CN109852692A - Circ_3480 and its preparing application and diagnostic reagent in nasopharyngeal carcinoma diagnosis preparation - Google Patents
Circ_3480 and its preparing application and diagnostic reagent in nasopharyngeal carcinoma diagnosis preparation Download PDFInfo
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Abstract
The invention belongs to oncomolecularbiology technical fields, and in particular to a kind of circular rna circ_3480 and its prepare application and corresponding preparation in nasopharyngeal carcinoma diagnosis preparation.The present invention collects many cases tissues of nasopharyngeal carcinoma and normal inflammatory nasopharyngeal epithelium tissue, and the expression of circ_3480 is had detected using qRT-PCR technology.As the result is shown, compared with 12 normal inflammatory nasopharyngeal epithelium tissues, circ_3480 obvious high expression in 28 tissues of nasopharyngeal carcinoma, circ_3480 expression is about 7 times of normal inflammatory nasopharyngeal epithelium in nasopharyngeal carcinoma group, and the difference of two groups of data has statistical significance (P=0.0006).Therefore, circRNA circ_3480 high expression, circ_3480 in tissues of nasopharyngeal carcinoma may have important biological function to the occurrence and development of nasopharyngeal carcinoma, can be used as nasopharyngeal carcinoma diagnosis molecular marked compound.
Description
Technical field
The invention belongs to oncomolecularbiology technical fields, and in particular to a kind of circular rna circ_3480 and its
Prepare the application in nasopharyngeal carcinoma diagnosis preparation and corresponding preparation.
Background technique
Circular rna (Circular RNA) is a research hotspot at present, and circRNA is mainly derived from protein coding gene
Exon 1, can also be by including sub-district, the area UTR, intergenic region, non-coding RNA site and the antisense position of known transcript
Point is formed.CircRNA is reversely spliced to form by Pre-mRNA (precursor messenger RNA, pre-mRNA)
One kind does not have 5 ' end cap and 3 ' end poly (A) tails and the non-coding RNA molecules that ring structure is formed with covalent bond.
CircRNA forming process can be divided into two major classes, i.e. exon cyclisation is (exon circularization) and interior
Containing the big mechanism of subring (intron circularization) two.CircRNA (the exonic in the proposition such as Jeck exon source
CircRNA, ecircRNA) lasso trick driving cyclisation (lariat-driven circularization) and introne can be divided into
Pairing driving cyclisation (intron-pairing-driven circularization) two kinds of generation types, lasso trick driving cyclisation
It is exon 3 ' it holds as 5 ' end acceptor splicing site (splice receptor) of donor splicing site (splice donor) attack, the area Alu
Covalent bond and form lasso structure, excision introne forms circRNA after lasso structure carries out internal splicing；Introne pairing
Driving cyclisation is to wipe out introne after two introne base pair complementarities form cyclic structure and form circRNA.It includes in fact
Son can also be cyclized itself, can form introne source circRNA (circular intronic RNA, ciRNA).
CircRNA is that one kind for being reversely spliced to form by Pre-mRNA does not have 5 ' end cap and 3 ' end poly (A) tails are simultaneously
The non-coding RNA molecule of the closed ring structure formed in the form of covalent bond.There are the stability, conservative, specificity of height,
And the high feature of content.
CircRNA most had found that subsequent Hsu MT et al. uses electron microscope skill for the first time early in 1976 in RNA virus
Art has found the presence of circRNA in the nephrocyte matter of monkey.In 1996, circRNA was found in human cell, with
The development of RNA-seq new-generation sequencing technology, more and more circRNA are found in recent years, known so far
CircRNA number has reached more than 30,000.CircRNA is also no longer regarded as the rna transcription sheet of mistake, but as non-volume
Dazzling star in code RNA research rises up slowly.Screening and more novel circRNA are verified as diagnosing tumor and prognosis
Biomarker and its application, and the protection that can be got well as early as possible in patent field can be obviously improved China in the technical field
International competitiveness.
Nasopharyngeal carcinoma (nasopharyngeal carcinoma, NPC) belongs to head and neck neoplasm, originates from pharynx nasalis epithelium group
It knits.It falls ill generally at the rear cornucopia of pharynx nasalis (Luo Senmiaole nest, Fossa of Rosenm ü ller), nasopharynx herein
Cancer cell easily invades neighbouring tissue and organ.Since the occurrence and development of nasopharyngeal carcinoma are the familial inheritance and body cell something lost by accumulating
Pass mutation and epigenetics be mutated caused by multistage complexity gene regulation process, be related to proto-oncogene and tumor suppressor gene
The epigenetic regulation etc. that activation and silencing and ncRNA are participated in.So the specific molecular mechanism of nasopharyngeal carcinoma occurrence and development is still
It is indefinite, and due to Tumor Heterogeneity and individual difference, the curative effect of traditional radiotherapy equipment system is not significant.Therefore pass through
CircRNA probes into itself and nasopharyngeal carcinoma occurrence and development mechanism, is further the diagnosis of nasopharyngeal carcinoma, and controls nasopharyngeal cardnoma proliferation, invades
Attacking and shifting will be the hot spot studied.
We carry out the circ_3480 that a length 425bp is detected in RNA-seq to nasopharyngeal carcinoma cell 5-8F.Pass through
Test finds that the circular rna exists with the occurrence and development of nasopharyngeal carcinoma and is associated with, it is possible to as nasopharyngeal carcinoma diagnosis marker, Yi Jizhi
The target spot for the treatment of.
Summary of the invention
The present invention has found the circ_3480 of a size 425bp from 5-8F cell RNA-seq data, and has found
Its existing relationship between nasopharyngeal carcinoma, it is possible to the target spot as nasopharyngeal carcinoma diagnosis marker and treatment.
The first purpose of the invention is to provide a kind of new circular rna circ_3480, sequence such as SEQ ID NO.1 institutes
Show.
A second object of the present invention is to provide the reagents of circular rna circ_3480 described in detection a kind of to examine in preparation
Application on disconnected or prognosis nasopharyngeal carcinoma tool.
Further, the reagent of the detection circular rna circ_3480 is PCR reagent.
Further, the reagent of the detection circular rna circ_3480 is PCR Real_time quantitative detection reagent.
Further, the primer in the PCR reagent are as follows:
Upstream primer: 5 '-CAACGTTCAGTGCCTCGAAA-3 '
Downstream primer: 5 '-CCTGCTCATACTGGTCAACG-3 '.
Further, the β-actin internal control primer in the PCR reagent are as follows:
Upstream primer: 5 '-TCACCAACTGGGACGACATG-3 '
Downstream primer: 5 '-GTCACCGGAGTCCATCACGAT-3 '.
Third object of the present invention is to provide a kind of preparations of diagnosis of nasopharyngeal carcinoma, comprising detecting the circular rna
The reagent of circ_3480.
Further, the reagent of the detection circular rna circ_3480 is PCR detection reagent.
Further, the primer in the PCR detection reagent are as follows:
Upstream primer: 5 '-CAACGTTCAGTGCCTCGAAA-3 '
Downstream primer: 5 '-CCTGCTCATACTGGTCAACG-3 '.
Further, the β-actin internal control primer in the PCR detection reagent are as follows:
Upstream primer: 5 '-TCACCAACTGGGACGACATG-3 '
Downstream primer: 5 '-GTCACCGGAGTCCATCACGAT-3 '.
The present invention collects many cases tissues of nasopharyngeal carcinoma and normal inflammatory nasopharyngeal epithelium tissue, is had detected using qRT-PCR technology
The expression of circ_3480.The results show that circ_3480 is in 28 noses compared with 12 normal inflammatory nasopharyngeal epithelium tissues
Obvious high expression in pharynx cancer tissue, circ_3480 expression is about the 7 of normal inflammatory nasopharyngeal epithelium in nasopharyngeal carcinoma group
Times, the difference of two groups of data has statistical significance (P=0.0006).Therefore, circRNA circ_3480 is in tissues of nasopharyngeal carcinoma
Middle high expression, circ_3480 may have important biological function to the occurrence and development of nasopharyngeal carcinoma, can be used as nasopharyngeal carcinoma and examine
Disconnected molecular marked compound.
Detailed description of the invention
Fig. 1 is the expression that qRT-RCR detects circ_3480 in tissues of nasopharyngeal carcinoma and non-tumour rhinitis epithelial tissue；
The expression analysis of circ_3480 turns to non-tumour nasopharyngeal epithelium organizational standard using β-actin as reference
1, N is non-tumour nasopharyngeal epithelium tissue, sample number 12；T is tissues of nasopharyngeal carcinoma, and sample number 28, n is sample size, is adopted
It is examined with t, P < 0.05 has statistical significance.
Fig. 2 is over-express vector map.
Fig. 3 is that qRT-PCR detects the circ_3480 overexpression effect in Nasopharyngeal Carcinoma Cell Line；
QRT-PCR detection circ_3480 in Nasopharyngeal Carcinoma Cell Line HONE1, HNE2 and CNE2 is overexpressed effect with β-
Actin is used as reference that pcDNA3.1 (+) group is standardized as 1, * P < 0.05, P < 0.001 * * P < 0.01, * * *.
Fig. 4 is the silence efficiency that qRT-PCR technology detects circ_3480siRNA in Nasopharyngeal Carcinoma Cell Line；
The silence efficiency of qRT-PCR detection circ_3480siRNA in Nasopharyngeal Carcinoma Cell Line HONE1, HNE2 and CNE2；
Circ_3480 expression is analyzed using β-actin as reference, and NCsiRNA group is standardized as 1, ns and represents nonsensical, * P
<0.05,**P<0.01,***P<0.001。
Fig. 5 is the external influence for being overexpressed circ_3480 and being proliferated to nasopharyngeal carcinoma cell；
A-c.qRT-PCR detection circ_3480 in Nasopharyngeal Carcinoma Cell Line HONE1, HNE2 and CNE2 is overexpressed efficiency；d-
F. MTT experiment is carried out to above-mentioned cell and detects ability of cell proliferation, the analysis of circ_3480 expression is using β-actin as ginseng
According to pcDNA3.1 (+) group is standardized as 1, ns and represents nonsensical, P < 0.001 * P < 0.05, * * P < 0.01, * * *.
Fig. 6 is the influence that external silencing circ_3480 is proliferated nasopharyngeal carcinoma cell；
A-c.qRT-PCR detection circ_3480 silence efficiency in Nasopharyngeal Carcinoma Cell Line HONE1, HNE2 and CNE2；d-f.
MTT experiment is carried out to above-mentioned cell and detects ability of cell proliferation, circ_3480 expression is analyzed using β-actin as reference,
PcDNA3.1 (+) group is standardized as 1, ns and represents nonsensical, P < 0.001 * P < 0.05, * * P < 0.01, * * *.
Fig. 7 is that qRT-PCR detects circ_3480 overexpression efficiency in scratch experiment cell；
A-c. human nasopharyngeal epithelioma 1 HONE1, HNE2 and CNE2 used in scratch experiment is overexpressed efficiency and tests inspection with qRT-PCR
It surveys, circ_3480 expression is analyzed using β-actin as reference, and pcDNA3.1 (+) group is standardized as 1, * P < 0.05, * *
P<0.01,***P<0.001。
Fig. 8 is the influence for being overexpressed circ_3480 to nasopharyngeal carcinoma cell HONE1, HNE2 and CNE2 scratch Healing Experiments；
A-c. Nasopharyngeal Carcinoma Cell Line HONE1, HNE2 and CNE2 transfects pcDNA3.1 (+) zero load, pcDNA3.1 (+)/circ_
3480, the scratch after cell density reaches 100% was taken pictures in observation in the 0th, 12,24 hour.
Fig. 9 is nasopharyngeal carcinoma HONE1, HNE2 and CNE2 the cell scratch experiment statistical chart for being overexpressed circ_3480；
A-c. 6 perimetry scratch widths are randomly selected for every group, 0 hour scratch width is standardized as 1, is taken statistics
Figure；It is each to test in triplicate, using Student's t-test method statistic.*P<0.05,**P<0.01,***P<0.001.
Figure 10 is that qRT-PCR detects circ_3480 jamming effectiveness in scratch experiment cell；
A-c. human nasopharyngeal epithelioma 1 HONE1, HNE2 and CNE2 jamming effectiveness used in scratch experiment is tested with qRT-PCR and is examined
It surveys, circ_3480 expression is analyzed using β-actin as reference, NC siRNA group is standardized as 1, * P < 0.05, * * P <
0.01,***P<0.001。
Figure 11 is the influence for interfering circ_3480 to nasopharyngeal carcinoma cell HONE1, HNE2 and CNE2 scratch Healing Experiments；
A-c. nasopharyngeal carcinoma HONE1, HNE2 and CNE2 cell transfecting NC siRNA/circ_3480siRNA, to cell density
Scratch after reaching 100% was taken pictures in observation in the 0th, 12,24 hour.
Figure 12 is nasopharyngeal carcinoma HONE1, HNE2 and CNE2 the cell scratch experiment statistical chart for interfering circ_3480；
A-c. 6 perimetry scratch widths are randomly selected for every group, 0 hour scratch width is standardized as 1, is taken statistics
Figure；It is each to test in triplicate, using Student's t-test method statistic.*P<0.05,**P<0.01,***P<0.001.
Figure 13 is the overexpression efficiency that qRT-PCR detects circ_3480 in the experiment of the cell Transwell matrigel invasion；
The cell a-c.Transwell matrigel invasion is tested human nasopharyngeal epithelioma 1 HONE1, HNE2 and CNE2 used and is overexpressed
Efficiency is tested with qRT-PCR and is detected, and circ_3480 expression is analyzed using β-actin as reference, by pcDNA3.1 (+) group
It is standardized as 1, * P < 0.05, P < 0.001 * * P < 0.01, * * *.
Figure 14 is the influence for being overexpressed circ_3480 to Nasopharyngeal Carcinoma Cell Line HONE1, HNE2 and CNE2 invasive ability；
HONE1, HNE2 and the CNE2 and respective cellular control unit for being overexpressed circ_3480 respectively are carried out
The experiment of the cell Transwell matrigel invasion.
Figure 15 is the cell the Transwell matrigel invasion experiment statistics figure for being overexpressed circ_3480；
Every group randomly selects 3 cell visuals field and carries out cell count, and take statistics figure；Each experiment in triplicate, uses
Student's t-test method statistic；Using β-actin as reference, by pcDNA3.1 (+) group be standardized as 1, * P <
0.05,**P<0.01,***P<0.001。
Figure 16 is the jamming effectiveness that qRT-PCR detects circ_3480 in the experiment of the cell Transwell matrigel invasion；
The cell a-c.Transwell matrigel invasion tests human nasopharyngeal epithelioma 1 HONE1, HNE2 and CNE2 interference effect used
Rate is tested with qRT-PCR and is detected, and circ_3480 expression is analyzed using β-actin as reference, and NC group is standardized as 1, * P
<0.05,**P<0.01,***P<0.001。
Figure 17 is the influence for interfering circ_3480 to Nasopharyngeal Carcinoma Cell Line HONE1, HNE2 and CNE2 invasive ability；
HONE1, HNE2 and the CNE2 and respective cellular control unit for interfering circ_3480 respectively carry out Transwell
The experiment of cell matrigel invasion.
Figure 18 is the cell the Transwell matrigel invasion experiment statistics figure for interfering circ_3480；
Every group randomly selects 3 cell visuals field and carries out cell count, and take statistics figure；Each experiment in triplicate, uses
Student's t-test method statistic；Using β-actin as reference, NC group is standardized as 1, * P < 0.05, * * P <
0.01,***P<0.001。
Specific embodiment
It is intended to further illustrate the present invention below in conjunction with specific embodiment, is not intended to limit the present invention.
It is attached swollen that normal inflammatory nasopharyngeal epithelium tissue and tissues of nasopharyngeal carcinoma sample used in the present invention are all from Central South University
The first visit patient of tumor hospitalize, without chemicotherapy and operative treatment.After fresh tissues of nasopharyngeal carcinoma is collected, it is immediately placed in liquid nitrogen
It is saved in tank, all experiment tissue samples acquisitions of relevant clinical data for then collecting all patients obtain Central South University's human relations
It manages committology and patient agrees to.
Tri- plants of Nasopharyngeal Carcinoma Cell Lines of HONE1, HNE2 and CNE2 are Tumour Inst., Zhongnan Univ.'s molecule used in the present invention
Genetic laboratory is saved.Cell culture condition are as follows: containing 10% fetal calf serum (FBS) and 1% dual anti-(penicillin, streptomysin)
RPMI1640 fluid nutrient medium, 37 DEG C, 95% humidity, 5%CO2Adherent growth in the constant incubator of concentration.
The primer of circular rna of the present invention is different from the design of linear rna primer, is designed according to splicing site two sides,
The Photographing On-line on the website Primer3.0, final primer synthetic work are ordered goods by sending Email, and the commission section of holding up is raw
The synthesis of object company Changsha combining unit.
(1)β-actin
Upstream primer: 5 '-TCACCAACTGGGACGACATG-3 ', sequence is as shown in SEQ ID NO.2；
Downstream primer: 5 '-GTCACCGGAGTCCATCACGAT-3 ', sequence is as shown in SEQ ID NO.3.
(2) circular rna circ_3480 real-time quantitative PCR primer
Upstream primer: 5 '-CAACGTTCAGTGCCTCGAAA-3 ', sequence is as shown in SEQ ID NO.4；
Downstream primer: 5 '-CCTGCTCATACTGGTCAACG-3 ', sequence is as shown in SEQ ID NO.5.
(3) circ_3480 overall length primer is expanded
Upstream primer: 5 '-CCATCGATGGATAACATCCCTGAGGACCTC-3 ', sequence is as shown in SEQ ID NO.6；
Downstream primer: 5 '-TCCCCGCGGGGACTTGTTGATCCAGAACACCA-3 ', sequence such as SEQ ID NO.7 institute
Show.
The present invention expresses to specifically strike low circular rna without influencing its glm gene, is designed according to splicing site
SiRNA, targeted silent circ_3480.
Circ_3480siRNA sequence:
Positive-sense strand (5'-3') CUGGAUCAACAAGAUAACAUU, sequence is as shown in SEQ ID NO.8；
Antisense strand (5'-3') UGUUAUCUUGUUGAUCCAGUU, sequence is as shown in SEQ ID NO.9.
Negative control:
Positive-sense strand (5'-3') UUCUCCGAACGUGUCACGUUU, sequence is as shown in SEQ ID NO.10；
Antisense strand (5'-3') ACGUGACACGUUCGGAGAAUU, sequence is as shown in SEQ ID NO.11.
Test result of the present invention is all made of statistical analysis: t, which is examined, to be used to evaluate the difference between two groups.Chi-square Test is used
To assess gene expression or not express in terms of the clinical parameters such as gender, age, tumor stage, clinical stages and transfer case
Difference.Survival analysis is examined using in terms of Kaplan-Meier.For indicating significance,statistical, all p values make for p < 0.05
Use two-sided test.Statistical analysis is carried out using SPSS13.0 and 5.0 software of Graphpad.
Expression of the embodiment 1:circ_3480 in tissues of nasopharyngeal carcinoma and cell
1, according to master sample acquisition scheme, we collect Nasopharyngeal Carcinoma Patients tissue samples 40 from Hunan Provincial Tumour Hospital
Example.All cases are that (time interval: in January, 2016 was to 2016 11 by the first visit patient of Hunan Provincial Tumour Hospital's Head and neck tumour
Month).Make a definite diagnosis 28 Cases Nasopharyngeal Carcinoma through pathology department, nasopharynx inflammatory patients 12 (excluded tumor disease, no active infectious disease,
Serious immunity disease and other major diseases).
Record complete personal information and clinical data during acquisition, including name, gender, the age, outpatient service number,
Number, histological type, case by stages and EBV infection conditions etc., are detailed in Excel electrical form screenshot in hospital.All samples
Patient's permission is all obtained in acquisition, and signs written agreement with patient, establishes the more complete sample storehouse of data.
2, nasopharyngeal carcinoma or normal inflammatory nasopharyngeal tissue RNA are extracted
(1) preparation: mortar is soaked in 3% hydrogen peroxide (H after being cleaned with detergent2O2) in 4 hours or more, wash away
For several times, primary with distillation washing, with masking foil covering mortar when taking-up (help to be heated evenly, prevent from polluting), it is placed in 180 DEG C
It is done in drying box 8 hours or more roasting.After reaching the dry roasting time, drying box is closed, takes out and grinds when box temperature degree to be dried is down to room temperature
Alms bowl deposits in clean area.
(2) liquid nitrogen grinding: being added Liquid nitrogen precooler in mortar, then clamps the pharynx nasalis tissue saved in cryopreservation tube, fastly
Speed grinding, grinds on one side, continuously adds a small amount of liquid nitrogen on one side, then grind, and clays into power shape until by nasopharyngeal tissue.According to best
Ratio, i.e., every 50-100mg is normal or nasopharyngeal carcinoma sample (NPC) adds 1ml Trizol.In our experimentations, a nasopharyngeal carcinoma
Tissue samples about 200mg or so, it is therefore desirable to 2ml Trizol.Then it is further ground, is placed in 4 DEG C of about 5- of refrigerator
10min allows tissue to crack completely.2ml Tube is transferred to when pyrolysis product is melted into pink liquid.Each sample can be with
Separation is saved to 2 Tube to -80 DEG C.
(3) aqueous phase separation: Tissue lysates of every 1000 μ l containing trizol add the chloroform of 4 DEG C of 200 μ l pre-coolings, concussion
About 30s is mixed.Low temperature environment is placed after five minutes, and 25 minutes centrifugally operateds (12,000rpm, 4 DEG C) are carried out.Centrifugation finishes,
Liquid in pipe can be observed and be divided into 3 layers, there are RNA in the hyaline layer of upper layer, middle layer is membranaceous white precipitate, lower layer's pink.
Therefore, the upper strata aqueous phase containing RNA is further drawn into 1.5ml Tube, with 100 μ l rifle gentle aspirations, avoids being drawn onto as far as possible
Middle layer and lower layer's substance, cause RNA to pollute.
(4) RNA precipitate: being added the isometric about 500 μ l of isopropanol of 1:1 in supernatant, and movement is gently turned upside down mixed
It is even for several times, be placed in -20 DEG C of refrigerator put 30 minutes after, be centrifuged 30 minutes (4 DEG C, 12,000rpm), it is seen that tube bottom has RNA precipitate, use
The suction of 100 μ l liquid-transfering guns discards supernatant, as far as possible reservation RNA precipitate.
(5) RNA is washed: 75% ethyl alcohol that 1ml is prepared with no enzyme water, mild turned upside down is added in every pipe RNA precipitate sample
Centrifuge tube, to wash RNA precipitate.It is then centrifuged for 5 minutes (4 DEG C, 7,600rpm), is discarded supernatant as far as possible with the liquid-transfering gun of 100 μ l,
Room temperature is dried 10~15 minutes.
(6) re-dissolve RNA and save: 15-30 μ l DECP water is added in every pipe sample, is stored in -80 DEG C.
3, cell total rna extracts
Preparation: after sterilizing without RNase water, 75% ethyl alcohol (no RNase prepare), chloroform, isopropanol, 1 × PBS,
No enzyme tip are managed with EP, and high speed low temperature centrifugal machine is cooled to 4 DEG C in advance, and experiment will first test table top and liquid-transfering gun with 75% before starting
Alcohol wipe.
(1) cell for taking RNA to be extracted, is cleaned twice with 1 × PBS or D-hanks；
Every hole adds 500 μ l Trizol lysates in (2) 12 orifice plates, lysis at room temperature 1-2 minutes, is gently blown down with liquid-transfering gun
Cell, upper and lower gentle inversion 10 times are stored at room temperature 5 minutes；
(3) chloroform (pressing 1mlTrizol:0.2ml chloroform: 0.5ml isopropanol) of 100 μ l is added, firmly shakes 15-30s,
It places 5 minutes on ice；
(4) 4 DEG C, 12000rpm/20min；
(5) it takes upper strata aqueous phase in the Tube pipe of pre-cooling, 250 μ l isopropanols is added, it is mixed with eddy mixer or liquid-transfering gun
Even (- 20 DEG C > 1h)；
(6) 4 DEG C, 12000rpm/30min, abandon supernatant；
(7) 75% ethyl alcohol (pre-cooling) 1ml is added, mixes；
(8) 4 DEG C, 7600rpm/5min；Abandoning supernatant, repetition step 8,9；
(9) it dodges from 10s, as far as possible exhaustion supernatant, is inverted 10 minutes dry；
(10) 20-30 μ l DEPC is added, surveys RNA concentration and OD value.
4, gene circRNA reverse transcription PCR reacts
(according to 5 × All-In-OneRTMasterMix of abm company (withAccuRTGenomicDNARemovalKit)
The description of test handbook of (#G492) operates)
Configure following reaction system:
Machine response procedures are as follows on reverse transcription PCR:
25 DEG C of 10min,
42 DEG C of 15min,
85℃ 5min。
To which after reaction, -20 DEG C of preservation products are spare.
5, real-time fluorescence quantitative PCR
Reverse transcription reaction product is first diluted 5 times, then according to abm company EvaGreen qPCR MasterMix
(MasterMix-R) description of test handbook operation, configures following reaction system:
Response procedures are as follows on real-time fluorescence quantitative PCR machine: (Cycle × 39)
Bio-RadIQ5 real-time fluorescence quantitative PCR instrument carry out it is above-mentioned after the reaction was completed, and reference gene β-actin markization,
With 2-ΔΔThe relative expression quantity of CT value displaying target gene, determines the differential expression of gene.It is examined using unpaired t-test
Calculate P value.
As a result: collecting many cases tissues of nasopharyngeal carcinoma and normal inflammatory nasopharyngeal epithelium tissue, had detected using qRT-PCR technology
The expression of circ_3480.The results show that circ_3480 is in 28 noses compared with 12 normal inflammatory nasopharyngeal epithelium tissues
Obvious high expression in pharynx cancer tissue, circ_3480 expression is about the 7 of normal inflammatory nasopharyngeal epithelium in nasopharyngeal carcinoma group
Times, the difference of two groups of data has statistical significance (P=0.0006), the result is shown in Figure 1.Therefore, circRNA circ_3480 exists
High expression, circ_3480 may have important biological function to the occurrence and development of nasopharyngeal carcinoma in tissues of nasopharyngeal carcinoma.
Embodiment 2: circ_3480 is overexpressed effect detection in Nasopharyngeal Carcinoma Cell Line
We select restriction enzyme site first, and circ_3480 full length sequence is put into the online website NEB cutter 2.0 point
Analysis, display ClaI and SacII restriction enzyme site is the site being not present in circ_3480 full length sequence, while in pcDNA3.1 matter
Single existing DNA restriction enzyme in grain carrier (being purchased from Invitrogene company).Over-express vector is constructed accordingly；Fig. 2
For the over-express vector map of drafting.
In order to detect the cyclic efficiency of circ_3480, we are by the pcDNA3.1/circ_3480 eukaryon mistake of building first
Expression vector is expressed in nasopharyngeal carcinoma cell.Good third and fourth generation nasopharyngeal carcinoma cell HONE1, the HNE2 of upgrowth situation
With CNE2 kind into 12 orifice plates, when cell fusion degree reaches 60%-80%, with liposome method lipofectamine 3000
Endotoxin-free plasmid pcDNA3.1 empty carrier and pcDNA3.1/circ_3480 over-express vector transiently transfect nasopharyngeal carcinoma cell
HONE1, HNE2 and CNE2 continue to cultivate to after 48 hours.Cell is collected, detects circ_ using Real-Time Fluorescent Quantitative PCR Technique
3480 expression and cyclic efficiency.QPCR turns pcDNA3.1/ the results show that compared with pcDNA3.1 empty plasmid group cell
The expression that circ_3480 is overexpressed circ_3480 in the cell of plasmid group significantly increases, and in HONE1, HNE2 and CNE2
Its in cell line expresses multiple at 20 times or more, sees Fig. 3, as a result has statistical significance.
Embodiment 3: the effect detection that silencing circ_3480 is expressed in Nasopharyngeal Carcinoma Cell Line
The SI sequence of circ_3480 is devised according to splicing site, siRNA is that a kind of length is 21-25 nucleotide, energy
It is complementary with cognate rna to combine, selective degradation purpose RNA, thus the double stranded rna molecule for inhibiting it to express.Currently, siRNA is
Develop into the important tool of gene functional research.In order to probe into effect of the circ_3480 in tumor development, Wo Mengen
SiRNA is devised according to the splicing site of circ_3480, it is using Hiperfect reagent that siRNA and siNC (blank control) is instantaneous
It is transfected into the expression of silencing circ_3480 in HONE1, HNE2 and CNE2 cell line.Continue to collect for culture 48 hours after transfection thin
Born of the same parents are detected the transfection efficiency of siRNA using the expression of Real-Time Fluorescent Quantitative PCR Technique detection circ_3480, confirm this
Secondary transfection efficiency is reduced to 50% hereinafter, result is shown in Fig. 4.
Embodiment 4: the external circ_3480 that is overexpressed promotes nasopharyngeal carcinoma cell proliferation
We use liposome method lipofectamine 3000 endotoxin-free plasmid pcDNA3.1 and pcDNA3.1/ first
Circ_3480 over-express vector transiently transfects nasopharyngeal carcinoma cell HONE1, HNE2 and CNE2, after continuing culture 48 hours, utilizes
QRT-PCR ensures that circ_3480 after expression raising, carries out MTT experiment in Nasopharyngeal Carcinoma Cell Line, verifies it and increases to cell
The influence grown.Based on first to the 5th day testing result, we simultaneously had found pcDNA3.1 empty plasmid group and pcDNA3.1/
Circ_3480 is overexpressed the cell Proliferation between plasmid group, and there are notable differences, are overexpressed circ_3480 to condition of in vitro culture
Under nasopharyngeal carcinoma cell proliferation have facilitation.(result is shown in Fig. 5)
Embodiment 5: external silencing circ_3480 inhibits the proliferation of nasopharyngeal carcinoma cell
It is using Hiperfect reagent that siRNA and siNC (blank control) transient transfection is thin to HONE1, HNE2 and CNE2
The expression of silencing circ_3480 in born of the same parents system.Continue to cultivate 48 hours collection cells after transfection, utilizes real-time fluorescence quantitative PCR skill
Art detects the expression of circ_3480 to detect the transfection efficiency of siRNA.The results show that siRNA is imitated with good silencing
Fruit.After ensuring that circ_3480 is disturbed, we silencing circ_3480 Nasopharyngeal Carcinoma Cell Line HONE1, HNE2 and
MTT experiment is carried out in CNE2, verifies the influence of its cell proliferation.Based on first to the 5th day testing result, relative to NC
The growth rate of group, siRNA group cell obviously slows down, i.e. silencing circ_3480 inhibits the proliferation of nasopharyngeal carcinoma cell.By positive and negative
The verifying of both direction, we can say that circ_3480 has facilitation to the nasopharyngeal carcinoma cell proliferation under condition of in vitro culture.
(result is shown in Fig. 6)
Embodiment 6: the healing migration experiment of cell scratch:
(1) photo cell platform: the D-Hank ' s of 1000 μ l/10 μ l Tip, autoclave sterilization, ruler, 1000 μ l/10 μ l
Liquid-transfering gun, marker etc., ultraviolet irradiation 30 minutes in super-clean bench are placed on after disinfecting in alcohol again；
(2) siRNA and NC group or transfected plasmids are transfected respectively to cell length to 50%~70% or so；
(3) 10 μ l pipette tips second day beginning scratch after cell covers with tiling board bottom: are compared into ruler perpendicular to 6 orifice plates bottom
Portion simply quickly carries out cross or well stroke trace, not tilt, strength is consistent, to ensure scratch broadband as far as possible；
(4) it inhales and discards culture solution, gently washed with D-hanks 3 times, it is thin to wash off the fragment due to caused by scratch as far as possible
Born of the same parents；
(5) 1640 culture mediums of 1% dual anti-2% fetal calf serum are added；
(6) scratch width beside cross at this time is photographed to record, 0h is denoted as；
(7) 6 orifice plates are put back into incubator culture, is spaced 12 hours and takes out, shoots the position of clapped picture when 0h, be denoted as
12h；
(8) same position is clapped again when being spaced for 24 hours, until scratch healing, arranges all pictures, and for statistical analysis.
The external migration for being overexpressed circ_3480 and promoting nasopharyngeal carcinoma cell
After determining that circular rna circ_3480 has facilitation to the proliferative capacity of nasopharyngeal carcinoma cell, we are again in nasopharynx
Scratch experiment is carried out in cancerous cell line, to verify circ_3480 to the migration of Nasopharyngeal Carcinoma Cell Line either with or without influence.Utilize rouge
Plastid method lipofectamine 3000 is overexpressed endotoxin-free plasmid pcDNA3.1 and pcDNA3.1/has_circ_3480
Carrier transient transfection nasopharyngeal carcinoma cell HONE1, HNE2 and CNE2 continue to cultivate to after 48 hours.Collect cell, using in real time it is glimmering
Fluorescent Quantitative PCR technology detects the expression and cyclization efficiency of circ_3480.Circ_3480 overexpression plasmid is being determined
After being overexpressed good result, we have carried out cell scratch Healing Experiments in Nasopharyngeal Carcinoma Cell Line HONE1, HNE2 and CNE2.It draws
Multiple time points (HONE1 0h, 12h, for 24 hours of the trace Healing Experiments in these cells；HNE2 is 0h, 12h, for 24 hours；CNE2 is
0h, 12h, for 24 hours) confirm: relative to unloaded pcDNA3.1 (+) plasmid group, pcDNA3.1/circ_3480 is overexpressed plasmid group
The transfer ability of cell is remarkably reinforced.Scratch width differs greatly, and has statistical significance.The above results show that being overexpressed
The expression of circ_3480 in Nasopharyngeal Carcinoma Cell Line can promote the migration of nasopharyngeal carcinoma cell HONE1, HNE2 and CNE2 in vitro
Ability.(result see Fig. 7,8,9)
External silencing circ_3480 inhibits the migration of nasopharyngeal carcinoma cell
SiRNA and NC is transiently transfected into the silencing into HONE1, HNE2 and CNE2 cell line using Hiperfect reagent
The expression of circ_3480.Continue to cultivate 48 hours collection cells after transfection, detects circ_ using Real-Time Fluorescent Quantitative PCR Technique
3480 expression is to detect the transfection efficiency of siRNA.The results show that siRNA has good silencing efficiency.Ensuring
After circ_3480 is disturbed, we carry out in Nasopharyngeal Carcinoma Cell Line HONE1, HNE2 and CNE2 of silencing circ_3480
Scratch experiment verifies its influence to cell migration.Multiple time points of the scratch Healing Experiments in these cells, (HONE1 was
0h,12h,24h；HNE2 is 0h, 12h, for 24 hours；CNE2 is 0h, 12h, for 24 hours) confirm: relative to NC group, siRNA group cell
Transfer ability obviously weakens.Scratch width difference is obvious, and has statistical significance.The above results show that silencing nasopharyngeal carcinoma is thin
The expression of circ_3480, is able to suppress the transfer ability of nasopharyngeal carcinoma cell HONE1, HNE2 and CNE2 in vitro in born of the same parents system.Pass through
Positive and negative both direction verifying proves that circ_3480 can promote the migration of nasopharyngeal carcinoma cell.(the result is shown in Figure 1 0,11,12)
Embodiment 7: cell transwell Matrigel:
(1) matrigel prepares: mentioning the previous day is placed in 4 DEG C of refrigerators in -20 DEG C of BDMatrigel glue and is melted into liquid freezing
Tip head, the EP pipe of releasing glue are placed in -20 DEG C overnight by state, and Matrigel glue will not mistake when spreading glue when operation in such second day
Fast solidification；
(2) matrigel dilutes: BDMatrigel glue: anteserum-less substrate=1:8, i.e. 20 μ l matrigels add 160 μ l 1640 to train
Base featheriness mixes；
(3) 100 μ l of the cell transwell is added in the matrigel diluted, then 80 μ l is sucked out along side, successively completed and be put into
It is incubated for 2-3 hours in 37 DEG C of incubators, when it is white for seeing paving glue-line, shows that liquid Matrigel glue has been in solid-state；
(4) experimental cell after digestion transfection is washed 2 times with anteserum-less substrate, then outstanding thin using the training base weight of serum-free
Born of the same parents, carry out cell count, and adjustment cell concentration is 2,0000 cell in every 200 μ l；
(5) 1640 culture mediums that 800 μ l contain 20%FBS are added to bottom chamber, tilts 24 orifice plates when being put into cell
45° angle generates bubble to avoid being put into during cell between cell and liquid level；
(6) every room adds indoor on the unified cell suspension to transwell counted of 200 μ l, and 24 orifice plates are put back to 37 DEG C
In incubator, according to cell state and cell invasion speed, it is incubated for about 24~48 hours.
(7) 24 orifice plates are taken out, are washed twice with PBS or D-hanks, 4% paraformaldehyde embathes 10 minutes, washes 3 with clear water
Time.
(8) it dyeing: being added drop-wise to the bottom of the cell transwell with 0.1% crystal violet, 5-10min is placed in room temperature peace and quiet,
It is cleaned 2-3 times with PBS, the matrigel above cell is carefully wiped with cotton swab；
(9) it is added in 800 μ l, transwell upper chamber of distilled water in 24 orifice plates and about 200 μ l of distilled water is added, then existed
It under inverted microscope, is observed, the different visuals field are taken pictures, and count with image J software aobvious with statistical analysis difference
Work property.
The external invasion for being overexpressed circ_3480 and promoting nasopharyngeal carcinoma cell
We have carried out the experiment of the cell Transwell matrigel invasion in Nasopharyngeal Carcinoma Cell Line HONE1, HNE2 and CNE2,
To observe the influence for being overexpressed circ_3480 to cell invasion ability.We are also with liposome method lipofectamine
3000 endotoxin-free plasmid pcDNA3.1 and pcDNA3.1/circ_3480 over-express vectors transiently transfect nasopharyngeal carcinoma cell
HONE1, HNE2 and CNE2 continue to cultivate to after 48 hours.Cell is collected, detects circ_ using Real-Time Fluorescent Quantitative PCR Technique
3480 expression and cyclic efficiency.After being determined that circ_3480 is overexpressed the overexpression good result of plasmid, we will
For cell inoculation into the cell Transwell of paving matrigel, discovery is overexpressed the cell invasion of plasmid group to small chamber lower surface
Number is obviously more than zero load group, and the trend of two plants of cell line results is consistent.Random shooting 3 opens photo and records cell number,
There is notable difference in each cell line between two group data, and there is statistical significance.The above result shows that being overexpressed nasopharynx
The expression of circ_3480 in cancerous cell line can promote the invasive ability of nasopharyngeal carcinoma cell HONE1, HNE2 and CNE2 in vitro.
(the result is shown in Figure 1 3,14,15)
The expression of external silencing circ_3480 influences the invasion of nasopharyngeal carcinoma
It is overexpressed phenotypic alternation brought by circ_3480 in order to probe into after silencing circ_3480 whether to reverse, I
Matrigel invasion experiment in the cell Transwell has been carried out in three plants of cell lines again, using Hiperfect reagent by circ_
3480siRNA and NC transiently transfects the expression of the silencing circ_3480 into HONE1, HNE2 and CNE2 cell line.Continue after transfection
48 hours collection cells are cultivated, using the expression of Real-Time Fluorescent Quantitative PCR Technique detection circ_3480 to detect siRNA
Transfection efficiency.The results show that circ_3480siRNA has good silencing efficiency.Ensuring that circ_3480 is disturbed
Afterwards, we carry out the cell Transwell matrix in Nasopharyngeal Carcinoma Cell Line HONE1, HNE2 and CNE2 of silencing circ_3480
Glue Matrigel, the results show that siRNA group can be obviously few in the tumor cell number that the small chamber lower surface of Transwell is observed
In NC group, and the trend of two plants of cell line results is consistent.Random shooting 6 opens photo and records cell number, in each cell line
There is notable difference between two group data, and there is statistical significance.The above result shows that in silencing Nasopharyngeal Carcinoma Cell Line
The expression of circ_3480 is able to suppress the invasive ability of nasopharyngeal carcinoma cell HONE1, HNE2 and CNE2 in vitro.Pass through positive and negative two
A direction proves that circular rna circ_3480 can promote the invasion of nasopharyngeal carcinoma cell.(the result is shown in Figure 1 6,17,18).
Sequence table
<110>Central South University
<120>circ_3480 and its application and diagnostic reagent in nasopharyngeal carcinoma diagnosis preparation are being prepared
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 425
<212> RNA
<213>homo sapiens (Homo sapiens)
<400> 1
agugcccaca uggcaauggu ggaugcccug augauggccu acacugugga gaugaucagc 60
aucgagaagg ugguggccag ugucaagcgc uucucaacgu ucagugccuc gaaagaacuu 120
ccguacgacc ucgaggaugc caugguguuc uggaucaaca agauaacauc ccugaggacc 180
ucagagaccc uuucuacguu gaccaguaug agcaggagca cauuaagccg ccuguuauca 240
agcuucuccu guccagcgag cuguacugcc gugucugcag ccucauccug aaaggggacc 300
agguggccgc cuuacaggga caccagucug ucauccaggc ccugucccgg aaagggaucu 360
augugaugga gagugaugac acccccguga cagaguccga ccucagucgc gcacccauaa 420
aaaug 425
<210> 2
<211> 20
<212> DNA
<213>unknown (Unknown)
<400> 2
tcaccaactg ggacgacatg 20
<210> 3
<211> 21
<212> DNA
<213>unknown (Unknown)
<400> 3
gtcaccggag tccatcacga t 21
<210> 4
<211> 20
<212> DNA
<213>unknown (Unknown)
<400> 4
caacgttcag tgcctcgaaa 20
<210> 5
<211> 20
<212> DNA
<213>unknown (Unknown)
<400> 5
cctgctcata ctggtcaacg 20
<210> 6
<211> 30
<212> DNA
<213>unknown (Unknown)
<400> 6
ccatcgatgg ataacatccc tgaggacctc 30
<210> 7
<211> 32
<212> DNA
<213>unknown (Unknown)
<400> 7
tccccgcggg gacttgttga tccagaacac ca 32
<210> 8
<211> 21
<212> RNA
<213>unknown (Unknown)
<400> 8
cuggaucaac aagauaacau u 21
<210> 9
<211> 21
<212> RNA
<213>unknown (Unknown)
<400> 9
uguuaucuug uugauccagu u 21
<210> 10
<211> 21
<212> RNA
<213>unknown (Unknown)
<400> 10
uucuccgaac gugucacguu u 21
<210> 11
<211> 21
<212> RNA
<213>unknown (Unknown)
<400> 11
acgugacacg uucggagaau u 21
Claims (10)
1. a kind of circular rna circ_3480, sequence is as shown in SEQ ID NO.1.
2. the reagent for detecting circular rna circ_3480 described in claim 1 is preparing the application on diagnosis of nasopharyngeal carcinoma tool.
3. application according to claim 2, which is characterized in that detection circular rna described in claim 1
The reagent of circ_3480 is PCR reagent.
4. application according to claim 3, which is characterized in that detection circular rna described in claim 1
The reagent of circ_3480 is PCR Real_time quantitative detection reagent.
5. application according to claim 3, which is characterized in that the primer in the PCR reagent are as follows:
Upstream primer: 5 '-CAACGTTCAGTGCCTCGAAA-3 '
Downstream primer: 5 '-CCTGCTCATACTGGTCAACG-3 '.
6. application according to claim 3, which is characterized in that the β-actin internal control primer in the PCR reagent are as follows:
Upstream primer: 5 '-TCACCAACTGGGACGACATG-3 '
Downstream primer: 5 '-GTCACCGGAGTCCATCACGAT-3 '.
7. a kind of preparation of diagnosis of nasopharyngeal carcinoma, which is characterized in that comprising detecting circular rna circ_ described in claim 1
3480 reagent.
8. preparation according to claim 7, which is characterized in that detection circular rna described in claim 1
The reagent of circ_3480 is PCR detection reagent.
9. preparation according to claim 7, which is characterized in that the primer in the PCR detection reagent are as follows:
Upstream primer: 5 '-CAACGTTCAGTGCCTCGAAA-3 '
Downstream primer: 5 '-CCTGCTCATACTGGTCAACG-3 '.
10. preparation according to claim 7, which is characterized in that reference in the β-actin in the PCR detection reagent
Object are as follows:
Upstream primer: 5 '-TCACCAACTGGGACGACATG-3 '
Downstream primer: 5 '-GTCACCGGAGTCCATCACGAT-3 '.
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Citations (3)
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|---|---|---|---|---|
| CN107557472A (en) * | 2017-10-27 | 2018-01-09 | 中南大学湘雅医院 | Diagnosis of glioma mark circ9:135881633 | 135883078 and application |
| CN107586844A (en) * | 2017-10-27 | 2018-01-16 | 中南大学湘雅医院 | Glioma prognostic marker Circ9:135881633 | 135883078 application |
| CN108660214A (en) * | 2018-05-28 | 2018-10-16 | 中南大学 | Detect application and kit of the reagent of circ_CLASP2 in preparing nasopharyngeal carcinoma diagnosis preparation |
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2019
- 2019-01-24 CN CN201910066566.7A patent/CN109852692B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107557472A (en) * | 2017-10-27 | 2018-01-09 | 中南大学湘雅医院 | Diagnosis of glioma mark circ9:135881633 | 135883078 and application |
| CN107586844A (en) * | 2017-10-27 | 2018-01-16 | 中南大学湘雅医院 | Glioma prognostic marker Circ9:135881633 | 135883078 application |
| CN108660214A (en) * | 2018-05-28 | 2018-10-16 | 中南大学 | Detect application and kit of the reagent of circ_CLASP2 in preparing nasopharyngeal carcinoma diagnosis preparation |
Non-Patent Citations (7)
| Title |
|---|
| JECK等: "has_cir_0001900", 《CIRCBASE DATABASE》 * |
| JECK等: "hsa_circ_0004338", 《CIRCBASE DATABASE》 * |
| JECK等: "hsa_circ_0006814", 《CIRCBASE DATABASE》 * |
| JORDAN L PLIESKATT等: "Methods and matrices: approaches to identifying miRNAs for Nasopharyngeal carcinoma", 《JOURNAL OF TRANSLATIONAL MEDICINE》 * |
| JULIA SALZMAN等: "Cell-Type Specific Features of Circular RNA Expression", 《PLOS GENETICS》 * |
| XIN SUN等: "MicroRNA-126 modulates the tumor microenvironment by targeting calmodulin-regulated spectrin-associated protein 1 (Camsap1)", 《INTERNATIONAL JOURNAL OF ONCOLOGY》 * |
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