain non-coding RNA markers for diagnosis and treatment osteosarcoma.Profit of the invention
With chip and QPCR experiments have shown that expressions of the LINP1 in osteosarcoma tissue is significantly lower than the level in cancer beside organism,
It therefore can be using LINP1 as the molecular marker of diagnosis osteosarcoma.
In order to test above-mentioned purpose, present invention employs following technical solutions：
The present invention provides the reagents of detection long-chain non-coding RNA expression to prepare osteosarcoma auxiliary diagnosis or prognosis prediction
Application in product；The Gene ID of the long-chain non-coding RNA：108570035.
The long-chain non-coding RNA of the present invention is named as LINP1 in NCBI.The Genbank accession number NR_ of LINP1
138480.1, the length with 838bp, corresponding DNA sequence dna is as shown in SEQ ID NO.1.
Further, the reagent including the use of SYBR Green, TaqMan probe, molecular beacon, double cross probe or is answered
Close the PCR amplification primer used when probe in detecting LINP1 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 present invention provides a kind of for osteosarcoma auxiliary diagnosis or the product of prognosis prediction, the product include detection
The reagent of LINP1 expressions.
Further, the reagent includes SYBR Green, TaqMan probe, molecular beacon, double cross probe or compound spy
Needle detects the PCR amplification primer used when LINP1 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 not limited to chip, kit, test paper or high-flux sequence platform；It is high
Flux microarray dataset is a kind of tool of special diagnosis osteosarcoma, with the development of high throughput sequencing technologies, to people's
The structure of rna expression spectrum, which will become, very easily to work.By comparing the rna expression spectrum of Disease and normal population, hold
The exception for easily analyzing which RNA is related to disease.Therefore, the exception and osteosarcoma of non-LINP1 are known in high-flux sequence
Correlation also belongs to the purposes of LINP1, equally within protection scope of the present invention.
The kit includes detecting the reagent of LINP1 expression quantity, and the reagent includes and LINP1 or its DNA sequence dna knot
The nucleic acid of conjunction, the nucleic acid include SYBR Green, TaqMan probe, molecular beacon, double cross probe or combined probe detection
The PCR amplification primer used when LINP1 expression quantity.
The chip includes the reagent for detecting LINP1 expression quantity, and the reagent includes being combined with LINP1 or its DNA sequence dna
Nucleic acid, the nucleic acid includes the probe that can detect LINP1 expression quantity.
The test paper includes the reagent for detecting LINP1 expression quantity, and the reagent includes being combined with LINP1 or its DNA sequence dna
Nucleic acid, the nucleic acid includes the probe that can detect LINP1 expression quantity.
The present invention provides a kind of pharmaceutical compositions for treating osteosarcoma, and described pharmaceutical composition includes LINP1's
Activator.
Further, the activator is unrestricted, as long as can increase LINP1 expressions or improve LINP1 functions
Activity.
The activator includes that LINP1 itself, promoted type miRNA, promoted type transcription regulatory factor or promoted type targeting are small
Molecular compound.
The pharmaceutical composition of the present invention can be administered alone as medicine or be applied together with other medicines.It 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 dosage forms as needed.Including but not limited to, percutaneous, mucous membrane, nose,
Buccal, the sublingual or oral tablet used, solution, granule, patch, paste, capsule, aerosol or suppository.
The administration method of the pharmaceutical composition of the present invention is unrestricted, as long as it can play desired therapeutic effect or prevention
Effect, including but not limited to intravenously, in peritonaeum, intraocular, intra-arterial, intrapulmonary takes orally, in vesicle, intramuscular, and tracheae
Interior, subcutaneous, local by pleura by skin, sucking, by mucous membrane, skin, stomach is intra-articular, intra-ventricle, directly
Intestines, vagina, in skull, in urethra, in liver, in tumor.In some cases, it 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, gender, age etc..Medicine composition or the prophylactic agent combination of the present invention
The dosage of object can use therapeutic effect for example to disease or preventive effect to be determined as index.
The present invention also provides application of the foregoing activator in the drug for preparing treatment osteosarcoma.
The present invention also provides a kind of methods of diagnosis osteosarcoma, and described method includes following steps：
(1) sample of subject is obtained；
(2) expression of LINP1 in Samples subjects is detected；
(3) it is associated whether by the expression of the LINP1 measured with the illness of subject.
(4) compared with the control, the expression of LINP1 reduces, then the subject is judged with osteosarcoma or should be by
Risk of the examination person with osteosarcoma is high or Patients with Osteosarcoma is judged as prognosis mala.
The present invention also provides a kind of therapies of osteosarcoma, and the method includes increasing LINP1 expression quantity or increasing
The adjusting activity of strong LINP1.
The present invention also provides a kind of screening techniques of bone and flesh tumor medicine, can be by adding test to osteosarcoma cell
After drug or swollen to measure using the expression of some period measurement LINP1 after testing drug to osteosarcoma animal pattern
Tumor medicine improves the effect of tumor prognosis.More specifically, it is risen after adding or applying testing drug when the expression of LINP1
When high or when restoring normal level, the drug may be selected as the medicine for improving tumor prognosis.
In the context of the present invention, " diagnosis osteosarcoma " includes judging whether subject has suffered from osteosarcoma, judged
Subject, which whether there is, to be suffered from the risk of osteosarcoma, judges Patients with Osteosarcoma to the reactivity of drug therapy or judge bone and flesh
The prognosis situation of tumor 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 include：
(1) prevent disease or morbid state occurs in mammals, especially when the mammal is susceptible in the disease
Diseased state, but when being not yet diagnosed with this morbid state；
(2) inhibit disease or morbid state, that is, prevent its generation；Or
(3) alleviate disease or morbid state, even if disease or morbid state subside.
Term " treatment " is usually directed to treatment mankind or animal (for example, being applied by animal doctor), wherein can reach certain pre-
The therapeutic effect of phase, for example, inhibiting the development (including reduce development speed, development is made to stop) of illness, improving illness and healing
Illness.It further include the treatment as precautionary measures (such as prevention).To developing into illness not yet but developing into illness danger
The purposes of the patient of danger, is also included in term " treatment ".
The advantages of the present invention：
The present invention's is found that a kind of molecular marker of diagnosis osteosarcoma, can be in osteosarcoma using the molecular marker
The early stage of generation can be used as judging, provide the survival rate of patient.
The medicine of the activator including LINP1 of the present invention can be used as the medicine of new osteosarcoma.
Description of the drawings
Fig. 1 shows the statistical chart that situation is overexpressed using QPCR detections LINP1；
Fig. 2 shows that LINP1 is overexpressed the statistical chart influenced on human osteosarcoma cell proliferation.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually 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 normal condition proposed by manufacturer.
Embodiment 1 screens the non-long-chain coding RNA of differential expression
1, research object：
It chooses the classifiable tumor sample of 3 Patients with Osteosarcoma that hospital orthopedics row operative treatment is finely cut off and 3 normal
Cancer beside organism, tumor specimen pathological diagnosis turns out to be osteosarcoma.It is quick-frozen that the taken sample of operation is in charge of loading cryopreservation tube immediately,
It is in vitro to being respectively less than 30min between quick-frozen.
2, tissue RNA is always extracted
2.1 tissue homogenate
In take about 50mg tissue samples to be put into high-temperature sterilization treated mortar, the Trizol solution of the 1ml of addition, fully
Tissue abrasion is moved into liquid-transfering gun in the centrifuge tube of 1.5ml, is overturned under mixing 10, is stored at room temperature 10 minutes, nucleic acid-protein is made to answer
Object is closed to be kept completely separate.
2.2 separation phase
4 DEG C, 12000rpm is centrifuged 5-10 minutes, takes supernatant.It includes epicyte, polysaccharide, high score to centrifuge obtained precipitation
Son contains RNA in supernatant.It takes clear even dress solution that the chloroform of 0.2ml is added, covers pipe lid, acutely concussion 15 seconds, make it
Fully mixed Uniform, is stored at room temperature 5 minutes.4 DEG C again, 12000rpm centrifuges 10min, and sample can be divided into three layers, and RNA is mainly on upper layer
In water 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 middle layer and lower liquid, it is no
It will cause DNA and protein contamination.
2.3RNA precipitation
The isopropanol that 0.5ml is freezed in advance is added in new pipe, is positioned in -20 DEG C 30 minutes after mixing, 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 tube bottom after centrifugation
Precipitation.
2.4RNA washing
Supernatant carefully is outwelled, leaves and takes precipitation.75% ethyl alcohol (being prepared with DEPC water) of 1ml, vortex oscillation mixing sample is added
Product washing precipitation RNA, cannot such as be vortexed mixing, can use manually acutely reverse mixing 2 minutes.Then 4 DEG C, 7000rpm centrifuges 5 points
Clock.
2.5RNA being 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 that RNA precipitate is allowed to be completely dried in order to avoid reducing the dissolubility of RNA.Finally with suitable
DEPC water dissolutions precipitate, and are divided into -70 DEG C of aliquot until completely dissolved and preserve or carry out immediately reverse transcription.
3, RNA quality testings
The measurement of total tissue RNA purity and concentration
The concentration and purity of total tissue RNA are measured using NanoDropND-1000 type ultraviolet specrophotometers, 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 measurement automatically, RNA concentration and quality it is various
Parameter will automatically generate Parameter File in computer；
(3) after having measured 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：40 μ of A260* extension rates *
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 the DEPC water of 495 μ l, measure A260=
0.21, RNA concentration=0.21*100*40 μ g/ml=840 μ g/ml
After taking 5 μ l to be used for 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, ideal pure RNA its
The ratio of OD260/A280 is 1.8-2.1, and purity is higher closer to 2.0
The ratio of OD260/A280>2.1<1.8 are regarded as RNA pollutions.
4, total tissue RNA integrity mensuration'
Through 1% denaturing formaldehyde agarose gel electrophoresis, is observed under ultraviolet transmission light, detect the integrality of RNA.
5, array experiment
Applicant selects the human LncRNA Microarray of the 12*135K of Arraystar companies of U.S. offer
(v2.0) chip, which almost contains all authority's 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.
5.1LncRNA chip hybridization：
Key step is as follows：
(1) synthesis double-strand complementation cRNA：
Using Invitrogen SuperScript ds-cDNA synthetic agent box, 5 μ g total serum IgEs is taken to be added 100pmol's
Oligo dT primers (special primer) are closed according to Invitrogen SuperScript ds-cDNA synthetic agent box handbooks
At；
(2) label purifying double-strand complementation cDNA
Ds-cDNA is purified and is marked in strict accordance with Nimblegen gene expression analyisis operation manuals,
In simple terms, first the RNase of 4 μ g is added in ds-cDNA in 37 DEG C and is incubated 10 minutes, then use phenol chloroform-isoamyl
The cleaning of alcohol mixed liquor is then precipitated with ice-cold ethanol；Mark ds-cDNA according to Nimblegen gene expression
Analyisis operation manuals use Nimblegen One-Color DNA marker kits, first take the ds-cDNA of 1 μ g, are added
The Cy3 primers of 1OD are incubated 10 minutes in 98 DEG C, and 100pmol triphosphoric acids DNA and archaeal dna polymerase carboxylic is then added
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
It answers, is finally purified with isopropanol ethanol precipitation；
(3) labeling effciency quality testing：With NanoDropND-1000 to ds-cDNA into line efficiency quality testing；
(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, it is incubated 16-20 hours for 42 DEG C in hybridization cultivating chamber, the chip after hybridization is existed
It is cleaned according to Nimblegen wash buffer kits manuals under ozone-free environment；
5.2 Image Acquisition 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 steady multi-chip, 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 plots of differential expression and volcano figure (mark, while application software Agilent
The clustering chart of genespring GX softwares carries out clustering to data.
13, result
The data that mRNA and LncRNA have been obtained after Image Acquisition and data analysis set the standard of differential expression：
Value≤0.05 Fold chang >=2.0, P.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 a, lower 306.
2 large sample of embodiment verifies the difference expression gene filtered out
LINP1 is selected to be verified according to the size of P value based on the selection result of embodiment 1.
1, sample collection
Osteosarcoma tissue and each 50 of normal cancer beside organism are collected according to the method for embodiment 1.
2, it is 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 Takara companies of Japan.
2.1 extraction tissue RNA
Step is the same as embodiment 1.
2.2 design of primers
According to LINP1 transcript sequences, pass through design of primers tool (Primer BLAST) design primer of NCBI, upstream
Primer：5’-GAATAAGACAGACCAACT-3’(SEQ ID NO.2)；Downstream primer：5’-GCTGACAATATCTAAGGA-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, specially：Buffer
4 μ L,1 μ L, Oligo dT Primer (50 μM) of RT Enzyme Mix, 1 μ L, Random 6mers (100 μM)
1 μ L, with the ddH of no RNA enzyme2O supplies reaction volume for 20 μ L.Above-mentioned mixed liquor is placed in 37 DEG C of 15min, 85 DEG C of 5s, i.e.,
Obtain cDNA.The cDNA can be used for lncRNA Real-time PCR detections.
2.4Real-time PCR
By Japanese Takara companiesPremix Ex TaqTMWhat (Tli RNaseH Plus) kit was recommended
Primer optimum concentration (10 μM), by LINP1 primers with deionized water dissolving, and establishes following reaction system：SYBR Premix
Ex TaqTM1 μ L, PCR sense primers (10 μM) of (2 ×) 25 μ L, ROX Reference Dye (50 ×), 1 μ L, PCR downstream primers
4 μ L of (10 μM) 1 μ L, cDNA, sterilize ddH2O 18μL.With 95 DEG C of 20s pre-degenerations, anneal by 95 DEG C of 10s denaturation, 60 DEG C of 20s,
70 DEG C of 10s extend process and recycle 40 times, obtain Ct values.As a result relative quantification method, formula 2 are used-△△ctIt calculates.Experiment weight
It is 3 times multiple.
3, result
The results show that with LINP1 relative expression levels in normal structure for 1, LINP1 expressions are in osteosarcoma tissue
0.42 ± 0.14, difference has statistical significance (P<0.05).
Embodiment 3LINP1 is overexpressed
1, plasmid construction
The cDNA of LINP1 is obtained by the method for embodiment 2, extension increasing sequence LINP1 as shown in SEQ ID NO.1
CDNA sequence.Above-mentioned cDNA sequence is inserted into eukaryotic expression vector pcDNA3.1, the recombinant vector of acquisition is connected
PcDNA3.1-LINP1 is used for subsequent experimental.
2, the culture and transfection of osteosarcoma cell
2.1 cell culture
Osteosarcoma cell line 143B is used and contains 10% fetal calf serum, the RPMI1640 of each 100U/ml of penicillin and streptomycin is in 37
DEG C, 5%CO2Saturated humidity case in cultivate.
2.2 cell transfecting
(1) day before transfection is by 0.5-2*105A tumour cell is suspended in the not antibiotic culture mediums of 500 μ l, inoculation
To 24 well culture plates.
(2) transfection same day cell density should reach 80%-90%, prepare following compound A：1 μ g Plasmid DNA is diluted in nothing
In blood serum medium, gently mixing；Compound B：It takes 4 μ l Lipofectamine2000 to be diluted in serum free medium, mixes
It is even.
(3) compound A and B are mixed, gently mixing, incubation at room temperature.
(4) 100 μ l liposome compounds are added in tumour cell, up and down gently mixing, cell is put into 37 DEG C
Containing 5%CO2Incubator is incubated 5-7 hours.
(5) it is small to continue culture cell 18-24 for the growth medium for adding 1ml to contain 2 times of normal serums and antibiotic concentration
When.
3, the overexpression situation of detection pcDNA3.1-LINP1 is tested using QPCR
3.1 extraction cell total rna is operated using conventional method.
3.2 reverse transcription
Step is the same as embodiment 2.
3.3QPCR
Step is the same as embodiment 2.
3.4 result
The results are shown in Figure 1, and pcDNA3.1-LINP1 can be successfully overexpressed, and difference has statistical significance (P<
0.05)。
Measurement of the expression of embodiment 4LINP1 to human osteosarcoma cell proliferation ability
1, step：
Osteosarcoma cell 143B after transfecting 24 hours is inoculated in 96 porocyte culture plates, per hole 2*103A 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-LINP1.
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) measures cell proliferation rate.
2, statistical method
Experiment is all completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD,
Using SPSS13.0 statistical softwares come for statistical analysis, difference between the two is examined using t, it is believed that works as P<Have when 0.05
It is statistically significant.
3, result
The results are shown in Figure 2, and compared to experimental group 1, the cells proliferation slowed down of experimental group 2, difference has statistical significance
(P<0.05).It is above-mentioned the experimental results showed that, LINP1 expression can inhibit 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, 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 domain of the claims in the present invention.
Sequence table
<110>Bo Jian Bioisystech Co., Ltd of Guan County
<120>Long-chain non-coding RNA marker for diagnosis and treatment osteosarcoma
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 838
<212> DNA
<213>People source (Homo sapiens)
<400> 1
ccctccgtct ccttgactct gggtgggctg tgtgactttt ccttgctcaa tagaatgaga 60
aggaggtggc gcaccggcta caaggctaga ccgggggctc gcatatctcc acttgcagct 120
gccactgcca ttagaagaac ttgctcagcc agctcctggg ccccaggaaa aggacaagtg 180
tcacccagaa cagagccacc tccagctgca ctcagctcca gaagctggcc cagccggtcc 240
agtacacctt ttaaataatg tcctctacgt gccggtgtgg aagtagcccg gatgcaattg 300
aatgaacaac agacggtgct ttccaggacg gcgctgtgct ttccaggatg gtgctgtgct 360
ttcattcatt tgggtagctc ctctgtgagc ctcccagcgc cgactgcaga gcccccactc 420
tccagcctgc aagaccccga aattcaagcc acacaaagaa aggaggaggg ggccgttggc 480
atttactgaa ccttataaaa ctgtcagcaa aacagccctt aggcttggac tccctgctag 540
ccgggtttta cggtgctgaa gtcagcatct tgattcagct gcataaataa tctcctgcag 600
tcctgcaagg cctggggtag gagagggtat ggggaccagg gcactctgta agggctggga 660
taggaacccc agggaataag acagaccaac tgcgggactt cagactccac tgcagccggg 720
atcgggttgt tgttaatttc ttaagcaatt tctaaattct gtattgactc tctcatgcat 780
gtaactgatc cttagatatt gtcagccaaa tgactaaagg atttagaaat aaaaaaaa 838
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
gaataagaca gaccaact 18
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gctgacaata tctaagga 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 (8)
1. detecting application of the reagent of long-chain non-coding RNA expression in preparing osteosarcoma auxiliary diagnosis or prognosis prediction product；
The long-chain non-coding RNA is LINP1, and the corresponding DNA sequence dna of the long-chain non-coding RNA is as shown in SEQ ID NO.1.
2. application according to claim 1, which is characterized in that the reagent is visited including the use of SYBR Green, TaqMan
Needle, molecular beacon, double cross probe or combined probe detect the pcr amplification primer used when the long-chain non-coding RNA expression quantity
Object.
3. application according to claim 2, which is characterized in that the primer sequence such as SEQ ID NO.2 and SEQ ID
Shown in NO.3.
4. application according to any one of claim 1-3, which is characterized in that the product include kit, chip or
Test paper.
5. application according to claim 1, which is characterized in that the product includes that test right requires any one of 1-4
The reagent of the long-chain non-coding RNA expression.
6. application according to claim 5, which is characterized in that the reagent includes SYBR Green, TaqMan probe, divides
Sub- beacon, double cross probe or combined probe detect the PCR amplification primer used when the long-chain non-coding RNA expression quantity.
7. application according to claim 6, which is characterized in that the primer sequence such as SEQ ID NO.2 and SEQ ID
Shown in NO.3.
8. the activator of the long-chain non-coding RNA described in any one of claim 1-4 is in the drug for preparing treatment osteosarcoma
Application, which is characterized in that the activator is the recombinant expression using the cDNA structures of LINP1 shown in SEQ ID NO.1
Carrier.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710723968.0A CN107475386B (en) | 2017-08-22 | 2017-08-22 | Long-chain non-coding RNA marker for diagnosis and treatment osteosarcoma |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710723968.0A CN107475386B (en) | 2017-08-22 | 2017-08-22 | Long-chain non-coding RNA marker for diagnosis and treatment osteosarcoma |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107475386A CN107475386A (en) | 2017-12-15 |
| CN107475386B true CN107475386B (en) | 2018-08-21 |
Family
ID=60602197
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710723968.0A Active CN107475386B (en) | 2017-08-22 | 2017-08-22 | Long-chain non-coding RNA marker for diagnosis and treatment osteosarcoma |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107475386B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109112212A (en) * | 2018-09-14 | 2019-01-01 | 北京泱深生物信息技术有限公司 | sepsis diagnosis marker |
| CN113789380B (en) * | 2021-09-13 | 2023-06-16 | 山东大学 | Application of long-chain non-coding RNA lncRNA JCX as osteosarcoma molecular marker |
| CN114350796B (en) * | 2021-12-17 | 2023-05-05 | 南方医科大学 | Application of LINP1 in diagnosis and treatment of skin squamous cell carcinoma |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| USRE48856E1 (en) * | 2014-02-07 | 2021-12-21 | Vib Vzw | Inhibition of NEAT1 for treatment of solid tumors |
| CN106148529B (en) * | 2016-07-13 | 2019-12-06 | 中国人民解放军总医院 | LncRNA marker related to gastric cancer, special detection primer, detection method, kit and application thereof |
-
2017
- 2017-08-22 CN CN201710723968.0A patent/CN107475386B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN107475386A (en) | 2017-12-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107475386B (en) | Long-chain non-coding RNA marker for diagnosis and treatment osteosarcoma | |
| Gao et al. | BZW2 gene knockdown induces cell growth inhibition, G1 arrest and apoptosis in muscle‐invasive bladder cancers: A microarray pathway analysis | |
| CN110423819A (en) | A kind of participation Human colorectal carcinoma proliferation and drug resistant lncRNA and its application | |
| CN107312865B (en) | Purposes of the LOC100130111 in preparation osteosarcoma diagnostic products, therapeutic agent | |
| CN107058480B (en) | Long-chain non-coding RNA marker for diagnosing adenocarcinoma of lung | |
| CN108559779A (en) | Diagnosis and treatment marker of the long-chain non-coding RNA as gastric cancer | |
| CN108504658A (en) | Purposes of the LINC01836 in preparing diagnosing gastric cancer product, medicine | |
| CN112430665B (en) | Molecular biomarker for diagnosing and treating triple negative breast cancer and application thereof | |
| CN109576371A (en) | Application of the long-chain non-coding RNA in adenocarcinoma of lung | |
| CN106420791B (en) | Application of miR-145-3p in preparation of medicine for preventing or treating multiple myeloma diseases | |
| CN107365859B (en) | Molecular markers of the LncRNA as diagnosis and treatment osteosarcoma | |
| CN110093416A (en) | Application of the biomarker in diagnosis orthopaedic disease | |
| CN111424092B (en) | Detection gene and application thereof | |
| CN105603117B (en) | MiR-3613 is used to distinguish lung squamous cancer transfer and non-diverting miRNA marker | |
| CN110042164B (en) | Lung cancer diagnosis and treatment lncRNA marker | |
| CN108118093A (en) | Purposes of the LINC00426 in bone and flesh tumor metastasis diagnostic products are prepared | |
| CN112553342B (en) | Biomarker for diagnosing lung adenocarcinoma and application thereof | |
| CN110042160B (en) | Digestive system malignant tumor marker and method for preparing medicine for inhibiting digestive system malignant tumor | |
| CN108753983A (en) | The diagnosis and treatment marker of cervical carcinoma | |
| CN107385100A (en) | Purposes of the MCM8 as sdenocarcinoma of stomach Metastatic Marker | |
| CN108624694A (en) | Purposes of the CMC2 as cervical carcinoma diagnosis and treatment marker | |
| Liang et al. | lncRNA NR2F2-AS1 inhibits the methylation of miR-494 to regulate oral squamous cell carcinoma cell proliferation | |
| CN110025793B (en) | Application of ion channel gene KCNQ1 in preparation of medicine for treating endometrial cancer | |
| WO2017054759A1 (en) | Prevention, diagnosis and treatment of cancer overexpressing gpr160 | |
| CN107653319B (en) | Glioma diagnosis marker circ8:61680968|61684188 and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |