CN106086214A - MiR 149 5p is as clinical diagnosis or the application of suppression hepatocarcinoma - Google Patents
MiR 149 5p is as clinical diagnosis or the application of suppression hepatocarcinoma Download PDFInfo
- Publication number
- CN106086214A CN106086214A CN201610656288.7A CN201610656288A CN106086214A CN 106086214 A CN106086214 A CN 106086214A CN 201610656288 A CN201610656288 A CN 201610656288A CN 106086214 A CN106086214 A CN 106086214A
- Authority
- CN
- China
- Prior art keywords
- mir
- hepatocarcinoma
- mirna
- application
- expression
- 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.)
- Pending
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
-
- 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)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Pathology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Animal Behavior & Ethology (AREA)
- Biotechnology (AREA)
- Hospice & Palliative Care (AREA)
- Veterinary Medicine (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Public Health (AREA)
- Oncology (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to biomedicine field, be specifically related to miR 149 5p as clinical diagnosis or the application of suppression hepatocarcinoma.By fluorescence quantitative PCR detection miR 149 5p expression in different tissues, using miR 149 5p as the molecular marker of hepatocarcinoma early diagnosis.Gene for the purpose of the miRNA of miR 149 5p, builds the over-express vector of miR 149 5p, and the preparation medicine containing miR 149 5p expression vector, by using in vitro or the internal administration used.Present invention discover that miR 149 5p expression in liver cancer tissue to occur substantially to lower.MiR 149 5p plays important regulating and controlling effect in the generation and evolution of hepatocarcinoma, and miR 149 5p can significantly inhibit propagation and the migration of hepatoma carcinoma cell, and miR 149 5p is expected to prevent and treat the medicine of hepatocarcinoma for preparation.
Description
Technical field
The invention belongs to biomedicine field, be specifically related to miR-149-5p and as clinical diagnosis or suppress answering of hepatocarcinoma
With.
Background technology
MicroRNA(miRNA) being the non-coding small RNA molecular of a class high conservative, length is about 18-25 nucleotide.
The regulation and control of gene expression in miRNA wide participation protokaryon and eukaryote body, to biological growth, the differentiation of cell, propagation with
Apoptosis, and the generation of disease, develop and lapse to all play an important role.MiRNAs is considered as a main Gene regulation
Device.
The generation of functional miRNA is broadly divided into the following continuous print stage, first transcribes generation length in core about
It is 80 nucleotide the initial miRNA(pri-miRNA with loop-stem structure), by under the common shear action of complex,
Pri-miRNA is processed into the precursor miRNA (pre-miRNA) of about 70 length of nucleotides.Pre-miRNA is by RAN-GTP
White with outer turning egg(s) 5 jointly transport in Cytoplasm, and cut loop-stem structure by nuclease Dicer and generate length and be about 22 nucleoside
The miRNA:miRNA double-strand of acid.Subsequently under the effect of the silencing complex of miRNA induction, ripe strand miRNA passes through alkali
3 ' the non-coding regions (3 ' UTR) of the one or more gene mRNA of base reverse complemental targeting, specifically degraded or reticent mRNA
Level, the carrying out of suppression translation, thus reach the regulating and controlling effect to gene expression.Substantial amounts of evidence shows, miRNA is in tumor group
There will be unconventionality expression in knitting, the generation and evolution of tumor play promotion or suppresses tumorigenic effect.
Hepatocarcinoma has become the most the fifth-largest tumor, occupies the 3rd in the death caused because of tumor.Primary hepatocarcinoma
Pathogenic factor and definite molecular mechanism be still not clear, be considered at present the knot of the multiple factors effects such as E&H more
Really.Hepatocarcinoma onset is hidden, and grade malignancy is higher and progress is rapid, and patient is many middles and advanced stage that advanced to when there is symptom, treatment difficulty
Degree is greatly and survival rate is low, and therefore the effective biomarker of examination has important meaning for early diagnosis and the prognosis of hepatocarcinoma
Justice.Research in recent years finds, miRNA can be from each stages such as cell cycle, apoptosis, angiogenesis, metaplasia, transfers
Participate in generation and the development of hepatocarcinoma.Such as miR-21, miR-221/222, miR-155 etc. are up-regulated in hepatocarcinoma, points out them
Proto-oncogene may be played a part；And miR-122, miR-101, miR-200 family, let-7 miRNA family etc. is in hepatocarcinoma
Middle lower, point out them may play a part antioncogene.Therefore the new miRNA relevant to hepatocarcinoma generation is found,
Study its mechanism of action and use it for hepatocarcinoma diagnosis and treatment will be the most meaningful.
Recently, miR-149-5p is found to suppress the generation of kinds of tumors, such as glioma, gastric cancer, Colon and rectum
Cancer, breast carcinoma, small cell lung cancer, prompting miR-149-5p is an antioncogene.MiR-149-5p is as diagnosing cancer of liver mark
As the purposes of a kind of hepatocellular carcinoma supressor gene, thing and miR-149-5p have no that document is reported.
Summary of the invention
For solve hepatocarcinoma can not early diagnosis, what hepatocarcinoma can not get effectively suppressing technical barrier, the invention discloses
MiR-149-5p is as clinical diagnosis or the application of suppression hepatocarcinoma.
For solve above-mentioned technical barrier, the present invention by the following technical solutions:
MiR-149-5p is as the application of clinical diagnosis hepatocarcinoma, by fluorescence quantitative PCR detection miR-149-5p at different tissues
In expression, using miR-149-5p as the molecular marker of hepatocarcinoma early diagnosis.
MiR-149-5p, as the application of suppression hepatocarcinoma, gene for the purpose of the miRNA of miR-149-5p, builds miR-
The over-express vector of 149-5p, preparation is containing the medicine of miR-149-5p expression vector, by using in vitro or internal using
Administration.
Described miRNA is precursor miRNA or the miR-of maturation of initial miRNA, miR-149-5p of miR-149-5p
One or more in 149-5p.
The miR-149-5p of described maturation is the RNA sequence as shown in SEQ ID NO:1, adds chemical group modification
One or more in RNA sequence or the DNA sequence as shown in SEQ ID NO.2.
Described over-express vector is virus expression carrier or carrier for expression of eukaryon.
Described viral vector is adenovirus vector, gland relevant viral vector, retroviral vector or herpesvirus vector.
Described carrier for expression of eukaryon is pCMV-myc expression vector, pcDNA3.0, pcDNA3.1 or the base at expression vector
Carrier engineered on plinth.
Described medicine is granule, slow releasing agent, microinjection agent, transfection dosage form or surfactant.
Described medicine uses (ex vivo approach) in vitro；By miR-149-5p or containing the DNA as shown in SEQ ID NO:2
Carrier import or be transfected into human body self or variant cell (or heterogenous cell), after vitro cell expansion, defeated time in vitro
Human body.
(in vivo approach) is used in described medicine body；By miR-149-5p or containing the DNA as shown in SEQ ID NO:2
Carrier be introduced directly into internal, described carrier is virus type or non-viral type, it is possible to for naked DNA or RNA.
The beneficial effects of the present invention is:
First: the present invention passes through reliable miRNA fluorescence quantifying PCR method, screening finds new miRNA marker miR-
149-5p expression in liver cancer tissue occurs substantially to lower.Compared with traditional protein marker, the sensitivity of this mark
Height, high specificity, can be as the clinical diagnosis mark of hepatocarcinoma early diagnosis.
Second: the present invention is proved by substantial amounts of experimental data, miR-149-5p are in the generation and evolution of hepatocarcinoma
Playing important regulating and controlling effect, miR-149-5p can significantly inhibit propagation and the migration of hepatoma carcinoma cell, and miR-149-5p is expected
The medicine of hepatocarcinoma is prevented and treated for preparation.
Accompanying drawing explanation
Fig. 1 is that quantitative fluorescent PCR analyzes miR-149-5p in mankind's different tissues or organ, normal liver cell and hepatocarcinoma
Differential expression analysis in cell；Wherein L-02 cell line is normal liver cell, HepG2 and CRL-8024 is hepatoma carcinoma cell
System.
Fig. 2 is that quantitative fluorescent PCR is analyzed miR-149-5p(and schemed A) and PHLPP2(figure B) expression in primary hepatocarcinoma；
Wherein normal structure derives from the normal liver tissue (n=5) of liver cancer patient；Cancer beside organism derives from the other liver of cancer of liver cancer patient
Dirty tissue (n=5)；Cancerous tissue (n=5), U6 rRNA compares as the internal reference of miR-149-5p.β-actin is as in PHLPP2
Ginseng comparison.
Fig. 3 is the targeting binding site of miR-149-5p Yu PHLPP2 of diagram different software prediction；Wherein
TargetScan software (figure A), miRanda software (figure B) and Diana Tools software (figure C) on-line analysis show
3 ' the UTR region of PHLPP2 mRNA and the binding site of miR-149-5p seed zone.
Fig. 4 is PHLPP2 Dual-Luciferase reporter assay in the hepatoma carcinoma cell of miR-149-5p process LAN.
Fig. 5 is quantitative fluorescent PCR analysis, A: in hepatoma carcinoma cell the ripe miR-149-5p analogies of transfection (mimic) or
After miR-149-5p inhibitor (inhibitor), the change of intracellular mature miR-149-5p level；B: turn in hepatoma carcinoma cell
After dying ripe miR-149-5p analogies or miR-149-5p inhibitor, the change of intracellular PHLPP2 level.
Fig. 6 is that cell proliferation tracing analysis transfects the miR-149-5p impact on hepatoma cell proliferation.
Fig. 7 is that scratch experiment analysis transfects the miR-149-5p impact on fucosylation；A: hepatoma carcinoma cell transfects into
Ripe miR-149-5p analogies are after 24,48 hours, and the migration situation of hepatoma carcinoma cell, B: hepatoma carcinoma cell transfects ripe miR-149-
After 5p analogies 24,48 hours, the transfer ability schematic diagram of hepatoma carcinoma cell.
Detailed description of the invention
" miR-149-5p " of the present invention unless otherwise stated, when mentioning miR-149-5p, it includes initially
MiR-149-5p(pri-miRNA), miR-149-5p precursor (pre-miRNA) and ripe miR-149-5p.
Term used herein " processes " the whole bioprocess referring to obtain ripe miR-149-5p from DNA, although
The concrete mechanism of the course of processing is not yet fully apparent from, but does not hinder the realization of " processing ".In eukaryotic cell, the course of processing can
Complete and produce pri-miRNA, pre-miRNA and ripe miRNA voluntarily.Wherein said DNA is not limited to it and specifically originates,
Chromosomal DNA and carrier DNA can be included, but be not limited to above-mentioned 2 kinds.
Further describe the present invention by the following examples, including using material and specifically originating.It is to be understood that
, these are exemplary, and the unrestricted present invention.With such as undertissue, cell, reagent, the type of instrument and model or
Character or functional similarity or identical material may be incorporated for the enforcement of the present invention.
Method in following example is commonsense method if no special instructions.
Main material:
| Human hepatoma cell line HepG2 | Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences |
| Human embryonic kidney cell line 293T | Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences |
| Tri reagent | Jiangsu En Moasai Bioisystech Co., Ltd |
| Nuclease free water | Ambion company of the U.S. |
| MiRNA and inhibitor | Rui Bo bio tech ltd, Guangzhou |
| Liposome 2000 | American I nvitrogen company |
| BioTeke miRNA cDNA the first chain synthetic agent box | Beijing hundred Tyke Bioisystech Co., Ltd |
| Restricted enzyme | TaKaRa company of Japan |
| Dual-Luciferase Reporter Assay System | Promega company of the U.S. |
| 2×SYBR Green qPCR Mixture | American AB I company |
| Primer | Shanghai Sheng Gong biological engineering limited company |
Note: unless otherwise stated, the reagent used in the present invention can be any suitable commercial reagent；Cell line all can be led to
Cross commercially available acquisition.
One, the tissue expression analysis detection of miR-149-5p
1. the collection of hepatocarcinoma clinical sample
Normal liver tissue and hepatocarcinoma are provided by department of general surgery of attached Huaihe River hospital of He'nan University.Whole collection and after
Continuous experimentation meets medical ethical moral requirement and follows strictly the principle of secrecy of case-data.Tissue sample underwent operative is taken out
After, being cut into small pieces rapidly to be placed in liquid nitrogen in cryopreservation tube saves backup.
2. RNA extracting
Every 100 mg tissues add 1 mL Tri reagent, are placed in the most fully homogenate disrupting tissue block, left at room temperature 5 points
Zhong Hou, add 1/10 times of Tri reagen volume BCP solution, vortex mixes that 15 seconds rear chamber are gentle and quiet puts 10 minutes.4 DEG C,
13400g room temperature is centrifuged 15 minutes；Supernatant is transferred to 1.5 new mL centrifuge tubes, adds the isopropanol of equimultiple supernatant volume,
Gently after reverse mixing for several times, room temperature stands 10 minutes, 4 DEG C, after 13400g is centrifuged 10 minutes, absorbs supernatant, adds 500 μ L
75% ethanol solution (without RNase water Fresh), featheriness suspend clean RNA, 4 DEG C, 13400g be centrifuged 5 minutes precipitation RNA.
After absorbing supernatant, it is placed in room temperature ventilation and dries, be dried about 5 minutes.Add appropriate nuclease free water and be placed in 55 DEG C of water-baths
10 minutes, after fully dissolving, measure OD260 and OD280 absorption value.It is generally acknowledged that A260/A280 is the most permissible
Preliminary judgement total serum IgE quality is preferable.
3. fluorescence quantitative PCR detection miR-149-5p level
Take 2 μ g RNA as template, use the miRNA cDNA the first chain synthetic agent box (BioTeke) for miRNA
MiRNA is added Poly (A) tail and reverse transcription becomes cDNA.With cDNA as template, use for miR-149-5p's
Primer and PCR 2 × SYBR Green qPCR Mixture, expand on ABI 7500 quantitative real time PCR Instrument.PCR bar
Part is: 50 ° of C 20 seconds；95 ° of C 10 minutes；95 ° of C 1 minute；60 ° of C 1 minute, repeat 40 circulations；Record sample
The CT value of miR-149-5p amplification, is standardized correction with the CT value of reference gene U6；Amplification target gene PHLPP2 simultaneously, with
Beta-actin is that reference gene is corrected.Gained CT value use 2-Δ∆CTMethod calculates, relatively miR-between different samples
The difference of 149-5p content.
Result: as shown in Figure 1 compared with normal liver tissue, miR-149-5p expression in hepatocarcinoma is bright
Aobvious reduction, declines about 50%(P=0.01).Show that the exception of miR-149-5p reduces closely related with the generation of hepatocarcinoma.
Two, the prediction of miR-149-5p target gene
Owing to miR-149-5p expression in liver cancer tissue occurs substantially to reduce, therefore can speculate that miR-149-5p is normally
Liver organization plays the function of antioncogene.Utilize bioinformatics software can predict target base potential for miR-149-5p
Cause.For improving the accuracy of prediction, utilize three kinds of conventional miRNA on-line prediction softwares, miRanda, Diana Tools and
TargetScan, finds the miR-149-5p target gene relevant to hepatocarcinoma.
Result: as it is shown on figure 3, setting is predicted by two or more analysis software is successfully defined as positive target gene simultaneously.
Finally determine that the 3 ' UTR region of PHLPP2 mRNA and the seed zone of miR-149-5p have potential binding site.
Three, miR-149-5p can effectively identify target gene PHLPP2 and affect its expression
1. cell is cultivated
Human hepatoma cell line HepG2 cultivates in DMEM culture medium (Thermo, USA).Containing 10% tire cattle in culture medium
Serum (Gibco, USA), penicillin (100 U/mL) and streptomycin.All cells is placed in 37 ° of C incubators, 5% CO2Condition
Lower cultivation.
2. cell transfecting
MiRNA is purchased from Guangzhou Rui Bo company, and HepG2 plating cells starts transfection, transfection after about 20 hours to about 60% density
Arrange ripe miR-149-5p group, the ripe general negative control group of miRNA, miR-149-5p inhibitor group, miRNA inhibitor lead to
Use negative control group.Transfection reagent used is liposome 2000(Invitrogen), transfection method is carried out with reference to description.
3. miR-149-5p level and the detection of target gene level
Continue after transfection to cultivate 48 hours, collect cell.According to the method in embodiment 1, the total serum IgE of extraction cell, reverse transcription
The rear method utilizing quantitative fluorescent PCR, after detection transfection, the change of HepG2 intracellular mature miR-149-5p level, examines simultaneously
Survey the situation of change of target gene PHLPP2 level.PHLPP2 forward primer used is as described in SEQ ID NO:3；PHLPP2 reversely draws
Thing is as described in SEQ ID NO:4.Internal reference comparison β-actin forward primer is as described in SEQ ID NO:5；Internal reference comparison β-actin
Reverse primer is as described in SEQ ID NO:6.
Result: normal structure derives from the normal liver tissue (n=5) of liver cancer patient as shown in Figure 2；Cancer beside organism originates
The other liver organization (n=5) of cancer in liver cancer patient；Cancerous tissue (n=5).U6 rRNA compares as the internal reference of miR-149-5p.β-
Actin compares as the internal reference of PHLPP2.Quantitative fluorescent PCR analysis shows, transfecting 200 pmol maturations miR-149-5p can show
Write improve the intracellular miR-149-5p of HepG2 expression (P, and can substantially suppress endogenous PHLPP2 gene=0.01)
Transcriptional level (P=0.048)；Owing to miRNA inhibitor is simply by reverse complemental competitive binding endogenous miRNA, do not change
Becoming its Level of Expression of Retinoic Acid, after therefore transfecting the miR-149-5p inhibitor of 400 pmol, HepG2 is intracellular and is not detected by
The change of miR-149-5p expression (P=0.78), but the mRNA level in-site of endogenous PHLPP2 occur substantially to raise (P=
0.047) as shown in Figure 5.This experiment proves to be improved by exogenous method or reduced the level of cylinder mature miR-149-5p
It is practicable, it is possible to effectively change the expression of its target gene PHLPP2 mRNA.
Four, luciferase reporting experiment proves the miR-149-5p direct regulation effect to target gene PHLPP2
3 ' UTR region of people source PHLPP2 gene carry out PCR amplification with following primer:
PHLPP2-3 ' UTR-F is as described in SEQ ID NO:7；PHLPP2-3 ' UTR-R is as described in SEQ ID NO:8.PCR expands product
Thing is cloned into the downstream of pmirGLO carrier (Promega) luciferase gene, builds wild type recombinant vector PHLPP2-WT-
pmirGLO；Simultaneously with this carrier as template, use site-directed mutagenesis kit (QuikChange II XL Site-Directed
Mutagenesis Kit, Agilent Technologies), sudden change PHLPP2 gene 3 ' UTR region and miR-149-5p kind
The sequence that sub-district combines, builds saltant type recombinant vector PHLPP2-mut-pmirGLO.Mutant primer is: mut-PHLPP2-3 '
UTR forward primer: as described in SEQ ID NO:9；Mut-PHLPP2-3 ' UTR reverse primer: as described in SEQ ID NO:10.
Above two plasmid vector respectively with ripe miR-149-5p and maturation miRNA general negative control cotransfection 293T
Cell, transfection reagent used is Lipofectamine 2000(Invitrogen), report according to Dual-Luciferase after 48 hours
The method that gene detection system (Dual-Luciferase Reporter Assay System, Promega) provides processes thin
Born of the same parents, detect fluorescent value in multi-functional microplate reader, and all transfection group all contain three multiple holes, and experimental result is three independent repetitions
Experiment gained.
Result: as shown in Figure 4, compared with negative control cotransfection group, miR-149-5p can make PHLPP2-WT-
The uciferase activity of pmirGLO decline about 35% (P=0.03), but after the 3 ' UTR region of PHLPP2 are carried out point mutation, glimmering
Light element enzyme activity recovery is normal,P＜ 0.01.This result shows, in hepatoma carcinoma cell miR-149-5p by directly in conjunction with
Sequence in PHLPP2 mRNA 3 ' UTR region disturbs its translation process, and may thus affect the propagation of hepatoma carcinoma cell and move
Move as shown in Figure 6.
Five, miR-149-5p can suppress the propagation of hepatocellular carcinoma H22
Use the proliferative conditions of direct counting method detection cell.By 5 × 104Individual HepG2 cell is laid in 6 hole culture dishs, presses
According to the method in embodiment 3, the ripe miR-149-5p(200pmol of transfection) and the ripe general negative control of miRNA
(200pmol).In transfecting peptic cell one day after, use the trypan blue solution dyeing of 0.05% that every hole living cells is counted
Number, continuous counter 6 days.Each transfection group arranges three repeating holes.
Result: as shown in Figure 7 compared with matched group, the ripe miR-149-5p of transfection is after mono-day, and the growth of hepatoma carcinoma cell is fast
Degree i.e. generation substantially reduction (P=0.01), and prolongation over time, the most obvious to the depression effect of hepatoma cell proliferation.Table
Bright process LAN miR-149-5p can effectively suppress the propagation of hepatoma carcinoma cell.
Six, miR-149-5p can suppress the transfer ability of hepatocellular carcinoma H22
Being laid on by HepG2 cell in 6 hole culture dishs, the front cell of transfection grows to about 60% density and is advisable, according in embodiment 3
Method, the ripe miR-149-5p of transfection and the ripe general negative control of miRNA.With 200 μ L aseptic shifting liquid after transfecting 24 hours
Rifle rifle head is in " one " stroke trace on cell monolayer, and the cell that PBS is removed under drawing for three times, addition does not contains the culture medium of serum
Continue to cultivate cell.By 0, within 24,48 hours, take pictures under inverted microscope.With Image Pro Plus 6.0 software measurement cut
Area and width.The healing rate of cell: (width 1-width 2)/width 1, width 1 and width 2 were respectively cut at 0 hour
With the width of 24/48 hour, scratch width was the ratio of scratch area and length.
Result: compared with matched group, miR-149-5p is after 24 hours in transfection, and the transfer ability of hepatoma carcinoma cell does not occur
Significant change (P=0.23), but after transfection 48 hours the transfer ability of hepatoma carcinoma cell substantially reduce (P=0.04), miR-is shown
149-5p can significantly inhibit the transfer ability of hepatoma carcinoma cell.
Statistical analysis: all data take the meansigma methods independently repeating experiment three times, and standard deviation (SD) utilizes GraphPad
Method in Prism 5 carries out data analysis.P < 0.05 thinks have statistical significance.
The scope that the present invention is claimed is not limited to the description of detailed description of the invention.
Claims (8)
1.miR-149-5p is as the application of clinical diagnosis hepatocarcinoma, it is characterised in that: by fluorescence quantitative PCR detection miR-149-
5p expression in different tissues, using miR-149-5p as the molecular marker of hepatocarcinoma early diagnosis.
2.miR-149-5p is as the application of suppression hepatocarcinoma, it is characterised in that: gene for the purpose of the miRNA of miR-149-5p,
Building the over-express vector of miR-149-5p, the preparation medicine containing miR-149-5p expression vector, by using in vitro or body
The administration inside used.
3. miR-149-5p as claimed in claim 2 is as the application of suppression hepatocarcinoma, it is characterised in that: described miRNA is
One or more in the precursor miRNA of initial miRNA, miR-149-5p of miR-149-5p or the miR-149-5p of maturation.
4. miR-149-5p as claimed in claim 3 is as the application of suppression hepatocarcinoma, it is characterised in that: the miR-of described maturation
RNA sequence that 149-5p is the RNA sequence as shown in SEQ ID NO:1, add chemical group modifies or such as SEQ ID NO.2 institute
One or more in the DNA sequence shown.
5. miR-149-5p as claimed in claim 2 is as the application of suppression hepatocarcinoma, it is characterised in that: described over-express vector
For virus expression carrier or carrier for expression of eukaryon.
6. the miR-149-5p as described in claim 2 or 5 is as the application of suppression hepatocarcinoma, it is characterised in that: described virus carries
Body is adenovirus vector, gland relevant viral vector, retroviral vector or herpesvirus vector.
7. the miR-149-5p as described in claim 2 or 5 is as the application of suppression hepatocarcinoma, it is characterised in that: described eucaryon table
Reaching carrier is pCMV-myc expression vector, pcDNA3.0, pcDNA3.1 or carrier engineered on the basis of expression vector.
8. miR-149-5p as claimed in claim 2 is as the application of suppression hepatocarcinoma, it is characterised in that: described medicine is granule
Agent, slow releasing agent, microinjection agent, transfection agents or surfactant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610656288.7A CN106086214A (en) | 2016-08-11 | 2016-08-11 | MiR 149 5p is as clinical diagnosis or the application of suppression hepatocarcinoma |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610656288.7A CN106086214A (en) | 2016-08-11 | 2016-08-11 | MiR 149 5p is as clinical diagnosis or the application of suppression hepatocarcinoma |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106086214A true CN106086214A (en) | 2016-11-09 |
Family
ID=57455772
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610656288.7A Pending CN106086214A (en) | 2016-08-11 | 2016-08-11 | MiR 149 5p is as clinical diagnosis or the application of suppression hepatocarcinoma |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106086214A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113604569A (en) * | 2021-08-04 | 2021-11-05 | 广州医科大学 | Application of miR-6883-3p in preparation of anti-liver cancer or liver cancer prognosis evaluation product |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630224A (en) * | 2015-01-08 | 2015-05-20 | 江苏大学 | Micro RNA related to BafilomycinA1 liver cancer and ovarian cancer inhibition cell line |
| WO2015075557A2 (en) * | 2013-11-22 | 2015-05-28 | Mina Alpha Limited | C/ebp alpha compositions and methods of use |
-
2016
- 2016-08-11 CN CN201610656288.7A patent/CN106086214A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015075557A2 (en) * | 2013-11-22 | 2015-05-28 | Mina Alpha Limited | C/ebp alpha compositions and methods of use |
| CN104630224A (en) * | 2015-01-08 | 2015-05-20 | 江苏大学 | Micro RNA related to BafilomycinA1 liver cancer and ovarian cancer inhibition cell line |
Non-Patent Citations (2)
| Title |
|---|
| 曹亚玲: "miR-149-5p抑制肝癌侵袭转移的实验研究", 《中国优秀硕士学位论文全文数据库（电子期刊）》 * |
| 曹亚玲等: "miR-149-5p在肝细胞癌中的表达及其临床意义", 《第三军医大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113604569A (en) * | 2021-08-04 | 2021-11-05 | 广州医科大学 | Application of miR-6883-3p in preparation of anti-liver cancer or liver cancer prognosis evaluation product |
| CN113604569B (en) * | 2021-08-04 | 2023-11-17 | 广州医科大学 | Application of miR-6883-3p in preparation of anti-liver cancer or liver cancer prognosis evaluation product |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6441372B2 (en) | Application of alphavirus to the production of antitumor drugs | |
| CN103097535A (en) | Method for producing novel hipsc by means of sirna introduction | |
| Zhang et al. | Low expression of lncRNA MEG3 promotes the progression of oral squamous cell carcinoma by targeting miR-21. | |
| CN104388556A (en) | Application of long non-coding RNA in preparation of medicament for diagnosing or treating ovarian cancer | |
| CN106701900A (en) | Long-chain noncoding RNA HERC2P3 gene and application thereof in gastric cancer | |
| CN107630092A (en) | The 3p of miR 505 are applied to diagnosis, prognosis and the treatment of prostate cancer with osseous metastasis | |
| CN104774929B (en) | The application of diagnosis, treatment and prognosis of the 3p of miR 455 in esophageal squamous cell carcinoma | |
| CN104208723B (en) | MiR-638 application in anti-acute myeloid leukemia | |
| CN108374048A (en) | A kind of lncRNA markers for diagnosing and treating hepatocellular carcinoma | |
| CN108277279A (en) | A kind of application of lncRNA in diagnosing and/or treating breast cancer | |
| CN101353656B (en) | siRNA inhibiting expression of epidermal growth factor receptor genes and use thereof | |
| CN105441566B (en) | Kit and chemotherapy of hepatocellular carcinoma sensitizer for prognosis evaluation after Liver Cancer Operation | |
| CN106086214A (en) | MiR 149 5p is as clinical diagnosis or the application of suppression hepatocarcinoma | |
| CN111096962A (en) | Application of fatty acid oxidation inhibitor in preparation of medicine for treating colorectal cancer | |
| CN113549697B (en) | Gastric cancer heat chemotherapy sensitive marker and application thereof | |
| Gao et al. | miRNA-217 inhibits proliferation of hepatocellular carcinoma cells by regulating KLF5. | |
| CN109536497A (en) | The application of schistosoma japonicum infection and its component in human tumor prevention and treatment | |
| CN110960546B (en) | Application of MicroRNAs in preparation of reinforcing agent for treating liver cancer by sorafenib | |
| CN105497916B (en) | Small molecule non-coding RNA miR-125b is preparing the application in the drug for treating the wrap-around vascular group liver cancer of tumour | |
| CN111407758A (en) | Application of cantharidin as miR-214-3p/Wnt/β -Catenin signal pathway inhibitor in preventing and treating osteosarcoma | |
| CN109295220A (en) | Application of the miR-495-5p in the product of preparation diagnosis, prognosis, prevention or treatment cancer of pancreas | |
| CN109224076A (en) | Gene miR-140-3P and its mimics relevant to lung cancer diagnosis and treatment and application | |
| CN104031987B (en) | MiRNA application in myocardial fibrosis disease treatment | |
| CN107582525A (en) | TRIM31 inhibitor magnetic target drug bearing microspheres are preparing the application in suppressing PDAC multiplication capacity medicines | |
| CN107723367A (en) | The 3p of miRNA 885 application and apply its product |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20161109 |