CN108277225A - A kind of application of circular rna in diagnosing and/or treating ameloblastoma - Google Patents
A kind of application of circular rna in diagnosing and/or treating ameloblastoma Download PDFInfo
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- CN108277225A CN108277225A CN201810409975.8A CN201810409975A CN108277225A CN 108277225 A CN108277225 A CN 108277225A CN 201810409975 A CN201810409975 A CN 201810409975A CN 108277225 A CN108277225 A CN 108277225A
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- 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
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The present invention provides a kind of application of circular rna in diagnosing and/or treating ameloblastoma, and the circbase ID of the circular rna are：Has circRNA 100375, sequence such as SEQ NO:Shown in 1, finished up the cyclic structure that connects by the nucleotide sequence as shown in the sequence.The cyclic DNA can be used as the molecular marker that ameloblastoma diagnoses and/or treats.This research is started in molecular mechanism level from non-coding RNA hsa circRNA 100375, its effect played in ameloblastoma invasive growth is demonstrated, and new developing direction is provided for the diagnosing and treating of ameloblastoma.
Description
Technical field
The invention belongs to biomedicine technical fields, are related to a kind of circular rna, and in particular to a kind of circular rna is diagnosing
And/or the application in treatment ameloblastoma.
Background technology
Ameloblastoma（Ameloblastoma, AB）It is common odontogenic tumor, occupies in mouth neoplasm important
Status.Ameloblastoma is classified as benign tumour by the classification of World Health Organization head and neck neoplasm, and ROSAI ＆ ACKERMAN surgeries
Pathology（Tenth edition）Ameloblastoma is classified as borderline（Low potential malignancy）Tumour, at present therapeutic modality be with operative treatment
It is main, but due to its biological behaviour with invasive growth and canceration potential, Postoperative recurrent rate is higher, or even can occur remote
Every organ metastasis, the life quality of patient has been seriously affected.Research about ameloblastoma invasion biological behaviour has become
The conclusion not yet reached an agreement for the mechanism of hot spot, but ameloblastoma invasive growth.Therefore, further investigated ameloblastoma
Invasion mechanism is of great significance.
Circular rna（Circular RNA, circRNA）A nova of RNA research fields in recent years, be one kind by spy
Different alternative splicing generate and in eukaryocyte wide expression annular endogenous RNA molecule, there is closed hoop structure,
Its special structure feature makes it have special biological function.The study found that circular rna can play microRNA molecule
Sponge acts on, while being also possible to adjust host gene expression, with rna binding protein interaction regulation and control translation process, plays suitable
Formula transcriptional control acts on, it might even be possible to regulate and control alternative splicing etc., comparatively or a fresh things due to circular rna,
Still is in for the research of circular rna, research conclusion is both needed to lot of experiment validation the starting stage.Part cyclic RNA is rich in
Micro RNA binding sites can play competitive endogenous RNA effects, be released to its target base as miRNA molecule sponge
The depression effect of cause.For this purpose, effect of the research circular rna during invasive growth of ameloblastoma is to ameloblastoma
Diagnose and/or treat and be of great significance.
Invention content
In view of the problems of the existing technology, a kind of circular rna of present invention offer is diagnosing and/or treating ameloblastoma
In application.The technical scheme is that：
The first aspect, the present invention provide a kind of circular rna, and the circbase ID of the circular rna are：has- circRNA-
100375, sequence such as SEQ ID NO:It is the cyclic annular knot connected by the nucleotide sequence ending as shown in the sequence shown in 1
Structure.There has been no any reports about 100375 functions of circular rna has- circRNA- at present.
The second aspect, the present invention also provides a kind of kits of diagnosis ameloblastoma, and the kit includes detection
The reagent of the circular rna expression quantity.
Further, the reagent includes the primer for expanding the circular rna.
Further, the primer sequence such as SEQ ID NO:Shown in 2.
Further, the kit further includes the reagent of extraction and purifying RNA, progress from tissue by cytoma and tumor
The reagent of reverse transcription and quantitative fluorescent PCR.
In terms of third, the present invention also provides a kind of pharmaceutical composition for treating ameloblastoma, described pharmaceutical compositions
At least one of reagent including the circular rna and the downward circular rna expression quantity.
4th aspect, the present invention also provides a kind of expression vector for treating ameloblastoma, the expression vector expression
The circular rna.
5th aspect, the present invention also provides the circular rnas to prepare the purposes in diagnosing ameloblastoma kit.
6th aspect, the present invention also provides the circular rnas in preparing or screening the drug for the treatment of ameloblastoma
Purposes.
7th aspect, the present invention also provides the reagents for lowering the circular rna expression quantity to prepare treatment ameloblast
Purposes in the drug of tumor.
Compared with prior art, the features of the present invention and advantageous effect are：We combine Human Circular RNA tables
Up to spectrum chip technology and bioinformatics technique, it is found that a new circular RNA molecule for the first time（hsa_circRNA_
100375）, the high expression in ameloblastoma, expression and the ameloblastoma of hsa_circRNA_100375 recur close phase
It closes.The expression for lowering hsa_circRNA_100375 can significantly inhibit people and immortalize ameloblastoma cell line AM-1
Cell migration and invasive ability prompt hsa_circRNA_100375 may be mainly by promoting the invasion of ameloblastoma raw
Length plays a role, and molecular basis is provided for the diagnosing and treating of ameloblastoma.
Description of the drawings
Fig. 1 is relative expressions of the hsa_circRNA_100375 in tissue by cytoma and tumor in the embodiment of the present invention 1
Spirogram.
Fig. 2 is the relative expression of primary group of cytoma and recurrence group hsa_circRNA_100375 in the embodiment of the present invention 1
Spirogram.
Fig. 3 is the qRT-PCR verifications of hsa-circRNA-100375-siRNA transfections in the embodiment of the present invention 2
Figure, wherein Control represents blank control group；NC represents negative control group（SiRNA control groups）；Si-1, Si-2, Si-3
Respectively represent 3 siRNA groups of transfection synthesis；* * represent p value<0.0001, difference highly significant.
Fig. 4 is to inhibit hsa-circRNA-100375 expression to AM-1 cell migrations and invasion energy in the embodiment of the present invention 2
The influence diagram of power, wherein Fig. 4-1 are the cell migration of blank control group as a result, Fig. 4-2 is the cell migration knot of negative control group
Fruit, Fig. 4-3 are the cell migration of hsa-circRNA-100375-siRNA transfection groups as a result, Fig. 4-4 is the thin of blank control group
Born of the same parents invade as a result, Fig. 4-5 is the cell invasion of negative control group as a result, Fig. 4-6 is hsa-circRNA-100375-siRNA turns
The cell invasion of dye group is as a result, the transfer ability that Fig. 4-7 is three groups influences the comparison diagram of result, the invasion energy that Fig. 4-8 is three groups
Power influences the comparison diagram of result.
Specific implementation mode
For the object, technical solutions and advantages of the present invention are better described, with reference to specific embodiment to the present invention
It is described in further details, the explanation of the invention is not limited.
Embodiment 1：Expressions of the hsa_circRNA_100375 in ameloblastoma
1.1 tissue specimen
Collect 26 AB operation Operated Specimens of Chinese Medical Sciences University's stomatological hospital decorative sursery in January, 2014 in December, 2015
(including NOM by AB and tumor), patient age 8-82 Sui, wherein male 16, women 10；Maxilla 14, mandibular 12.
Histopathology parting：Capsule full mold 26.Primary group 15, recurrence group 11, follow up time 6-30 months.Wherein 6 AB and
Tissue is used for circular rna chip analysis by tumor, and tumor patient relevant clinical data refers to table 1.All patients, which do not go, preoperative puts
Chemotherapy.The sample cut off of performing the operation is immediately placed in -80 DEG C of refrigerators after precooling normal saline flushing is clean and freezes.This research obtains
The approval of Ethics Committee of Chinese Medical Sciences University.
1 ameloblastoma patient clinical data of table
1.2 main agents
1.2.1 RNA extractions and purified reagent
TRIzol ：Invitrogen companies of the U.S.
Chloroform：Beijing Xin Dingpengfei developments in science and technology Co., Ltd
Absolute ethyl alcohol：Beijing Rui Zekang Science and Technology Ltd.s
Isopropanol：Thing instrument（Beijing）Science and Technology Ltd.
1.2.2 real-time quantitative PCR reagent
iScript cDNA synthesis kit：BIO-RAD companies of the U.S.
ABI Power SYBR Green PCR Master Mix：American AB I companies
Primer synthesizes：Upper Haikang is at biological Co., Ltd
1.2.3 PCR primer sequence（Hsa_circRNA_100375 primer sequences such as SEQ ID NO:Shown in 2, β-actin primers
Sequence such as SEQ ID NO:Shown in 3）
Table 2
1.3 key instrument
Nanodrop ND-1000 ultraviolet specrophotometers：Nanodrop companies of the U.S.
High speed low temperature centrifugal machine：Sigma companies of the U.S.
7500 type real-time PCR systems of ABI：American AB I companies
The extraction of 1.4 total tissue RNAs, quality and Concentration Testing
The TRIZOL reagents of 1 ml are added in 100mg tissue samples, electric homogenizer is homogenized.30 DEG C incubation 5 min, every 1
The chloroform of 0.2 ml is added in the sample of the TRIZOL reagents homogenate of ml, acutely vibrates 15 s of tube body, 30 DEG C are incubated 2-3 min.4
DEG C 12,000 g centrifuge 15 min.Mixing liquid is classified into the red phenol chloroform phase of lower layer, the nothing of middle layer and upper layer after centrifugation
The water phase of color.RNA is all distributed in water phase.Water phase is transferred in new centrifuge tube.Water phase is mixed with isopropanol to precipitate
RNA adds 0.5 ml isopropanols while 1 ml TRIZOL reagents are added.After mixing after 30 DEG C of 10 min of incubation, 4 DEG C 12,
000 g centrifuges 10 min.The RNA of precipitation is the gelatinous precipitate block formed on bottom of the tube and side wall.Supernatant is removed, is added 1
75% ethyl alcohol of ml cleans RNA precipitate, and 4 DEG C 7,500 g centrifuge 5 min.Remove ethanol solution, 5-10 min of air drying.
When dissolving RNA, the water that no RNA enzyme is first added is blown and beaten several times repeatedly with rifle, 55-60 DEG C of 10 min of incubation.The RNA solution of acquisition is protected
It is stored in -80 DEG C.OD value of the cell total rna at 280 nm and 260 nm is measured using ultraviolet specrophotometer
（OD）, detect the purity and concentration of RNA.
1.5 reverse transcriptions synthesize cDNA
Reaction solution is prepared in 0.2 ml PCR pipes（Table 2）；
3 20ul reverse transcription reaction solution of table prepares system
PCR pipe is placed in 25 DEG C of 5 min, 42 DEG C of 30 min of incubation, 85 DEG C of 5 min of denaturation, 4 DEG C keep the temperature.
1.6 SYBR Green RT-PCR
1) it is carried out according to ABI Power SYBR Green PCR Master Mix operating processes；
2) reaction system is prepared（Table 3）；
3) mixing said mixture, the of short duration centrifugations of 5000 rpm；
4) PCR pipes are placed in quantitative PCR apparatus and are reacted, 95 DEG C, 10 min；Then 40 cycles are carried out：95 DEG C,
15 s；60 DEG C, 1 min annealing/extension.
4 20 μ l PCR of table react preparation system
| Component | Volume |
| 1×SYBR green PCR master mix | 10ul |
| Forward primer(200nM) | 2ul |
| Reverse primer(200nM) | 2ul |
| Template (10ng) | 2ul |
| ddH2O | 4ul |
| Total volume | 20ul |
QPCR after reaction, into melt program：95 DEG C of 15sec, 60 DEG C of 1min, 95 DEG C of 15sec add and do melting curve,
To distinguish amplified production and primer dimer.
1.7 result criterions
Real-time quantitative PCR testing result is divided using Rotorgene Real-Time Analysis Software softwares
Analysis.β-actin are used as internal reference, with 2-△△CTMethod indicates relative quantification as a result, indicating target gene by tumor sample and tumor
The opposite variation of expression between Normal oral mucosa sample.
Experimental result shows that relative expression quantities of the Hsa_circRNA_100375 in 26 AB is significantly higher than NOM by tumor
（P<0.01）；And the relative expression quantity of AB recurrence groups hsa_circRNA_100375 is significantly higher than primary group（P<0.01）（Respectively
As illustrated in fig. 1 and 2）.
Embodiment 2：Influences of the hsa_circRNA_100375 to ameloblastoma cell behaviors
Cell line used in 2.1
People immortalizes ameloblastoma cell line AM-1, is given by Japanese scholars.
2.2 primer
Hsa_circRNA_100375 primers and-actin primers are synthesized by upper Haikang at biological Co., Ltd, primer sequence point
Not such as SEQ NO:2 and SEQ NO:Shown in 3.
2.3 main agents
Defined keratinocyte-SFM（1X）Liquid：Match Mo Feishier companies in the U.S.
1640 culture mediums：Match Mo Feishier companies in the U.S.
Hsa_circRNA_100375 siRNA and Control-siRNA：The Guangzhou bio tech ltd Ji Sai
Lipofectamine TM 2000：Invitrogen companies of the U.S.
Cell Counting Assay Kit-8：Eastern Renhua Science and Technology Ltd.
Crystal violet：He Peng biology Science and Technology of Shanghai Co., Ltd
FITC Annexin V Apoptosis Detection Kit I：U.S. company BD
PI：U.S. company BD
The cells Transwell：U.S. company BD
Matrigel matrigels：U.S. company BD
2.4 key instrument
Superclean bench：The conspicuous Teco Corp. in Shenyang
C02 cell incubators：Thermo companies
Constant temperature water bath：Shanghai standing grain work
Inverted biologic microscope：Japanese OLYMPUS companies
Stream type cell analyzer：U.S. company BD
Multi-function microplate reader：BioTEK companies
2.5 qRT-PCR methods
With 1.7 sections of embodiment 1.
2.6 cell recoveries, passage, culture
Cryopreservation tube is taken out from liquid nitrogen container, is directly immersed in 37 DEG C of warm water, constantly shakes and it is enabled to melt as early as possible.From 37 DEG C of water-baths
Middle taking-up cryopreservation tube is sucked out cell suspension, is added in centrifuge tube and instills 10 times or more Defined keratinocyte-SFM,
Mixing.Centrifugation（1000rpm, 5min）, liquid is discarded supernatant, culture solution is added, cell is resuspended, count, adjusts cell density, inoculation
Culture bottle, 37 DEG C of incubator stationary cultures.Extinction culture medium is added 1ml 0.25%EDTA- trypsin solutions and stands 5 min.
The Defined keratinocyte-SFM of 6 times of volumes are added, terminate digestion, piping and druming, suspension cell.Centrifugation（1000 rpm, 5
min）, supernatant is abandoned, culture medium, piping and druming precipitation is added to be formed and cell is resuspended.Take appropriate cell inoculation in new tissue culture plate,
In 5% CO2It is cultivated in cell incubator.
The design and synthesis of 2.7 Hsa_circRNA_100375 siRNA
Hsa_circRNA_100375_siRNA sequences are designed and synthesized by upper Haikang at bioengineering Co., Ltd, and are passed through
QRT-PCR detects expression quantity, judges whether transfection is effective.For the sequence of hsa_circRNA_100375,3 siRNA are designed
Sequence, it is as follows：（SEQ NO are seen respectively:4、SEQ NO:5、SEQ NO:Shown in 6）
Hsa-circRNA-100375 siRNA-1：
target sequence: 5'-ATTCCCGTCTTCCGTGTGT-3'
hsa-circRNA-100375 siRNA-2：
target sequence: 5'-GTCTCCCATTCCCGTCTTC -3'
hsa-circRNA-100375 siRNA-3：
target sequence: 5'-TCCCATTCCCGTCTTCCGT-3'
2.8 experiment packet
It is divided into 3 groups：Experimental group（SiRNA groups）For hsa-circRNA-100375 siRNA/ are added when AM-1 cell transfectings
2000 compounds of Lipofectamine TM；Negative control group（SiRNA control groups）To be added when AM-1 cell transfectings
SiRNA NC/Lipofectamine TM2000 compounds；Blank control group（Control groups）Not carry out any intervention
AM-1 cells.
2.9 cell transfecting
1）Pancreatin digests the AM-1 cells in culture dish, counts, and not antibiotic 1640 culture medium of use spreads 6 hole cell culture
Plate, per 1500 μ l of hole culture medium.
2）It is put into CO2 incubators and cultivates, when cell covers with 80%, start to transfect.
3）10 ul siRNA or siRNA-NC are dissolved in 1640 culture mediums of 250 μ l serum-frees, after soft mixing
It is placed at room temperature for 5 min.
4）5 μ l lipofectamine TM 2000 are dissolved in 1640 culture mediums of 250 μ l serum-frees, soft mixing
After be placed at room temperature for 5 min.
5）Above-mentioned 2 pipe is mixed, 20 min are placed at room temperature for after soft mixing.
6）Above-mentioned mixed liquor is added in 6 orifice plates hole to be transfected, gently mixing.It is trained in 37 DEG C, 5 % CO2 incubators
It supports.
7）Complete medium is changed after 6 h, is continued after cultivating 24 h, cell is collected and is used for subsequent experimental.
2.10 real-time quantitative PCRs detect transfection efficiency
1) cell total rna is extracted:1 ml trizol mixings are added per hole cell, are stored at room temperature 5 min；Every 1 ml trizol
Add 0.2 ml chloroforms；Acutely 15 s of oscillation, are stored at room temperature 2-3 min；4 DEG C of 12,000g centrifuge 15 min；Take the colourless water in upper layer
Mutually managed to another Eppendorf；0.5 ml isopropanols, mixing is added to be stored at room temperature 10 min；4 DEG C of 12,000g centrifugations 15
min；Supernatant is abandoned, 1 ml, 75% ethyl alcohol washing precipitation, the mixing that turns upside down 6-8 times are added；4 DEG C of 12,000g centrifuge 5min；It sucts
Clearly, room temperature is dried in the air 10-15 min；Precipitation is dissolved in 20-50ul DEPC water；Survey the concentration and purity of RNA；RNA is placed on standby on ice
With, or -80 DEG C of long-term preservations.
2) reverse transcription, real-time quantitative PCR measure hsa_circRNA_100375 expressions, and step is the same as embodiment 1
1.6 section.
2.11 Transwell migration experiments
After siRNA transfects 24 h, cell suspension is made in vitellophag, detects cell migration rate.
1）Take 100 μ l of cell suspension（Cell density 1x106）The cells Transwell are added.
2）500 culture mediums of the μ l containing FBS are added in room under 24 orifice plates.
3）Cultivate cell：24 h of routine culture.
4）Upper indoor cell is wiped with cotton swab, paraformaldehyde fixes 10 min, 0.5% violet staining, 15 min, carries out
It observes and takes pictures.
2.11 Transwell Matrigels
After siRNA transfects 24 h, cell suspension is made in vitellophag, detects cell migration rate.
1）Take 100 μ l of cell suspension（Cell density 1x106）The cells Transwell containing matrigel are added.
2）500 culture mediums of the μ l containing FBS are added in lower room.
3）24 h of routine culture cell.
4）Cotton swab wipes matrigel and upper indoor cell, and paraformaldehyde fixes 10 min, 0.5 % violet stainings 15
Min is observed and is taken pictures.
2.13 statistical analysis
Using 13.0 softwares of SPSS carry out statistical procedures, measurement data, with means standard deviation (± SD) it is described,
Using independent samples t test（Independent-Sample T Test）And one-way analysis of variance（One-Way ANOVA）Into
Row statistical analysis；Inspection level selects α=0.05.
2.14 result
The ameloblastoma cellular identification of 2.141 interference
By 3 interference sequences of structure（siRNA）With a control sequence（siRNA-NC）It is each separately transfected into AM-1 cells, qRT-
PCR examines transfection efficiency.Experimental result is shown, has transfected the AM-1 cells of Si-1, Si-2 and Si-3 sequence, hsa-circRNA-
Apparent downward has occurred compared with cellular control unit in 100375 expression（As shown in Figure 3）.Wherein, the interference effect of Si-2 sequences
Fruit is best.
Influences of 2.142 hsa-circRNA-100375 to ameloblastoma cell migration and invasive ability
By carrying out Transwell migrations, Matrigel, hsa_circRNA-100375 is inquired into AM-1 cell invasions and migration
The influence of ability.Experimental result shows that compared with the control group, after inhibiting hsa-circRNA-100375 expression, AM-1 cells move
It moves and invasive ability is remarkably decreased（P<0.05, Fig. 4）, show the migration and invasion of has-circRNA-100375 and AM-1 cells
Ability is closely related.
To sum up, the specific embodiment of the invention demonstrates hsa-circRNA-100375 significantly high tables in ameloblastoma
Reach and biological functions of the has-circRNA-100375 in ameloblastoma, can be used as ameloblastoma diagnosis and/
Or the molecular marker for the treatment of, possibility is provided for the gene therapy of ameloblastoma, is also the diagnosing and treating of ameloblastoma
New developing direction is provided.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changes, replacing and modification.
Sequence table
<110>The attached stomatological hospital of Chinese Medical Sciences University
<120>A kind of application of circular rna in diagnosing and/or treating ameloblastoma
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 257
<212> RNA
<213>Homo sapiens (homo sapiens)
<400> 1
<210> 2
<211> 42
<212> RNA
<213>" artificial sequence " ()
<400> 2
<210> 3
<211> 43
<212> RNA
<213>" artificial sequence " ()
<400> 3
<210> 4
<211> 19
<212> RNA
<213>" artificial sequence " ()
<400> 4
<210> 5
<211> 19
<212> RNA
<213>" artificial sequence " ()
<400> 5
<210> 6
<211> 19
<212> RNA
<213>" artificial sequence " ()
<400> 6
Claims (10)
1. a kind of circular rna, which is characterized in that the circbase ID of the circular rna are：Has- circRNA- 100375,
Its sequence such as SEQ NO:Shown in 1, finished up the cyclic structure that connects by the nucleotide sequence as shown in the sequence.
2. a kind of kit of diagnosis ameloblastoma, which is characterized in that the kit includes detecting the circular rna expression
The reagent of amount.
3. a kind of kit of diagnosis ameloblastoma according to claim 2, which is characterized in that the reagent includes expanding
Increase the primer of the circular rna.
4. a kind of kit way of diagnosis ameloblastoma according to claim 3, which is characterized in that the primer sequence
Such as SEQ NO:Shown in 2.
5. a kind of kit of diagnosis ameloblastoma according to claim 2, which is characterized in that the kit is also wrapped
Include the reagent of the reagent of extraction and purifying RNA, progress reverse transcription and quantitative fluorescent PCR from tissue by cytoma and tumor.
6. a kind of pharmaceutical composition for treating ameloblastoma, which is characterized in that described pharmaceutical composition includes the circular rna
With at least one of the reagent for lowering the circular rna expression quantity.
7. a kind of expression vector for treating ameloblastoma, which is characterized in that the expression vector expresses the circular rna.
8. circular rna described in claim 1 is preparing the purposes in diagnosing ameloblastoma kit.
9. purposes of the circular rna described in claim 1 in preparing or screening the drug for the treatment of ameloblastoma.
10. purposes of the reagent of circular rna expression quantity described in claim 1 in the drug for preparing treatment ameloblastoma.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109207425A (en) * | 2018-09-29 | 2019-01-15 | 中国医科大学附属口腔医院 | Porphyromonas gingivalis inducing macrophage excretion body rna expression research method |
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