CN108384805B - Promote application of the reagent of circular rna circRNF13 expression on preparation treatment Dendritic cell drug - Google Patents
Promote application of the reagent of circular rna circRNF13 expression on preparation treatment Dendritic cell drug Download PDFInfo
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Abstract
The invention discloses promote application of the reagent of circular rna circRNF13 expression on preparation treatment Dendritic cell drug.It confirms to be overexpressed circRNF13 in Tca8113 cells by studying, the proliferation of Tca8113 cells can be inhibited.Therefore, it is used for circRNF13 overexpression preparation to treat Dendritic cell, there is far-reaching clinical meaning and important popularization and application foreground.
Description
Technical field
The invention belongs to oncomolecularbiology fields, and in particular to promote the reagent of circular rna circRNF13 expression
Application on preparation treatment Dendritic cell drug.
Background technique
The Human Genome Project and its subsequent DNA element encyclopedia plan (The Encyclopedia of DNA
Elements Project, ENCODE) research achievement shows that protein coding gene sequence only accounts for the 1- of human genomic sequence
3%, and the transcribed sequence of the overwhelming majority is non-coding RNA (non-coding RNA, ncRNA) in human genome.Non- volume
Code RNA is although non-coding protein, due to wide participation genes within cells expression regulation, in field of biomedical research one
Directly it is concerned.Study more is linear ncRNA molecule, from RNA sequence length can be divided into Microrna (microRNA,
MiRNA, 19-23nt) and long-chain non-coding RNA (long non-coding RNA, lncRNAs, > 200nt), they are including
Important function is played in the occurrence and development of a variety of mankind's common diseases including malignant tumour.Recently, a kind of completely new
NcRNA molecule, circular rna (circular RNA, circRNA) is due to its unique configuration and the important biomolecule increasingly found
Function is learned, the new forward position of field of biomedicine and hot spot are had become.
CircRNA had once once been taken as the wrong product during genetic transcription post-processing.With the lines such as mRNA and lncRNA
Property RNA process it is much like, the formation of circRNA is also raw when carrying out montage processing to RNA precursor by splicing complex
At, only in this course, the loop-forming sequences of flank combine cyclization first, and subsequent splicing complex carries out montage, most
Flanking sequence cuts off to form circRNA at last.Recently as new-generation sequencings technologies such as sequencing technologies especially RNA-seq
Development, more and more circRNA are by it has been found that simultaneously having passed through research discloses part circRNA in numerous life
It all plays an important role in activity.Additionally due to its unique structure causes circRNA insensitive to nuclease, in tumour
CircRNA also has a clear superiority in terms of the development and application of marker, therefore very likely as a kind of important molecular marker
The target spot of object and emerging drug development.But since this field has just emerged, most of circRNA is not yet found or there is no
Research.
Squamous cell carcinoma of tongue (tongue squamous cell carcinoma, TSCC) is that the highest oral cavity of disease incidence is disliked
Property tumour, Dendritic cell majority is squamous carcinoma, mostly occurs in lingual margin, is often ulcer type or leaching secondly for the tip of the tongue, back and root of the tongue etc.
Profit type.General grade malignancy is higher, and growth is fast, and wellability is stronger, and ordinary wave and lingualis cause tongue limitation of movement, makes to speak, feed
And it swallows and difficulty occurs.The chemotherapy that operative treatment is aided with based on suitable platinum medicine is Dendritic cell clinically main treatment side
Case.However, finding that the therapeutic effect of Dendritic cell patient is often not satisfactory in clinic, survival rate is only 55-65% within 5 years, studies carefully it
Reason essentially consists in the remaining cancer cell of operation, and (for example to have invaded the circulating tumor being transferred in blood vessel or lymphatic vessel thin
Born of the same parents) generally existing drug resistance, oncogenic recurrence and transfer are led, treatment failure is caused.Therefore, Tca8113 cells are found out to chemotherapy
The resistance mechanisms of drug screen new drug target, final to improve survival and life quality, are to be worth clinical section
The important topic that the workers of grinding study.
In order to study circRNA in Tca8113 cells to the effect and mechanism in cis-platinum class Drug-resistant, we are used
The circRNA genetic chip Arraystar Human Circular RNA Microarray V2.0 of Agilent company is to two
The expression of circRNAs in Tca8113 cells strain (Tca8113 and Cal27) before and after cisplatin treated is analyzed.I
Find circRNF13 expression variation it is the most significant, prompt circRNF13 may be related to the cisplatin resistance of Dendritic cell, and
Up to the present, the biological function of circRNF13 and mechanism of action have no any report.
Since genetic chip can only be based on circRNA ring-type splicing site upstream and downstream sequence design probe, visited by these
Needle specificity captures circRNA, to detect the gene expression abundance of circRNA in cell or tissue;Pass through particularly for us
CircRNA chip discovery circRNF13 for, by genetic chip provide technical data we can find its corresponding spy
The total 60bp of needle, the last period is the sequence of 8 exon, 3 ' the end 30bp of RNF13 gene, and latter section is then RNF13 gene 2
5 ' end 30bp of exon, prompting it is to be joined end to end to add by No. 2 of the precursor RNA of RNF13 genetic transcription and 8 exons
Does work form, but specifically RNF13 which exon processing generates (whether 2~8 exons are included entirely within? whether
Also include intron sequences? because some circRNA just include intron sequences) it does not know.Then, our design primers are simultaneously
Pass through the PCR successful clone nested twice full length sequence of circRNF13, it was confirmed that circRNF13 is strictly by being located at dye
The 2-8 extra of RNF13 gene (ring finger protein 13, NM_183381.2) on the region colour solid 3q25.1 is aobvious
Subring is spliced to form, overall length 716bp.
In order to further study the biological function of circRNF13, it is dry that we devise the small molecule for circRNF13
The expression that RNA (siRNA) sequence-specific has struck circRNF13 low is disturbed, while successfully constructing circRNF13 over-express vector
CircRNF13 is overexpressed in Tca8113 cells.MTT and flow cytometry detection confirm that up-regulation circRNF13 can be bright
Aobvious to inhibit Tca8113 cells proliferation, mechanism is arresting cell cycle and induces cell apoptosis, and strikes low circRNF13 and then obtain
Opposite result.Further, we successfully obtain Dendritic cell chemotherapy resistance cell strain, in real time by Fiber differentiation
The expression of circRNF13 in quantitative PCR detection cell finds expression of the circRNF13 in drug-resistant cell strain and former
Beginning cell strain is lowered compared to significant;And we are overexpressed circRNF13 in mdr cell, can obviously reverse Dendritic cell thin
The drug-resistant phenotype of born of the same parents, and the expression of low circRNF13 is struck in initial cell strain, then Tca8113 cells can be significantly improved to suitable
The tolerance of platinum.
Finally, we are had detected in Dendritic cell clinical sample by real-time fluorescence quantitative PCR and the method for in situ hybridization
The expression of circRNF13, discovery circRNF13 expresses significant downward in Dendritic cell, and circRNF13 expresses low trouble
Its life span of person is shorter than circRNF13 and expresses high patient, therefore can be used for tongue for the detection preparation of the lncRNA
The auxiliary diagnosis and Index for diagnosis of squamous carcinoma.
Summary of the invention
The object of the present invention is to provide promote the reagent of circular rna circRNF13 expression to treat Dendritic cell drug in preparation
On application, the sequence of the circular rna circRNF13 is as shown in SEQ NO:1.
The reagent for promoting circular rna circRNF13 expression is circRNF13 over-express vector.
The building process of the circRNF13 over-express vector is as follows:
(1) it according to the fundamentum of the Forming Mechanism of circRNA and eucaryote RNA montage, designs and is suitable for
The loop-forming sequences of circRNA expression;According to the sequence information of commercial carrier pcDNA3.1, suitable multiple cloning sites are designed,
It is as follows to obtain the DNA sequence dna for completely realizing that circRNA is overexpressed:
GTGCTGGGATTACAGGTGTGAGCTACCACCCCCGGCCCACTTTTTCTTAAGCTTGGTACCGAGCTCGG ATCCACATCGATTGGTGGAATTCTGCAGATATCCACCGCGGTGGCGGCCGCTCGAGTCTAGAGAAAAGAATTAGGC
TCGGCACGGTAGCTCACACCTGTAATCCCAGCA, intermediate underscore part are multiple cloning sites, and both ends are respectively upper and lower
Swim loop-forming sequences;
(2) above-mentioned synthesis is realized that NheI restriction enzyme site sequence is added in DNA sequence dna one end that circRNA is overexpressed, separately
It adds ApaI restriction enzyme site sequence and obtains pcCirc by being built into pcDNA3.1 carrier after NheI and ApaI digestion in one end
Empty plasmid, sequencing confirmation plasmid is correct, and sequence is as shown in SEQ NO:13;
(3) selection Cla I and Sac II restriction enzyme site is used for digestion pcCirc empty plasmid, and by circRNF13 sequence
It is inserted between the upstream and downstream loop-forming sequences of carrier.
Detailed process is as follows for step (3):
1) using Tca8113 cells Tca8113cDNA as template, TaKaRa LA is utilizedEnzyme carries out PCR amplification overall length
CircRNF13 sequence;CircRNF13 full length sequence amplimer is as follows:
Upstream primer: 5 '-GTGATTTTACAACGAGAT-3 ';
Downstream primer: 5 '-CTTTCTTGAATTTATGTA-3 ';
In upstream and downstream, 5 ' ends of primer are respectively plus restriction enzyme Cla I and Sac II recognition site and protection alkali
After base, primer sequence is as follows:
Upstream primer: 5 '-AGGAATCGAT GTGATTTTACAACGAGAT-3 ', underscore part are that Cla I identifies position
Point;
Downstream primer: 5 '-ATGCCCGCGG CTTTCTTGAATTTATGTA-3 ', underscore part are that Sac II identifies position
Point;
2) PCR amplification, circRNF13 full length sequence, PCR reaction condition are as follows:
PCR reaction step
5 return to step 2, carry out 39 secondary response circulations altogether;
3) by, through Cla I and Sac II double digestion rear electrophoresis, glue returns again after PCR product electrophoresis, glue recycling target fragment
It receives;
4) pcCirc empty plasmid recycles target fragment through Cla I and Sac II double digestion rear electrophoresis glue;
5) the carrier matter of overexpression circRNF13 3) is arrived with 4) step glue recovery product with the connection of T4 DNA ligase
Grain;
6) by the eukaryon expression plasmid transformed competence colibacillus large intestine bar comprising circRNF13 full length sequence that 5) step obtains
Bacterium, to expand plasmid.
After we transfect circRNF13 carrier for expression of eukaryon, had detected in cell by real-time fluorescence quantitative PCR
The expression of circRNF13, discovery circRNF13 expression are significantly raised;And transfect the siRNA of targeting circRNF13
(siRNA) after, the expression of intracellular circRNF13 is obviously lowered;Compared with the cell of transfection empty carrier, it is overexpressed
The speed of growth of the Tca8113 cells Tca8113 and Cal27 of circRNF13 significantly slow down, and are struck using siRNA interference sequence low
After the expression of circRNF13, vitro growth rates are accelerated.Flow cytometry analysis shows to transfect in Tca8113 cells
After circRNF13, G2/M phase cell proportion is dramatically increased, and S phase cell proportion is reduced, and shows that circRNF13 can be by Dendritic cell
Cell block makes its cell division slow speed in the G2/M phase.Meanwhile apoptosis after circRNF13 is transfected in Tca8113 cells
Cell proportion obviously increases, this is also one of the reason of circRNF13 inhibits Tca8113 cells growth.And strike low circRNF13
Opposite result is then obtained.Therefore, it is used for circRNF13 overexpression preparation to treat Dendritic cell, with far-reaching clinical meaning
Adopted and important popularization and application foreground.
Detailed description of the invention
Fig. 1: circRNF13 is demonstrated by qRT-PCR and expresses significant up-regulation (left side), sequencing result card after cisplatin treated
Real qRT-PCR detected is strictly circRNF13 (right side).Arrow represents splicing site, is 8 extra of RNF13 on the left of arrow
Aobvious son 3 ' is held, and is 5 ' terminal sequences of 2 exon of RNF13 gene on the right side of arrow.
Fig. 2: being sequenced by nesting PCR- twice, for the first time the successful clone full length sequence of circRNF13 molecule, it was demonstrated that
CircRNA is the 2-8 exon ring of the RNF13 gene transcripts NM_183381.2 by being located on the region chromosome 3q25.1
Change is spliced to form, overall length 716bp.
Fig. 3: design simultaneously successfully construct circRNF13 over-express vector, using pcDNA3.1 carrier as basic framework, by
Two sections of loop-forming sequences and restriction enzyme site is added in its CMV promoter downstream, and the 2-8 exon of RNF13 is passed through
PCR, digestion are connected in carrier (left side), are then transfected into Tca8113 cells, are successfully overexpressed in Tca8113 cells
CircRNF13 (right side).
Fig. 4: it is devised for circRNF13 ring-type splicing site (exon 8 and exon 2 junction) selectively targeted
The siRNA sequence (left side) of the circular rna, is then transfected into Tca8113 cells, successfully strikes in Tca8113 cells low
The expression (right side) of circRNF13.
Fig. 5: MTT proliferation experiment shows compared with control group (NC), and being overexpressed circRNF13 (OE-circRNF13) can be with
Inhibit the proliferation of Tca8113 cells, and the expression (si-circRNF13) for striking low circRNF13 can promote Tca8113 cells
Proliferation.
Fig. 6: flow cytometry analysis is found compared with control group (NC), is overexpressed circRNF13 (OE-circRNF13)
Tca8113 cells G2/M phase distribution proportion obviously increases afterwards, shows that cell-cycle arrest in the G2/M phase, and strikes low circRNF13's
After expressing (si-circRNF13), the S phase, which is distributed, significantly increases, and shows that cell cycle progression accelerates.
Fig. 7: flow cytometry analysis Apoptosis situation is found compared with control group (NC), is overexpressed circRNF13
(OE-circRNF13) Tca8113 cells apoptosis ratio obviously increases afterwards, in the case of normal culture, apoptosis of tumor cells ratio compared with
Low, so striking the expression of low circRNF13, apoptotic cell ratio is in a slight decrease.
Fig. 8: circRNF13 expression significantly reduces in chemotherapy resistance cell (CR) compared with control group (NC).
Fig. 9: the expression (si-circRNF13) of low circRNF13 is struck in original Tca8113 cells (NC) or in drug resistance
After being overexpressed circRNF13 (OE-circRNF13) in cell (CR), with the cisplatin treated cell of same concentrations, then streaming is thin
Born of the same parents' instrument detects Apoptosis situation, and the expression that low circRNF13 is struck in discovery can increase tolerance of the cell to cis-platinum, and (apoptosis subtracts
It is few), and be overexpressed circRNF13 then and the drug resistance of mdr cell can be significantly reduced.
Figure 10: it is had detected in 28 pairs of fresh Dendritic cell biopsies (T) and cancer side control tissue (N) by qRT-PCR
The expression of circRNF13, discovery circRNF13 express significant downward in Dendritic cell biopsy.
Figure 11: it is had detected in Dendritic cell (right side) and cancer the side control tissue (left side) of archive by hybridization in situ technique
The expression of circRNF13 further demonstrates circRNF13 and expresses lower or do not express in Dendritic cell biopsy.
Figure 12: we have collected 88 Dendritic cell tissue specimens for having Clinical Follow-up data, and in situ hybridization has detected
The expression of circRNF13, wherein 21 can't detect the expression (Negative) of circRNF13, the existence of these patients
Time is shorter compared with other patients's (although circRNF13 expression is not high, can detecte to obtain, Positive), and prognosis is more
Difference.
Specific embodiment
The present invention is further illustrated below in conjunction with specific embodiment, is not intended to limit the present invention.
Embodiment 1, PCR sequencing have determined the full length sequence of circRNF13 in Tca8113 cells
1. materials and methods
1.1 cell line
Tca8113 cells Tca8113 and Cal27 are purchased from Central South University's cell centre, and normal condition is cultivated.
1.2 reagents and kit
TRIZOLTMReagent(Invitrogen);Plastic recovery kit (OMEGA);Reverse Transcriptase kit
(Promega);Proteinase K, DNase I, RNAsin, RNase A (GBICOL company);LAEnzyme (Takara).
1.3 fluorescence quantitative PCR detection circRNF13 are expressed in Tca8113 cells
Extracted total RNA, 1 μ g RNA carry out real-time fluorescence quantitative PCR after reverse transcription is at cDNA.CircRNF13 forward direction is drawn
Object is 5-GTCCAGGATAGACATAGAGC-3, as shown in SEQ NO:2 and reverse primer 5-GTGTAGACTTGTGTGGCTGA-
3.As shown in SEQ NO:3.
GAPDH forward primer for control is 5 '-ACCACAGTCCATGCCATCAC-3 ' as shown in SEQ NO:4, and
Reverse primer 5 '-TCCACCACCCTGTTGCTGTA-3 ', as shown in SEQ NO:5.
Real-time fluorescence quantitative PCR reaction system
Real-time fluorescence quantitative PCR reaction step
5 Plate read
6 82℃ 30sec
7 Plate read
8 Go to step 2for more 39times
9 55.0 DEG C of from of Perform melting curve 95.0 DEG C of to, 0.2 DEG C of read every, hold
for 1sec.
The amplification curve and melting curve of real-time fluorescence quantitative PCR, the expression intensity root of each gene are confirmed after reaction
After CT value (threshold cycle values), reference gene (GAPDH) markization, using group t-test checking computation
P value.
It is sequenced finally, real-time fluorescence quantitative PCR is amplified the segment come by us, sequence is carried out to the segment expanded
Column compare, confirm we amplification be exactly circRNF13, and obtain circRNF13 cyclisation splice specific site.
1.4 reverse transcription PCRs obtain circRNF13 overall length
1) it using Tca8113 cells Tca8113 cDNA as template, by designing two pairs of primers, is overlapped mutually twice embedding
The PCR amplification overall length circRNF13 sequence of set (sequence obtained by design of primers and PCR amplification is as shown in Figure 2).CircRNF13 is complete
Long sequence amplification primer is as follows:
It expands for the first time, upstream primer: 5-GCTAGAAGAAACAGACTTCGTAAA-3;As shown in SEQ NO:6;
Downstream primer: 5-CTAATGAGGTCATCAGAATCAACA-3, such as SEQ NO:, shown in 7;
Second of amplification, upstream primer: 5-AATGTTGATTCTGATGACCT-3, as shown in SEQ NO:8,
Downstream primer: 5-AGATTGTGTAGACTTGTGTGG-3, as shown in SEQ NO:9.
2) PCR amplification, circRNF13 full length sequence, PCR reaction condition are as follows:
PCR reaction step
5 return to step 2, carry out 39 secondary response circulations altogether;
3) sequencing analysis is carried out after PCR product glue being recycled target fragment.
2. result
2.1 circRNF13 express significant raising in the Tca8113 cells after cisplatin treated
After cis-platinum (P) processing, we are had detected in cell Tca8113 cells by real-time fluorescence quantitative PCR
The expression of circRNF13, discovery circRNF13 expression compared with negative control (NC) are significantly raised (Fig. 1, left);We
In order to which that confirm real-time fluorescence quantitative PCR detection is strictly circRNF13, we carry out real-time fluorescence quantitative PCR product
Sequencing analysis, discovery amplified fragments are strictly that the 8 exons 3 ' end of RNF13 and 2 exons 5 ' end are spliced, strictly
One new circRNA molecule.
2.2 we expand sequencing and confirm the full length sequence of circRNF13
Because circRNF13 is a new circular RNA molecule, specific sequence is especially expressed in Dendritic cell
Transcript sequence need further confirmation.We design two pairs of primers and by PCR successful clones nested twice
The full length sequence of circRNF13, it was confirmed that circRNF13 is strictly the RNF13 gene by being located on the region chromosome 3q25.1
The 2-8 exon cyclisation of (ring finger protein 13, NM_183381.2) is spliced to form (Fig. 2), and overall length is
716bp (as shown in SEQ NO:1).
Embodiment 2, circRNF13 inhibit Tca8113 cells Cycle Arrest, induce cell apoptosis
1. materials and methods
1.1 reagents and kit
Restriction enzyme Cla I and Sac II and T4DNA ligase etc. is purchased from TakaRa company;TRIZOLTM
Reagent(Invitrogen);Plasmid extraction kit, plastic recovery kit (OMEGA);Reverse Transcriptase kit (Promega);
Proteinase K, DNase I, RNAsin, RNase A (GBICOL company);Methyl thiazoly tetrazolium assay (MTT, Sigma);Antibiotic
G418(Ameresc);Cell cycle detection kit (Invitrogen), cell apoptosis detection kit (Invitrogene).
The Fiber differentiation of 1.2 Dendritic cell mdr cells
In cell culture medium, the cis-platinum of low dosage is added, and cis-platin concentrations are gradually increased, is trained by long-time induction
It supports, is finally obtained the drug-resistant cell strain to cis-platinum tolerance.
The building of 1.3circRNF13 carrier for expression of eukaryon
We are inserted into upstream and downstream at multiple cloning sites in pcDNA3.1 carrier (deriving from Invitrogen company) first
Loop-forming sequences, i.e. GTGCTGGGATTACAGGTGTGAGCTACCACCCCCGGCCCACTTTTT are as shown in SEQ NO:10, GAAA
AGAATTAGGCTCGGCACGGTAGCTCACACCTGTAATCCCAGCA is as shown in SEQ NO:11) it is used to help Insert Fragment turn
Record is processed as circular rna;Here it is circular rna eukaryotic expression blank expression vectors;Specific building process is as follows:
According to the fundamentum of the Forming Mechanism of circRNA and eucaryote RNA montage, design suitable for circRNA
The loop-forming sequences of expression;According to the sequence information of commercial carrier pcDNA3.1, suitable multiple cloning sites are designed, to obtain
The complete DNA sequence dna realizing circRNA and being overexpressed:
GTGCTGGGATTACAGGTGTGAGCTACCACCCCCGGCCCACTTTTTCTTAAGCTTGGTACCGAGCTCGG ATCCACATCGATTGGTGGAATTCTGCAGATATCCACCGCGGTGGCGGCCGCTCGAGTCTAGAGAAAAGAATTAGGC
TCGGCACGGTAGCTCACACCTGTAATCCCAGCA, as shown in SEQ NO:12;
Intermediate underscore part is multiple cloning sites, and both ends are respectively upstream and downstream loop-forming sequences;
Above-mentioned synthesis is realized that NheI restriction enzyme site sequence, the other end are added in DNA sequence dna one end that circRNA is overexpressed
It adds ApaI restriction enzyme site sequence and obtains pcCirc blank by being built into pcDNA3.1 carrier after NheI and ApaI digestion
Plasmid (sequence is as shown in SEQ NO:13), sequencing confirmation plasmid are correct.
In order to construct circRNF13 over-express vector.We select Cla I and Sac II restriction enzyme site for digestion
PcCirc empty plasmid (the i.e. above-mentioned pcDNA3.1 carrier for inserting upstream and downstream loop-forming sequences built), and will
Between the upstream and downstream loop-forming sequences of circRNF13 sequence insertion vector (Fig. 3, left).
Constructing circRNF13 over-express vector (i.e. carrier for expression of eukaryon), steps are as follows:
1) using Tca8113 cells Tca8113cDNA as template, TaKaRa LA is utilizedEnzyme carries out PCR amplification overall length
CircRNF13 sequence.CircRNF13 full length sequence amplimer is as follows:
Upstream primer: 5 '-GTGATTTTACAACGAGAT-3 ' are as shown in SEQ NO:14;
Downstream primer: 5 '-CTTTCTTGAATTTATGTA-3 ' are as shown in SEQ NO:15;
In upstream and downstream, 5 ' ends of primer are respectively plus restriction enzyme Cla I and Sac II recognition site and protection alkali
After base, primer sequence is as follows:
Upstream: 5 '-AGGAATCGAT GTGATTTTACAACGAGAT-3 ' (underscore part is Cla I recognition site) is such as
Shown in SEQ NO:16;
Downstream: 5 '-ATGCCCGCGG CTTTCTTGAATTTATGTA-3 ' (underscore part is Sac II recognition site)
As shown in SEQ NO:17;
2) PCR amplification, circRNF13 full length sequence, PCR reaction condition are as follows:
PCR reaction step
5 return to step 2, carry out 39 secondary response circulations altogether;
3) by, through Cla I and Sac II double digestion rear electrophoresis, glue returns again after PCR product electrophoresis, glue recycling target fragment
It receives;
4) pcCirc empty plasmid recycles target fragment through Cla I and Sac II double digestion rear electrophoresis glue;
5) it can be obtained 3) with 4) step glue recovery product with the connection of T4 DNA ligase and can be used for eukaryotic expression
The vector plasmid of circRNF13;
6) by the eukaryon expression plasmid transformed competence colibacillus large intestine bar comprising circRNF13 full length sequence that 5) step obtains
Bacterium, to expand plasmid.
1.4 circRNF13 tiny RNA interference sequences (siRNA)
The splice site that we are directed to circRNF13 devises specific siRNA, and (siRNA, Fig. 4, a left side are shown
The position of siRNA) sequence is as follows:
5-AGAAAGGUGAUUUUACAACGA-3 as shown in SEQ NO:18,
Control of Scramble sequence of the simultaneous selection in human genomic sequence there is no target spot as siRNA:
Scramble sequence is as follows:
5'-GACACGCGACUUGUACCAC-3'.As shown in SEQ NO:19,
The sequence is synthesized by Invitrogen company.
1.5 prepare the coated nano silicon particles of poly-D-lysine
The coated nano silicon particles of poly-D-lysine are carried out with OP-10/ hexamethylene/ammonia microemulsion self-assembling technique
The synthesis of nano silicon particles (silica nanoparticle, SiNP), and can and pass through ion using the surface of nano silicon particles
Electrostatic interaction prepares the nano silicon particles of polylysine modification;The nano particle can be prepared by following methods:
1) OP-10 (nonylphenol polyoxyethylene ether), hexamethylene and ammonium hydroxide are mixed, positive silicic acid is added after being stirred at room temperature uniformly
Different ester (TEOS), continue stirring to polymerization complete, be added equal-volume acetone, ultrasonic disperse, centrifugation, distilled water wash three times, from
Heart collection is deposited in 80 DEG C of dryings, finely ground to obtain nano silicon particles (SiNP, particle size range 10-50nm).Wherein H2O and OP-10 and
H2The molar ratio of O and TEOS is 2~10, ammonia concn is 1.6~28%, molar concentration of the TEOS in hexamethylene be 0.1~
3mol/L。
2) SiNP is resuspended in 0.6M NaCO by 0.1~10mg/ml3In solution, supernatant is abandoned in ultrasonic disperse, centrifugation, then
Sediment is resuspended in PBS (pH 7.4) by 0.1~10mg/ml, ultrasonic disperse, add polylysine (final concentration of 4~
15nmol/mL), it mixes well, room temperature is mixed to shake;Centrifugation, abandons supernatant, and precipitating is resuspended in distilled water by 0.1~10mg/ml, is obtained
To the nano silicon particles of polylysine modification.
3) by modified nano silicon particles ultrasonic disperse, 10~50ug circRNF13 mistake is added in every milliliter of nano particle suspension
Expression vector or 10-100pmol siRNA, mixing, are stored at room temperature and make it combine.
1.6 cell culture and transfection
The good Tca8113 cells Tca8113 and Cal27 of growth conditions or mdr cell are pressed 2 × 105A cells/well
It is inoculated in 6 orifice plates, 6 orifice plates is placed in 37 DEG C, 5%CO2In incubator, cell to be cultivated grows to 50-70% density
Start circRNF13 over-express vector or the transfection of siRNA;Transfection process is as follows:
The poly that carrying circRNF13 eukaryon expression plasmid or siRNA that 100 μ l are prepared are added in sterile EP tube relies
The nano silicon particles suspension of propylhomoserin modification, mildly mixes with 100 μ l serum free mediums;It is washed cell 3 times with D-Hank's liquid;
800 μ l serum free mediums (antibiotic-free) will be added in said mixture, 1 hole in 6 orifice plates is added after mild mixing;It will
6 orifice plates are placed in CO2In incubator, 37 DEG C are cultivated 6 hours, and supernatant is then abandoned, and complete medium is added and continues overnight incubation.With sky
The nano silicon particles of carrier or the polylysine modification of Scramble sequence are as experiment contrast.1.7 real time fluorescent quantitative
PCR detects the expression of intracellular circRNF13
After cell processing, in suitable time point collecting cell, extracted total RNA, 1 μ g RNA after reverse transcription is at cDNA,
Carry out real-time fluorescence quantitative PCR.CircRNF13 forward primer is 5-GTCCAGGATAGACATAGAGC-3, such as SEQ NO:2 institute
Show and reverse primer 5-GTGTAGACTTGTGTGGCTGA-3.As shown in SEQ NO:3.
GAPDH forward primer for control is 5 '-ACCACAGTCCATGCCATCAC-3 ' as shown in SEQ NO:4, and
Reverse primer 5 '-TCCACCACCCTGTTGCTGTA-3 ', as shown in SEQ NO:5.
Real-time fluorescence quantitative PCR reaction system
Real-time fluorescence quantitative PCR reaction step
5 Plate read
6 82℃ 30sec
7 Plate read
8 Go to step 2for more 39times
9 55.0 DEG C of from of Perform melting curve 95.0 DEG C of to, 0.2 DEG C of read every, hold
for 1sec.
The amplification curve and melting curve of real-time fluorescence quantitative PCR, the expression intensity root of each gene are confirmed after reaction
After CT value (threshold cycle values), reference gene (GAPDH) markization, using group t-test checking computation
P value.
1.8 MTT cell proliferation experiments
1) digest cell obtained in the previous step, cell counted with cell counter, will transfection circRNF13 and
The cell inoculation of pcDNA3.1 empty carrier is in 96 orifice plates, and every hole is inoculated with 1000 cells, as a result every kind of 5 hole of cell inoculation takes
Its mean value.
2) 37 DEG C, 5%CO2Incubator culture 6 hours, after cell is adherent, every hole added 20 μ l of MTT liquid (5mg/ml).After
It is continuous to be incubated for 4 hours, culture is terminated, culture solution is discarded.Every hole adds 150 μ l DMSO, vibrates 10 minutes, dissolves crystal.
3) 490nm wavelength is selected, zeroing hole is concurrently set, on enzyme-linked immunosorbent assay instrument, measures each hole absorbance value simultaneously
Record result.
4) it is same as above every 24 hours repetition steps, detects 6 days altogether.Using absorbance value as ordinate, interval time is horizontal seat
Plotting MTT curve.
5) it tests in triplicate.Using each time point as abscissa, absorbance value is ordinate, draws cell growth curve.
1.9 flow cytometry analysis cell cycles
1) trypsin digestion cell when culture cell reaches 85% fusion, 1200rpm/min are centrifuged 5min, and it is heavy to collect cell
It forms sediment.
2) cell is resuspended in 1xPBS, and 1200rpm/min is centrifuged 5min, collects cell.Repeat this step 2 time.
3) cell is resuspended in 1xPBS, and the fixed cell pellet overnight of 70% ethyl alcohol of pre-cooling is added.
4) 1000rcf/min is centrifuged 5min, collects cell precipitation.
5) PH7.4PBS is washed cell 1 time, PBS is added after centrifugation, cell is resuspended, and adjust cell concentration to Ix 106/
ml。
6) propidium iodide (PI) dyeing liquor (PI containing 50mg/L, 1g/L Triton X-100,100g/L RNase) is added
It mixes, 4 DEG C are protected from light incubation 30min.
7) flow cytometer FACStar (U.S. company BD) is detected.Received signal is handled through Cellquest software, right
The fluorescence intensity of detection cell is analyzed.Experiment is repeated 3 times.
1.10 flow cytometry analysis natural death of cerebral cells rates
1) when culture cell reaches 85% fusion, group of cells is digested with pancreatin and is collected in centrifuge tube, is collected simultaneously
Each group supernatant suspension cell.Pay attention to gently blowing and beating cell, pancreatin is avoided excessively to digest.
2) merge group of cells respectively and be transferred in centrifuge tube, 1000rpm is centrifuged 5min, abandons supernatant and collects cell, PBS
After being gently resuspended, cell count.
3) 5~100,000 resuspension cells are taken, 1000rpm is centrifuged 5min, abandons supernatant, and 195ul Annexin V-FITC knot is added
It closes liquid and cell is gently resuspended.
4) 5ul Annexin V is added, mixes gently.It is protected from light incubation at room temperature 10min.
5) 1000rpm is centrifuged 5min, abandons supernatant, 190ul Annexin V-FITC combination liquid is added, cell is gently resuspended.
6) 10ul PI dyeing liquor is added, mixes gently, ice bath is protected from light.
7) flow cytomery is carried out immediately, and Annexin V-FITC is green fluorescence, and PI is red fluorescence.
2. result
The growth of 2.1 circRNF13 inhibition Tca8113 cells
After transfecting circRNF13 carrier for expression of eukaryon, we are had detected in cell by real-time fluorescence quantitative PCR
The expression of circRNF13, discovery circRNF13 expression are significantly raised (Fig. 3);And transfect the small dry of targeting circRNF13
After disturbing RNA (siRNA), the expression of intracellular circRNF13 obviously lowers (Fig. 4);
Compared with the cell of transfection empty carrier, it is overexpressed the life of the Tca8113 cells Tca8113 and Cal27 of circRNF13
Long speed significantly slows down, and after striking the expression of low circRNF13 using siRNA interference sequence, vitro growth rates accelerate (figure
5)。
2.2 circRNF13 pass through arresting cell cycle, induce cell apoptosis the growth for inhibiting Tca8113 cells.
Flow cytometry analysis shows after transfecting circRNF13 in Tca8113 cells that G2/M phase cell proportion significantly increases
Add, and S phase cell proportion is reduced, and shows that circRNF13 can block Tca8113 cells in the G2/M phase, make its cell division slow
Speed (Fig. 6).Meanwhile apoptotic cell ratio obviously increases (Fig. 7) after transfection circRNF13 in Tca8113 cells, this is also
CircRNF13 inhibits one of the reason of Tca8113 cells growth.And it strikes low circRNF13 and has then obtained opposite result.
2.3 circRNF13 express downward in Dendritic cell mdr cell
We by some months are cultivated, successfully induction obtains by being stepped up cis-platin concentrations in the medium
Tca8113 and Cal27 has detected the expression of circRNF13 in cell to the cell of cisplatin resistance, real-time quantitative PCR, sends out
Existing expression of the circRNF13 in drug-resistant cell strain is significant compared with initial cell strain to have lowered (Fig. 8).
2.4 overexpression circRNF13 can reverse Tca8113 cells drug-resistant phenotype
The expression (si-circRNF13) of low circRNF13 is struck in original Tca8113 cells (NC) or in mdr cell
(CR) after being overexpressed circRNF13 (OE-circRNF13) in, with the cisplatin treated cell of same concentrations, then flow cytometer
Apoptosis situation is detected, the tolerance (apoptosis reduction) that the expression of low circRNF13 can increase cell to cis-platinum is struck in discovery, and
Being overexpressed circRNF13 then can be significantly reduced the drug resistance (Fig. 9) of mdr cell.
Embodiment 3, quantitative real-time PCR detection confirm that circRNF13 expresses downward in Dendritic cell
1. materials and methods:
Collect by 28 Dendritic cells and 28 Dendritic cell tissues, extracted total RNA, 1 μ g RNA after reverse transcription is at cDNA, into
Row real-time fluorescence quantitative PCR.CircRNF13 forward primer is 5-GTCCAGGATAGACATAGAGC-3, as shown in SEQ NO:2,
With reverse primer 5-GTGTAGACTTGTGTGGCTGA-3.As shown in SEQ NO:3.
GAPDH forward primer for control is 5 '-ACCACAGTCCATGCCATCAC-3 ' as shown in SEQ NO:4, and
Reverse primer 5 '-TCCACCACCCTGTTGCTGTA-3 ', as shown in SEQ NO:5.
Real-time fluorescence quantitative PCR reaction system
Real-time fluorescence quantitative PCR reaction step
5 Plate read
6 82℃ 30sec
7 Plate read
8 Go to step 2for more 39times
9 55.0 DEG C of from of Perform melting curve 95.0 DEG C of to, 0.2 DEG C of read every, hold
for 1sec
The amplification curve and melting curve of real-time fluorescence quantitative PCR, the expression intensity root of each gene are confirmed after reaction
After CT value (threshold cycle values), reference gene (GAPDH) markization, using group t-test checking computation
P value.
2. result
CircRNF13 expresses by the cancer higher in control tissue, and expression significantly reduces P < 0.01 in Dendritic cell tissue
(Figure 10).
Embodiment 4, low expression of the in situ hybridization detection discovery circRNF13 in Dendritic cell are related to patient's poor prognosis
1. MATERIALS METHODS
1.1 design and synthesize hybridization probe
In order to which using the expression of in-situ hybridization method detection circRNF13, we are for circRNF13 cyclisation splicing
Site (namely 2 exon of RNF13 gene and 8 exon stitching portions) devises oligonucleotide probe 1, and is used as
The in situ hybridization oligonucleotide probe of positive control 3.
Oligonucleotide probe in situ hybridization detection circRNF13 expression:
TCGTTGTAAAATCACCTTTCTTGAATTTAT as: shown in SEQ NO:20,
Positive control probe (detection house-keeping gene GAPDH):
GAPDH probe 1:5 '-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3 ', as shown in SEQ NO:21,
GAPDH probe 2:5 '-CAGTAGAGGCAGGGATGATGTTCTGGAGAG-3 ', as shown in SEQ NO:22,
GAPDH probe 3:5 '-GTCAGAGGAGACCACCTGGTGCTCAGTGTA-3 ', as shown in SEQ NO:23.
Each gene specific oligonucleotides probe sequence of above-mentioned design is synthesized using chemical synthesis process.
1.2 oligonucleotide probe labelling kits and in situ hybridization detection reagent
Digoxin oligonucleotides tailing reagent (Dig Oligonucleitide Tailing Kit 2ndGeneration,
Roche company), anti-digoxin-horseradish peroxidase complex detection kit (Anti-Digoxigenin-POD, Fab
Fragments, Roche company), enhance the TSA signal amplifying system (TSA of detection of expression signal in situTMBiotin System,
NEL700 kit, PerkinElmer company), DAB staining kit (Beijing Zhong Shan company), 20x sodium citrate buffer
(saline sodium citrate, SSC), dextran sulfate (Dextran sulphate), deionized formamide
(Deionized Formamide), polyadenylic acid (polyadenylic acid, Poly A), poly deoxyadenylic acid
(polydeoxyadenylic acid, Poly dA) is denaturalized frog essence DNA (the denatured and sheared of shearing
Salmon sperm DNA, ssDNA), yeast transfer RNA (yeast t-RNA, tRNA), dithiothreitol (DTT) (DTT), 50x Deng Han
Family name's buffer (Denhardts ' s solution), phosphate buffer (PBS buffer), pepsin K, bovine serum albumin(BSA)
(BSA), triethanolamine (TEA), TNB Buffer (0.1M Tris-HCl, pH7.5,0.15M NaCL, 0.5%Blocking
Reagent), TNT Buffer (0.1M Tris-HCl, pH7.5,0.15M NaCL, 0.05%Tween 20), acetic anhydride, resistance
Disconnected reagent (Blocking reagent agent, Roche company).
1.3 other main agents and material
Dehydrated alcohol, 90% alcohol, 70% alcohol, 50% alcohol, turpentine oil, distilled water, PBS buffer solution (pH7.2~
7.4, NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO44.3mmol/L, KH2PO41.4mmol/L);3% methanol-
Hydrogen peroxide solution (80% methanol and the configuration of 30% hydrogen peroxide);0.01mol/L citrate buffer (citrate buffer,
CB, pH6.0 ± 0.1,9ml 0.1M citric acid solution and 41ml 0.1M sodium citrate solution are added interim in 450ml distilled water
With postponing correction work liquid pH value again);0.1% trypsase;Haematoxylin;1% hydrochloride alcohol (1ml concentrated hydrochloric acid+99ml70% wine
Essence configuration);Mounting glue (PTS Cure Mount II);Dedicated coverslip (480 × 240mm2) customize in Zhengzhou Glassware Factory.
Leica low melting point (58 DEG C) paraffin, domestic beeswax, absolute alcohol, dimethylbenzene, 10% neutrality paraformaldehyde (0.01mol/L,
PH7.4, DEPC distilled water and PBS buffer solution are prepared), haematoxylin, Yihong, neutral mounting natural gum, coverslip, glass slide.
1.4 label probe
Oligonucleotide probe label is carried out using 3-tailing DIG Olignucleutide Kit, reaction system is such as
Under.
100pmol oligonucleotide+ddH2O=9 μ l (5 μ of control:control oligonucleutide
l+ddH2O4μl)
It mixes, is slightly centrifuged.37 DEG C of water-bath 30min add 2 μ l EDTA (0.2M, PH 8.0) stopped reactions.
1.5 being purified after oligonucleotide probe label
In order to increase the purity of label probe, marked probe need to be purified, concrete operations are as follows:
1) cold ethyl alcohol (- 20 DEG C) of+2.5 μ l 4M LiCL+75 μ l of probe reaction mixture (22 μ l) 100%
2) -70 DEG C of precipitatings 60min, or-20 DEG C of 2h.
3) 13.000 × g, 4 DEG C of centrifugation 15min.
4) supernatant is abandoned, with 70% (V/V) ethanol washing that 50 μ l are ice-cold.
5) 4 DEG C of 13.000xg are centrifuged 5min.
6) supernatant, 4 DEG C of dryings of vacuum are abandoned.
7) with the molten probe of aseptic double-distilled water weight.
1.6 in situ hybridizations detection achieves the expression of circRNF13 in paraffin section
Paraffin section hybridizes pre-treatment
1) paraffin section of 4 DEG C of preservations is placed in 58 DEG C of roasting piece 30min, melted surface paraffin.
2) dimethylbenzene successively dewaxes 3 × 5min.
3) step ethanol wash, 100% 1 × 5min of the alcohol of 2 × 2min of alcohol → 95% alcohol of 1 × 5min → 70% →
50% 1 × 5min of alcohol → 2 × 3min of DEPC water washing → DEPC-PBS washs 2 × 5min.
4) 300 μ l pepsin K (10 μ g/ml) are added dropwise on slice, 37 DEG C of digestion 20min.
5) it is sliced and washs 1min, stopped reaction into PBS (0.1M PBS+2mg/ml glutamic acid).
6) it is sliced into 0.2N HCL, in 37 DEG C of reaction 20-30min, increases the permeability of tissue.
7) slice fixes 10min, room temperature with 4% paraformaldehyde (0.1M PBS dissolution) afterwards.
8) in order to increase tissue positive intensity for hybridization, acetyl processing is carried out to slice.It is sliced into 0.25% acetic anhydride
Buffer I (0.1M triethanolamine), room temperature 10min.
9) 1M PBS washs 2 × 5min.
Prehybridization and hybridization
Prehybridization: the prehybridization solution of -20 DEG C of preservations is first placed in 37 DEG C of incubation 60min, and the dosage of prehybridization solution is 50 μ l,
Parafilm is carried out lid and is sliced, prehybridization 2 hours in 37 DEG C of wet box.(prehybridization solution composition includes: 2XSSC, 10%Dextran
Sulphate, 1X Denhardt ' s solution, 50mM Phosphate Buffer (PH 7.0), 50mM DTT, 250 μ l,
100 μ g/ml poly A, 5 μ g/ml poly dA, 250 μ g/ml yeast t-RNA, 500 μ g/ml ssDNA, 47%
Deionized formamide)。
1) parafilm is removed, prehybridization solution is got rid of, slice is placed in 5min in 2 × SSC.
2) hybridization reaction: 37 DEG C of hybridized overnights (18-20h).Each slice is added 250 μ l hybridization solutions and is carried out with parafilm
Lid.Corresponding probe is added in prehybridization solution just becomes hybridization solution.Hybridization solution is prepared in prehybridization, is placed 37 DEG C of incubations, is made
Probe is completely dissolved in hybridization solution, this experiment is mixed with a plurality of oligonucleotide probe, is matched by each probe 500ng/ml concentration
Probe hybridization solution is made.Digoxin tailing labelling kit label probe concentration calculation foundation: the concentration of each probe by its with
Colour developing is compared when detection reaction when positive quantitative probe and the naked probe of 30 bases of 100pmol marks reaction theory to visit
Needle yield is that two kinds of standards of 900ng carry out the concentration that COMPREHENSIVE CALCULATING goes out label probe.
3) post-hybridization washing, slice immerse 2 × SSC, 10min, throw off parafilm.Successively washed in shake on shaking table, 2 ×
SSC (0.5%SDS), 2 × 15min → 0.25 × SSC (0.5%SDS), 2 × 15min.
Color developing detection is reacted after hybridization
1) digoxigenin-probe compound in conjunction with mRNA is detected using Anti-Digoxigenin-POD;TSA amplification system
Enhance the positive signal of in situ hybridization reaction solution reaction, DAB colour developing.
2) slice is gone in TNT buffer, 3 × 5min.
3) TNB is added dropwise and blocks buffer, 300 μ l/TMAs, room temperature, 30min.
4) extra blocking agent, the diluted Anti-Digoxigenin-POD of 1:100 (TBS+0.1%Triton X- are sucked
100+1% blocking agent), room temperature 4 hours.
5) TNT Buffer (0.1M Tris-CL, pH7.5,0.15M NaCL, 0.05%Tween 20) is washed,
3x5min。
6) signal is added dropwise on slice and amplifies reagent Biotinyl Tyamid, 300 μ l/TMAs, (Biotinyl Tyramid
Store liquid: Biotinyl Tyramid is dissolved in 0.2ml DMSO, Biotinyl Tyramid working solution: 1 × dilution, 1:50
Dilute Biotinyl Tyramid and store liquid), room temperature 10 minutes.
7) TNT is washed, 3 × 5min.
8) SA-HRP (strepto- avidin-horseradish peroxidase) is added dropwise in slice, 300 μ l/TMAs, room temperature 30min.
9) TNT is washed, 3 × 5min.
10) distilled water washing, 1 × 1min.
11) DAB develops the color, and controls chromogenic reaction under microscope.
12) haematoxylin is redyed,
13) alcohol step is dehydrated, chip drying.
14) mounting glue, the coverslip cover plate of dimension, crosslinking slice 1min under ultraviolet lamp is added dropwise.
The judgement of 1.7 results and standard
Application Optics microscope is observed under low power and high power lens respectively, looks first at the positive expression of target RNA
The signal positioning intracellular in object observing: it is located at nucleus, cytoplasm or cell membrane.
It is carried out respectively with two kinds of standards of the intensity of the detection rna expression position positive signal and the cell number of positive expression again
Comprehensive score, judgment criteria are as follows: (1) judge according to positive cell dyeing intensity: a. cell dye-free remembers 0 point;B. cell is dyed
Light brown is weakly positive, remembers 1 point;C. cell dyes brown and dyes dark-brown without background coloration or cell and have light brown back
Scape is moderate positive, remembers 2 points;D. cell dyes dark-brown and is strong positive without background coloration, remembers 3 points.(2) according to positive cell
Express number score: the no positive cell expression of a. remembers 0 point;B. positive expression cell number≤25% remembers 1 point;C.25% < is positive thin
Born of the same parents' number < 50% remembers 2 points;D. positive expression cell number >=50% remembers 3 points.
In order to reduce the subjective factor of appraisal result as far as possible, it is respective that You Liangwei pathology expert presses one of above-mentioned standard respectively
Judged and scored, then the two is scored and is multiplied, as a result are as follows: 1. 0 point of person is finally calculated as 0 point, it is believed that feminine gender expression;2. 1 point
1 point is finally calculated as with 2 points of persons, it is believed that weakly positive expression;3. 3 points and 4 points of persons are finally calculated as 2 points, it is believed that moderate positive expression;④
6, which assign to 9 points of persons, is finally calculated as 3 points, it is believed that strong positive expression.
1.8 analyses and statistical software
Statistical analysis is carried out to experimental result using SPSS13.0 statistical software, compares use χ two-by-two2Test or Fisher
Exact test, correlation analysis use Spearmen correlation method;P < 0.05 is that difference is statistically significant.
Survivorship curve analysis uses Kaplan-Meier method and log-rank test;Multi-variables analysis uses Cox ' s
proportional hazards model;P < 0.05 is that difference is statistically significant.
2 results
Expression of 2.1 circRNF13 in Dendritic cell is significantly increased than the expression in normal control tissue
CircRNF13 is not expressed in Dendritic cell tissue or expression is lower, and the normal control by Dendritic cell cancer
There is expression (Figure 11) in tissue, there is apparent statistical difference between the two.
Dendritic cell patient's prognosis of 2.2 circRNF13 low expressions is worse
Effect of follow-up visit by telephone has been carried out to 88 Dendritic cell patients, inquired in detail they start time, treatment condition, whether there is or not
Recurrence, whether there is or not suffering from other diseases, recurrence and death time etc. again, and register life span and state, and to Dendritic cell tissue
The survival analysis that the expression of middle circRNF13 and the life span of patient and state carry out finds not express in Dendritic cell tissue
The patient of circRNF13 is significantly shorter than circRNF13 the mean survival time and expresses higher patient (Figure 12).Explanation
CircRNF13 is a molecular labeling relevant to Dendritic cell prognosis, and circRNA expression is low or does not express, patient's poor prognosis.
Sequence table
<110>Central South University
<120>promote application of the reagent of circular rna circRNF13 expression on preparation treatment Dendritic cell drug
<160> 23
<170> SIPOSequenceListing 1.0
<210> 1
<211> 716
<212> RNA
<213>homo sapiens (Homo sapiens)
<400> 1
gugauuuuac aacgagaugc ugcucuccau agggaugcuc augcugucag ccacacaagu 60
cuacaccauc uugacugucc agcucuuugc auucuuaaac cuacugccug uagaagcaga 120
cauuuuagca uauaacuuug aaaaugcauc ucagacauuu gaugaccucc cugcaagauu 180
ugguuauaga cuuccagcug aagguuuaaa ggguuuuuug auuaacucaa aaccagagaa 240
ugccugugaa cccauagugc cuccaccagu aaaagacaau ucaucuggca cuuucaucgu 300
guuaauuaga agacuugauu guaauuuuga uauaaagguu uuaaaugcac agagagcagg 360
auacaaggca gccauaguuc acaauguuga uucugaugac cucauuagca ugggauccaa 420
cgacauugag guacuaaaga aaauugacau uccaucuguc uuuauuggug aaucaucagc 480
uaauucucug aaagaugaau ucacauauga aaaagggggc caccuuaucu uaguuccaga 540
auuuagucuu ccuuuggaau acuaccuaau ucccuuccuu aucauagugg gcaucugucu 600
caucuugaua gucauuuuca ugaucacaaa auuuguccag gauagacaua gagcuagaag 660
aaacagacuu cguaaagauc aacuuaagaa acuuccugua cauaaauuca agaaag 716
<210> 2
<211> 20
<212> DNA
<213>unknown (Unknown)
<400> 2
gtccaggata gacatagagc 20
<210> 3
<211> 20
<212> DNA
<213>unknown (Unknown)
<400> 3
gtgtagactt gtgtggctga 20
<210> 4
<211> 20
<212> DNA
<213>unknown (Unknown)
<400> 4
accacagtcc atgccatcac 20
<210> 5
<211> 20
<212> DNA
<213>unknown (Unknown)
<400> 5
tccaccaccc tgttgctgta 20
<210> 6
<211> 24
<212> DNA
<213>unknown (Unknown)
<400> 6
gctagaagaa acagacttcg taaa 24
<210> 8
<211> 24
<212> DNA
<213>unknown (Unknown)
<400> 8
ctaatgaggt catcagaatc aaca 24
<210> 8
<211> 20
<212> DNA
<213>unknown (Unknown)
<400> 8
aatgttgatt ctgatgacct 20
<210> 9
<211> 21
<212> DNA
<213>unknown (Unknown)
<400> 9
agattgtgta gacttgtgtg g 21
<210> 10
<211> 45
<212> DNA
<213>unknown (Unknown)
<400> 10
gtgctgggat tacaggtgtg agctaccacc cccggcccac ttttt 45
<210> 11
<211> 47
<212> DNA
<213>unknown (Unknown)
<400> 11
gaaaagaatt aggctcggca cggtagctca cacctgtaat cccagca 47
<210> 12
<211> 177
<212> DNA
<213>unknown (Unknown)
<400> 12
gtgctgggat tacaggtgtg agctaccacc cccggcccac tttttcttaa gcttggtacc 60
gagctcggat ccacatcgat tggtggaatt ctgcagatat ccaccgcggt ggcggccgct 120
cgagtctaga gaaaagaatt aggctcggca cggtagctca cacctgtaat cccagca 177
<210> 13
<211> 5515
<212> DNA
<213>unknown (Unknown)
<400> 13
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtgctgggat tacaggtgtg agctaccacc cccggcccac ttttttttaa acttaagctt 960
ggtaccgagc tcggatccac atcgattggt ggaattctgc agatatccac cgcggtggcg 1020
gccgctcgag tctagagaaa agaattaggc tcggcacggt agctcacacc tgtaatccca 1080
gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 1140
gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 1200
tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 1260
ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg 1320
gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat 1380
ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 1440
accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 1500
gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 1560
tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt 1620
gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 1680
agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat 1740
ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa 1800
tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct 1860
ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa 1920
agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 1980
ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt 2040
ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct 2100
ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc 2160
tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc 2220
gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat 2280
tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt 2340
cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac 2400
tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg 2460
tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc 2520
aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa 2580
tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc 2640
gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg 2700
aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg 2760
acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa 2820
atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg 2880
acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct 2940
tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc 3000
ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa 3060
cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat 3120
cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt 3180
cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 3240
aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 3300
caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg 3360
gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 3420
cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 3480
gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 3540
cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 3600
tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 3660
aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 3720
gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 3780
ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 3840
ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 3900
gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 3960
ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 4020
cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 4080
cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 4140
gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 4200
aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 4260
tagctcttga tccggcaaac aaaccaccgc tggtagcggt ttttttgttt gcaagcagca 4320
gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 4380
cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 4440
cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 4500
gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 4560
tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 4620
gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 4680
agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 4740
tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 4800
agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 4860
gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 4920
catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 4980
ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 5040
atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 5100
tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 5160
cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 5220
cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 5280
atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 5340
aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 5400
ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 5460
aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc 5515
<210> 14
<211> 18
<212> DNA
<213>unknown (Unknown)
<400> 14
gtgattttac aacgagat 18
<210> 15
<211> 18
<212> DNA
<213>unknown (Unknown)
<400> 15
ctttcttgaa tttatgta 18
<210> 16
<211> 28
<212> DNA
<213>unknown (Unknown)
<400> 16
aggaatcgat gtgattttac aacgagat 28
<210> 17
<211> 28
<212> DNA
<213>unknown (Unknown)
<400> 17
atgcccgcgg ctttcttgaa tttatgta 28
<210> 18
<211> 21
<212> RNA
<213>unknown (Unknown)
<400> 18
agaaagguga uuuuacaacg a 21
<210> 19
<211> 19
<212> RNA
<213>unknown (Unknown)
<400> 19
gacacgcgac uuguaccac 19
<210> 20
<211> 30
<212> DNA
<213>unknown (Unknown)
<400> 20
tcgttgtaaa atcacctttc ttgaatttat 30
<210> 21
<211> 30
<212> DNA
<213>unknown (Unknown)
<400> 21
ccactttacc agagttaaaa gcagccctgg 30
<210> 22
<211> 30
<212> DNA
<213>unknown (Unknown)
<400> 22
cagtagaggc agggatgatg ttctggagag 30
<210> 23
<211> 30
<212> DNA
<213>unknown (Unknown)
<400> 23
gtcagaggag accacctggt gctcagtgta 30
Claims (3)
1. promoting application of the reagent of circular rna circRNF13 expression on preparation treatment Dendritic cell drug, the ring-type
The sequence of RNA circRNF13 is as shown in SEQ NO:1;The reagent of the described promotion circular rna circRNF13 expression is
CircRNF13 over-express vector.
2. application according to claim 1, which is characterized in that the building process of circRNF13 over-express vector is as follows:
(1) it according to the fundamentum of the Forming Mechanism of circRNA and eucaryote RNA montage, designs and is suitable for
The loop-forming sequences of circRNA expression;According to the sequence information of commercial carrier pcDNA3.1, multiple cloning sites are designed, thus
It is as follows to the complete DNA sequence for realizing that circRNA is overexpressed: GTGCTGGGATTACAGGTGTGAGCTACCACCCCCGG
CCCACTTTTTCTTAAGCTTGGTACCGAGCTCGGATCCACATCGATTGGTGGAATTCTGCAGATATCCACCGCGGTG GCGGCCGCTCGAGTCTAGAGAAAAGAATTAGGCTCGGCACGGTAGCTCACACCTGTAATCCCAGCA, intermediate underscore
Part is multiple cloning sites, and both ends are respectively upstream and downstream loop-forming sequences;
(2) above-mentioned synthesis is realized that NheI restriction enzyme site sequence is added in DNA sequence one end that circRNA is overexpressed, it is another
End addition ApaI restriction enzyme site sequence obtains pcCirc by being built into pcDNA3.1 carrier after NheI and ApaI digestion
Empty plasmid, sequencing confirmation plasmid is correct, and sequence is as shown in SEQ NO:13;
(3) selection Cla I and Sac II restriction enzyme site is used for digestion pcCirc empty plasmid, and circRNF13 sequence is inserted
Enter between the upstream and downstream loop-forming sequences of carrier.
3. application according to claim 2, which is characterized in that detailed process is as follows for step (3):
1) using Tca8113 cells Tca8113 cDNA as template, PCR amplification overall length is carried out using TaKaRa LA Taq enzyme
CircRNF13 sequence;CircRNF13 full length sequence amplimer is as follows:
Upstream primer: 5 '-GTGATTTTACAACGAGAT-3 ';
Downstream primer: 5 '-CTTTCTTGAATTTATGTA-3 ';
At 5 ' ends of upstream and downstream primer respectively plus restriction enzyme Cla I and Sac II recognition site and after protecting base,
Primer sequence is as follows:
Upstream primer: 5 '-AGGAATCGAT GTGATTTTACAACGAGAT -3 ', underscore part are that Cla I identifies position
Point;
Downstream primer: 5 '-ATGCCCGCGG CTTTCTTGAATTTATGTA -3 ', underscore part are that Sac II identifies position
Point;
2) PCR amplification, circRNF13 full length sequence, PCR reaction condition are as follows:
1 μ l of TaKaRa LA Taq enzyme
10 μm of 0.4 μ l of upstream primer
10 μm of 0.4 μ l of downstream primer
2.0 μ l of cDNA template
dH2O 12.2μl
4 μ l of 5x PCR reaction buffer
20 μ l of Total,
PCR reaction step
1 94℃ 5 min
2 95℃ 10 sec
3 58℃ 30 sec
4 72℃ 60 sec
5 return to step 2, carry out 39 secondary response circulations altogether;
3) by, through Cla I and Sac II double digestion rear electrophoresis, glue recycles again after PCR product electrophoresis, glue recycling target fragment;
4) pcCirc empty plasmid recycles target fragment through Cla I and Sac II double digestion rear electrophoresis glue;
5) vector plasmid of overexpression circRNF13 3) is arrived with 4) step glue recovery product with the connection of T4 DNA ligase;
6) by the eukaryon expression plasmid transformed competence colibacillus Escherichia coli comprising circRNF13 full length sequence that 5) step obtains, with
Expand plasmid.
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CN112574992A (en) * | 2020-12-17 | 2021-03-30 | 湖南丰晖生物科技有限公司 | Circular RNA over-expression cyclization vector DNA sequence and construction method and application thereof |
Citations (1)
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CN104419704A (en) * | 2013-09-05 | 2015-03-18 | 中国科学院上海生命科学研究院 | Intron-source circular RNA molecules and application of cyclization key nucleotide sequences of intron-source circular RNA molecules |
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CN104419704A (en) * | 2013-09-05 | 2015-03-18 | 中国科学院上海生命科学研究院 | Intron-source circular RNA molecules and application of cyclization key nucleotide sequences of intron-source circular RNA molecules |
Non-Patent Citations (2)
Title |
---|
RNF13: a novel RING-type ubiquitin ligase over-expressed in pancreatic cancer;Qiang Zhang,et al;《Cell Research》;20091231;348-357页 * |
RNF13: an emerging RING finger ubiquitin ligase important in cell proliferation;Xianglan Jin,et al;《the FEBS Journal》;20111231;78-84页 * |
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