CN108310001A - MiR-23a is preparing the application in treating TSGA10 low expression medicine for nasopharyngeal - Google Patents
MiR-23a is preparing the application in treating TSGA10 low expression medicine for nasopharyngeal Download PDFInfo
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- CN108310001A CN108310001A CN201810086673.1A CN201810086673A CN108310001A CN 108310001 A CN108310001 A CN 108310001A CN 201810086673 A CN201810086673 A CN 201810086673A CN 108310001 A CN108310001 A CN 108310001A
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Abstract
The invention discloses a kind of miR 23a to prepare the application in treating TSGA10 low expression medicine for nasopharyngeal.MiR 23a of the present invention are a kind of miRNA molecules of newfound regulation and control TSGA10 genes, can inhibit proliferation, the migration of nasopharyngeal carcinoma cell；It is precisely treated for nasopharyngeal carcinoma and provides a novel targets.
Description
Technical field
The present invention relates to a kind of medicinal usages.
Background technology
Nasopharyngeal carcinoma（Nasopharyngeal carcinoma, NPC）It is most common head-neck malignant tumor.It sends out in China
The hat in the worlds Bing Shuaiju accounts for about global 80%.Although as the rapid development of chemicotherapy technology, NPC prognosis obtains bright
It is aobvious to improve.But still there is the patient of 38%-87% to shift, and transfer poor prognosis once occurs, survival rate only has 25- within 5 years
30%.Therefore, the related mechanism of research NPC transfers, and it is significant based on this searching therapy.
Testicle specificity gene antigen（TSGA10）On No. 2 chromosomes, it is about 3K base.Studies have shown that
TSGA10 low expressions in the cancer of the esophagus, and the TSGA10 of low expression is related with the malignant progression of the cancer of the esophagus.Separately some researches show that
TSGA10 can hypoxia inducible factor HIF-1 α TAD-C（CBP）The combination of structural domain induces the destruction of HIF-1 α axis, to press down
Tumor Angiongesis processed and transfer.Currently, adjusted for TSGA10 expression in nasopharyngeal carcinoma and its by which kind of miRNA, it is unclear.
MiRNA is a kind of small non-coding single stranded RNA, mainly combines mRNA by targeting（mRNA）3' untranslateds
Area（3UTR）To play Gene regulation function.It is estimated that miRNA has adjusted the generation of the human mRNA more than one third to join
With extensive biological processes.Research finds that miR-23a 27a 24-2 families play during the occurrence and development of cancer and focuses on
Big effect：MiR-23a 27a 24-2 family member's height is expressed in endothelial cell（ECs）With very vascular tissue,
MiR-23/27, which can be targeted, adjusts Sprouty2 and Sema6A protein expressions to play promotion angiogenesis.Ruan W et al.
A nearest research finds to increase the endothelial cell apoptosis for reducing TNF-α induction when miR-23a expression.In view of tumour is given birth to
It is long, shift to blood transport there is an urgent need to study miR-23a before the expression of nasopharyngeal carcinoma and function show good clinical treatment
Scape.
Invention content
The purpose of the present invention is to provide a kind of good miR-23a of effect to prepare treatment TSGA10 low expression nasopharyngeal carcinoma medicines
Application in object.
Technical solution of the invention is：
A kind of miR-23a is preparing the application in treating TSGA10 low expression medicine for nasopharyngeal.
The nasopharyngeal carcinoma is related to TSGA10 expression, and miR-23a acts on the 3 ' areas the UTR of TSGA10
Inhibit the proliferation and transfer ability of nasopharyngeal carcinoma cell by low expression miR-23a, improves Nasopharyngeal Carcinoma Patients prognosis.
MiR-23a of the present invention is a kind of miRNA molecule of newfound regulation and control TSGA10 genes, and it is thin to can inhibit nasopharyngeal carcinoma
The proliferation of born of the same parents, migration；It is precisely treated for nasopharyngeal carcinoma and provides a novel targets.
Description of the drawings
The invention will be further described with reference to the accompanying drawings and examples.
Fig. 1 is expression of results figures of the TSGA10 in tissues of nasopharyngeal carcinoma.
Fig. 2 is the result figure that miR-23a targetings adjust TSGA10.Wherein：（a）The miR-23a of TSGA10-3'-UTR predictions
The schematic diagram of binding sequence.（b）MiR-23a is overexpressed reduces TSGA10-3'-UTR uciferase activity schematic diagrames in vitro
(c）After miR-23a mimic/nc/inhibitor transfections HUVEC, the protein expression figure of TSGA10.(d）TSGA10 tables
The statistical results chart reached.（e）Transwell migration experiments are carried out, assessment miR-23a becomes TSGA10 induced cell migration abilities
The redemption situation schematic diagram of change.（f）Transwell statistical results charts.
Fig. 3 is that miR-23a adjusts nasopharyngeal carcinoma cell proliferation and the result figure of transfer ability.Wherein（a）QRT-PCR verifications are thin
The transfection efficiency schematic diagram of born of the same parents.（b）The growth ability schematic diagram of CCK8 assay verification cells.（c）Streaming is surveyed the cell cycle and is shown
It is intended to.（d）Cut shows cell migration ability schematic diagram.（e）Scratch experiment statistical results chart.（f）Transwell
Migration assays verify cell migration ability schematic diagram.（g）Transwell statistical results charts.
Fig. 4 be miR-23a nasopharyngeal carcinoma expression of results figure and its with Prognostic significance figure.Wherein（a）Representative original position is miscellaneous
Intersection graph (nasopharynx inflammatory tissue and tissues of nasopharyngeal carcinoma).（b）Representative in situ hybridization figure (non-diverting tissues of nasopharyngeal carcinoma and transfer nose
Pharynx cancer tissue).（c）The statistics credit schematic diagram of miR-23a tissue expressions.（d）MiR-23a is in Nasopharyngeal Carcinoma Patients expression and prognosis
Relational graph.（e）QRT-PCR shows the cellular expression levels schematic diagram of miR-23a.
Specific implementation mode
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.
A kind of miR-23a is preparing the application in treating TSGA10 low expression medicine for nasopharyngeal.
Key instrument used in following embodiment is：Full-automatic high-pressure steam disinfection cabinet（Japanese TOMY companies）；Water jacket
Formula carbon dioxide incubator（Thermo companies of the U.S.）；Medical purification workbench（Suzhou Decontamination Equipment Plant）；It is inverted fluorescent phase-contrast
Microscope（German Leica companies）；Cryogenic freezing centrifuge（Beckman companies of the U.S.）；Thermostat water bath（The U.S.
Pharmacia companies）；Microplate reader（Thermo companies of the U.S.）；Fluorescence quantitative PCR instrument（Eppendoff companies of the U.S.）；Image point
Analysis system（Alpha innotech corp CA companies of the U.S.）
Embodiment 1：Patient and tissue specimen
Tissues of nasopharyngeal carcinoma sample and blood sample are all from Hospital Attached to Nantong Univ.'s ENT ＆ HN Surgery Dept..150 nasopharynxs
Cancer patient is pressed from both sides during being on October 1, -2008 years on the 30th June in 2006 in Hospital Attached to Nantong Univ.'s ear-nose-throat department outpatient service biopsy
The sample taken, without chemicotherapy, operation or other treatment before biopsy.Through follow-up as long as five years, we, which obtain, completely faces
Bed information and pathological data.Nasopharyngeal carcinoma chip 150/51 is super prepared for Shanghai core.The pathological diagnosis of sample and be by stages according to
According to the World Health Organization in 2005（WHO）Criteria for classification and 1997 nasopharyngeal carcinoma UICC parting systems.
We have obtained the agreement of patient before the study, and obtain batch of Ethics Committee of Hospital Attached to Nantong Univ.
It is accurate（Approval number：2016--101）
Embodiment 2：Cell origin
Human nasopharynx's cancer cell（CNE-1、CNE-2、5-8F、6-10B）And normal nasopharyngeal immortalizes epithelial cell（NP-69）Train
It supports in ENT ＆ HN Surgery Dept. laboratory.
Embodiment 3：Paraffin section in situ hybridization
Sample dewaxes（This step carries out in draught cupboard）：3 x 5min of dimethylbenzene；Remove dimethylbenzene：About ethyl alcohol 100%
It embathes 10 times, about 100% ethyl alcohol embathes 10 times, and 100% 5min of ethyl alcohol, about 96% ethyl alcohol embathes 10 times, ethyl alcohol 96%
5min, about 70% ethyl alcohol embathe 10 times, ethyl alcohol 70% 5min, PBS 2-5min
Protease digestion sample：Proteinase K 15ug/mL at 37oC X40min；PBS 2 times；Dehydration of alcohol；It is placed on clean
15min is air-dried on paper.
Hybridize (Tm -21oC of hybridization temperature=probe)
Stringent develops a film
Immune detection：1, slide is dried, room temperature is put into confining liquid, closes 15min；2, after blotting confining liquid with paper, it is placed on wet box
1:800（1：500-1：Between 2000）Anti-DIG-AP Fab fragments, 4 spend night；3, room temperature, PBST 3 times
X3min；
Color reaction (room temperature is protected from light)：NBT/BCIP.
In situ hybridization result judgement result：Assessment is carried out by two pathology teachers in the case where not knowing slice data, is tied
Fruit determination method is as follows.Staining power：Strong dye（3 points）, middle dye（2 points）, understain（1 point）, do not contaminate（0 point）；Area：≤5 %（0
Point）, 6-25 %（1 point）, 26-50%（2 points）, 51-75%（3 points）, >=76（4 points）.Staining power result of calculation is multiplied by area meter
Calculate obtained by result is final result.≤ 4 point as a result it is low expression group,>4 points are high expression group.
Embodiment 4：CCK-8
The inoculating cell suspension in 96 orifice plates（100 μ L bases trainings/hole, attached cell at least need 1000 cells per hole）.It will training
Foster plate puts preculture in the incubator（37℃,5% CO2）.
CCK-8 solution is added to every hole（+ 90 μ L basigamys of 10 μ L stostes）（To avoid influencing the reading of OD values, it is careful not to
Bubble is generated in hole）.
37 DEG C are incubated culture plate 90min.
The absorbance at 450nm is measured with microplate reader.
Embodiment 5：Transwell is migrated
Reagent and cell are placed in 37 DEG C of incubations.
Cell culture to be measured to exponential phase, pancreatin digestion obtains suspension cell, and PBS is washed 2 times, uses free serum culture
Base（Basigamy）Suspension cell counts, adjustment a concentration of 1 × 105/mL。
In lower room（That is in 24 orifice plates）The 500 complete trainings of μ L are added（Culture medium containing 10% serum）, upper chamber（Common 8.0um films）
100-150 μ L cell suspensions, incubator culture 12-24h is added.
It is careful to take out chamber, upper chamber liquid is blotted, paraformaldehyde room temperature fixes 30 min.Upper chamber fixer is blotted, is used
Cotton swab gently wipes indoor cell, fixes dyeing with 0.1% crystal violet, room temperature dyes 15-30 min.
Chamber is taken out, is rinsed for several times with clear water, sucks upper chamber liquid.It waits for that chamber dries, cell is moved to load glass
Piece takes several visuals field to take pictures, count under microscope, statistical result.
Embodiment 6：qRT-PCR (miRNA)
The preparation of total serum IgE：1, Trizol (tissues：50mg-100mg tissues plus 1ml trizol;Cell：1ml/ bottles)；2, it receives
Collect dissolved matter, move to centrifuge tube, chlorination is imitative（Ratio：1ml Trizol add 0.2ml chloroforms）, 15s is acutely got rid of, 2-3min is stood
(room temperature) centrifuges 4 DEG C/12000r/15min；3,3 layers of liquid point, takes three layers of top layer：Upper colourless aqueous phase/middle DNA phases/
Lower phenol chlorine phase（Supernatant layer recycles total serum IgE with isopropanol precipitating, and middle layer recycles DNA, lower layer's isopropyl alcohol precipitation with ethanol precipitation
Form sediment recycling albumen.）；4,500 μ l isopropanols stand 10min；5,4 DEG C/12000r/10min is centrifuged；6, supernatant is abandoned, 75% second is added
Alcohol 1ml (twice)；7,4 DEG C/7500r/5min is centrifuged；8, supernatant is abandoned, it is dry（It dries）；9, add DEPC water dissolutions（ 20μl ）
；10,60 DEG C of water-bath 10min；
Reverse transcription；QRT-PCR reacts.
Embodiment 7：Cell cycle
（1）Collect cell：Cell arrives suitable time point through processing culture, and pancreatin thoroughly digests（Operating process will subtract as far as possible
Few cell mechanical damage prevents that cell DNA is caused to lose and influence result accuracy）, it is finished training and terminates digestion, is collected into
In the pipe of 1.5ml.
（2）Centrifugation, 1200 turns of 5min abandon supernatant, cell are washed with 1mL PBS.
（3）Repeat step（2）.
（4）It is fixed：Cell is thoroughly resuspended in 1mL fixed solutions.（70% ethyl alcohol, -20 DEG C at least for 24 hours）（One month it
Interior detection is effective）.
（5）The fixed cell of centrifugation, abandons supernatant, and PBS washs cell 3 times to remove remaining ethyl alcohol.
（6）With 4 DEG C of penetrating 10min of PBS of 1%Triton X-100, every pipe 200ul.
（7）Using the PBS of the X-100 containing Triton containing RNase A（1：1000）Cell is resuspended, is protected from light 4 DEG C of reactions
20 min, often pipe 300-400ul.
（8）200ul PI coloring agents are added, are protected from light 4 DEG C of dyeing 20min（After the dyestuff can be combined with nucleic acid chains, it is excited
Send out fluorescence）.
（9）Filtration cell.
（10）The cell cycle is measured using flow cytometer.It is general to count ten thousand cells of 2-3.
Embodiment 8：Western Blot
The extraction of protein and assay：1, cell to be measured abandons culture medium, is placed on ice bath, and precooling PBS is rinsed 3 times, is exhausted
PBS.2, the protein cleavage liquid of 100u1 precoolings is added in every bottle of cell（RIPA:PMSF=100:1）, fully scraped with cell scraper
Cell is taken, and is moved in EP pipes.3, every 5 min spirals concussion EP pipes 5s × 3 time, abundant crack protein.4, it centrifuges：4 DEG C,
12000rpm/min 15 minutes, abandons precipitation, and BCA methods measure after albumen concentration boiling water boiling 15 minutes, packing, -80 DEG C of preservations.
PAGE gel electrophoresis：1, offset plate is cleaned, distilled water leak detection.It hangs down with the injection of 10% resolving polyacrylamide gel
In the glass plate interlayer of straight electrophoresis tank, isopropanol moulding is placed at room temperature for about 1h to separation gel solidification.2,5% polyacrylamide is prepared
(30% deposit glue 0.67 ml, 1.0M Tri-HCL 0.5ml, 10%SDS 0.04 is added in ddH2O 2.7ml to amine spacer gel
Ml, 10% 0.004 μ l of Ammonium Persulfate 98.5 0.04ml, TEMED).The isopropanol on solidification separation gel is removed, deionized water is gently
It rinses, sucks excessive moisture, spacer gel, quick insertion Telfon combs, room temperature solidification is added.3, gel slab is fixed on electricity
Swimming slot, extracts Telfon combs, electrophoretic buffer is added in electrophoresis tank.4, loading albumen is taken out（20 ug）, by what is measured
Albumen concentration determines loading volume, adjusts electrophoresis time.5, electrode is installed, connected with the mains, 80 V of constant pressure makes protein go to together
On one starting line, 120 V are changed to when protein is run to separation gel.6, glue is cut, suitable offset plate block merging transferring film buffer solution is taken.
Transferring film liquid will be put into after the polarization of pvdf membrane methanol, distillation water hydratable simultaneously.7, by glue, cotton pad and filter paper pvdf membrane be fabricated to " three
Mingzhi " structure, cotton pad-filter paper-glue-PVDF- filter paper-cotton pad clamp.8, " sandwich " is put into transferring film slot（It is black to black）,
Transferring film liquid and ice chest is added, transferring film slot is integrally placed in the basin for filling ice water.Installation electrode connects power supply, and transferring film condition is set
It is set to constant current 300 mA, 90 min.9, pvdf membrane is taken out after transferring film, carries out positive and negative label, 5% skim milk closing 1
h.10, TBST washes 5 min × 3 time of film.11, it is incubated primary antibody antibodies against p-ERK, ERK,
Flotillin-1, CD63, β-actin (Santa Cruz Biotechnology, CA, USA), TSGA10
(Sigma-Aldrich, America), CD9 (Abcam, MA, USA).12, BST washes 10 min × 3 time of film.13, it incubates
Educate secondary antibody（Anti-rabbit）, 2 h of room temperature.14, TBST washes 10 min × 3 time.15, develop in darkroom.16, film gives scanner scanning
Afterwards, Image J analyze its gray scale.The ratio of the gray value of comparative purpose albumen and β-actin, gained is the opposite of purpose albumen
Expression quantity.
Embodiment 9：Scratch experiment
5 × 105 cells are added per hole to be laid on 6 orifice plates and cultivate 16-24h, make to form cell monolayer（Quantity is with adherent energy overnight
Being paved with is advisable, and quantity can cultivate a period of time when few low to plate is paved with）.
With 100 μ L pipette tips perpendicular to 6 orifice plates, it is in " one " stroke trace on cell monolayer, is cleaned 3 times, incubated with sterile PBS
It educates and changes the RMPI.1640 culture solutions containing 10% fetal calf serum into afterwards for 24 hours, be put into 37 DEG C of 5% CO2 culture.
It observes and takes pictures：Culture medium was sucked by 0,6,12,24 hour, after cleaning 3 times with PBS, in inverted fluorescence microscope
Lower observation is simultaneously taken pictures.
It calculates, compare, analyze.
Embodiment 10：Statistical procedures
Statistical analysis, measurement data mean ± standard deviation (Mean ± SD) are carried out to data using GraphPad Prism
It indicates.WithP<0.05 is significant difference.Every group of experiment is at least in triplicate.
Claims (1)
1. a kind of miR-23a is preparing the application in treating TSGA10 low expression medicine for nasopharyngeal.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115317490A (en) * | 2021-12-24 | 2022-11-11 | 南通大学附属医院 | Application of compound BML-275 in preparation of medicine for improving nasopharyngeal carcinoma prognosis |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2112235A1 (en) * | 2008-04-24 | 2009-10-28 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Compositions and methods for microRNA expression profiling of nasopharyngeal carcinoma |
-
2018
- 2018-01-30 CN CN201810086673.1A patent/CN108310001A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2112235A1 (en) * | 2008-04-24 | 2009-10-28 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Compositions and methods for microRNA expression profiling of nasopharyngeal carcinoma |
Non-Patent Citations (3)
| Title |
|---|
| JIA YUN ZHU ET AL.: "Identification of Novel Epstein-Barr Virus MicroRNA Genes from Nasopharyngeal Carcinomas", 《JOURNAL OF VIROLOGY》 * |
| JIA-QUAN QU ET AL.: "MiR-23a sensitizes nasopharyngeal carcinoma to irradiation by targeting IL-8/Stat3 pathway", 《ONCOTARGET》 * |
| LILI BAO ET AL.: "Metastasis-associated miR-23a from nasopharyngeal carcinoma carcinoma-derived target gene TSGA10", 《ONCOGENE》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115317490A (en) * | 2021-12-24 | 2022-11-11 | 南通大学附属医院 | Application of compound BML-275 in preparation of medicine for improving nasopharyngeal carcinoma prognosis |
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Application publication date: 20180724 |