CN107312865B - Purposes of the LOC100130111 in preparation osteosarcoma diagnostic products, therapeutic agent - Google Patents
Purposes of the LOC100130111 in preparation osteosarcoma diagnostic products, therapeutic agent Download PDFInfo
- Publication number
- CN107312865B CN107312865B CN201710725181.8A CN201710725181A CN107312865B CN 107312865 B CN107312865 B CN 107312865B CN 201710725181 A CN201710725181 A CN 201710725181A CN 107312865 B CN107312865 B CN 107312865B
- Authority
- CN
- China
- Prior art keywords
- osteosarcoma
- rna
- expression
- long
- seq
- 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
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Pathology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Oncology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Hospice & Palliative Care (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses new application of the LOC100130111 in diagnosis and treatment osteosarcoma.It can detect the expression quantity of LOC100130111 in biological sample for the primer and probe of LOC100130111 by designing.Judge whether subject suffers from osteosarcoma, judges risk height of the subject with osteosarcoma, or the prognosis situation of prediction Patients with Osteosarcoma by the height of the expression quantity of LOC100130111.The studies above achievement according to the present invention can prepare the product of diagnosis osteosarcoma.In addition, cell in vitro proliferation experiment of the invention proves, inhibits LOC100130111 expression that can inhibit the proliferation of osteosarcoma cell, the drug for the treatment of osteosarcoma can be developed accordingly.
Description
Technical field
The invention belongs to fields of biomedicine, are related to LOC100130111 in preparation osteosarcoma diagnostic products, therapeutic agent
In purposes.
Background technique
Osteosarcoma is derived from the malignant tumour of mesenchymal tissue, and principal causative feature is that the tumour cell that is 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 disease 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 disease 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 judgement etc..But the diagnosis of osteosarcoma is carried out using above-mentioned clinical means, frequently result in patient's
State of an illness delay, 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 account for the 2% of human genome in human body, remaining 98% is not compiled
The genomic DNA of code protein is initially considered as 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 mature 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 research 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 usually 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 discovery 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 gene cloned is 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 for it is subsequent further investigation experiment basis and research direction are provided.
Summary of the invention
The purpose of the present invention is to provide a kind of long-chain non-coding RNA markers for diagnosis and treatment osteosarcoma.Benefit of the invention
With experiments have shown that expression of the LOC100130111 in osteosarcoma tissue is apparently higher than the level in cancer beside organism, therefore
It can be using LOC100130111 as the molecular marker of diagnosis and treatment 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 in preparation osteosarcoma auxiliary diagnosis or prognosis prediction
Application in product.
Long-chain non-coding RNA of the invention is named as LOC100130111 in NCBI, gene I/D: 100130111,
The transcript sequence of LOC100130111 is Genbank accession number NR_135221.1 (length with 2580bp, corresponding DNA
Sequence 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
The PCR amplification primer used when closing probe in detecting LOC100130111 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 LOC100130111 expression.
Further, the reagent includes SYBR Green, TaqMan probe, molecular beacon, double cross probe or compound spy
Needle detects the PCR amplification primer used when LOC100130111 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, mentioned-above 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 tool of special diagnosis osteosarcoma, with the development of high throughput sequencing technologies, to people's
The building of rna expression spectrum will become very easily work.By the rna expression of comparison Disease and normal population spectrum, hold
The exception for easily analyzing which RNA is related to disease.Therefore, know in high-flux sequence the exception of non-LOC100130111 with
Osteosarcoma correlation also belongs to the purposes of LOC100130111, equally within protection scope of the present invention.
The kit includes the reagent for detecting LOC100130111 expression quantity, and the reagent includes and LOC100130111
Or its DNA sequence dna combine nucleic acid, the nucleic acid include SYBR Green, TaqMan probe, molecular beacon, double cross probe,
Or the PCR amplification primer used when combined probe detection LOC100130111 expression quantity.
The chip include detect LOC100130111 expression quantity reagent, the reagent include with LOC100130111 or
The nucleic acid that its DNA sequence dna combines, the nucleic acid includes the probe for being able to detect LOC100130111 expression quantity.
The test paper include detect LOC100130111 expression quantity reagent, the reagent include with LOC100130111 or
The nucleic acid that its DNA sequence dna combines, the nucleic acid includes the probe for being able to detect LOC100130111 expression quantity.
The present invention provides a kind of for treating the pharmaceutical composition of osteosarcoma, and described pharmaceutical composition includes
The inhibitor of LOC100130111.
Further, the inhibitor is unrestricted, as long as can inhibit LOC100130111 expression or inhibition
LOC100130111 functional activity.
The inhibitor includes the siRNA or shRNA of LOC100130111.In specific embodiments of the present invention, institute
The siRNA sequence of LOC100130111 is stated as shown in SEQ ID NO.6 and SEQ ID NO.7.
Pharmaceutical composition of the invention can be used as medicine and be administered alone or apply 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 therapeutic or preventative medicine of the invention
The effect of compositions.
Pharmaceutical composition of the 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 pharmaceutical composition of the invention is unrestricted, as long as it can play desired therapeutic effect or prevention
Effect, including but not limited to intravenously, in peritonaeum, intraocularly, intra-arterial, intrapulmonary is taken 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 pharmaceutical composition of the 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..Therapeutic agent composition of the invention or prophylactic agent combination
The dosage of object, which can be used, for example determines the therapeutic effect of disease or preventive effect as index.
The present invention also provides application of the mentioned-above inhibitor in the drug of preparation treatment osteosarcoma.
The present invention also provides a kind of methods for diagnosing osteosarcoma, and described method includes following steps:
(1) sample of subject is obtained；
(2) expression of LOC100130111 in Samples subjects is detected；
(3) it associates whether by the expression of the LOC100130111 measured with the illness of subject.
(4) compared with the control, LOC100130111 expression increase, 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.
The present invention also provides a kind for the treatment of methods of osteosarcoma, and the method includes reducing LOC100130111 expression quantity
Or inhibit the functional activity of LOC100130111.
The present invention also provides a kind of screening techniques of bone and flesh tumor medicine, can be by adding test to osteosarcoma cell
The expression of some period measurement LOC100130111 after drug or after applying testing drug to osteosarcoma animal pattern
Improve the effect of tumor prognosis to measure tumour medicine.More specifically, when the expression of LOC100130111 addition or
When reducing after application testing drug or when restoring normal level, the drug may be selected as the medicine for improving tumor prognosis
Object.
In the context of the present invention, " diagnosis osteosarcoma " includes judging whether subject has suffered from osteosarcoma, judgement
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 related 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, stop development) of illness, improving illness and healing
Illness.It further include the treatment as precautionary measures (such as prevention).To not yet development be illness but have development be the illness endanger
The purposes of the patient of danger, is also included in term " treatment ".
The advantages of the present invention:
Of the invention has found 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, provide the survival rate of patient.
The therapeutic agent of inhibitor including LOC100130111 of the invention can be used as the therapeutic agent of new osteosarcoma.
Detailed description of the invention
Fig. 1 shows the statistical chart using QPCR detection LOC100130111 expression in osteosarcoma tissue；
Fig. 2 shows the statistical chart using QPCR detection LOC100130111 expression inhibiting situation；
Fig. 3 display inhibits LOC100130111 to express 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 performing the 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 sample to be put into high-temperature sterilization treated mortar, the Trizol solution of the 1ml of addition, sufficiently
Tissue abrasion is moved into liquid-transfering gun in the centrifuge tube of 1.5ml, is mixed by inversion under 10, is stored at room temperature 10 minutes, answers nucleic acid-protein
Object is closed to be kept completely separate.
2.2 separation phase
4 DEG C, 12000rpm is centrifuged 5-10 minutes, takes supernatant.Being centrifuged obtained precipitating includes epicyte, polysaccharide, high score
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
It mixes well, is stored at room temperature 5 minutes.4 DEG C again, 12000rpm is centrifuged 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.
2.3 RNA precipitate
The isopropanol that 0.5ml is freezed in advance is added in new pipe, is placed in 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 being centrifuged, and forms glue in pipe side and tube bottom after centrifugation
Precipitating.
2.4 RNA washing
Supernatant carefully is outwelled, leaves and takes precipitating.75% ethyl alcohol (being prepared with DEPC water) of 1ml is added, vortex oscillation mixes sample
Product washing precipitating RNA, cannot such as be vortexed mixing, can be with being acutely mixed by inversion manually 2 minutes.Then 4 DEG C, 7000rpm is centrifuged 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 precipitating.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 dissolution precipitating, is divided into -70 DEG C of aliquot until completely dissolved and saves or carry out immediately reverse transcription.
3, RNA quality testing
The measurement of total tissue RNA purity and concentration
Use the concentration and purity of NanoDropND-1000 type ultraviolet specrophotometer measurement 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 automatically and be automatically performed measurement, 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 calculation:
Readings is 1 40 μ g RNA/ml of expression under A260.Sample RNA concentration (calculation formula are as follows: 40 μ of A260* extension rate *
g/ml.Specific calculated example is as follows:
RNA is dissolved in 40 μ l DEPC water, is taken 5 Μ l 1:100 to be diluted in the DEPC water of 495 μ l, is measured A260=
0.21, RNA concentration=0.21*100*40 μ g/ml=840 μ g/ml
After taking 5 μ l to be used to measure, remaining sample RNA is 35 μ l, remaining RNA total amount are as follows: 35 μ l*840 μ g/ml=29.4 μ
g；
Purity detecting:
The A260/A280 ratio of RNA solution is a kind of common method of RNA purity detecting, ideal pure RNA its
The ratio of OD260/A280 is 1.8-2.1, and purity is higher closer to 2.0
Ratio>2.1 of OD260/A280 or<1.8 be regarded as RNA pollution.
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
The human LncRNA Microarray for the 12*135K that applicant selects Arraystar company in the U.S. to provide
(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 number
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 is taken to be added 100pmol's
Oligo dT primers (special primer) is closed according to Invitrogen SuperScript ds-cDNA synthetic agent box handbook
At；
(2) label purifying double-strand complementation cDNA
Ds-cDNA is purified and is marked in strict accordance with Nimblegen gene expression analyisis operation manual,
In simple terms, first the RNase of 4 μ g is added in ds-cDNA in 37 DEG C and is incubated for 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 manual uses Nimblegen One-Color DNA marker kit, first takes the ds-cDNA of 1 μ g, is added
The Cy3 primer of 1OD is incubated for 10 minutes in 98 DEG C, and 100pmol triphosphoric acid DNA and archaeal dna polymerase carboxylic is then added
Cardinal extremity large fragment (and be sufficiently mixed and be incubated for 2 hours in 37 DEG C.The 0.5MEDTA reaction solution for being eventually adding 0.1 volume terminates instead
It answers, is finally purified with isopropanol ethanol precipitation；
(3) efficiency quality testing labeling effciency quality testing: is carried out to ds-cDNA with NanoDropND-1000；
(4) chip hybridization:
Put the 4 μ g ds-cDNA and Microarrays chip for being marked with Cy3 fluorescent dye into Nimblegen
In hybridization buffer liquid, 42 DEG C incubation 16-20 hours, the chip after hybridization is existed in hybridization cultivating chamber
It is cleaned under ozone-free environment according to Nimblegen wash buffer kits manuals；
5.2 Image Acquisition and data analysis
Chip after cleaning is put into Axon genepix 4000B chip scanner, opens genepix6.0 software with 5 μm
The resolution ratio of pixel is scanned, and the scan image of acquisition is input to NimbleScan software with tiff format and carries out Grid Align
It is analyzed with data.These data need to standardize by quantile and the Average normalized analysis of steady multi-chip, to be marked
Chip expression data of standardization, then enter data into Agilent genespring GX software 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 screening, mRNA the and LncRNA data for filtering out differential expression remake into mRNA the and LncRNA data of differential expression,
Differential expression huge mRNA and LncRNA scatter plot and volcano figure mark, while application software Agilent
The clustering chart of genespring GX software carries out clustering to data.
13, result
The data of mRNA and LncRNA have been obtained after Image Acquisition and data analysis, have 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 tissue, 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
LOC100130111 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) SYBR Premix Ex Taq used in quantitative PCR (polymerase chain reaction)TM(Tli RNaseH
Plus) kit is the production of Takara company, Japan.
2.1 extract tissue RNA
Step is the same as embodiment 1.
2.2 design of primers
According to LOC100130111 transcript sequence, drawn by design of primers tool (Primer BLAST) design of NCBI
Object, upstream primer: 5 '-GGAAGAATGAACGAATGG-3 ' (SEQ ID NO.2)；Downstream primer: 5 '-
ATTACCTATGAAGGAGAAGAA-3’(SEQ ID NO.3)。
According to GAPDH (reference gene) primers, upstream primer: 5 '-CTCTGGTAAAGTGGATATTGT-3 '
(SEQ ID NO.4)；5'-GGTGGAATCATATTGGAACA-3'(SEQ ID NO.5).
2.3 cDNA synthesis
With the total serum IgE (1 μ g) of extraction for template, following reaction system is added, specifically:Buffer
4 μ L,1 μ L, Oligo dT Primer (50 μM) of RT Enzyme Mix, 1 μ L, Random 6mers (100 μ
M) 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,
Obtain cDNA.The cDNA can be used for lncRNA Real-time PCR detection.
2.4 Real-time PCR
By Japanese Takara companyPremix Ex TaqTMWhat (Tli RNaseH Plus) kit was recommended
Primer optimum concentration (10 μM), by LOC100130111 primer with deionized water dissolving, and establishes following reaction system: SYBR
Premix Ex TaqTMUnder 1 μ L, PCR upstream primer (10 μM) of (2 ×) 25 μ L, ROX Reference Dye (50 ×) 1 μ L, PCR
4 μ L of primer (10 μM) 1 μ L, cDNA is swum, sterilize ddH2O 18μL.With 95 DEG C of 20s initial denaturations, by 95 DEG C of 10s denaturation, 60 DEG C of 20s
Annealing, 70 DEG C of 10s extend process and recycle 40 times, obtain Ct value.As a result relative quantification method, formula 2 are used-△△ctIt calculates.It is real
It tests and is repeated 3 times.
3, result
As a result as Fig. 1 shows that compared with normal tissue, LOC100130111 expression is significantly risen in osteosarcoma tissue
Height, difference have statistical significance (P < 0.05).
Embodiment 3 inhibits LOC100130111 expression
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, siRNA is designed
The design of siRNA (small interfering RNA): it is retrieved by BLAST, in the special of LOC100130111
Property sequence area design siRNA sequence:
siRNA-LOC100130111
Positive-sense strand: 5 '-UCCAUUAGCAGCAUUUGGCAC-3 ' (SEQ ID NO.6)；
Antisense strand: 5 '-GCCAAAUGCUGCUAAUGGACA-3 ' (SEQ ID NO.7).
Above-mentioned siRNA is synthesized by Shanghai JiMa pharmacy Technology Co., Ltd, while the said firm provides negative control siRNA
(siRNA-NC)。
3, cell transfecting
The osteosarcoma cell line 143B cell for taking logarithmic growth, is inoculated in culture plate, adds culture solution, (cell is long for 24 hours for culture
It is full to 7 0-9 0%), transfection experiment is carried out by lipofectamine2000 (invitrogen) specification, RNA is received after 48h and uses
Real-time PCR detection.
4, the disturbed condition of detection siRNA is tested using QPCR
4.1 extraction cell total rnas are operated using conventional method.
4.2 cDNA synthesis
Step is the same as embodiment 2.
4.3 QPCR
Step is the same as embodiment 2.
4.4 result
As a result as shown in Fig. 2, LOC100130111 is inhibited to express successfully, difference has statistical significance (P < 0.05).
Embodiment 4 inhibits LOC100130111 to express the measurement to human osteosarcoma cell proliferation ability
1, step:
Osteosarcoma cell 143B after transfecting 24 hours is inoculated in 96 porocyte culture plates, every hole 2*103A cell/
Hole/200 μ l, cell are grouped as follows:
Experimental group 1 (control group): osteosarcoma cell transfects siRNA-NC；
Experimental group 2: osteosarcoma cell transfects siRNA-LOC100130111.
By cell in 37 DEG C, 5%CO2After incubator is incubated for 24 hours again, according to Brd U cell proliferation reagent box
The specification of (Chemicon International) measures cell proliferation rate.
2, statistical method
Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD,
Using SPSS13.0 statistical software come for statistical analysis, difference between the two is examined using t, it is believed that is had as P < 0.05
It is statistically significant.
3, result
As a result as shown in figure 3, 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, inhibit LOC100130111 expression can inhibit human osteosarcoma cell proliferation.
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.
Sequence table
<110>Guan County Bo Jian Bioisystech Co., Ltd
<120>purposes of the LOC100130111 in preparation osteosarcoma diagnostic products, therapeutic agent
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2580
<212> DNA
<213>source of people (Homo sapiens)
<400> 1
gggatccgcc cctccgccag ccccgctggc ttcccctctc cgccaccccg gactcagaac 60
ctcccctccc ccgccggggc ctgggcgcgc ctgcccttcg ctccccgacc tcctgctgca 120
gctctgggcc cctcggccgc tccctcccct cctcccttgc tcaccccacg gttggccgcg 180
gatctccagc acgccgcccc agtcctcggc gtcccgcagc gcctgccagg gtcgccgagc 240
ctcccctgcc tcctggccgc ctctgcagtg ccgtcctccc ggggctggct ctctccccag 300
gcccccgctt tggccaccca ggcactttcc gccctgagcg tggcagctgc tgccgggggt 360
cctgggaccg cgggatggga actcccacgc agcgtcccac aggctcctgg ccgccgcgag 420
cccgcgcgac tcgcacgagc atgtccaccc gcagcgaggg gccggtcact gctacggagc 480
ccgccagaag gcaaagcccc agacagcaaa tagggaggcg gctggcaccc agggaggccg 540
aggcgggagg atcgctggag cccaggagtt cgagacctgc cttggcaaca tactccctcc 600
agcgtgccaa atgctgctaa tggacagccg ctcctgccgc tctgatgttg ctagacctcc 660
tcagcactca gcccaaggca gccaggaaga atgaacgaat ggtgattcag agggaaagga 720
ggaagctgcg tggcatttca gagatactta ttttaaaact gctggtggcc atgtcttctt 780
ttcatatcag agctaatgag ttttgatatg tgaacatttc ttctccttca taggtaatgt 840
gttgatttgg cactttaatc ttagaataat catttctaaa accaacccaa ctgcttttct 900
ttacaatttt ggccttagca aacagatgag tccgttgact tgagcattgc ttgggcactg 960
tctgtgtgca ggtcaggcag gggatgctgg tggctgggaa acatagagga tggaggtgtc 1020
ttgggtgtac atgaaggggt ggggtcaagt ccacaggggg tcggcccctg gcagcagtcc 1080
tggagggctt cgtggaggaa gggagaattg agtgagaaag tggtccccag gtaagaggga 1140
agagcatcca gggagaggag actgcttctg ggaagaccag aggcatcacc tcgcaggggc 1200
tggtatggtg gtggctcagg tgggaggaag agagacctca gggaaaataa caggttccat 1260
tgacaggagg cctgctctgg tggggaggca ggctggtggt ggagtcatgg ctgcaggcca 1320
gggagatgcc tgaccaacag gtgtgttggc cagggtccct gtagcaaaaa cacggagctg 1380
aggacctcat ggcagggacc ccatgcagag ggagccgcgg gtggcatggg gatagtcacc 1440
tggctctcct gctctcggcc tcctgcagct cccctcactc cctgccctgc cacacaaggt 1500
caaacccacc aggaggcaga aggcgaaggg cgaggtccca gggtgaggag cagggaggag 1560
atggtggcag cagggaaggt gatgcctgct gcagagtgag acctgggact gccaaacaca 1620
cagtgacggg agctgaggag gcctgccggg ggaggaagac agtggctggc agtgacagga 1680
agggagggtc agcatggggc ggcagtccca gagctcaggg ctccctgcca ggccagcctt 1740
tgtggcctgt ggggtggagg tggcaattgc tgtcatgtca taaacacgtc aagcttacaa 1800
tgaaatctcc agtgtggctt gggagcaaat acatgtgcag gtgtttgagt atctgtgctc 1860
ttacctaacc tcagggagca ggaaaacgaa agcttatcat gtaaacacat cagcagcagg 1920
gcttatgtgt gtctccacac tggaaccccc aagacaagag actggaggca gagttgattt 1980
ccaagcccag agtgctgggc agcctttggg ttttatgtct gcttgcttgg agctcagtga 2040
tagaaatcta ggccccagaa gaggaaagga ctgtcctgca taaccagcac acccggggaa 2100
tgcttcaggt gcagctggat ccaggcatcc tcaaatagtg tgattaggac tccactttcc 2160
tttgtcacct tggcttggct cttccctgct ctgtgttgtt ttcagacgag gtggggcccc 2220
agcagctctg gtcctcctga catcctagag aagagccagc ccttccctcc tggggtctac 2280
accgtcccct aagggacctc agactgggct gatggaaggt ctcaagcagg ccatgtgaag 2340
ggcactgcac aggaaacaca gaaacgggaa tcctataagg tcagcagagc tctgagccct 2400
ttgttcttgg gcctgttttt atgatcctcc ctctacatgt agctcacact tccctgcttc 2460
tgtgcaggca ccaggtgagg gggttgtcat ggcctcctgc cactcttgag gctgtcagat 2520
attacctgtg ctgtccctgc attctcaagc tccaaattgt caactaagaa atggtcactt 2580
<210> 2
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggaagaatga acgaatgg 18
<210> 3
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
attacctatg aaggagaaga a 21
<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
<210> 6
<211> 21
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 6
uccauuagca gcauuuggca c 21
<210> 7
<211> 21
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gccaaaugcu gcuaauggac a 21
Claims (6)
1. detecting application of the reagent of long-chain non-coding RNA expression in preparation osteosarcoma auxiliary diagnosis product；The long-chain is non-
Coding RNA is LOC100130111, 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, 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. a kind of for treating the pharmaceutical composition of osteosarcoma, which is characterized in that described pharmaceutical composition includes claim 1-4
Any one of described in long-chain non-coding RNA inhibitor, the inhibitor be inhibit LOC10013011 expression RNA interfering.
6. application of the inhibitor described in claim 5 in the drug of preparation treatment osteosarcoma.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710725181.8A CN107312865B (en) | 2017-08-22 | 2017-08-22 | Purposes of the LOC100130111 in preparation osteosarcoma diagnostic products, therapeutic agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710725181.8A CN107312865B (en) | 2017-08-22 | 2017-08-22 | Purposes of the LOC100130111 in preparation osteosarcoma diagnostic products, therapeutic agent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107312865A CN107312865A (en) | 2017-11-03 |
| CN107312865B true CN107312865B (en) | 2019-06-11 |
Family
ID=60176428
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710725181.8A Active CN107312865B (en) | 2017-08-22 | 2017-08-22 | Purposes of the LOC100130111 in preparation osteosarcoma diagnostic products, therapeutic agent |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107312865B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108118093A (en) * | 2018-03-06 | 2018-06-05 | 谢琳 | Purposes of the LINC00426 in bone and flesh tumor metastasis diagnostic products are prepared |
| CN111424082A (en) * | 2019-01-09 | 2020-07-17 | 上海中医药大学附属龙华医院 | Application of lncRNA-SNHG6 gene in preparation of medicine for treating osteosarcoma |
| CN114627969A (en) * | 2022-03-23 | 2022-06-14 | 中国医学科学院肿瘤医院 | Application of complement-associated-gene-based prognosis prediction model and kit for sarcoma patient |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106191264A (en) * | 2016-07-21 | 2016-12-07 | 杨祚璋 | Osteosarcomatous miRNA diagnosis marker |
| CN106434864A (en) * | 2016-07-08 | 2017-02-22 | 吉林大学 | Tumor marker LIMK1 and application thereof |
-
2017
- 2017-08-22 CN CN201710725181.8A patent/CN107312865B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106434864A (en) * | 2016-07-08 | 2017-02-22 | 吉林大学 | Tumor marker LIMK1 and application thereof |
| CN106191264A (en) * | 2016-07-21 | 2016-12-07 | 杨祚璋 | Osteosarcomatous miRNA diagnosis marker |
Non-Patent Citations (5)
| Title |
|---|
| Homo sapiens uncharacterized LOC100130111 (LOC100130111), long non-coding RNA;GenBank;《GenBank Database》;20170316;Accession NO: NR_135221.1 |
| Overexpression of long non-coding RNA MFI2 promotes cell proliferation and suppresses apoptosis in human osteosarcoma;Yin Z等;《ONCOLOGY REPORTS》;20160811;第36卷(第4期);第2033-2040页 |
| 长链非编码RNA在骨肉瘤中表达的研究进展;顾宗欣;《实用骨科杂志》;20170430;第23卷(第4期);第336-339页 |
| 长链非编码RNA在骨肉瘤患者中的差异表达研究;李金平;《中国博士学位论文全文数据库 医药卫生科技辑》;20140215(第2期);摘要 |
| 长链非编码RNA对骨肉瘤的调控作用;郭健民等;《中国生物化学与分子生物学报》;20170531;第33卷(第5期);第456-460页 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107312865A (en) | 2017-11-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111004850B (en) | Application of circRNAs molecules in preparation of liver cancer diagnosis kit and kit applying molecules | |
| JP5209272B2 (en) | Liver cancer-related gene and method for determining liver cancer risk | |
| EP2910948B1 (en) | Novel cancer marker and utilization thereof | |
| CN107312865B (en) | Purposes of the LOC100130111 in preparation osteosarcoma diagnostic products, therapeutic agent | |
| US20210404001A1 (en) | Biomarker for diagnosing or predicting reactivity of ovary to fsh and use thereof | |
| CN107475386B (en) | Long-chain non-coding RNA marker for diagnosis and treatment osteosarcoma | |
| US20220228223A1 (en) | Squamous cell carcinoma diagnostic or prognosis prediction marker and use thereof | |
| CN107881239B (en) | miRNA marker related to colorectal cancer metastasis in plasma and application thereof | |
| CN108559779A (en) | Diagnosis and treatment marker of the long-chain non-coding RNA as gastric cancer | |
| CN109055555A (en) | A kind of lung cancer transfer diagnosis marker and its kit and application in early days | |
| CN108504658A (en) | Purposes of the LINC01836 in preparing diagnosing gastric cancer product, medicine | |
| CN107058480B (en) | Long-chain non-coding RNA marker for diagnosing adenocarcinoma of lung | |
| CN107586842A (en) | A kind of biomarker for clear cell carcinoma of kidney diagnosis and treatment | |
| CN112430665B (en) | Molecular biomarker for diagnosing and treating triple negative breast cancer and application thereof | |
| CN110317867A (en) | Application of the TARS as the molecular marker of diagnosis Male Osteoporosis | |
| CN106929577A (en) | A kind of lncRNA biomarker related to adenocarcinoma of lung | |
| CN109576371A (en) | Application of the long-chain non-coding RNA in adenocarcinoma of lung | |
| CN107365859B (en) | Molecular markers of the LncRNA as diagnosis and treatment osteosarcoma | |
| CN110093416A (en) | Application of the biomarker in diagnosis orthopaedic disease | |
| CN105603117B (en) | MiR-3613 is used to distinguish lung squamous cancer transfer and non-diverting miRNA marker | |
| CN105506169B (en) | Fibroid diagnosis and treatment marker | |
| CN107937514A (en) | Applications of the circZFY in pulmonary tuberculosis biomarker | |
| CN108841961B (en) | Application of the LINC01702 in the diagnostic kit of preparation hepatocellular carcinoma | |
| CN110042164B (en) | Lung cancer diagnosis and treatment lncRNA marker | |
| CN108165546A (en) | A kind of miRNA biomarker, composition and application thereof |
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 |