CN105112553B - Application of the miRNA-924 in cirrhosis diagnosis and treatment - Google Patents
Application of the miRNA-924 in cirrhosis diagnosis and treatment Download PDFInfo
- Publication number
- CN105112553B CN105112553B CN201510629683.1A CN201510629683A CN105112553B CN 105112553 B CN105112553 B CN 105112553B CN 201510629683 A CN201510629683 A CN 201510629683A CN 105112553 B CN105112553 B CN 105112553B
- Authority
- CN
- China
- Prior art keywords
- mirna
- expression
- cirrhosis
- reagent
- application
- 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
-
- 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
- C12Q2600/158—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
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Pathology (AREA)
- Molecular Biology (AREA)
- Pharmacology & Pharmacy (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Veterinary Medicine (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses application of the miRNA-924 in cirrhosis diagnosis and treatment, and the expression by detecting miRNA-924 can be used for diagnosing whether patient suffers from cirrhosis.The invention discloses miRNA-924 to prepare the application in diagnostic kit for liver cirrhosis, also discloses application of the miRNA-924 in the drug of preparation treatment cirrhosis.
Description
Technical field
The invention belongs to biomedicine field, it is related to a kind of application of miRNA-924 in cirrhosis diagnosis and treatment.
Background technique
Cirrhosis is clinical common chronic progressive hepatopathy, is various cause of disease long terms and the diffusivity liver damage formed
Evil, is the terminal stage of a variety of chronic liver disease progression of the disease, and main pathogenesis is the proliferation of hepatic stellate cells, activation.Liver
Onset concealment is hardened, progression of the disease is slow, and late complication is more, and poor prognosis, case fatality rate are high.The liver after most of China is hepatitis
Hardening, small part are alcoholic cirrhosis and Cirrhosis In Schistosomiasis.
MiRNA is naturally present in the non-coding RNA molecule of intracorporal 21-22nt, is a kind of heavy by posttranscriptional gene
The silent RNA that expression of target gene is adjusted.It is estimated that in organism, there are about 1/3 genes by the regulation of 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 inhibited
Protein translation, or cause mRNA degradation, thus the expression of negative regulation target gene.
The expression of detection miRNA can provide reference for the clinical diagnosis of cancer.And the unconventionality expression of miRNA is direct
Lead to some abnormal expressions that related gene occurs with disease, induces the generation of cancer.Having been reported proves that miRNA can pass through
The expression for regulating and controlling target gene mRNA, plays an important role in cirrhosis.Present invention firstly discovers that miRNA-924's suffers from cirrhosis
Expression quantity in person is well below normal group of control, and the abnormal expression of miRNA-924 and the occurrence and development of cirrhosis are related, this is
The clinical treatment of the miRNA of cirrhosis provides the foundation.
Summary of the invention
In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide miRNA-924 in liver cirrhosis diagnosis and to control
Application in treatment.Compared to the diagnostic method of traditional cirrhosis, diagnosed using miRNA cirrhosis with timeliness, sensitivity,
Specificity, so that patient be made to understand the state of an illness, and then take corresponding prevention and treatment measure.
To achieve the goals above, present invention employs following technical solutions:
The present invention provides application of the miRNA-924 in the product of preparation diagnosis cirrhosis, the miRNA-924 is selected from
With at least one of the following group: initial miRNA, miRNA-924 precursor miRNA of miRNA-924, maturation miRNA-924；It is described
The initial miRNA of miRNA-924 can be sheared in people's cell and be expressed as mature miRNA-924；The miRNA-924 precursor
MiRNA can be sheared in people's cell and be expressed as mature miRNA-924.
Further, the miRNA-924 is mature miRNA-924, the SEQ ID NO.1 in nucleotide sequence such as sequence table
It is shown.
It should be known that miRNA-924 of the invention includes the functional equivalent of composing type nucleic acid molecules, i.e. variant, show
Show the identical function of complete miRNA-924 nucleic acid molecules, they may pass through the missing of nucleotide residue, displacement or insertion
Mutation.
Those skilled in the art are known, in order to guarantee the stability of miRNA, can increase in the one or both ends of miRNA and protect
Shield property base, such as TT, can also modify miRNA base, but not influence the function of miRNA.Therefore, those skilled in the art
Member is known, under conditions of not influencing miRNA-924 function, carries out base modification to miRNA-924 or increases alkali at both ends
The sequence that base obtains is also contained within protection scope of the present invention.
Although being mature miRNA-924 used in some specific embodiments, those skilled in the art can
With expection, initial miRNA (pri-miRNA-924), precursor miRNA (pre-miRNA-924) can be obtained and maturation
The same technical effect of miRNA-924, because cell is had the ability further by initial miRNA (pri-miRNA-924), precursor
MiRNA (pre-miRNA-924) is processed as mature miRNA-924.
MiRNA-924 nucleic acid molecules of the invention can exist in single-stranded or double-stranded form.Mature miRNA-924 master
It to be in single stranded form, and miRNA-924 precursor is part from complementation, to form duplex structure.Nucleic acid molecules of the invention can
In the form of being RNA, DNA, PNA, LNA.
The present invention provides application of the miRNA-924 in high-flux sequence platform.By high-flux sequence can know to
The expression of miRNA-924 easily determines to be measured by the result of sample to be tested compared with the sample of Healthy People in detection sample
Whether sample suffers from cirrhosis.Therefore, the occurrence and development of miRNA-924 expression and cirrhosis are known by high-flux sequence
Relevant application is also contained within protection scope of the present invention.
The present invention provides a kind of product for diagnosing cirrhosis, the product can pass through the expression of detection miRNA-924
Level diagnoses cirrhosis.
Further, product recited above includes chip or kit；Wherein, the chip includes solid phase carrier；And
The oligonucleotide probe being fixed on the solid phase carrier, the oligonucleotide probe include specifically corresponding to described above
Some or all of miRNA-924 sequence.The kit includes the expression water for detecting miRNA-924 recited above
Flat reagent, the reagent of the detection miRNA-924 expression include the primer and/or probe for miRNA-924.
Further, oligonucleotide probe recited above may also include in the prior art it has been reported that can be used for examining
The oligonucleotide probe of the miRNA of disconnected cirrhosis.The detection probe of a variety of miRNA is placed more by detecting on the same chip
The case where kind miRNA index Combining diagnosis cirrhosis, is also contained within protection scope of the present invention.Reagent described above also wraps
Include in the prior art it has been reported that diagnosis cirrhosis miRNA primer and/or probe.The detection of a variety of miRNA is drawn
Object and/or probe are placed in same reagent box also includes by the case where detection a variety of miRNA index Combining diagnosis cirrhosis
Within protection scope of the present invention.
Further, the solid phase carrier includes the various common used materials that genetic chip field can be used in the solid phase carrier,
Such as, but not limited to nylon membrane, slide or silicon wafer through active group (such as aldehyde radical, amino) modification, unmodified slide, modeling
Tablet etc..
The conventional manufacturing method of biochip known in the art can be used in the preparation of the miRNA chip, for example, such as
Fruit solid phase carrier is gone here and there using modification slide or silicon wafer, 5 ' ends of probe containing amido modified poly- dT, can be by oligonucleotides
Probe is configured to solution, then uses point sample instrument that its point on modification slide or silicon wafer, is arranged in scheduled sequence or array,
Then it is fixed by standing overnight, so that it may obtain miRNA chip of the invention.If nucleic acid is without amido modified, system
Preparation Method can also refer to: " the gene diagnosis technology-on-radiation operation manual " of Wang Shenwu chief editor；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 edit biology core
The Beijing piece: Chemical Industry Press, 2000,1-130.
The present invention provides application of the miRNA-924 recited above in preparation treatment cirrhosis drug.
Further, the drug includes the reagent for promoting the miRNA-924 expression or enhancing miRNA-924 function.Institute
It states and the target of the reagent of miRNA-924 expression or enhancing miRNA-924 function is promoted to be not limited to miRNA-924 itself, further include
The upstream and downstream of miRNA-924, such as: the genome sequence of coding miRNA -924, miRNA-924 target gene, regulation miRNA-
924 albumen or gene.
Further, to promote the reagent of miRNA-924 expression or enhancing miRNA-924 function include albumen, oligonucleotides, small
Molecular compound.
Preferably, the reagent be the analogies of miRNA-924, the agonist of miRNA-924, precursor miRNA -924,
Carry the carrier of miRNA-924.
Go out its analogies or agonist according to miRNA-924 sequence design, the analogies of miRNA-924 are chemical syntheses
Tiny RNA, the agonist of miRNA-924 is the double-strand tiny RNA by special marking and chemical modification, and miRNA-924 is simulated
After object or agonist transfer into the human body, they can obviously raise the expression of miRNA-924.
The present invention provides a kind of drug for treating cirrhosis, the drug includes promotion miRNA-924 recited above
The reagent of expression or enhancing miRNA-924 function.
The present invention also provides the reagents of promotion miRNA-924 expression recited above or enhancing miRNA-924 function to exist
Application in preparation treatment cirrhosis drug.
MiRNA-924 of the invention can be natural or artificial synthesized, or use can express miRNA-924
DNA fragmentation carrier transfection cell obtain.The carrier includes viral vectors, carrier for expression of eukaryon.
Viral vectors can be any carrier appropriate, including but not limited to retroviral vector, adenovirus vector, gland
Viral related viral vectors, herpesviral (such as herpes simplex virus, vaccinia virus and Epstein-Barr virus) carrier, alphavirus vectors.
Carrier for expression of eukaryon can be any expression vector appropriate, including but not limited to pCMV-Myc expression vector,
PcDNA3.0 expression vector, pcDNA3.1 expression vector, pEGFP expression vector, pEF Bos expression vector, pTet expression vector,
PTRE expression vector or modified carrier on the basis of known expression vector, such as pBin438, pCAMBIA1301
Deng.
The DNA fragmentation that miRNA-924 can be expressed can obtain in the following way: from miRNA database
(http://microrna.sanger.ac.uk/sequences/) finds the position in the genome miRNA-924 and specific
Sequence information determines the position of the initial miRNA of miRNA-924 according to genome sequence, in the initial position miRNA miRNA-924
The section upstream and downstream 500-800bp in design specific primer, the sequence among amplimer can be obtained expression miRNA-924
DNA fragmentation.
The drug for the treatment of cirrhosis of the invention also includes pharmaceutically acceptable carrier, and the carrier includes but not
It is limited to: diluent, buffer, suspension, emulsion, granule, encapsulation agents, excipient, filler, adhesive, spray, transdermal
Absorbent, wetting agent, disintegrating agent, sorbefacient, surfactant, colorant, corrigent or absorption carrier.
Including but not limited to microinjection agent, the dosage form suitable for transfection, injection, tablet, powder can be made in the drug
Agent, granula, capsule.The drug of above-mentioned various dosage forms can be prepared according to the conventional method of pharmaceutical field.
The drug that the drug can be administered alone or be able to suppress cirrhosis with other is combined application.
The drug can be applied in vitro: by miRNA-924 analogies, the agonist of miRNA-924, precursor miRNA-
The expression vector of 924 or miRNA-924 imports or transfects in vitro human body itself or variant cell (or heterogenous cell), through body
After outer cell amplification, defeated the Huis' body.
The drug can be applied in vivo: by miRNA-924 analogies, the agonist of miRNA-924, precursor miRNA-
The expression vector of 924 or miRNA-924 is introduced directly into vivo.This carrier can be virus type or non-viral, even
Naked DNA or RNA.
The subject can be the mankind or other mammals.More specifically, subject be organ, it is tissue, thin
Born of the same parents.
Nucleic acid molecules of the invention can be the form of RNA, DNA, PNA, LNA.
The advantages of the present invention:
Present invention firstly discovers that miRNA-924 expression is to the occurrence and development of cirrhosis related, it is tested by detecting
The expression of person miRNA-924, it can be determined that whether subject suffers from cirrhosis, so that clinician be instructed to mention to subject
For prevention scheme or therapeutic scheme.
Detailed description of the invention
Fig. 1 shows the expression using QPCR detection miRNA-924 in liver cirrhosis patient biopsy；
Fig. 2 shows the facilitation that miRNA-924 analogies express miRNA-924.
Specific embodiment
Below with reference to specific embodiment further illustrate the present invention, the embodiment of the present invention for explaining only the invention,
It is not intended to limit protection scope of the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The screening of the miRNA relevant to cirrhosis of embodiment 1
1, sample acquisition: the biopsy sample of each biopsy for collecting 10 Healthy Peoples and liver cirrhosis patient.It is above-mentioned
The acquirement of all samples passes through the agreement of the committee, organizational ethics.
2, the extraction of sample total serum IgE
Shift to an earlier date total serum IgE using the tissue RNA extracts kit of QIAGEN company.Specific step is as follows:
1) in the clear area of less RNase interference, biopsy sample about 20mg is weighed using the mortar containing appropriate liquid nitrogen,
It is ground to pestle powdered；
2) sample is transferred in the centrifuge tube of a 2ml without RNA enzyme；
3) 300 μ l Lysis solution are added, is placed in homogenizer, is fully ground 1-5min；
4) 12000g, is centrifuged 10min by 4 DEG C, shifts supernatant into the centrifuge tube of new 1.5ml；
5) 600 μ l RNase-Free Water are added, are mixed with vortex device；
6) 20 μ l Proteinase Ks are added, the warm bath 15min in 55 DEG C of water-baths is constantly vortexed and mixes；
7) 14000g, room temperature are centrifuged 1min, make pellet cell debris in centrifugation bottom of the tube, supernatant is taken to be transferred to other one
In a centrifuge tube without RNA enzyme 1.5ml；
8) 95% ethyl alcohol of 450 μ l is added, is vortexed and mixes；
9) 650 lysates of the μ l containing ethyl alcohol are taken to be added in centrifugal column, 14000g is centrifuged 1min；Lower layer is abandoned, again by pillar
It is placed in collecting pipe；
10) according to the capacity of lysate, step 9) is repeated；
11) 400 μ l Wash solution, 14000g are added and are centrifuged 2min；Lower layer is abandoned, column is placed in a new collecting pipe
In；
12) 100 μ l Enzyme Incubation Buffer are added and 15 μ l DNase I, 14000g are centrifuged 1min, it will
Solution in collecting pipe moves into column again, is placed at room temperature for 15min；
13) 400 μ l Wash solution, 14000g are added and are centrifuged 1min, abandons lower layer, pillar is replaced in collecting pipe
In；
14) 400 μ l Wash solution, 14000g are added and are centrifuged 2min, abandons collecting pipe, pillar is put into 1.7ml
In Elution pipe；
15) 30 μ l Elution Buffer, 200g centrifugation 2min are added, make solution sufficiently in conjunction with column；
16) 14000g is centrifuged 1min, by RNA use without RNA deionized water dissolving, for use.
3, the quality analysis (NanoDrop1000 spectrophotometer) of RNA sample
NanoDrop1000 spectrophotometer detects RNA sample, the sample requirement of RNA-seq sequencing, and: OD260/OD280 is
1.8-2.2。
The RNA of said extracted is subjected to agarose gel electrophoresis, Agilent Technologies
2100Bioanalyzer detects RNA sample quality, observes, takes pictures in gel imager, saves image, it is considered that 28S:
It 18S >=2 can be preferable with preliminary judgement total serum IgE quality.
4, the extraction of miRNA and label
1) miRNA is obtained with the miRNAs extraction agent box extracting of Ambion company, concrete operations are according to corresponding instructions.
Sample is with T4RNA ligase markers step according to the method for Thomson.MiRNA labeling method is approximately as 1.4 μ g miRNA
With 500ng 5 '-phosphate-cytimidine-uracil cy3-3 ' (Dharmacon, Chicago, USA) and 2 unit T4RNA
Ligase (NEB, Ipswich, USA) is incubated for 2 hours in 4 DEG C.Every part of miRNA sample is all provided with the corresponding negative control of equivalent.
2) mark RNA precipitated with the ethyl alcohol of 0.3M sodium acetate and 2.5 times of volumes, then with 15 μ l contain 3 × SSC,
The hybridization solution of 0.2%SDS and 15% formamide is resuspended, and all hybridization is repeated twice, hybridization LifterSlipTM (Erie,
PA USA) to guarantee hybridization solution Uniform Flow between chip and cover plate.
3) hybridization chamber is placed on hybridization instrument BioMixerTMII (CapitalBio Corp, Beijing, China) in 42
DEG C water-bath is stayed overnight, and is washed twice with washing lotion.
5, miRNA chip operation:
MiRNA chip, using the miRNA chip of expression spectrum (single channel chip) of Boao Biological Co., Ltd, according to explanation
The instruction of book carries out the detection of miRNA express spectra.
6, result:
Analyze the testing result of miRNA chip expression spectrum, it is known that miRNA-924 is in liver cirrhosis patient biopsy and strong
There are significant differences for expression in the biopsy of health people, compared with the biopsy of Healthy People, in cirrhosis biopsy
The horizontal of miRNA-924 significantly reduces.
The miRNA-924 of 2 QPCR of embodiment verifying differential expression
1, miRNA-924 is selected to carry out large sample QPCR verifying according to the testing result of miRNA chip.According to embodiment 1
In sample collection mode select liver cirrhosis patient biopsy sheet and each 80, Healthy People biopsy sample.
2, RNA extraction process is the same as embodiment 1.
3, reverse transcription:
1) 2 μM of miRNA reverse transcriptase primers of the total serum IgE template of 2 μ g and 1 μ l, 1 μ l, 2 μM of U6snRNA reverse transcriptions are drawn
Object, 1 μ l dNTP (10mM) mixture and the deionized water mixing without RNase, final volume are 20 μ l.After mixing, it is incubated in 65 DEG C
5min。
2) it cools down on ice immediately.Sequentially add following component: 45 × buffers of μ l, 2 μ l DTT (0.1M), 1 μ l core
Ribonuclease T. (RNase) inhibitor, solution is mixed, 37 DEG C of incubation 5min.
3) 1 μ l M-MLV reverse transcriptase, 37 DEG C of reaction 1h after mixing is added.
4) be placed in 70 DEG C, 15min, terminate reaction, -20 DEG C freeze it is spare.In miRNA reverse transcriptase primer sequence such as sequence table
Shown in SEQ ID NO.2, U6snRNA reverse transcriptase primer sequence is as shown in SEQ ID NO.3 in sequence table.
4, QPCR reacts:
Using 25 μ l reaction systems, 3 parallel pipes are arranged in each sample, and all amplified reactions are repeated three times above to protect
Demonstrate,prove the reliability of result.Prepare following reaction system: 12.5 μ l of SYBR Green polymerase chain reaction system, forward primer (5
μM) 1 μ l, reverse primer (5 μM) 1 μ l, template cDNA 2.0 μ l, no 8.5 μ l of enzyme water.Operations are carried out on ice.Amplification
Program are as follows: 95 DEG C of 10min, (95 DEG C of 20s, 56 DEG C of 55s) × 50 circulations.Using SYBR Green as fluorescent marker,
PCR reaction is carried out on Light Cycler fluorescence real-time quantitative PCR instrument.Expand the forward primer sequence of miRNA-548a-3p such as
Shown in SEQ ID NO.4, reverse primer is as shown in SEQ ID NO.5.Using U6snRNA as reference gene, upstream primer
Sequence is shown in SEQ ID NO.6；Downstream primer sequence is shown in SEQ ID NO.7.It is true by melt curve analysis analysis and electrophoresis
Determine purpose band, Δ Δ CT method carries out relative quantification.
5, result
As shown in Figure 1, the expression of miRNA-924 is aobvious in liver cirrhosis patient tissue compared with Healthy People tissue samples
Writing reduces, consistent with miRNA chip results.
Embodiment 3 studies influence of the miRNA-924 to cirrhosis ability of cell proliferation
1, design synthesis is directed to the analogies of miRNA-924
MiRNA-924 is synthesized by the design of Dalian treasured biotinylated biomolecule Technology Co., Ltd. according to the sequence information of miRNA-924
Analogies and random controls sequence.
2, cell culture
By DMEM high glucose medium of the hepatic stellate cells LX-2 containing 10% fetal calf serum, in 37 DEG C, 5%CO2Culture
It is cultivated in case.
3, cell transfecting
LX-2 cell is divided into two groups, respectively control group, miRNA-924 analogies group.By control group and analogies group
Random controls sequence and miRNA-924 analogies are transfected respectively, with 2000 turns of transfection reagent LipofectamineTM
Dye, transfection method is referring to specification.Control group and the working concentration of miRNA-924 analogies group are 5 μM.48h is received after transfection
Collect group of cells and is used for subsequent experimental.
4, QPCR is tested
1) cell total rna extracts: the extraction of cell total rna is carried out using the RNA extracts kit of QIAGEN company, according to
Specification instruction carries out.
2) QPCR: step is the same as embodiment 2.
As a result as shown in Fig. 2, compared with the control group, on the level of the miRNA-924 of miRNA-924 analogies group is significant
It rises, shows that miRNA-924 analogies can effectively facilitate the expression of miRNA-924.
5, cell proliferation experiment
The LX-2 cell for transfecting 48h is digested to cell suspension using 0.25% pancreatin, with 5 × 104A/ml is inoculated in 96
Porocyte culture plates, after every hole 0.1ml, 60min, mtt assay surveys each hole 490nm wavelength absorbance value.With absorbance value size generation
The relative populations of table living cells.
6, result
MiRNA-924 analogies group relative optical density number is 0.342 ± 0.021, and control group relative optical density number is 1.317
±0.023.Compared with the control group, miRNA-924 analogies group absorbance value is remarkably decreased (P < 0.05).Above-mentioned experimental result table
Bright miRNA-924 analogies can significantly inhibit LX-2 cell Proliferation, while show that miRNA-924 is unfavorable for the increasing of LX-2 cell
It grows.
The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.
Claims (4)
1. detecting application of the reagent of miRNA-924 expression in the product of preparation diagnosis cirrhosis, which is characterized in that described
MiRNA-924 is selected from at least one of the following group: initial miRNA, miRNA-924 precursor miRNA of miRNA-924, maturation
miRNA-924；The initial miRNA of miRNA-924 can be sheared in people's cell and be expressed as mature miRNA-924；It is described
MiRNA-924 precursor miRNA can be sheared in people's cell and be expressed as mature miRNA-924.
2. application according to claim 1, which is characterized in that the miRNA-924 is mature miRNA-924.
3. application according to claim 1, which is characterized in that the product includes chip or kit；Wherein, the core
Piece includes solid phase carrier；And it is fixed on the oligonucleotide probe on the solid phase carrier；The oligonucleotide probe includes spy
Correspond to some or all of miRNA-924 described in claim 1 sequence anisotropicly；The kit includes being used for right to examin
Benefit require 1 described in miRNA-924 expression reagent；The reagent includes being directed to miRNA-924 described in claim 1
Primer and/or probe.
4. promoting the reagent of miRNA-924 expression described in claim 1 or enhancing miRNA-924 function hard in preparation treatment liver
Application in chemical drug object, which is characterized in that the reagent include: the analogies of miRNA-924, miRNA-924 precursor, take
Carrier with miRNA-924.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510629683.1A CN105112553B (en) | 2015-09-28 | 2015-09-28 | Application of the miRNA-924 in cirrhosis diagnosis and treatment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510629683.1A CN105112553B (en) | 2015-09-28 | 2015-09-28 | Application of the miRNA-924 in cirrhosis diagnosis and treatment |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105112553A CN105112553A (en) | 2015-12-02 |
| CN105112553B true CN105112553B (en) | 2019-03-01 |
Family
ID=54660655
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510629683.1A Active CN105112553B (en) | 2015-09-28 | 2015-09-28 | Application of the miRNA-924 in cirrhosis diagnosis and treatment |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105112553B (en) |
-
2015
- 2015-09-28 CN CN201510629683.1A patent/CN105112553B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| Changes in microRNAs associated with hepatic stellate cell activation status identify signaling pathways;Guo CJ et al.;《FEBS J》;20090930;5163-5176 |
| MicroRNA对原发性胆汁性肝硬化患者来源Th17细胞的调控机制研究;唐裕杰;《中国优秀硕士学位论文全文数据库（医药卫生科技辑）》;20091015;1-16 |
| Serum microRNAs as potential biomarkers of primary biliary cirrhosis;Youwen Tan et al.;《PLoS One》;20141027;e111424 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105112553A (en) | 2015-12-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2406402B1 (en) | Method to assess human allograft status from microrna expression levels | |
| WO2016186987A1 (en) | Biomarker micrornas and method for determining tumor burden | |
| CN105154541B (en) | Application of the miRNA in Diagnosing Acute Myeloid Leukemia and treatment | |
| CN107519193B (en) | Molecular diagnostic marker for early stage esophageal squamous carcinoma and application thereof | |
| CN107586842A (en) | A kind of biomarker for clear cell carcinoma of kidney diagnosis and treatment | |
| CN108624693B (en) | MiR-577 is preparing the application in diagnosis of nephropathy marker | |
| CN105441565B (en) | MiRNA as osteosarcoma diagnosis and treatment target | |
| CN105506156B (en) | Diagnose the molecular marker of osteosarcoma | |
| CN105063052B (en) | Acute myeloid leukemia miRNA markers | |
| CN106929577A (en) | A kind of lncRNA biomarker related to adenocarcinoma of lung | |
| CN109371124A (en) | Application of the miR-6802-3p in postmenopausal osteoporosis early diagnosis | |
| CN105349666B (en) | Cerebral arterial thrombosis miRNA markers | |
| CN107190005B (en) | Applications of the lncRNA as biomarker in adenocarcinoma of lung diagnosis and treatment | |
| CN105177149B (en) | Application of the miRNA in liver cirrhosis diagnosis and treatment | |
| CN106191264B (en) | The miRNA diagnosis marker of osteosarcoma | |
| CN104774966B (en) | Adenocarcinoma of lung miRNA labels | |
| CN110241211A (en) | Diagnose the molecular combinations marker and detection kit of colorectal cancer | |
| CN105603117B (en) | MiR-3613 is used to distinguish lung squamous cancer transfer and non-diverting miRNA marker | |
| CN105112553B (en) | Application of the miRNA-924 in cirrhosis diagnosis and treatment | |
| CN105148274B (en) | Applications of the 5p of miRNA 885 in acute myeloid leukemia diagnosis and treatment | |
| CN105169393B (en) | Application in the anti-acute myeloid leukemias of miRNA-548a-3p | |
| CN105349667A (en) | Cerebral-arterial-thrombosis diagnosis marker | |
| CN107184983B (en) | Diagnosis and treatment target for lung adenocarcinoma | |
| CN105821131B (en) | Osteosarcoma miRNA marker | |
| CN105603115B (en) | Lung squamous cancer shifts diagnosis and treatment marker |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |