CN108721319A - Inhibit application and preparation of the reagent of circ_CLASP2 in preparing treatment of nasopharyngeal carcinoma preparation - Google Patents
Inhibit application and preparation of the reagent of circ_CLASP2 in preparing treatment of nasopharyngeal carcinoma preparation Download PDFInfo
- Publication number
- CN108721319A CN108721319A CN201810525695.3A CN201810525695A CN108721319A CN 108721319 A CN108721319 A CN 108721319A CN 201810525695 A CN201810525695 A CN 201810525695A CN 108721319 A CN108721319 A CN 108721319A
- Authority
- CN
- China
- Prior art keywords
- circ
- clasp2
- nasopharyngeal carcinoma
- cell
- sirna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Molecular Biology (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a kind of applications and preparation inhibiting the reagent of circ_CLASP2 in preparing treatment of nasopharyngeal carcinoma preparation.It confirms to inhibit circ_CLASP2 in nasopharyngeal carcinoma cell by studying, proliferation and the invasion transfer of nasopharyngeal carcinoma cell can be inhibited.Therefore, circ_CLASP2 inhibitor had into far-reaching clinical meaning and important popularizing application prospect for treating nasopharyngeal carcinoma.
Description
Technical field
The invention belongs to oncomolecularbiology technical fields, and in particular to a kind of inhibition circular rna circ_CLASP2
Application and corresponding preparation of the reagent in preparing treatment of nasopharyngeal carcinoma preparation.
Background technology
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 areas UTR, intergenic region, non-coding RNA site and the antisense position of known transcript
Point is formed.CircRNA is that do not have 5 by one kind that precursor RNA (precursor RNA, pre-RNA) is reversely spliced to form
' end cap and 3 ' end poly (A) tails and the non-coding RNA molecule that loop configuration is formed with covalent bond.
CircRNA forming processes can be divided into two major classes, i.e. exon cyclisation is (exon circularization) and interior
Containing two big mechanism of subring (intron circularization).Jeck etc. proposes the circRNA (exonic in 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 sites (splice receptor) of donor splicing site (splice donor) attack, the areas Alu
Covalent bond and form lasso structure, excision introne forms circRNA after lasso structure carries out internal splicing；Introne matches
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 the one kind 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. use electron microscope skill for the first time early in 1976 in RNA virus
Art is found that 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 technologies, in recent years more and more circRNA be found, it is known so far
CircRNA numbers have 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 researchs rises up slowly.Screening and the more novel circRNA of verification are 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 nests, 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
Multistage complexity gene regulation process caused by mutation and epigenetics mutation is passed, proto-oncogene and tumor suppressor gene are related to
The epigenetic regulation etc. that activation and silence and non-coding RNA participate in.So the specific molecule machine of nasopharyngeal carcinoma occurrence and development
The effect of system is still not clear, and due to Tumor Heterogeneity and individual difference, traditional radiotherapy equipment system be not notable.Therefore
Itself and nasopharyngeal carcinoma occurrence and development mechanism are probed by circRNA, is further the diagnosis of nasopharyngeal carcinoma, and control nasopharyngeal carcinoma increases
It will be the hot spot studied to grow, invade and shift.
We detect the circ_CLASP2 of length 449bp in the RNA-seq of nasopharyngeal carcinoma cell 5-8F cells, are
The circular rna that CLASP2 genes 2,3,4,5,6 exon, five exons are reversely spliced to form.It is found by experiment that the ring-type
RNA exists with the occurrence and development of nasopharyngeal carcinoma to be associated with, it is possible to as nasopharyngeal carcinoma diagnosis marker, and the target spot for the treatment of.
Invention content
Due to the overall length 7145bp of CLASP2 genes, there are 40 exons, there are extremely complex alternative splicings；But this
Invention is only found that size is the circ_CLASP2 of 449bp from the RNA-seq of 5-8F cells, includes 2 to No. 6 of CLASP2
Exon is simultaneously verified in other human nasopharyngeal epithelioma 1s and Biopsies from Patients with Nasopharyngeal Carcinoma.And it is found that it between nasopharyngeal carcinoma
Existing relationship.Possible as the target spot for the treatment of of nasopharyngeal carcinoma.
The first purpose of the invention is to provide a kind of reagents inhibiting circ_CLASP2 to prepare treatment of nasopharyngeal carcinoma preparation
In application, the sequence of the circ_CLASP2 is as shown in SEQ No.1.
The reagent of the inhibition circ_CLASP2 includes siRNA.
Currently, siRNA has evolved into the important tool of gene functional research.In order to probe into circ_CLASP2 in tumour
Effect in occurrence and development, we are devised two couples of siRNA according to the splicing site of circ_CLASP2, are tried using Hiperfect
SiRNA and siNC is transiently transfected the expression to silence circ_CLASP2 in CNE2 and HNE2 cell lines by agent.Continue to train after transfection
It supports 48 hours and collects cell, the expression of circ_CLASP2 is detected to detect siRNA using Real-Time Fluorescent Quantitative PCR Technique
Transfection efficiency, while detecting the expression of circ_CLASP2, it is found that the siRNA of design can significantly inhibit circ_CLASP2
Expression.
SiRNA preferably includes following any pair or two pairs of combination (siRNA1+2)：
circ_CLASP2 siRNA1：
Positive-sense strand (5'-3') CUGCUAGGUAUCAUUAAUGUU
Antisense strand (5'-3') CAUUAAUGAUACCUAGCAGUU；
circ_CLASP2 siRNA2：
Positive-sense strand (5'-3') CCCCUGCUAGGUAUCAUUAUU
Antisense strand (5'-3') UAAUGAUACCUAGCAGGGGUU.
The reagent of the inhibition circ_CLASP2 includes negative control：
Positive-sense strand (5'-3') UUCUCCGAACGUGUCACGUUU
Antisense strand (5'-3') ACGUGACACGUUCGGAGAAUU.
But the present invention is not limited only to the siRNA and negative control of above-mentioned offer.
Treatment of nasopharyngeal carcinoma preparation further includes the reagent transfected needed for siRNA.
Second object of the present invention is to provide a kind for the treatment of of nasopharyngeal carcinoma preparation, contains the examination that can inhibit circ_CLASP2
Agent.A kind of new efficient treatment product is provided for the treatment of nasopharyngeal carcinoma.
The present invention confirms above-mentioned conclusion by largely testing：The reagent of circ_CLASP2 is inhibited to can be used in preparing
Treatment of nasopharyngeal carcinoma preparation.These experiments include：External overexpression circ_CLASP2 experiments find nasopharyngeal carcinoma cell can be promoted to increase
It grows, external silence circ_CLASP2 then inhibits the proliferation of nasopharyngeal carcinoma cell；The external circ_CLASP2 that is overexpressed promotes nasopharyngeal carcinoma
The migration of cell, external silence circ_CLASP2 inhibit the migration of nasopharyngeal carcinoma cell；The external circ_CLASP2 that is overexpressed promotes
The invasion of nasopharyngeal carcinoma cell, the expression of external silence circ_CLASP2 influence the invasion of nasopharyngeal carcinoma.
The reagent of circ_CLASP2 that can inhibit includes siRNA.
SiRNA includes following any pair or two pairs：
circ_CLASP2 siRNA1：
Positive-sense strand (5'-3') CUGCUAGGUAUCAUUAAUGUU
Antisense strand (5'-3') CAUUAAUGAUACCUAGCAGUU；
circ_CLASP2 siRNA2：
Positive-sense strand (5'-3') CCCCUGCUAGGUAUCAUUAUU
Antisense strand (5'-3') UAAUGAUACCUAGCAGGGGUU.
Further, the reagent of circ_CLASP2 that can inhibit includes negative control：
Positive-sense strand (5'-3') UUCUCCGAACGUGUCACGUUU
Antisense strand (5'-3') ACGUGACACGUUCGGAGAAUU.
Further, treatment of nasopharyngeal carcinoma preparation includes the reagent transfected needed for siRNA.
Since siRNA has good silencing efficiency.After ensuring that circ_CLASP2 is disturbed, we are in silence
It is real that MTT cell proliferation experiments, transwell cell invasions are carried out in the Nasopharyngeal Carcinoma Cell Line CNE2 and HNE2 of circ_CLASP2
Test with cut Healing Experiments, relative to NC groups, the growth rate of siRNA group cells obviously slows down, invasion and transfer ability reduce,
That is silence circ_CLASP2 inhibits proliferation and the invasion transfer of nasopharyngeal carcinoma cell.Inhibit circ_CLASP2 that can treat nasopharynx
Cancer has far-reaching clinical meaning and important popularizing application prospect.
Description of the drawings
Fig. 1 is that qRT-RCR detects circ_CLASP2 in tissues of nasopharyngeal carcinoma and non-tumour rhinitis epithelial tissue (normal control)
Expression；
The expression analysis of circ_CLASP2 is using β-actin as reference by non-tumour nasopharyngeal epithelium organizational standard
It is non-tumour nasopharyngeal epithelium tissue, sample number 12 for 1, N；T is tissues of nasopharyngeal carcinoma, and sample number 22, n is sample size,
It is examined using t, P<0.05 has statistical significance.
Fig. 2 is the expression water that qRT-RCR detects circ_CLASP2 in human nasopharyngeal epithelioma 1 and normal nasopharyngeal epithelial cell strain
It is flat；
Q-PCR results show circ_CLASP2 normal nasopharyngeal epithelial cell NP69 and nasopharyngeal carcinoma cell CNE2,
Expression in HK1, HNE2, HNE1, the analysis of circ_CLASP2 expressions will immortalize normal using β-actin as reference
Inflammatory nasopharyngeal epithelial cells NP69 is standardized as 1, * P<0.05,**P<0.01,***P<0.001.
Fig. 3 is circ_CLASP2 over-express vector collection of illustrative plates.
Fig. 4 is that qRT-PCR detects circ_CLASP2 overexpression effects and cyclic efficiency in Nasopharyngeal Carcinoma Cell Line；
A and c.qRT-PCR detections circ_CLASP2 in Nasopharyngeal Carcinoma Cell Line CNE2, HNE2 is overexpressed effect and cyclization
Efficiency；B and d.qRT-PCR detections are overexpressed circ_CLASP2 to CLASP2 mRNA in Nasopharyngeal Carcinoma Cell Line CNE2, HNE2
Horizontal influence；PcDNA3.1 (+) group is standardized as 1, ns as reference using β-actin and represents nonsensical, * P<
0.05,**P<0.01,***P<0.001。
Fig. 5 is the silence efficiency that qRT-PCR technologies detect circ_CLASP2 siRNA in Nasopharyngeal Carcinoma Cell Line；
The silence efficiency of a and c.qRT-PCR detections circ_CLASP2siRNA in Nasopharyngeal Carcinoma Cell Line CNE2, HNE2；b
With the d.qRT-PCR detections shadows of silence circ_CLASP2 to CLASP2 mRNA level in-sites in Nasopharyngeal Carcinoma Cell Line CNE2, HNE2
It rings；Circ_CLASP2 expressions are analyzed using β-actin as reference, and NCsiRNA groups, which are standardized as 1, ns representatives, does not anticipate
Justice, * P<0.05,**P<0.01,***P<0.001.
Fig. 6 is the external influence for being overexpressed Circ_CLASP2 and being proliferated to nasopharyngeal carcinoma cell；
A-b.qRT-PCR detections circ_CLASP2 in Nasopharyngeal Carcinoma Cell Line CNE2, HNE2 is overexpressed efficiency；C-d. right
The MTT experiment that above-mentioned cell carries out detects ability of cell proliferation, and circ_CLASP2 expressions are analyzed using β-actin as reference,
PcDNA3.1 (+) group is standardized as 1, ns and represents nonsensical, * P<0.05,**P<0.01,***P<0.001.
Fig. 7 is the influence that external silence Circ_CLASP2 is proliferated nasopharyngeal carcinoma cell；
A-b.qRT-PCR detections circ_CLASP2 in Nasopharyngeal Carcinoma Cell Line CNE2, HNE2 strikes poor efficiency；C-d. to upper
It states cell and carries out MTT experiment detection ability of cell proliferation, circ_CLASP2 expressions are analyzed using β-actin as reference, will
NCsiRNA groups are standardized as 1, ns and represent nonsensical, * P<0.05,**P<0.01,***P<0.001.
Fig. 8 is that qRT-PCR detects circ_CLASP2 overexpressions efficiency in scratch experiment cell；
A-b. human nasopharyngeal epithelioma 1 CNE2, HNE2 transfection efficiencies used in scratch experiment are tested with qRT-PCR and are detected, circ_
CLASP2 expressions are analyzed using β-actin as reference, and pcDNA3.1 (+) group is standardized as 1, * P<0.05,**P<
0.01,***P<0.001。
Fig. 9 is the influence for being overexpressed circ_CLASP2 to nasopharyngeal carcinoma cell CNE2, HNE2 cut Healing Experiments；
A-b. nasopharyngeal carcinoma CNE2 and HNE2 cell transfectings pcDNA3.1 (+) empty carrier and overexpression circ_CLASP2 carriers
(circ_CLASP2), the cut after cell density reaches 100% was taken pictures in observation in the 0th, 12,24 hour.
Figure 10 is nasopharyngeal carcinoma CNE2, the HNE2 cell scratch experiment statistical chart for being overexpressed circ_CLASP2；
A-b. 4 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 statistics.*P<0.05,**P<0.01,***P<0.001.
Figure 11 is that qRT-PCR detects circ_CLASP2 jamming effectiveness in scratch experiment cell；
A-b. human nasopharyngeal epithelioma 1 CNE2, HNE2 transfection efficiencies used in scratch experiment are tested with qRT-PCR and are detected, circ_
CLASP2 expressions are analyzed using β-actin as reference, and NC siRNA groups are standardized as 1, * P<0.05,**P<
0.01,***P<0.001。
Figure 12 is the influence for interfering circ_CLASP2 to nasopharyngeal carcinoma cell CNE2, HNE2 cut Healing Experiments；
A-b. nasopharyngeal carcinoma CNE2 and HNE2 cell transfecting negative control siRNA sequences (NC) and targeting interference circ_
The siRNA sequence (mixture of siRNA sequence 1 and siRNA sequence 2, i.e. siRNA1+2) of CLASP2, waits for that cell density reaches
Cut after 100% was taken pictures in observation in the 0th, 12,24 hour.
Figure 13 is nasopharyngeal carcinoma CNE2, the HNE2 cell scratch experiment statistical chart for interfering circ_CLASP2；
A-b. 4 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 statistics.*P<0.05,**P<0.01,***P<0.001.
Figure 14 is the overexpression effect that qRT-PCR detects circ_CLASP2 in the matrigel invasion experiment of the cells Transwell
Rate；
Matrigel invasion experiment human nasopharyngeal epithelioma 1 CNE2, HNE2 transfection efficiency used in the cells a-b.Transwell is used
QRT-PCR experiment detections, circ_CLASP2 expressions are analyzed using β-actin as reference, by pcDNA3.1 (+) group standard
Turn to 1, * P<0.05,**P<0.01,***P<0.001.
Figure 15 is the influence for being overexpressed circ_CLASP2 to Nasopharyngeal Carcinoma Cell Line CNE2, HNE2 invasive ability；
CNE2, the HNE2 and respective cellular control unit for being overexpressed circ_CLASP2 respectively carry out the cells Transwell
Matrigel invasion is tested.
Figure 16 is the cells the Transwell matrigel invasion experiment statistics figure for being overexpressed circ_CLASP2；
Every group randomly selects 4 cell visuals field and carries out cell count, and take statistics figure；Each experiment in triplicate, uses
Student's t-test method statistics；Using β-actin as reference, pcDNA3.1 (+) group is standardized as 1, * P<
0.05,**P<0.01,***P<0.001。
Figure 17 is the jamming effectiveness that qRT-PCR detects circ_CLASP2 in the matrigel invasion experiment of the cells Transwell；
Matrigel invasion experiment human nasopharyngeal epithelioma 1 CNE2, HNE2 transfection efficiency used in the cells a-b.Transwell is used
QRT-PCR experiment detections, circ_CLASP2 expressions are analyzed using β-actin as reference, and NC groups are standardized as 1, * P<
0.05,**P<0.01,***P<0.001。
Figure 18 is the influence for interfering circ_CLASP2 to Nasopharyngeal Carcinoma Cell Line CNE2, HNE2 invasive ability；
CNE2, HNE2 of interference circ_CLASP2 and respective cellular control unit carry out the cells Transwell base respectively
Matter glue Matrigel.
Figure 19 is the cells the Transwell matrigel invasion experiment statistics figure for interfering circ_CLASP2；
Every group randomly selects 4 cell visuals field and carries out cell count, and take statistics figure；Each experiment in triplicate, uses
Student's t-test method statistics；Using β-actin as reference, NC groups are standardized as 1, * P<0.05,**P<
0.01,***P<0.001。
Specific implementation mode
It is intended to further illustrate the present invention below in conjunction with specific implementation mode, 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 preserved in tank, then collects the relevant clinical data of all patients.All experiments are acquired with tissue samples and obtain Central South University
Ethics Committee authorizes and patient agrees to.
The normal nasopharyngeal epithelial cell of the Nasopharyngeal Carcinoma Cell Lines such as CNE2, HNE2, HK1, HNE1 used in the present invention and immortalization
NP69 is that Tumour Inst., Zhongnan Univ.'s molecular genetic laboratory is preserved.Cell culture condition is：Containing 10% fetal calf serum
(FBS) and the RPMI1640 fluid nutrient mediums of 1% dual anti-(penicillin, streptomysin), 37 DEG C, 95% humidity, 5%CO2Concentration
Adherent growth in constant incubator.
The primer of circular rna of the present invention is different from the design of linear rna primer, is designed according to splicing site both sides,
The Photographing On-line on the websites Primer3.0, final primer synthetic work are ordered goods by sending Email, the life of the commission section of holding up
The Changsha combining unit synthesis of object company.
(1)β-actin
Sense primer：5'-TCACCAACTGGGACGACATG-3'
Downstream primer：5'-GTCACCGGAGTCCATCACGAT-3'
(2) circular rna circ_CLASP2 real-time fluorescence quantitative PCRs primer
Sense primer：5'-ACAGTCAGGTGAGAGATGCT-3'
Downstream primer：5'-TGGCATCTCCCATTCTGTCT-3'
(3) amplification circ_CLASP2 overall lengths primer (for building circ_CLASP2 over-express vectors)
Sense primer：5'-CCATCGATGG GTATCATTAATGGGA-3'
Downstream primer：5'-TTCCCGCGGGGACTAGCAGGGGGAATT-3'
Above-mentioned primer amplification CLASP2 genes 2-6 exons are inserted into after digestion and are cut with same restriction enzyme
Circular rna is overexpressed in empty carrier.
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_CLASP2.
Circ_CLASP2 siRNA sequences 1：
Positive-sense strand (5'-3') CUGCUAGGUAUCAUUAAUGUU
Antisense strand (5'-3') CAUUAAUGAUACCUAGCAGUU.
Circ_CLASP2 siRNA sequences 2：
Positive-sense strand (5'-3') CCCCUGCUAGGUAUCAUUAUU
Antisense strand (5'-3') UAAUGAUACCUAGCAGGGGUU.
Negative control：
Positive-sense strand (5'-3') UUCUCCGAACGUGUCACGUUU
Antisense strand (5'-3') ACGUGACACGUUCGGAGAAUU.
Test result of the present invention is all made of statistical analysis：T, which is examined, to be used for evaluating 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 Kaplan-Meier aspects.p<0.05 for indicating that significance,statistical, all p values make
Use two-sided test.Statistical analysis is carried out using SPSS 13.0 and 5.0 softwares of Graphpad.
Embodiment 1：Expression of the circ_CLASP2 in tissues of nasopharyngeal carcinoma and cell
1, according to master sample acquisition scheme, we collect doubtful Nasopharyngeal Carcinoma Patients tissue samples from Hunan Provincial Tumour Hospital
34.All cases are first visit patient's (time interval of Hunan Provincial Tumour Hospital's Head and neck tumour：In January, 2016 was to 2016
November).Nasopharyngeal carcinoma 24 is made a definite diagnosis through pathology department, nasopharynx inflammatory patients 12 (have excluded tumor disease, no active infection
Sick, 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., refer to Excel electrical form sectional drawings 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 the RNA extractions of normal inflammatory nasopharyngeal tissue
(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：Liquid nitrogen precooler is added in mortar, then grips the pharynx nasalis tissue preserved in cryopreservation tube, soon
Speed grinding, grinds, continuously adds a small amount of liquid nitrogen on one side, then grind on one side, clays into power shape until by nasopharyngeal tissue.According to best
Ratio, i.e., per 50-100mg, normal or NPC samples add 1ml Trizol.In our experimentations, a tissues of nasopharyngeal carcinoma sample
About 200mg or so, it is therefore desirable to 2ml Trizol.Then it is further ground, is placed in 4 DEG C of about 5-10min of refrigerator, allow group
Cast off solution of totally cleaving.2ml Tube are transferred to when pyrolysis product is melted into pink liquid.Each sample can be detached to 2
Tube is preserved to -80 DEG C.
(3) aqueous phase separation：Tissue lysates of every 1000 μ l containing trizol add the chloroform of 200 μ l, 4 DEG C of precoolings, concussion
About 30s carries out mixing.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 level is membranaceous white precipitate, lower layer's pink.
Therefore, it further draws in the upper strata aqueous phase containing RNA to 1.5ml Tube, with 100 μ l rifle gentle aspirations, avoids being drawn onto as possible
Middle level and lower layer's substance, cause RNA to pollute.
(4) RNA precipitate：1 is added in supernatant:About 500 μ l of 1 isometric isopropanol, action are gently turned upside down mixed
It is even for several times, be placed in after -20 DEG C of refrigerator puts 30 minutes, centrifuge 30 minutes (4 DEG C, 12,000rpm), it is seen that tube bottom has RNA precipitate, uses
The suction of 100 μ l liquid-transfering guns discards supernatant, and retains RNA precipitate as possible.
(5) RNA is washed：Often 75% ethyl alcohol that 1ml is prepared with no enzyme water, mild turned upside down is added in pipe RNA precipitate sample
Centrifuge tube, to wash RNA precipitate.5 minutes (4 DEG C, 7,600rpm) are then centrifuged for, are discarded supernatant as possible with the liquid-transfering gun of 100 μ l,
Room temperature is dried 10~15 minutes.
(6) it re-dissolves RNA and preserves：Often 15-30 μ l DECP water is added in 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 (NP69 and CNE2, HK1, HNE2, HNE1) of RNA to be extracted, is cleaned with 1 × PBS or D-hanks
Twice；
500 μ l Trizol lysates, lysis at room temperature 1-2 minutes are added gently to be blown down with liquid-transfering gun per hole in (2) 12 orifice plates
Cell, upper and lower gentle inversion 10 times are stored at room temperature 5 minutes；
(3) chloroform that 100 μ l are added (presses 1mlTrizol:0.2ml chloroforms:0.5ml isopropanols), 15-30s is firmly shaken,
It places 5 minutes on ice；
(4) 4 DEG C, 12000rpm/20min；
(5) it takes upper strata aqueous phase in the Tube pipes of precooling, 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 abandons supernatant；
(7) 75% ethyl alcohol (precooling) 1ml, mixing is added；
(8) 4 DEG C, 7600rpm/5min；Supernatant is abandoned, repeatedly step 8,9；
(9) it dodges from 10s, exhausts supernatant as possible, be inverted 10 minutes dry；
(10) 20-30 μ l DEPC are added, survey RNA concentration and OD values.
4, gene circRNA reverse transcription PCRs react
(according to abm companies
The experiment of 5 × All-In-OneRTMasterMix (withAccuRTGenomicDNARemovalKit) (#G492)
Instruction manual 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。
It waits for 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)
After the completion of Bio-RadIQ5 real-time fluorescence quantitative PCR instrument carries out above-mentioned reaction, and reference gene β-actin markizations,
With 2-△△The relative expression quantity of CT value display target genes, judges the differential expression of gene.It is examined using unpaired t-test
Calculate P values.
As a result：Compared with 12 nasopharynx inflammation epithelial tissues (being used as normal control), circ_CLASP2 is in 22 nasopharyngeal carcinoma
Apparent high expression in tissue, circ_CLASP2 expressions are about 3 times of normal inflammatory nasopharyngeal epithelium in nasopharyngeal carcinoma group,
The difference of two groups of data has statistical significance (P=0.0386), the result is shown in Figure 1.Therefore, circRNA circ_CLASP2 exist
High expression, circ_CLASP2 may have important biological function to the occurrence and development of nasopharyngeal carcinoma in tissues of nasopharyngeal carcinoma.
In order to further verify biological functions of the circ_CLASP2 in nasopharyngeal carcinoma, our normal noses in immortalization
It swallows and carries out qRT-PCR experimental verifications circ_CLASP2 in epithelial cell NP69 and nasopharyngeal carcinoma cell CNE2, HNE2, HK1, HNE1
Expression.The results show that circ_CLASP2 is above normal nasopharyngeal cancer epithelial cell NP69 in each Nasopharyngeal Carcinoma Cell Line
About 60 times, and the difference of two groups of data has statistical significance (P=0.008, P=0.0174, P=0.0003, P=
0.0402), as a result see Fig. 2.So circRNA circ_CLASP2 high expression in nasopharyngeal carcinoma cell, circ_CLASP2 is to nose
The occurrence and development of pharynx cancer may have important biological function.
The circ_CLASP2 in Nasopharyngeal Carcinoma Cell Line of embodiment 2 is overexpressed effect detection
We select restriction enzyme site first, and by circ_CLASP2 full length sequences, (i.e. 2 to 6 exon of CLASP2 genes is total
449bp sequences) 2.0 online web analytics of NEB cutter are put into, display ClaI and SacII restriction enzyme sites are circ_CLASP2
The site being not present in full length sequence, while the single existing DNA restriction enzymes in pcDNA3.1 plasmid vectors.Accordingly
Build over-express vector；Fig. 3 is over-express vector collection of illustrative plates.
In order to detect the overexpression efficiency of circ_CLASP2, the circ_CLASP2 eukaryons of structure are overexpressed by we first
Carrier is expressed in nasopharyngeal carcinoma cell.In good nasopharyngeal carcinoma cell CNE2, HNE2 kind to 12 orifice plates of upgrowth situation, wait for
When cell fusion degree reaches 60%-80%, with liposome lipofectamine 3000 endotoxin-free plasmid empty vectors and
Circ_CLASP2 over-express vectors transiently transfect nasopharyngeal carcinoma cell CNE2 and HNE2, continue to cultivate to after 48 hours.It collects thin
Born of the same parents detect the expression of circ_CLASP2 using Real-Time Fluorescent Quantitative PCR Technique.In order to identify circ_CLASP2 pairs of transfection
The mRNA of CLASP2 influences situation, we are detected using Real-Time Fluorescent Quantitative PCR Technique while detecting the expression of CLASP2
(see Fig. 4), for qPCR the results show that compared with pcDNA3.1 empty plasmid group cells, transfection circ_CLASP2 is overexpressed plasmid group
The expression of circ_CLASP2 significantly increases in cell, and it expresses multiple as 60 times and 20 in CNE2, HNE2 cell line
Times or more, as a result there is statistical significance.But the mRNA expressions of CLASP2 do not change significantly, you can illustrate to transfect
Circ_CLASP2 does not influence the mRNA level in-site of CLASP2, and the function in subsequent experimental result is played by circ_CLASP2
Rather than CLASP2 genes.
The effect detection that silence circ_CLASP2 is expressed in Nasopharyngeal Carcinoma Cell Line of embodiment 3
In order to ensure the preciseness of experiment, we devise the siRNA sequences of two circ_CLASP2 according to splicing site
Row, siRNA are that a kind of length is 21-25 nucleotide, can be combined with cognate rna complementation, selective degradation purpose RNA, to
The double stranded rna molecule for inhibiting it to express.Currently, siRNA has evolved into the important tool of gene functional research.In order to probe into
Effects of the circ_CLASP2 in tumor development, we devise two according to the splicing site of circ_CLASP2
SiRNA by siRNA (being respectively siRNA1, siRNA2 and their mixture siRNA1+2) and is made using Hiperfect reagents
The expression to silence circ_CLASP2 in CNE2 and HNE2 cell lines is transiently transfected for the unrelated sequences (NC) of experiment contrast.Turn
Continue after dye culture 48 hours collect cell, using Real-Time Fluorescent Quantitative PCR Technique detect circ_CLASP2 expression with
It detects the transfection efficiency of siRNA, while detecting the expression of CLASP2 (see Fig. 5).Target the siRNA sequences of circ_CLASP2
Row the expression of circ_CLASP2 can be reduced to 50% hereinafter, and the mRNA expressions of CLASP2 not by shadow
Ring, you can illustrate transfect circ_CLASP2 the mRNA level in-site of CLASP2 is not influenced, the function in subsequent experimental result by
Circ_CLASP2 is played rather than CLASP2 genes.
Embodiment 4：The external circ_CLASP2 that is overexpressed promotes nasopharyngeal carcinoma cell proliferation
We use liposome method lipofectamine 3000 endotoxin-free plasmid pcDNA3.1 and pcDNA3.1/ first
Has_circ_CLASP2 over-express vectors transiently transfect nasopharyngeal carcinoma cell CNE2 and HNE2, after continuing culture 48 hours, utilize
QRT-PCR ensures that circ_CLASP2 after expression raising, carries out MTT experiment, verify it to cell in Nasopharyngeal Carcinoma Cell Line
The influence of proliferation.Based on first to the 5th day testing result, we simultaneously had found pcDNA3.1 empty plasmids group and circ_CLASP2
The cell Proliferation between plasmid group is overexpressed there are notable difference, circ_CLASP2 is to the nose under condition of in vitro culture for overexpression
Pharynx cancer cell Proliferation has facilitation.(result is shown in Fig. 6)
Embodiment 5：External silence circ_CLASP2 inhibits the proliferation of nasopharyngeal carcinoma cell
The two siRNA mixtures (siRNA 1+2) and siRNA of circ_CLASP2 will be targeted using Hiperfect reagents
Compare expression of (NC) transient transfection to silence circ_CLASP2 in CNE2 and HNE2 cell lines.It is small to continue culture 48 after transfection
When collect cell, using Real-Time Fluorescent Quantitative PCR Technique detect circ_CLASP2 expression to detect the transfection of siRNA
Efficiency.The results show that siRNA has good silencing efficiency.After ensuring that circ_CLASP2 is disturbed, we use silence
The Nasopharyngeal Carcinoma Cell Line CNE2 and HNE2 of circ_CLASP2 carries out MTT experiment, verifies the influence of its cell proliferation.It is based on
First to the 5th day testing result, relative to NC groups, the growth rate of siRNA group cells obviously slows down, i.e. silence circ_
CLASP2 inhibits the proliferation of nasopharyngeal carcinoma cell.By the verification of positive and negative both direction, we can say that circ_CLASP2 is to external
Nasopharyngeal carcinoma cell proliferation under condition of culture has facilitation.(result is shown in Fig. 7)
Embodiment 6：The healing migration experiment of cell cut：
(1) photo cell platform：D-Hank ' the 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) wait for that cell grows the siRNA for transfecting targeting circ_CLASP2 respectively to 50%~70% or so and control siRNA
Sequence (NC), or it is overexpressed plasmid and empty plasmid；
(3) start cut within second day after cell covers with tiling board bottom：10 μ l pipette tips 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 cut 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 caused by cut 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 cut healing, arranges all pictures, and for statistical analysis.
The external migration for being overexpressed circ_CLASP2 and promoting nasopharyngeal carcinoma cell
After determining that circular rna circ_CLASP2 has facilitation to the proliferative capacity of nasopharyngeal carcinoma cell, we are again in nose
Scratch experiment is carried out in pharynx cancer cell line, to verify circ_CLASP2 to the migration of Nasopharyngeal Carcinoma Cell Line either with or without influence.Profit
It is with liposome method lipofectamine 3000 that endotoxin-free empty plasmid pcDNA3.1 and CLASP2 over-express vector is instantaneous
Nasopharyngeal carcinoma cell CNE2 and HNE2 are transfected, continues to cultivate to after 48 hours.Cell is collected, Real-Time Fluorescent Quantitative PCR Technique is utilized
Detect the expression of circ_CLASP2 and cyclic efficiency (Fig. 8).In the table excessively that circ_CLASP2 overexpression plasmids are determined
Up to after good result, we have carried out cell cut Healing Experiments in Nasopharyngeal Carcinoma Cell Line CNE2 and HNE2.Cut Healing Experiments
Multiple time points (12h and for 24 hours) in these cells confirm：Relative to unloaded pcDNA3.1 (+) plasmid group, it is overexpressed
The cut prognosis speed of the nasopharyngeal carcinoma cell of circ_CLASP2 faster, shows that cell migration ability is remarkably reinforced (Fig. 9).Cut
Width difference is larger, and has statistical significance (Figure 10).The above results show that being overexpressed circ_ in Nasopharyngeal Carcinoma Cell Line
The expression of CLASP2 can promote the transfer ability of nasopharyngeal carcinoma cell CNE2, HNE2 in vitro.
External silence circ_CLASP2 inhibits the migration of nasopharyngeal carcinoma cell
2 siRNA sequence mixtures (siRNA1+2) of circ_CLASP2 and right will be targeted using Hiperfect reagents
The expression to silence circ_CLASP2 in CNE2 and HNE2 cell lines is transiently transfected according to unrelated sequences (NC).Continue to train after transfection
It supports 48 hours and collects cell, the expression of circ_CLASP2 is detected to detect siRNA using Real-Time Fluorescent Quantitative PCR Technique
Transfection efficiency.The results show that siRNA has good silencing efficiency (Figure 11).Ensuring that circ_CLASP2 is disturbed
Afterwards, we carry out scratch experiment in the Nasopharyngeal Carcinoma Cell Line CNE2 and HNE2 of silence circ_CLASP2, verify it to cell
The influence of migration.Multiple time points (12h and for 24 hours) of the cut Healing Experiments in these cells confirm：Relative to NC groups,
The transfer ability of siRNA group cells obviously weakens (Figure 12).Scratch width difference is apparent, and has statistical significance (Figure 13).
The above results show that the expression of circ_CLASP2 can inhibit nasopharyngeal carcinoma cell CNE2, HNE2 to exist in silence Nasopharyngeal Carcinoma Cell Line
External transfer ability.Being verified by positive and negative both direction proves, circ_CLASP2 can promote the migration of nasopharyngeal carcinoma cell.
Embodiment 7：Cell transwell Matrigels：
(1) matrigel prepares：Carrying the previous day is placed in 4 DEG C of refrigerators in -20 DEG C of BD Matrigel glue and is melted into liquid freezing
State；
(2) matrigel dilutes：BD Matrigel glue：Anteserum-less substrate=1:8, i.e. 20 μ l matrigels add 160 μ l 1640
Train base featheriness mixing；
(3) 100 μ l of the cells transwell are added in the matrigel diluted, then 80 μ l is sucked out along side, completed be put into successively
It is incubated 2-3 hours in 37 DEG C of incubators, when it is white to see 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 uses the training base weight of serum-free outstanding thin
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) add interior on the unified cell suspensions to transwell counted of 200 μ l per room, 24 orifice plates are put back to 37 DEG C
In incubator, according to cell state and cell invasion speed, it is incubated about 24~48 hours.
(12) 24 orifice plates are taken out, are washed twice with PBS or D-hanks, 4% paraformaldehyde embathes 10 minutes, and 3 are washed with clear water
Time.
(13) it dyes：The bottom of the cells transwell is added drop-wise to 0.1% crystal violet, 5- is placed in room temperature peace and quiet
10min is cleaned 2-3 times with PBS, and the matrigel above cell is carefully wiped with cotton swab；
(14) it is added in 800 μ l, transwell upper chambers 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 with image J softwares count aobvious with statistical analysis difference
Work property.
The external invasion for being overexpressed circ_CLASP2 and promoting nasopharyngeal carcinoma cell
We have carried out the matrigel invasion experiment of the cells Transwell in Nasopharyngeal Carcinoma Cell Line CNE2 and HNE2, with observation
It is overexpressed influences of the circ_CLASP2 to cell invasion ability.We are also with liposome method lipofectamine 3000
Endotoxin-free empty plasmid pcDNA3.1 and circ_CLASP2 over-express vector transiently transfect nasopharyngeal carcinoma cell CNE2 and
HNE2 continues to cultivate to after 48 hours.Cell is collected, the expression of circ_CLASP2 is detected using Real-Time Fluorescent Quantitative PCR Technique
Horizontal and cyclic efficiency (Figure 14).After being determined that circ_CLASP2 is overexpressed the good representation effect of plasmid, we are by cell
It is inoculated into the cells Transwell of paving matrigel, finds to be overexpressed the cell invasion of plasmid group to the number of small chamber lower surface
It is apparent more than zero load group, and the trend of two plants of cell line results is consistent (Figure 15).Random shooting 6 opens photo and records cell number
Mesh has notable difference in each cell line, and has statistical significance (Figure 16) between two group data.The above result shows that
It is overexpressed the expression of circ_CLASP2 in Nasopharyngeal Carcinoma Cell Line, the invasion of nasopharyngeal carcinoma cell CNE2, HNE2 in vitro can be promoted
Ability.
The expression of external silence circ_CLASP2 influences the invasion of nasopharyngeal carcinoma
The cell table opposite with circ_CLASP2 is overexpressed whether can be obtained after silence circ_CLASP2 in order to probe into
Type changes, we after siRNA silences circ_CLASP2 again with having carried out the cells Transwell matrigel in two plants of cell line
Matrigel.SiRNA sequence mixture (siRNA1+2) and the control of circ_CLASP2 will be targeted using Hiperfect reagents
Unrelated sequences (NC) transiently transfect the expression to silence circ_CLASP2 in CNE2 and HNE2 cell lines.Continue to cultivate after transfection
It collects cell within 48 hours, the expression of circ_CLASP2 is detected to detect siRNA's using Real-Time Fluorescent Quantitative PCR Technique
Transfection efficiency.The results show that circ_CLASP2siRNA has good silencing efficiency (Figure 17).Ensuring circ_CLASP2
After being disturbed, we carry out the cells Transwell in the Nasopharyngeal Carcinoma Cell Line CNE2 and HNE2 of silence circ_CLASP2
Matrigel invasion is tested, the results show that the tumor cell number that siRNA groups can be observed in the small chamber lower surfaces of Transwell is bright
It is aobvious to be less than NC groups, and the trend of two plants of cell line results is consistent (Figure 18).Random shooting 6 opens photo and records cell number, often
There is notable difference in one cell line between two group data, and there is statistical significance (Figure 19).The above result shows that silence nose
The expression of circ_CLASP2 in pharynx cancer cell line can inhibit the invasive ability of nasopharyngeal carcinoma cell CNE2, HNE2 in vitro.It is logical
Crossing positive and negative both direction proves, circular rna circ_CLASP2 can promote the invasion of nasopharyngeal carcinoma cell.
Sequence table
<110>Central South University
<120>Inhibit application and preparation of the reagent of circ_CLASP2 in preparing treatment of nasopharyngeal carcinoma preparation
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 449
<212> DNA
<213>Unknown (Unknown)
<400> 1
gtatcattaa tgggattgga aattttaagt gcctttgtgg acagattatc aacacgcttt 60
aaatcctatg tagcaatggt tattgtagct ttaatagaca gaatgggaga tgccaaagac 120
aaggttcgag atgaagctca gactctgata ttgaagttaa tggatcaagt agcaccacct 180
atgtacattt gggagcagtt ggcttctggt tttaaacaca agaattttcg atctcgagaa 240
ggcgtgtgtc tgtgtcttat tgaaacctta aacatttttg gggctcagcc actagtcatc 300
agcaaattga taccacattt gtgtatcctg tttggagact ccaacagtca ggtgagagat 360
gctgcaatat tggctatagt ggagatttat agacatgtgg gagaaaaagt gaggatggat 420
ctttataaga gaggaattcc ccctgctag 449
<210> 2
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 2
acagtcaggt gagagatgct 20
<210> 3
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 3
tggcatctcc cattctgtct 20
<210> 4
<211> 25
<212> DNA
<213>Unknown (Unknown)
<400> 4
ccatcgatgg gtatcattaa tggga 25
<210> 5
<211> 27
<212> DNA
<213>Unknown (Unknown)
<400> 5
ttcccgcggg gactagcagg gggaatt 27
<210> 6
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 6
tcaccaactg ggacgacatg 20
<210> 7
<211> 21
<212> DNA
<213>Unknown (Unknown)
<400> 7
gtcaccggag tccatcacga t 21
<210> 8
<211> 21
<212> RNA
<213>Unknown (Unknown)
<400> 8
cugcuaggua ucauuaaugu u 21
<210> 9
<211> 21
<212> RNA
<213>Unknown (Unknown)
<400> 9
cauuaaugau accuagcagu u 21
<210> 10
<211> 21
<212> RNA
<213>Unknown (Unknown)
<400> 10
ccccugcuag guaucauuau u 21
<210> 11
<211> 21
<212> RNA
<213>Unknown (Unknown)
<400> 11
uaaugauacc uagcaggggu u 21
<210> 12
<211> 21
<212> RNA
<213>Unknown (Unknown)
<400> 12
uucuccgaac gugucacguu u 21
<210> 13
<211> 21
<212> RNA
<213>Unknown (Unknown)
<400> 13
acgugacacg uucggagaau u 21
Claims (10)
1. inhibiting application of the reagent of circ_CLASP2 in preparing treatment of nasopharyngeal carcinoma preparation, the sequence of the circ_CLASP2
Row are as shown in SEQ No.1.
2. application according to claim 1, which is characterized in that the reagent of the inhibition circ_CLASP2 includes
siRNA。
3. application according to claim 2, which is characterized in that siRNA includes following any pair or two pairs：
circ_CLASP2siRNA1：
Positive-sense strand (5'-3') CUGCUAGGUAUCAUUAAUGUU
Antisense strand (5'-3') CAUUAAUGAUACCUAGCAGUU；
circ_CLASP2siRNA2：
Positive-sense strand (5'-3') CCCCUGCUAGGUAUCAUUAUU
Antisense strand (5'-3') UAAUGAUACCUAGCAGGGGUU.
4. application according to claim 2, which is characterized in that the reagent of the inhibition circ_CLASP2 includes feminine gender
Control：
Positive-sense strand (5'-3') UUCUCCGAACGUGUCACGUUU
Antisense strand (5'-3') ACGUGACACGUUCGGAGAAUU.
5. application according to claim 2, which is characterized in that treatment of nasopharyngeal carcinoma preparation includes the examination transfected needed for siRNA
Agent.
6. a kind for the treatment of of nasopharyngeal carcinoma preparation, which is characterized in that contain the reagent that can inhibit circ_CLASP2.
7. preparation according to claim 6, which is characterized in that the reagent of circ_CLASP2 that can inhibit includes
siRNA。
8. preparation according to claim 7, which is characterized in that siRNA includes following any pair or two pairs：
circ_CLASP2siRNA1：
Positive-sense strand (5'-3') CUGCUAGGUAUCAUUAAUGUU
Antisense strand (5'-3') CAUUAAUGAUACCUAGCAGUU；
circ_CLASP2siRNA2：
Positive-sense strand (5'-3') CCCCUGCUAGGUAUCAUUAUU
Antisense strand (5'-3') UAAUGAUACCUAGCAGGGGUU.
9. preparation according to claim 7, which is characterized in that the reagent of circ_CLASP2 that can inhibit includes the moon
Property control：
Positive-sense strand (5'-3') UUCUCCGAACGUGUCACGUUU
Antisense strand (5'-3') ACGUGACACGUUCGGAGAAUU.
10. preparation according to claim 7, which is characterized in that treatment of nasopharyngeal carcinoma preparation includes the examination transfected needed for siRNA
Agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810525695.3A CN108721319B (en) | 2018-05-28 | 2018-05-28 | Application of agent for inhibiting circ _ CLASP2 in preparation of nasopharyngeal carcinoma treatment preparation and preparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810525695.3A CN108721319B (en) | 2018-05-28 | 2018-05-28 | Application of agent for inhibiting circ _ CLASP2 in preparation of nasopharyngeal carcinoma treatment preparation and preparation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108721319A true CN108721319A (en) | 2018-11-02 |
| CN108721319B CN108721319B (en) | 2020-04-28 |
Family
ID=63935512
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810525695.3A Active CN108721319B (en) | 2018-05-28 | 2018-05-28 | Application of agent for inhibiting circ _ CLASP2 in preparation of nasopharyngeal carcinoma treatment preparation and preparation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108721319B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109652556A (en) * | 2019-02-28 | 2019-04-19 | 中南大学 | CircARHGAP12 and its preparing application and diagnostic preparation in nasopharyngeal carcinoma diagnosis preparation |
| CN109701025A (en) * | 2019-03-05 | 2019-05-03 | 中南大学 | CircCDYL2 is preparing application and treatment preparation in treatment of nasopharyngeal carcinoma preparation |
| CN109758472A (en) * | 2019-01-24 | 2019-05-17 | 中南大学 | Circ_2157 is preparing application and treatment preparation in treatment of nasopharyngeal carcinoma preparation |
| CN109762823A (en) * | 2019-03-04 | 2019-05-17 | 中南大学 | Circ_1892 and its preparing application in treatment of nasopharyngeal carcinoma preparation and treatment preparation |
| CN109847064A (en) * | 2019-01-24 | 2019-06-07 | 中南大学 | Circ_3480 is preparing application and treatment preparation in treatment of nasopharyngeal carcinoma preparation |
| CN109908351A (en) * | 2019-04-08 | 2019-06-21 | 中南大学 | Circ_ADARB1 is preparing application and treatment preparation in treatment of nasopharyngeal carcinoma preparation |
| CN115317483A (en) * | 2021-12-24 | 2022-11-11 | 南通大学附属医院 | Application of chloroquine and derivatives thereof in preparation of PGCCs (PGCCs) inhibitor |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100305188A1 (en) * | 2007-10-03 | 2010-12-02 | Kyowa Hakko Kirin Co., Ltd. | Nucleic acid capable of regulating the proliferation of cell |
| CN104293789A (en) * | 2014-10-14 | 2015-01-21 | 广西医科大学 | siRNA for inhibiting gene expression of S100A8 and application thereof |
| CN107653318A (en) * | 2017-10-19 | 2018-02-02 | 中山大学肿瘤防治中心 | One group is used for the mark and its application for predicting that nasopharyngeal carcinoma shifts risk |
-
2018
- 2018-05-28 CN CN201810525695.3A patent/CN108721319B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100305188A1 (en) * | 2007-10-03 | 2010-12-02 | Kyowa Hakko Kirin Co., Ltd. | Nucleic acid capable of regulating the proliferation of cell |
| CN104293789A (en) * | 2014-10-14 | 2015-01-21 | 广西医科大学 | siRNA for inhibiting gene expression of S100A8 and application thereof |
| CN107653318A (en) * | 2017-10-19 | 2018-02-02 | 中山大学肿瘤防治中心 | One group is used for the mark and its application for predicting that nasopharyngeal carcinoma shifts risk |
Non-Patent Citations (4)
| Title |
|---|
| 冯美卿: "《生物技术制药》", 31 January 2016, 北京：中国医药科技出版社 * |
| 张文玲等: "鼻咽癌分子标志物研究", 《生物化学与生物物理进展》 * |
| 无: "ACCESSION：NM_015097.2", 《GENBANK》 * |
| 李伍举: "《计算机辅助分子生物学实验设计与分析》", 30 April 2009, 北京：军事医学科学出版社 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109758472A (en) * | 2019-01-24 | 2019-05-17 | 中南大学 | Circ_2157 is preparing application and treatment preparation in treatment of nasopharyngeal carcinoma preparation |
| CN109847064A (en) * | 2019-01-24 | 2019-06-07 | 中南大学 | Circ_3480 is preparing application and treatment preparation in treatment of nasopharyngeal carcinoma preparation |
| CN109847064B (en) * | 2019-01-24 | 2021-02-02 | 中南大学 | Application of circ _3480 in preparation of nasopharyngeal carcinoma treatment preparation and treatment preparation |
| CN109758472B (en) * | 2019-01-24 | 2021-11-30 | 中南大学 | Application of circ _2157 in preparation of nasopharyngeal carcinoma treatment preparation and treatment preparation |
| CN109652556A (en) * | 2019-02-28 | 2019-04-19 | 中南大学 | CircARHGAP12 and its preparing application and diagnostic preparation in nasopharyngeal carcinoma diagnosis preparation |
| CN109762823A (en) * | 2019-03-04 | 2019-05-17 | 中南大学 | Circ_1892 and its preparing application in treatment of nasopharyngeal carcinoma preparation and treatment preparation |
| CN109701025A (en) * | 2019-03-05 | 2019-05-03 | 中南大学 | CircCDYL2 is preparing application and treatment preparation in treatment of nasopharyngeal carcinoma preparation |
| CN109908351A (en) * | 2019-04-08 | 2019-06-21 | 中南大学 | Circ_ADARB1 is preparing application and treatment preparation in treatment of nasopharyngeal carcinoma preparation |
| CN109908351B (en) * | 2019-04-08 | 2021-04-06 | 中南大学 | Application of circ _ ADARB1 in preparation of nasopharyngeal carcinoma treatment preparation and treatment preparation |
| CN115317483A (en) * | 2021-12-24 | 2022-11-11 | 南通大学附属医院 | Application of chloroquine and derivatives thereof in preparation of PGCCs (PGCCs) inhibitor |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108721319B (en) | 2020-04-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108721319A (en) | Inhibit application and preparation of the reagent of circ_CLASP2 in preparing treatment of nasopharyngeal carcinoma preparation | |
| CN108660214A (en) | Detect application and kit of the reagent of circ_CLASP2 in preparing nasopharyngeal carcinoma diagnosis preparation | |
| Ma et al. | miR-27a regulates the growth, colony formation and migration of pancreatic cancer cells by targeting Sprouty2 | |
| Valastyan et al. | RETRACTED: a pleiotropically acting microRNA, miR-31, inhibits breast cancer metastasis | |
| AU2020203590A1 (en) | Mirna biogenesis in exosomes for diagnosis and therapy | |
| CN102813940B (en) | The predictor of Micro Core ribosomal ribonucleic acid as cancer progression and the application of Therapeutic cancer thereof | |
| Zhang et al. | CircRNA_0000502 promotes hepatocellular carcinoma metastasis and inhibits apoptosis through targeting microRNA-124 | |
| CN109266743B (en) | A kind of cancer markers and application thereof | |
| CN109666674A (en) | CircCDYL2 and its preparing application and diagnostic preparation in nasopharyngeal carcinoma diagnosis preparation | |
| CN109971759A (en) | Circ_ADARB1 and its preparing application and diagnostic preparation in nasopharyngeal carcinoma diagnosis preparation | |
| CN109852692A (en) | Circ_3480 and its preparing application and diagnostic reagent in nasopharyngeal carcinoma diagnosis preparation | |
| CN114032236B (en) | shRNA of TMEM2 and application thereof | |
| CN110577952B (en) | Application of siRNA interfering long non-coding RNA in preparation of medicine for treating breast cancer | |
| US20220145395A1 (en) | Transpochimeric Gene Transcripts (TCGTS) As Cancer Biomarkers | |
| CN109847064A (en) | Circ_3480 is preparing application and treatment preparation in treatment of nasopharyngeal carcinoma preparation | |
| CN109758472A (en) | Circ_2157 is preparing application and treatment preparation in treatment of nasopharyngeal carcinoma preparation | |
| CN109762818A (en) | Circ_2157 and its preparing application and diagnostic reagent in nasopharyngeal carcinoma diagnosis preparation | |
| CN109908351A (en) | Circ_ADARB1 is preparing application and treatment preparation in treatment of nasopharyngeal carcinoma preparation | |
| CN109652556A (en) | CircARHGAP12 and its preparing application and diagnostic preparation in nasopharyngeal carcinoma diagnosis preparation | |
| CN109971760A (en) | Circ_3234 and its preparing application in treatment of nasopharyngeal carcinoma preparation and treatment preparation | |
| CN109762823A (en) | Circ_1892 and its preparing application in treatment of nasopharyngeal carcinoma preparation and treatment preparation | |
| Al-Shibli et al. | Association of miRNA and bone tumors: Future therapeutic inroads | |
| CN109700825A (en) | CircARHGAP12 is preparing application and treatment preparation in treatment of nasopharyngeal carcinoma preparation | |
| CN116637123B (en) | Application of reagent for knocking down or down expression of C15orf39 gene in preparation of medicines for treating gastric cancer | |
| CN114134230B (en) | Application of hsa_circ_0003310 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |