CN105506156B - Diagnose the molecular marker of osteosarcoma - Google Patents
Diagnose the molecular marker of osteosarcoma Download PDFInfo
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Abstract
The invention discloses purposes of 4753 3p of miR as osteosarcoma diagnosis marker.The present invention proves that contents of 4753 3p of miR in Patients with Osteosarcoma blood is reduced than the obvious of normal person by high-flux sequence and QPCR experiments, it is taken as that 4753 3p of miR can be as the molecular marker of diagnosis osteosarcoma.The studies above achievement according to the present invention, develops the diagnostic products for diagnosing osteosarcoma, which is clinically with a wide range of applications.
Description
Technical field
The invention belongs to biomedicine field, is related to purposes of the miR-4753-3p in osteosarcoma.
Background technology
MiRNA is naturally present in the non-coding RNA molecule of internal 21-22nt, is that one kind is sunk by posttranscriptional gene
The silent RNA that expression of target gene is adjusted.Regulated and controled it is estimated that there are about 1/3 gene in organism by miRNA.MiRNA with
The complex of RISC can be combined by base pairing with the complementary series in target gene mRNA5 '-UTR or 3 '-UTR, be suppressed
Protein translation, or trigger mRNA degradeds, so that the expression of negative regulation target gene.
The expression of detection miRNA can provide reference for the clinical diagnosis of disease.And the unconventionality expression of miRNA is direct
The abnormal expression for causing some that related gene occurs with disease, the generation to induce an illness.Having been reported proves that miRNA can pass through
Regulate and control the expression of target gene mRNA, play a significant role in disease occurs, develops and shifts.In future clinical treatment,
MiRNA can not only become new disease early diagnosis and the relevant label of disease process, and be expected to by varying
The expression treatment disease of the expression of miRNA or its target gene.Find and with disease relevant miRNA and its target base occur for identification
Because the clinical treatment of miRNA provides basis.
Osteosarcoma is derived from the malignant tumour of mesenchymal tissue, 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
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, cure 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 a kind of osteosarcoma early diagnosis marker is found to be a problem to be solved.
The content of the invention
It is an object of the present invention to provide a kind of Microrna label that can be used for early diagnosis osteosarcoma.
The second object of the present invention is the purposes for providing above-mentioned Microrna.
To achieve these goals, present invention employs following technical solution:
The present invention provides a kind of application of Microrna in osteosarcoma diagnostic tool is prepared, the Microrna be selected from
The following group:Initial miRNA, precursor miRNA, maturation miRNA;Initial miRNA can be sheared in people's cell and be expressed as maturation
miRNA;Precursor miRNA can be sheared in people's cell and be expressed as ripe miRNA;The Microrna is miR-4753-3p.
It should be known that the Microrna of the present invention includes the functional equivalent of composing type nucleic acid molecules, i.e. variation, it shows
The identical function of complete Microrna nucleic acid molecules, although they are dashed forward by the missing of nucleotide residue, displacement or insertion
Become.
Those skilled in the art, can be in one end of Microrna or two it should be appreciated that in order to ensure the stability of Microrna
End increase protectiveness base, such as TT, can also modify Microrna base, but above-mentioned modification does not influence the work(of Microrna
Energy.Therefore, those skilled in the art are known, and under conditions of miR-4753-3p functions are not influenced, miR-4753-3p is carried out
Base modification is also contained within protection scope of the present invention in the sequence that both ends increase base obtains.
In some specific embodiments of the present invention, the miR-4753-3p is ripe miR-4753-3p.
Although used in some specific embodiments is ripe miRNA, those skilled in the art can be pre-
Phase, initial miRNA, precursor miRNA will can obtain the technique effect same with maturation miRNA, because cell is had the ability into one
Initial miRNA, precursor miRNA are processed as ripe miRNA by step.
The Microrna nucleic acid molecules of the present invention can exist in the form of single-stranded or double-stranded.Ripe miRNA is mainly in single-stranded
Form, and precursor miRNA is that part is certainly complementary, to form duplex structure.The present invention nucleic acid molecules can be RNA, DNA,
The form of PNA, LNA.
Further, above-mentioned diagnostic tool includes but not limited to, chip, kit, test paper, high-flux sequence platform.It is described
Diagnostic tool includes being used for the reagent for detecting the expression of miR-4753-3p in sample.
Further, the source of the sample includes but not limited to blood, urine, tear, saliva, tissue fluid, cerebrospinal fluid, sweat
Liquid.In specific embodiments of the present invention, the source of the sample is blood.
Further, the kit includes the primer and/or probe for miR-4753-3p;The chip includes solid phase
Carrier;And the oligonucleotide probe on the solid phase carrier is fixed on, the oligonucleotide probe includes specifically corresponding to
In the part or all of sequence of miR-4753-3p;The test paper includes the primer and/or probe for miR-4753-3p;It is described
High-flux sequence platform includes the primer and/or probe for miR-4753-3p.
The present invention provides a kind of diagnostic tool of osteosarcoma, the diagnostic tool includes miR-4753- in detection sample
The reagent of 3p expressions.
Further, the source of the sample includes but not limited to blood, urine, tear, saliva, tissue fluid, cerebrospinal fluid, sweat
Liquid.In specific embodiments of the present invention, the source of the sample is blood.
Further, the diagnostic tool includes kit, chip, test paper, high-flux sequence platform.
Further, the kit includes the primer and/or probe for miR-4753-3p;The chip includes solid phase
Carrier;And the oligonucleotide probe on the solid phase carrier is fixed on, the oligonucleotide probe includes specifically corresponding to
In the part or all of sequence of miR-4753-3p;The test paper includes the primer and/or probe for miR-4753-3p;It is described
High-flux sequence platform includes the primer and/or probe for miR-4753-3p.
Further, the primer and/or probe for miR-4753-3p in the kit may also include for existing skill
In art it has been reported that can be used for detect the primer and/or probe of foregoing microrna expression level.Will be a variety of small
The detection primer and/or probe of RNA is placed in same reagent box by detecting a variety of Microrna index Combining diagnosis osteosarcoma
Situation be also contained within protection scope of the present invention.
Further, the oligonucleotide probe fixed on the chip may also include in the prior art it has been reported that
Can be used for detection miR-4753-3p expression oligonucleotide probe.The detection probe of a variety of miRNA is placed on together
It is also contained on one chip by detecting a variety of miRNA indexs Combining diagnosis osteosarcoma within protection scope of the present invention.
Further, the solid phase carrier includes the various common used materials that the solid phase carrier can use genetic chip field,
Such as, but not limited to nylon membrane, slide or silicon chip through active group (such as aldehyde radical, amino) modification, unmodified slide, modeling
Tablet etc..
The miRNA chips prepare the common manufacturing method that can use biochip known in the art, for example, such as
Fruit solid phase carrier is gone here and there using modification slide or silicon chip, 5 ' ends of probe containing amido modified poly- dT, can be by oligonucleotides
Probe is configured to solution, then using point sample instrument by its point modification slide or silicon chip on, be arranged in predetermined sequence or array,
Then fixed by standing overnight, so that it may obtain the miRNA chips of the present invention.If nucleic acid is without amido modified, its system
Preparation Method can also refer to:Wang Shenwu chief editors'《Gene diagnosis technology-on-radiation operation manual》;J.L.erisi,
V.R.Iyer, P.O.BROWN.Exploring the metabolic and genetic control of gene
Expression on a genomic scale.Science, 1997;278:680 and Ma Li people, Jiang Zhonghua chief editor's biology cores
Piece Beijing:Chemical Industry Press, 2000,1-130.
The miR-4753-3p of the present invention can be natural or artificial synthesized, or use can express miR-
The carrier transfectional cell of the DNA fragmentation of 4753-3p obtains.The carrier includes viral vector, eukaryotic vector.
Viral vector can be any appropriate carrier, include but not limited to retroviral vector, adenovirus vector, gland
Viral related viral vectors, herpesviral (such as herpes simplex virus, vaccinia virus and Epstein-Barr virus) carrier, alphavirus vectors.
Carrier for expression of eukaryon can be any appropriate expression vector, include but not limited to pCMV-Myc expression vectors,
PcDNA3.0 expression vectors, pcDNA3.1 expression vectors, pEGFP expression vectors, pEF Bos expression vectors, pTet expression vectors,
PTRE expression vectors or engineered carrier on the basis of known expression vector, such as pBin438, pCAMBIA1301
Deng.
The DNA fragmentation of Microrna, which can be expressed, to be obtained in the following way:(the http from miRNA databases://
Microrna.sanger.ac.uk/sequences/ Microrna position in the genome and particular sequence information, root) are found
The position of initial miRNA is determined according to genome sequence, is designed in the upstream and downstream 500-800bp sections of initial miRNA positions special
Specific primer, the sequence among amplimer can obtain the DNA fragmentation of expression Microrna.
" Microrna " and " miRNA ", " miR " used in the present invention is general.
" the diagnosis osteosarcoma " used in the present invention includes the anticipation to osteosarcoma, that is, judges that subject whether there is and suffer from
The risk of osteosarcoma, also includes the diagnosis to osteosarcoma, that is, judges whether subject suffers from osteosarcoma, also include to bone and flesh
The judgement of knurl prognosis, that is, judge subject with the presence or absence of the possibility of recurrence or judge that subject has been recurred.
The advantages of the present invention:
Present invention firstly discovers that miR-4753-3p is related to osteosarcoma, by detecting subject
The expression of miR-4753-3p, it can be determined that whether subject suffers from osteosarcoma or judge that subject whether there is
Risk with osteosarcoma, so as to instruct clinician to provide prevention scheme or therapeutic scheme to subject.
It is small to examine compared to traditional detection means present invention finds a kind of new molecular marked compound-miR-4753-3p
It is disconnected more timely, more special, sensitiveer, the early diagnosis of osteosarcoma can be realized, so as to reduce the death rate of osteosarcoma.
Brief description of the drawings
Fig. 1 is shown detects miR-4753-3p in normal person and Patients with Osteosarcoma blood using high-flux sequence method
Content;
Fig. 2 shows the content in normal person and Patients with Osteosarcoma blood using QPCR detections miR-4753-3p.
Embodiment
The present invention is specifically described below by embodiment, it is necessary to which indicated herein is that following embodiments are only used
It is further described in the present invention, it is impossible to be interpreted as limiting the scope of the invention, person skilled in art can
Some nonessential modifications and adaptations are made to the present invention according to the invention described above content.In following embodiments, if not specially
Show, reagent used is that analysis is pure, and agents useful for same can be obtained from commercial channel.The experiment of actual conditions is not specified in text
Method, what the Science Press usually write according to normal condition such as J. Pehanorm Brookers etc. published for 2002《Molecular cloning is real
Test guide》Condition described in one book, or according to the condition proposed by manufacturer.Unless otherwise defined, the institute used in text
There are professional and scientific terms to have the same meanings as commonly understood by one of ordinary skill in the art.In addition, it is any similar to described content or
Impartial method and material all can be applied in the present invention.
1 high-flux sequence of embodiment screens and the relevant miRNA of osteosarcoma
1st, sample collection
6 primary Patients with Osteosarcoma and 6 normal persons.Subject requires at least 12h on an empty stomach, in m seq 7:00~
8:00 at room temperature, extracts 10ml venous blood in ethylenediamine tetra-acetic acid (EDTA) anticoagulant tube, extracts peripheral blood mononuclear cells
PBMCs, adds 1ml Trizol reagents (Invitrogen companies), fully mixes, and -80 DEG C of preservation samples, carry for RNA
Take.All blood samples and pathological examination should be true and reliable, study and ratify through Ethics Committee, patient's informed consent.
2nd, blood mononuclear cell RNA is extracted
Cell in step (1) is melted, adds the chloroform of about 1/5 volume, turns upside down and fully mixes 1 minute or so,
Stand 5 minutes at room temperature.4 DEG C, careful transfer supernatant enters new 1.5ml centrifuge tubes after 12,000rpm centrifugations 15 minutes, adds
Isometric isopropanol, gently overturns and mixes, be stored at room temperature 10 minutes.4 DEG C, 12000rpm is centrifuged after ten minutes, removes supernatant, to
70% ethanol of 2/5 volume is added in precipitation, 4 DEG C, 12000rpm centrifuge washings precipitate 5 minutes.Supernatant is removed, precipitation room temperature is dried in the air
The water without RNase in right amount is added after dry fully to dissolve, and measures OD260 and OD280 values.Using the DNA enzymatic I processing of no RNase,
QIAGEN RNeasy kits total serum IgEs, detailed operating principle and method are shown in kit specification.
3rd, the purity analysis of RNA sample
NanoDrop1000 spectrophotometers detect RNA sample, sample purity requirement:OD260/280≧1.8.
4th, the quality analysis (2100 Bioanalyzer of Agilent Technologies) of RNA sample
2100 Bioanalyzer of Agilent Technologies detect RNA sample quality, observation 28S rRNA and
18S rRNA master tapes are obvious, cDNA texts are sequenced without degraded, the RNA-seq that meets that RNA Perfection Index is qualified, concentration reaches requirement
The requirement of storehouse structure, can be used for library construction and sequencing.28S/18S≧1;RNA integralities:RIN Zhi≤7.0.
5th, sRNA library constructions
Experiment recycles length 18- using Illumina TruSeq Small RNA kits structure library in total serum IgE
30nt RNA, by RT-PCR reverse transcriptions into singly-bound cDNA, after cDNA amplifications, recycle cDNA products, build up tiny RNA s libraries.
6th, it is sequenced
MiRNA is sequenced with llumina Hiseq2500/Miseq second generation high throughput sequencing technologies, by going
Connector, the process such as go low quality, depollute complete the processing of data, obtains final data.
7th, interpretation of result
Progress t-test obtains P values after background correction is carried out to miRNA initial data by transcript profile Data Analysis Software,
Then examined using Fisher and merge P values, screening differential expression miRNA.P values < 0.01 is set, miR-4753-3p is filtered out and exists
Expression is had differences in normal person and Patients with Osteosarcoma, the level of miR-4753-3p is decreased obviously in Patients with Osteosarcoma blood
(P < 0.05) (Fig. 1).
2 QPCR of embodiment verifies the miR-4753-3p of differential expression
1st, large sample QPCR verifications are carried out according to high-flux sequence result selection miR-4753-3p in embodiment 1.According to reality
Apply the sample collection mode in example 1 and select normal person and each 40 of Patients with Osteosarcoma, separation and the guarantor for carrying out blood mononuclear cell
Deposit.
2nd, RNA extraction process is the same as embodiment 1.
3rd, reverse transcription:By the total serum IgE template of 10pg-1 μ g and 2 μ l 10* buffer solutions, 2 μ l dATP (10mM), 0.5 μ l
PolyA polymerases, 0.5 μ l ribalgilases (RNase) inhibitor and deoxyribonuclease water (RNase free water) are mixed
Close, volume is finally 20 μ l, 37 DEG C of incubation 1h.Then 1 μ l, 0.5 μ g/ μ l Oligo (dT) specificity RT are added in reaction tube to draw
Thing, 70 DEG C be incubated 5min after be incubated at least 2min on ice at once, interrupt the secondary structure of RNA and primer.Finally, by above-mentioned 20 μ l
Reaction mixture and 4 μ l 5* buffer solutions, 1 μ l dNTP (10mM), 0.5 μ l M-MLV reverse transcriptases, 0.5 μ l ribalgilases
(RNase) inhibitor, 10 μ l polyA reaction mixtures and the mixing of 4 μ l deoxyribonucleases water (RNase free water),
42 DEG C of incubation 1h.
4th, QPCR reacts:Using 25 μ l reaction systems, each sample sets 3 parallel pipes, and all amplified reactions repeat
To ensure the reliability of result more than three times.Prepare following reaction system:12.5 μ of SYBR Green PCRs system
L, 1 μ l of forward primer (5 μM/μ l), reverse primer (5 μM/μ l) 1 μ l, template cDNA2.0 μ l, no 8.5 μ l of enzyme water.Operations
Carried out on ice.Amplification program is:95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 55s) * 45 circulations.Using SYBR Green as
Fluorescent marker, carries out PCR reactions on Light Cycler fluorescence real-time quantitative PCR instrument.Expand the forward direction of miR-4753-3p
Primer sequence is:5 '-TTCTCTTTCTTTAGCCTTGTGT-3 ' (SEQ ID NO.1), reverse primer are general reverse primer
(being purchased from Beijing Quanto Biotechnology Co., Ltd.).Using snRNA U6 as reference gene, its upstream primer sequence is:5’-
CTCGCTTCGGCAGCACA-3’(SEQ ID NO.2);Downstream primer sequence is:5’-AACGCTTCACGAATTTGCGT-3’
(SEQ ID NO.3).Purpose band is determined by melt curve analysis analysis and electrophoresis, Δ Δ CT methods carry out relative quantification.
5th, result
As shown in Fig. 2, compared with normal person, the expression of miR-4753-3p is remarkably decreased in Patients with Osteosarcoma blood,
(P < 0.05) consistent with high-flux sequence result.
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, 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.
Claims (6)
1. application of the Microrna in osteosarcoma diagnostic tool is prepared, it is characterised in that the Microrna is selected from the following group:Just
Beginning miRNA, precursor miRNA, maturation miRNA;Initial miRNA can be sheared in people's cell and be expressed as ripe miRNA;Precursor
MiRNA can be sheared in people's cell and be expressed as ripe miRNA;The Microrna is miR-4753-3p.
2. application according to claim 1, it is characterised in that the Microrna is ripe
miR-4753-3p。
3. application according to claim 1, it is characterised in that the diagnostic tool includes claim 1 in detection sample
The reagent of the microrna expression level.
4. application according to claim 3, it is characterised in that the source of the sample includes blood, urine, tear, saliva
Liquid, tissue fluid, cerebrospinal fluid, sweat.
5. according to the application any one of claim 1-4, it is characterised in that the diagnostic tool includes kit, core
Piece, test paper or high-flux sequence platform.
6. application according to claim 5, it is characterised in that the kit is included for micro- described in claim 1
The primer and/or probe of tiny RNA;The chip includes solid phase carrier, and the oligonucleotides being fixed on the solid phase carrier
Probe, the oligonucleotide probe include specifically corresponding to the part or all of sequence of the Microrna described in claim 1
Row;The test paper includes the primer and/or probe for the Microrna described in claim 1;The high-flux sequence platform bag
Include the primer and/or probe for the Microrna described in claim 1.
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CN105821131B (en) * | 2016-04-28 | 2019-05-24 | 中南大学湘雅医院 | Osteosarcoma miRNA marker |
CN106148535A (en) * | 2016-07-29 | 2016-11-23 | 北京泱深生物信息技术有限公司 | The miRNA 4512 application in osteosarcoma diagnoses |
CN106011294A (en) * | 2016-07-29 | 2016-10-12 | 北京泱深生物信息技术有限公司 | Use of miRNA in diagnosis on chondroblast type osteosarcoma |
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