CN104974987B - Purposes of the small molecule RNA-1247-5p as novel tumor treatment molecular target - Google Patents
Purposes of the small molecule RNA-1247-5p as novel tumor treatment molecular target Download PDFInfo
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Abstract
The present invention provides a kind of 1247 5p of small molecule miRNA and stablizes the method for building up for being overexpressed liver cancer cell lines and its regulating and controlling effect to hepatoma cell strain metabolism, it is likely that as the marker of prevention and treatment tumour.More and more evidences show that microRNA can be used as a kind of oncogene or tumor suppressor gene, participate in the occurrence and development process of regulation and control kinds cancer.The experiment that the present invention infects tumor models by building slow virus proves, 1247 5p of miRNA have obvious inhibiting effect to tumor cell line HepG2 proliferation, migration and invasion etc., there is cell cycle regulation and metabolism, therefore 1247 5p of miR are likely to become tumor prevention and the completely new target spot for the treatment of；And it confirms that 1247 5p of miR play cell regulating and controlling effect by microRNA target prediction database and Dual-luciferase reportor systerm, is realized by selectively targeted expression of the inhibition oncogene without wing MMTV integration site families member 3 (WNT3).
Description
Technical field
The present invention relates to the method for building up that a kind of endogenic non-coding microRNA s stablizes overexpressing cell system, especially
Be related to miR-1247-5p the foundation of hepatoma H22 cells cytotostatic overexpressing cell system and its in prevention and treatment
Purposes in being developed in liver cancer diseases as new tumor markers and antitumor drug.
Background technology
Liver cancer is the third-largest malignant tumour that current incidence in the world is only second to gastric cancer and cancer of the esophagus.The pernicious journey of liver cancer
Spend high, prognosis is poor, and the death rate occupies various malignant tumour forefront.Liver cancer is broadly divided into primary carcinoma of liver and metastatic hepatic carcinoma
Two major class, in China, the death rate of liver cancer accounts for the second of kinds of tumor.In the pathogenesis of liver cancer, Wnt/ β-catenin
Signal path mainly by activate the transcription of the target genes such as downstream c-myc, cyclingD1, c-iun and cause the generation of liver cancer with
Development, while dysfunction an important factor for being also in liver cancer progression of GSK3 β/APC/Axin degradation complexs.Therefore,
The generation and development of liver cancer can effectively be controlled by the method for molecular therapy, carry out grinding for onset of liver cancer mechanism and clinical treatment
It is the fundamental way for mitigating its harm to study carefully.
MicroRNA (miRNA) is a kind of endogenous non-coding single stranded RNA of discovered in recent years, length about 18~25
Nucleotide.MiRNA degrades to said target mrna or inhibits to translate to adjust by Watson-Crick base complementrity matching principles
The expression of albumen.It was predicted that the mankind participate in protein coding gene of the regulation and control more than 30%, in biology about containing 1000 miRNA
The growth of body, development, differentiation etc. play an important role.The unconventionality expression of miRNA participates in the various biology mistakes of tumour
Journey including tumor cell proliferation, apoptosis and is migrated, is invaded.More and more evidences show that microRNA can be used as a kind of cancer
Gene or tumor suppressor gene participate in the occurrence and development process of regulation and control kinds cancer.Infer according to this, miRNA can be used as a kind of cancer base
Cause or tumor suppressor gene.
Means using slow virus carrier as research small molecule MicroRNA have been used by a variety of researchs, are taken using slow virus
Band foreign gene can continue in host cell stablize expression the characteristics of, can continually and steadily express have to tumour cell it is certain
The gene of specific function, can preferably the biological function of this gene and its application value be carried by establishing stable expression system
For model basis.
Maturation miR-1247-5p sequence informations, TargetScan, miRanda etc. are retrieved by miRBase and carry out target gene
Prediction, miR-1247-5p targeted inhibitions predict the expression of target gene WNT3 genes.Without wing MMTV integration site families member 3
(winglesstype MMTV integration site family member3, WNT3) is present in Wnt/ β-catenin letters
Number circuit upstream.WNT signal paths are a highly conserved accesses being present in unicellular lower eukaryote to higher organism, access into
Member has high homology, has regulated and controled including biological processes such as animal embryo early development, orga- nogenesis, regenerations,
In multiple Vitro Tumor models there is activation in various degree in WNT signal paths.In numerous miRNA, miR-1247 is
It is studied to confirm the abnormal expression in kinds of tumors model, and it is likely to participate in the process of tumour, become potential tumor suppressor gene,
And regulating and controlling effect mechanism of the miR-1247-5p in tumor cells of hepatocellular carcinoma and the research influenced with Apoptosis process is proliferated on it at present
It is still unclear, therefore, develop using miR-1247-5p as strategy the prevention of liver cancer diseases, the basic research of diagnose and treat and
Antitumor drug research has great importance and application prospect.
Invention content
The purpose of the present invention is to provide a kind of small molecule RNA-1247-5p (miR-1247-5p) in liver cancer cells
The method for stablizing expression in HepG2 cells and the effect in prevention and treatment liver cancer diseases and antitumor drug, it is described
The nucleotide sequence of miR-1247-5p is as follows：5’-ACCCGUCCCGUUCGUCCCCGGA-3’.
The Research foundation of the present invention：
1. the lentiviral particle that can be overexpressed miR-1247-5p has successfully been obtained in the present invention, viral effective titre reaches
1x108TU/ml, the method infected repeatedly using slow virus, by subculture multiple to cell, fluorescence microscopy Microscopic observation,
The cell for carrying miR-1247-5p foreign genes accounts for more than 95%, real time fluorescent quantitative detection cell miR-1247-5p expression
Amount rises 630 times.
2. the present invention carries out microRNA target prediction by forecasting softwares such as TargetScan, miRanda to miR-1247-5p,
Demonstrate it by Dual-Luciferase has selectively targeted inhibiting effect to WNT3.
3. the present invention confirms that miR-1247-5p is overexpressed by western blot specific significantly to inhibit WNT3
Protein expression level.
4. the present invention proves that miR-1247-5p passes through inhibition simultaneously by CCK-8, Hoechst dyeing and flow cytometry
The expression of target WNT3 lowers its level expressed in tumour cell height, cell cycle regulation, and tumour cell is made to be transferred to trend
Increase and then effectively inhibit the increment of cell, promote the apoptosis of tumour cell.
5. the present invention tests simultaneously from transwell cell invasions, it was confirmed that miR-1247-5P can effectively inhibit tumour thin
Born of the same parents' invasive ability.
6. present invention demonstrates that microRNA has very important meaning in clinical practice, will be given birth to as the tumour of a new generation
Object diagnosis marker and the molecular target for oncotherapy.Especially open the tumor disease based on miR-1247-5p
The research of completely new target, also opens up new approach to develop anti-cancer agent.
Description of the drawings
After Fig. 1 shows that pSicoR/miR-1247-5p recombinant slow virus infects HepG2 cells, green fluorescence under microscope
Expression quantity.
Fig. 2 shows HepG2 cell of the extraction after repeatedly passing on, real-time fluorescence quantitative PCR detection miR-1247-5p
Expression still maintains higher level.
Fig. 3 show extraction formalin fix after RNA, reverse transcription cDNA in paraffin-embedded tissue, with glimmering in real time
Fluorescent Quantitative PCR method detects miR-1247-5p expressions in liver cancer cancer beside organism and cancerous issue.
Fig. 4 shows Dual-luciferase reportor systerm verification miR-1247-5p targeted inhibition WNT3 uciferase activities.
Fig. 5 shows that concentrating virus supernatant infects tumour cell western blot and confirms that miR-1247-5p is overexpressed
It being capable of the apparent inhibition WNT3 protein expression levels of specificity.
Fig. 6 shows that CCK-8 experimental verifications miR-1247-5p significantly inhibits the proliferation of tumour cell HepG2 cells.
Fig. 7 shows that hoechst Coloration experiments demonstrate miR-1247-5p and are obviously promoted withering for tumour cell HepG2 cells
It dies.
Fig. 8 shows flow cytomery as a result, miR-1247-5p can increase HepG2 Apoptosis trend.
Fig. 9 shows that Transwell experimental verifications miR-1247-5p significantly inhibits the migration of tumour cell HepG2 cells
And invasion.
Specific embodiment
1.miR-1247-5p is overexpressed the acquisition of slow virus and stablizes the foundation of expression miR-1247-5p cell lines.
About 1 × 10 is inoculated in 6 porocyte culture plates6A HEK-293T cells add in 2mL DMEM containing 10%FBS per hole
Complete medium, 37 DEG C, after 5%CO2 cultures for 24 hours, when cell density reaches 80%~90%, according to TransLipid
Transfection Reagent specifications, by three pUC pUCs (1.5 μ g of pSicoR-miR-1247-5p over-express vectors,
PVSVG0.45 μ g, A8.911.05 μ g) in cotransfection to HEK-293T cells, collect the supernatant after transfection 48h and 72h.4℃
3000g centrifuges 10min, removes cell fragment, and then by the PVDF membrane filtrations of supernatant 0.45tm, -80 DEG C of packing preserve.Fortune
Titer determination is carried out to virus with doubling dilution principle, infects tumor cell line HepG2.
About 1 × 10 is inoculated in 6 porocyte culture plates6A HEK-293T cells add in 2mL DMEM containing 10%FBS per hole
Complete medium, after 5%CO2 cultures for 24 hours, infects HepG2 cells with different infection multiplicities (MOI) using slow virus by 37 DEG C, and 72
The hole cell dissociation that selection cell state is preferable and luciferase expression ratio is higher is observed after hour and is transferred to 25cm2 culture bottles
In, continue, with the infected cell of identical infection multiplicity MOI=30, to replace within 72 hours cell culture medium, digestion after 24 hour cells are adherent
Cell expands culture.
2.miR-1247-5p stable expression cell lines expression quantity detects
Extraction inverts RNA according to promege reverse transcription reagent box specifications by the cell line 5S RNA repeatedly passed on
It records as cDNA.U6 is as reference gene, as a result using relative quantification method, formula 2-△△CT。
3.miR-1247-5p the detection of paraffin organization expression is collected pathology department of hospital general of Ningxia Medical University and is arrived for 2011
16, the tissue (FFPE) of paraffin embedding and cancer beside organism Fu Er after the postoperative formalin of hepatocarcinoma patient is fixed during 2012
10, the tissue (FFPE) of paraffin embedding after Malin fixes, using FFPE RNA Isolation Kit (OMEGA) from paraffin group
Middle extraction RNA is knitted, with q-RT PCR methods detection miR-1247-5p expressions by the liver cancer and in cancerous issue.Even
Continuous slice 4-6 pieces, 10um is thick, dimethylbenzene dewaxing,MicroElute pillars isolate and purify RNA, according to
RNA reverse transcriptions are cDNA by promege reverse transcription reagent box specification.U6 is as reference gene, as a result using relative quantification method,
Formula is 2-△△cT
The structure of 4.pMIR-Report-WNT3-3 ' UTR expression vectors and identification.It is limited by HindI II and Spe I
Property enzymes double zyme cutting pMIR-Report plasmids, run electrophoresis with the Ago-Gel of 12g/L and carry out glue time to purpose band
It receives, by pMIR-Report plasmids and WNT33 '-the UTR connection of linearisation, reaction condition is 22 DEG C, 2h.Connection product is converted
To Escherichia coli Top10 competent cells, picking single bacterium colony shakes bacterium, extracts Plasmid DNA, HindIII, Spe I restriction enzymes
Enzyme double digestion is identified and is sequenced.
5. Dual-luciferase reportor systerm.By HEK-293T cell inoculations in 96 orifice plates, cell density during transfection is made to reach 1
×107/mL.By pSicoR-miR-1247-5p over-express vectors and pMIR-Report-WNT3-3 ' UTR carriers cotransfection extremely
In 293T cells, while cotransfection pMIR-Report-WNT3-3 ' UTR mut carriers and miR-1247-5p inhibitor are done
Control.For every group of cotransfection sea clam luciferase (PRL-TK) as internal reference, transfection reagent is Tranglipid Transfection
Regent.Green fluorescence is observed after transfecting 48h, under fluorescence inverted microscope to shine situation, and lytic cell, according to promega
Dual-Luciferase kit specification is operated, and instrument is microwell plate luminescence analyzer.
6.Western blot analysis.Slow virus infects routine culture cell, and the expression feelings of GFP are observed after 48h
Condition puies forward holoprotein kit specification according to Kai Ji and carries holoprotein, and -80 DEG C of packing preserve.It is legal that the albumen of extraction carries out BCA
Amount.SDS-PAGE is detached, half-dried to go to pvdf membrane, and the closing of 50g/L skimmed milk powers after 4 DEG C are stayed overnight, adds in the WNT3 antibody of rabbit-anti
(1: 100) room temperature rock be incubated 2h after 4 DEG C overnight, after TBST washes film, add in horseradish peroxidase (HRP) mark goat resist
Rabbit igg (1: 5000) is incubated 2h, fully washes after film and carries out darkroom exposure using ECL chemoluminescence methods.
7.CCK-8 detects cell proliferative conditions.Slow virus infected cell is same as above, 24,48, stimulation is terminated after 72h.Replace nothing
Serum free culture system liquid per 90 μ L of hole, adds in 10ul CCK-8 solution, continues to cultivate 2h.Enzyme-linked immunosorbent assay instrument detects the suction of 450nm
Luminosity (A450) value.Zeroing hole and control wells, every group of 3 multiple holes experimental result block diagram of setting or different time sections are set simultaneously
Line chart represents.
8.Hoechst dyeing detection Apoptosis situations.6 orifice plates place steril cell creep plate, are inoculated with the swollen of proper density
Oncocyte, slow virus infect rear 48h, add in fixer, after PBS cleanings, 0.5ml Hoechst33258 dyeing liquors are added in per hole,
It is protected from light dyeing 5min.Dye liquor is removed, PBS is washed twice, is dripped anti-fluorescent quenching mounting liquid on glass slide, is covered and post cell
Creep plate allows cell to contact mounting liquid, avoids bubble as possible.Fluorescence microscope detects, excitation wavelength 350nm or so, launch wavelength
460nm or so.Count nucleus (apoptotic cell) quantity of blue and white.
9.Transwell experiments detection cell migration and invasion situation.Cell invasion is tested：Matrigel matrigels source
In BD companies, it will dispense to freeze first and thaw on ice and according to volume ratio 1 in -20 DEG C of fresh Matrigel：8 and serum-free
DMEM culture mediums are configured to suspension, are coated with Transwell (aperture Sum) cell：Transwell cells are put with aseptic nipper
Enter 24 porocyte culture plates, the Matrigel suspensions prepared are added in into 24 orifice plates by 70ul/ rooms, are put into cell incubation case
30min air-dries 30min on superclean bench after taking-up, the lower indoor addition 500ul in Transwell cells contains 10%FBS
DMEM culture mediums, add in the cell suspension 10 prepared in upper chamber4/ hole, cell routine culture 48h carefully will with tweezers
Transwell cells take out, and upper chamber culture medium are discarded, with aseptic cotton carrier gently by the cell of upper indoor surface remnants and Matrigel glue
It scrapes off, lateral surface of 90% ethyl alcohol in cell is added dropwise, fixed 10min, 0.1% violet staining 5min are just being put under microscope mirror
Photo is acquired, randomly selects 5 high power field of view, counts the cell number for wearing film, and for statistical analysis.Cell migration assay class
Seemingly, in addition to not having to spread Matrigel matrigels, upper chamber inoculation 2x104/ hole cell, routine culture is for 24 hours.
Claims (2)
1. the reagent of tumor markers expression quantity is detected in prevention and treatment liver cancer diseases diagnostic reagent or kit is prepared
Using, which is characterized in that the tumor markers are MiRNA-1247-5p, and the MiRNA-1247-5p can inhibit oncogene
The expression of WNT3.
2.MiRNA-1247-5p is overexpressed application of the slow virus in the drug for preparing treatment liver cancer.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009130479A2 (en) * | 2008-04-23 | 2009-10-29 | Isis Innovation Limited | Virus |
| CN101821391A (en) * | 2007-10-04 | 2010-09-01 | 桑塔里斯制药公司 | Micromirs |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101821391A (en) * | 2007-10-04 | 2010-09-01 | 桑塔里斯制药公司 | Micromirs |
| WO2009130479A2 (en) * | 2008-04-23 | 2009-10-29 | Isis Innovation Limited | Virus |
Non-Patent Citations (3)
| Title |
|---|
| Concordant hypermethylation of intergenic microRNA genes in human hepatocellular carcinoma as new diagnostic and prognostic marker;Sumadi Lukman Anwar等;《International Journal of Cancer》;20131231;第133卷;第660-671页，尤其是摘要、第662页右边栏第2-3段 * |
| MicroRNA-Regulated Protein-Protein Interaction Networks and Their Functions in Breast Cancer;Chia-Hsien Lee等;《Int. J. Mol. Sci.》;20130530;第14卷;第11560-11606页 * |
| miR-1247 Functions by Targeting Cartilage Transcription Factor SOX9;Aida Martinez-Sanchez等;《THE JOURNAL OF BIOLOGICAL CHEMISTRY》;20131025;第288卷(第43期);第30802–30814页 * |
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